Susana Rodrigues Vacas - run.unl.ptrun.unl.pt/bitstream/10362/13251/1/Vacas Susana TD 2014.pdf · BACKGROUND Impairment of cognition after surgery is a disturbing reality. Postoperative

Understanding postoperative cognitive dysfunction: Novel insights

Susana Rodrigues Vacas

Dissertation presented to obtain the PhD degree in the Faculdade de Ciências

Médicas, Universidade Nova de Lisboa.

The experimental work was conducted in the Department of Anesthesiology

and Perioperative Care, University of California San Francisco, USA under

the scientific supervision of Mervyn Maze, MB, ChB, FRCP, FRCA, FMedSci.

The internal co-supervisor at Faculdade de Ciências Médicas was Professor

Mª Emília Monteiro.

1

This work was financially supported by:

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Table of contents Table of contents 3

List of abbreviations 4

BACKGROUND 6 Surgery 8 Sleep 11 Cerebral Ischemia 13 Metabolic Syndrome 14

AIMS OF THE THESIS 15

METHODS AND RESULTS 16 Surgery 17

Depletion of Bone Marrow-derived Macrophages Perturbs the Innate Immune Response to Surgery and Reduces Postoperative Memory Dysfunction 18 HMGB1 Initiates Postoperative Cognitive Decline by Engaging Bone Marrow-derived Macrophages 29 The Neuroinflammatory Response of Postoperative Cognitive Decline 38

Sleep 57 Consequences of Sleep Fragmentation in the Perioperative Setting 58 Sleep and Anesthesia: Common Mechanisms of Action 70

Cerebral Ischemia 80 Bone Fracture Exacerbates Murine Ischemic Cerebral Injury 81

Metabolic Syndrome 93 Surgery Results in Exaggerated and Persistent Cognitive Decline in a Rat Model of the Metabolic Syndrome 94

DISCUSSION AND CONCLUSION 103

TABLES 108 Table 1 – Risk Factors for Postoperative Delirium 108 Table 2 – Risk Factors for Postoperative Cognitive Dysfunction 109

REFERENCES 111

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List of abbreviations AD Alzheimer’s disease

BBB blood brain barrier

BZD benzodiazepines

BM-DM bone marrow-derived macrophage

CCR2 C-C chemokine receptor type 2

CSF cerebral spinal fluid

DAMP damage-associated molecular pattern

ELISA enzyme-linked immunosorbent assay

HMGB1 high-molecular group box 1 protein

ICU Intensive Care Unit

IKKβ Ikappa B kinase

IL interleukin

LPS lipopolysaccharide

MCP-1 monocyte chemotactic protein-1

MetaS metabolic syndrome

nAchR nicotinic acetylcholine receptor

qPCR quantitative polymerase chain reaction

NF-κB nuclear factor kappa B

NREM non-rapid eye movement

PAMP pathogen-associated molecular pattern

POCD postoperative cognitive dysfunction

POD postoperative delirium

PRR pattern recognition receptors

REM rapid eye movement

SF sleep fragmentation

TLR toll-like receptor

TNF tumor necrosis factor

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The scientific content of the present thesis has been included in the publication of the following international scientific periodicals with referees:

• The neuroinflammatory response of postoperative cognitive decline,

Vacas S., Degos V., Feng X., Maze M., British Medical Bulletin,

2013;106:161-78.

http://www.ncbi.nlm.nih.gov/pubmed/23558082

• Sleep and Anesthesia: Common Mechanisms of Action, Vacas S.,

Kurien P., Maze M., Sleep Medicine Clinic, Volume 8, Issue 1, March 2013

http://dx.doi.org/10.1016/j.jsmc.2012.11.009

• Surgery Results in Exaggerated and Persistent Cognitive Decline in a

Rat Model of the Metabolic Syndrome, Feng X., Degos V., Koch L.G., Britton

S.L., Zhu Y., Vacas S., Terrando N., Nelson J., Su X., Maze M.,

Anesthesiology. 2013 May;118(5):1098-105.


• Depletion of Bone Marrow-derived Macrophages Perturbs the Innate

Immune Response to Surgery and Reduces Postoperative Memory

Dysfunction, Degos V., Vacas S., Han Z., van Rooijen N., Gressens P., Su

H., Young W.L., Maze M., Anesthesiology. 2013 Mar;118(3):527-536.


• Bone Fracture Exacerbates Murine Ischemic Cerebral Injury, Degos

V., Maze M., Vacas S., Hirsch J., Guo Y., Shen F., Jun K., van Rooijen N.,

Gressens P., Young W.L., Su H., Anesthesiology. 2013 Jun;118(6):1362-72.


• HMGB1 Initiates Postoperative Coginitive Decline by Engaging Bone

Marrow-derived Machrophages, Vacas S., Degos V., Tracey K.J., Maze M.,

Anesthesiology, October 2013


• Consequences of Sleep Fragmentation in the Perioperative Setting,

Vacas S., Degos, V., Maze, M., submitted

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BACKGROUND

Impairment of cognition after surgery is a disturbing reality. Postoperative

delirium (POD), listed in the Diagnostic and Statistical Manual of Mental

Disorders, DSM-52, is characterized by inattention, disorganized thinking and

altered level of consciousness with acute onset and fluctuating course. While

some patients develop POD, others develop a later onset form of

postoperative cognitive decline known as postoperative cognitive dysfunction

(POCD). POCD is diagnosed by the International Society of Postoperative

Cognitive Dysfunction as subtle deficits in one or more discrete domains of

cognition, which include attention, concentration, executive function, verbal

memory, visuospatial abstraction and psychomotor speed3. The diagnosis

requires sensitive presurgical and postsurgical neurocognitive testing4. This

condition typically develops over weeks to months and is long-lasting. As a

consequence, patients can lose their livelihood or independence, which can

seriously reduce their quality of life5-7.

It is estimated that POCD occurs in more than 10% of non-cardiac surgical

patients8 over 60 years old9 and is independently associated with poor short-

term and long-term outcomes including an increased risk of mortality6,10.

Some reports describe cognitive decline persisting for up to one year after

surgery. Both because the number of major surgical interventions (requiring

anaesthesia) exceeds 230 million world-wide11 and because of the increasing

prevalence of surgical interventions in patients with comorbidities, if current

rates hold steady, we can expect that many millions of patients will run the

risk of developing POCD. This possibility raises the stakes considerably: not

only on an individual level, but also on a societal scale.

Although several risk factors have been identified (table 1 and 2), the exact

pathophysiology that underlies POCD remains undefined. Data from

preclinical studies support the concept that inflammation is a possible

pathogenic mechanism for post-operative cognitive dysfunction12-16. Increased

expression of interleukins in mice hippocampus following surgery was

associated with cognitive decline12,17, corroborating the view that surgery-

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induced neuroinflammation can result in cognitive impairment. Surgical

patients exhibit elevations of proinflammatory cytokines in both the central

nervous system and the systemic circulation, the extent of which may relate to

the degree of cognitive decline18-20. Assuming that neuroinflammatory

changes noted postoperatively in rodents also occur in humans, reasons must

be sought why POCD is a relatively infrequent clinical event (± 10%)5

whereas neuroinflammation always occurs12,19. Among the possibilities

include the fact that the neuroinflammatory changes are usually evanescent

and do not normally cause a long-lasting consequence in the animal

models21. Several clinical conditions can transform the self-limiting post-

surgical neuroinflammatory response into one that is persistent22-24. Causes

for the persistence in neuroinflammation may be due to dysfunction in the

inflammation initiation or resolving mechanisms.

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Surgery Tissue trauma, such as in surgical insult, releases damage-associated

molecular patterns (DAMPs) that are recognized by pattern recognition

receptors (PRR), which then trigger an immune response in a manner

remarkably similar to that of microbial-derived pathogen-associated molecular

patterns (PAMPs)25-27. Among PRRs, Toll-like receptors (TLRs) are of critical

importance, recognizing various ligands (including PAMPs and DAMPs) and

activating TLR signals along different pathways, thereby increasing the

synthesis and release of pro-inflammatory mediators. Although the function of

TLR4 during lipopolysaccharide (LPS) endotoxemia28 has been deeply

explored, the pathways of infection-mediated neuroinflammation and cognitive

decline seem to be distinct from that of aseptic surgical trauma14. One of the

most important DAMPs (released from dead or dying cells through non-

apoptotic processes29) is high-mobility group box 1 protein (HMGB1). HMGB1

can bind and signal through a family of PRRs that are evolutionarily

conserved30. Clinical conditions such as sepsis, arthritis, and stroke, all

release massive amounts of HMGB131. Both DAMPs and PAMPs converge

on NF-κB to increase synthesis and release of pro-inflammatory cytokines32,

including TNF-α, which disrupt blood brain barrier (BBB) integrity12,14,15,33.

Early activation of the innate immunity through DAMPs (HMGB1 and

cytokines) will introduce the initial response to surgery resulting in

neuroinflammation and concomitant cognitive decline (figure 1)14.

Figure 1. Neuroinflammatory response to surgery1

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Following injury, a transient inflammation is necessary for tissue repair

processes that promote healing. Neuroinflammation after surgery is likely to

include a pro-inflammatory phase and an anti-inflammatory phase (neural and

humoral pathways mediate the switch between these two phases)34,35.

Regarding the resolution phase of the inflammatory state, this is mediated by

neural factors, termed the cholinergic anti-inflammatory pathway. Release of

acetylcholine mediates inhibition of macrophage NF-κB activity by signalling

through the α7 subtype of nicotinic acetylcholine receptors (α7 nAChR).

Ultimately it inhibits synthesis and release of pro-inflammatory cytokines from

circulating immunocompetent cells32,36,37. The neural cholinergic reflex is very

important in resolving the inflammatory pathogenesis of several diseases

including sepsis38, rheumatoid arthritis39, and colitis40. Furthermore, the

cholinergic anti-inflammatory pathway also modulates the function of T

regulatory cells41, which influences the production of anti-inflammatory

cytokines (IL-10 and IL-4)42 and alternative macrophage activation that

promotes the resolution of inflammation43. Abnormalities of the switching

mechanism may cause a non-resolving chronic inflammatory state that could

create the circumstances for persistent cognitive decline.

Studies have shown the importance of this reflex for resolving DAMP-induced

neuroinflammation, pro-inflammatory cytokine release, neuroinflammation,

and cognitive decline; stimulating the α7 nAChR in macrophages, inhibited

NF-κB activity while in vivo, α7 nAChR agonists prevented postoperative

monocyte migration into the hippocampus as well as memory impairment44.

Advanced age is associated with decline in cholinergic function45, which may

be relevant in explaining the high prevalence of postoperative cognitive

dysfunction in elderly patients44.

When inflammation does not subside, it can contribute to the pathogenesis of

diseases46. Through a permeable BBB, CCR2-expressing bone marrow-

derived macrophages (BM-DM) are attracted, by the newly-expressed

chemokine, MCP-1, into the brain parenchyma. The macrophages synthesize

and release a variety of pro-inflammatory cytokines that interfere with

processes required for memory. Macrophage-specific Ikappa B kinase (IKK)β

coordinates activation of NF-κB; when it is deleted it prevents BBB disruption

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and BM-DM infiltration into the hippocampus following surgery44. Transgenic

mice that overexpress Hsp72 and inhibit NF-κB activity have attenuation of

postoperative neuroinflammation and cognitive decline21,47.

Learning and memory processes rely on the hippocampus, a region of the

brain that contains a large number of proinflammatory cytokine receptors48,49.

The hippocampus has the highest density of IL-1 receptors, and although IL-

1β is required for normal learning and memory processes, higher levels can

also produce diminished cognitive function50,51. Recent studies have

suggested a role for cytokines such as IL-1 as well as IL-6 in the genesis of

POCD12,15,16. The relative prevalence of the TNF-α receptor, and other PRR,

on the endothelium of this brain region may account for its vulnerability to

systemic proinflammatory cytokines52. Surgical trauma in animal models is

associated with the persistent activation of macrophages in the CNS12, as well

as, high hippocampal levels of IL-1β, TNF-α and IL-612. These changes are

correlated with cognitive dysfunction seen in animal models (contextual fear

memory12,14,16, spatial learning27,53 or reversal learning26). Sub-clinical

inflammation following administration of LPS substantially increases IL1-β

levels and cognitive deterioration after surgery15. In addition, several studies

suggest that the marked and sustained expression of inflammation-related

enzymes, such as cyclooxygenase-2, plays an important role in secondary

events that amplify cerebral injury after ischemia54. Patients also exhibit a

robust neuroinflammatory response to peripheral surgery with an initial rise in

pro-inflammatory cytokines in the CSF19,20,55.

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Sleep Sleep is crucial for the repair of many types of injury and disease, especially

with regard to the central nervous and immune systems; it also has anabolic,

restorative properties that improve both neurocognitive and immune function.

During non rapid eye movement (NREM) sleep slow wave activity performs a

homeostatic function to reduce the strength of synapses that has been

acquired during wakeful activity56. This synaptic homeostasis improves

subsequent cognitive function by allowing new changes in synaptic strength.

For example, both NREM and REM sleep are necessary for the consolidation

of learning and memory while sleep deprivation results in cognitive

dysfunction57.

Sleep disturbance is commonly observed in the hospital setting and include

changes to sleep patterns and quality (especially sleep fragmentation), as well

as sleep architecture. Polysomnographic studies revealed extreme sleep

disruption in Intensive Care Unite (ICU) patients with decreases in total sleep-

time, altered sleep architecture (predominance of stage 1 and 2 sleep,

decreased or absent stage 3 NREM and REM sleep), and sleep

fragmentation58,59; also, up to 50% of the total sleep-time occurred during

daytime. Studies have shown that fragmented sleep is prevalent due to

frequent arousals and awakenings, and that sleep architecture is altered with

an increase in light sleep, and a decrease in restorative slow wave sleep60.

Environmental factors and health care practices further contribute to sleep

disruption in critically ill patients; these include disturbances like

inappropriately high noise levels, continuous ambient light, and the near

constant performance of medical tests procedure and procedures. Lack of

sleep hygiene results in cognitive dysfunction61,62, contributes to delirium63,

adversely affects immunity64,65, and independently increases both morbidity

and mortality66. Sleep disruption during hospital care has the potential to

adversely impact patients’ outcome and also provides a direct financial cost

with respect to the length of hospital stay and depletion of healthcare

resources.

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Additionally, many sedative and analgesic agents potently suppress slow

wave sleep. Sedative practices have also shown to be a main causative factor

for this disruption67,68. Anaesthetics have different action targets and

ultimately different consequences. The pivotal work of the MENDs trial68-70

indicated the benefits of a specific sedative agent, dexmedetomidine, in the

outcome of ICU population. α2 adrenergic agonists converge on sleep

pathways within the brainstem while those that act by modulating the

GABAA receptor converge at the level of the hypothalamus. Several studies

have now demonstrated the association between the use of benzodiazepine

(BZD) and increased incidence69 and duration71 of delirium in ICU patients. A

recent prospective study has also shown that patients with sleep disorders

have an increased likelihood of exhibiting postoperative delirium24.

Despite the common occurrence of both ICU delirium and sleep disruption in

critically ill patients, a causal relationship has, as of yet, not been well

described. Still, the question remains if we are doing all in our power to avoid

the development of POCD.

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Cerebral Ischemia

Stroke is the leading cause of disability in adults72 and an important risk

factor for bone fracture73. In the United States, ≈ 70,000 stroke victims suffer

from bone fracture within the first year after their stroke74,75. A small proportion

of these stroke patients experience bone fracture within the first 24h after the

ischemic stroke, but the impact of bone fracture on acute stroke lesion is

unknown. Symptomatic pre-operative neurologic diseases, including dementia

and any disease of the central nervous system, are often considered as

exclusion criteria for POCD studies5. Interestingly, cerebral vascular accidents

(without residual deficit) were associated with risk factors for POCD,

suggesting a pre-operative ischemic brain insult could influence the possibility

of POCD5.

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Metabolic Syndrome Roughly 25% of the 45 million surgical patients in the US have MetaS76, a

constellation of conditions whose precise definition and diagnostic criteria

continue to evolve77. MetaS, comprising of insulin resistance, visceral obesity,

hypertension, and dyslipidemia increases the risk of postoperative

complications contributing to a significant higher mortality rate78-81. While each

of the subphenotypes that define the MetaS have a strong genetic

component, lifestyle factors that contribute to this cluster of conditions include

sedentary behaviour and a diet with a high caloric content from saturated fats

and/or simple carbohydrates82. Many complications of MetaS (including

atherosclerosis) are inflammatory in nature and the pathologic metabolism in

adipose stores may be the source of pro-inflammatory adipokines83.

Conversely, with little adiponectin to attenuate activation of the transcription

factor NF-κB in macrophages, expression of genes for pro-inflammatory

cytokines are increased84; up-regulated NF-κB activity in morbid obesity can

be rectified with adiponectin85. Recent evidence indicates patients suffering

from MetaS may be particularly susceptible to POCD23,80.

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AIMS OF THE THESIS Studies have sought to identify factors that may contribute to POCD, which

include surgery, in-patient care factors, and patient-related factors. This work

considers the possible role of inflammation in the development of

postoperative cognitive dysfunction in the setting of underlying systemic

diseases/conditions.

The specific aims are:

1) To identify the role of hippocampal recruitment of BM-DM in the

pathogenesis of POCD.

2) To determine the mechanism by which HMGB1 regulates the activation

and trafficking of circulating BM-DM to the brain.

3) To investigate the contribution of perioperative SF to the neuroinflammatory

and cognitive responses of surgery.

4) To determine whether bone fracture, shortly after ischemic stroke,

enhances stroke-related injuries by augmenting the neuroinflammatory

response.

5) To investigate whether surgery induces a more severe and persistent form

of cognitive decline in a rat model of MetaS.

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METHODS AND RESULTS

16

Surgery

17

Depletion of Bone Marrow-derived Macrophages Perturbs the Innate Immune Response to Surgery and Reduces Postoperative Memory Dysfunction

Participated in study design, conducted qPCR and ELISA experiments and participated in

data analysis and interpretation and final preparation of the manuscript.

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ABSTRACT

Background: According to rodent models of postoperative cognitive decline, activation of the innate immune response following aseptic surgical trauma results in the elaboration of hippocampal proinflammatory cytokines, which are capable of disrupting long-term potentiation, the neurobiologic cor-relate of memory. The authors hypothesize that hippocampal

recruitment of bone marrow–derived macrophages plays a causal role in these processes, resulting in memory dysfunction.Methods: Clodrolip injection (liposomal formulation of clodronate) before stabilized tibial fracture under general anesthesia was used to deplete bone marrow–derived mac-rophages. Systemic inflammation and neuroinflammation were studied on postoperative day 1, and memory in a fear-trace conditioning paradigm was assessed on postoperative day 3. CX3CR1GFP/+ CCR2RFP/+ mice were used to identify bone marrow–derived macrophages.Results: Clodrolip effectively depleted splenic CCR2+ bone marrow–derived macrophages. It also attenuated the surgery-induced increase of interleukin-6 in the serum and the hippocampus, and prevented hippocampal infiltration of CCR2+ cells without affecting the number of CX3CR1+ microglia. It did not alter the surgery-induced increase in hippocampal monocyte chemoattractant protein-1, the recruitment signal for CCR2+ cells. Clodrolip prevented surgery-induced memory dysfunction, as evidenced by a sig-nificant increase in freezing time (29% [95% CI, 21–38%] vs. 48% [95% CI, 38–58%], n = 20, P = 0.004), but did not affect memory in nonsurgical mice.Conclusion: Depletion of bone marrow–derived mac-rophages prevents hippocampal neuroinflammation and

Depletion of Bone Marrow–derived Macrophages Perturbs the Innate Immune Response to Surgery and Reduces Postoperative Memory Dysfunction

Vincent Degos, M.D., Ph.D.,* Susana Vacas, M.D.,† Zhenying Han, M.D.,‡ Nico van Rooijen, Ph.D.,§ Pierre Gressens, M.D., Ph.D.,‖ Hua Su, M.D.,# William L. Young, M.D.,** Mervyn Maze, M.B., Ch.B.‡

* Associate Researcher, Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, California, INSERM, U676, Hôpital Robert Debré, and Department of Anesthesiology and Criti-cal Care, Groupe Hospitalier Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Université Pierre et Marie Curie, Paris, France. † Postdoctoral Fellow, Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, Cali-fornia, and Program for Advanced Medical Education, Lisbon, Por-tugal. ‡ Assistant Researcher, Center for Cerebrovascular Research, Departments of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, California. § Professor, Vrije Universiteit, VUMC, Department of Molecular Cell Biology, Faculty of Medicine, Amsterdam, The Netherlands. ‖ Professor, INSERM, U676, Hôpital Robert Debré, Paris, France. # Associate Professor, Center for Cerebrovascular Research, Departments of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, California. ** Professor and Vice Chair, Center for Cere-brovascular Research, Departments of Anesthesia and Perioperative Care, Neurological Surgery, and Neurology, University of California, San Francisco, San Francisco, California. †† Professor and Chair, Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, California.

Received from the Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, Cali-fornia. Submitted for publication July 26, 2012. Accepted for pub-lication December 7, 2012. Supported by grant Nos. R01NS027713, P01NS044155, and R21NS070153 from the National Institutes of Health, Bethesda, Maryland; and AHA10GRNT3130004 from the American Heart Association, Dallas, Texas. Additional support was received from the University of California, San Francisco, San Francisco, California; INSERM, Paris, France; Société Française d’Anesthésie Réanimation, Paris, France; Fondation des Gueules Cassées, Paris, France; and Institut Servier, Paris, France. Dr. van Rooijen provided complimentary clodronate and control liposomes.

Address correspondence to Dr. Degos: Department of Anes-thesia and Perioperative Care, University of California, San Fran-cisco, 1001 Potrero Avenue, Box 1363, San Francisco, California 94110. [email protected]. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. ANESTHESIOLOGY’S articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.

Copyright © 2013, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins. Anesthesiology ; 118:527–

What We Already Know about This Topic

Animal models of postoperative cognitive dysfunction implicate an innate immune response, with increased circulating inflammatory mediators and migration of bone marrow–derived cells into the brainInhibitors of inflammatory mediators reduce postoperative memory deficits in mice but also reduce wound healing

What This Article Tells Us That Is New

In mice, treatment with a drug that depletes bone marrow–derived macrophages reduced circulating inflammatory mediators after surgical orthopedic injury, reduced migration of immune cells into the brain, and reduced postoperative memory deficits

ALN

Macrophages and Postoperative Memory Dysfunction

Degos et al.

March

10.1097/ALN.0b013e3182834d94

3

Frank

2013

◆ This article is accompanied by an Editorial View. Please see: Sanders RD, Avidan MS: Postoperative cognitive trajectories in adults: The role of inflammatory processes. ANESTHESIOLOGY 2013; 118:484–6.

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memory dysfunction after experimental tibial fracture. These data suggest that the hippocampal recruitment of bone marrow–derived macrophages is a necessary mechanism in murine postoperative cognitive dysfunction. Interventions designed to prevent its activation and/or migration into the brain may represent a feasible preemptive strategy.

ACUTE postsurgical memory deterioration leads to per-sistent cognitive decline1 that can result in considerable

morbidity and increased mortality.2 The specter of memory dysfunction, including acute delirium, postoperative decline, and dementia, is a source of anxiety for patients and their fam-ilies.3 Knowledge about the molecular and cellular pathways involved in postoperative memory dysfunction may provide a launching pad for the development of biomarkers to identify the most vulnerable patients as well as preventive strategies.

Using a murine model of aseptic surgical trauma with a long-bone fracture, we previously demonstrated that postop-erative cognitive decline requires the engagement of the innate immune response. This engagement includes increased sys-temic expression of alarmins and proinflammatory cytokines such as interleukin (IL)-6 in the blood4,5; increased ratio of CD11b+ cells corresponding to macrophages/microglia cells,4 and specifically the ratio of CCR2+ bone marrow-derived macrophages6; and elaboration of proinflammatory cytokines that are capable of disrupting hippocampal long-term poten-tiation, a neurobiologic correlate of learning and memory.3,7–10

Strategies designed to block the effect of proinflamma-tory cytokines with IL-1 receptor antagonist (anakinra) or tumor necrosis factor (TNF)-α antibody (etanercept) pre-vented murine postoperative memory dysfunction.4,5 These interventions also prevented inflammation-dependent wound healing.11,12

In this study, we tested the hypothesis that mediation of postoperative memory decline requires recruitment of sys-temic bone marrow–derived macrophages into the brain, using a specific pharmacologic strategy to acutely deplete systemic phagocytes before an aseptic surgical trauma with an experimental tibial fracture.

Materials and Methods

AnimalsAll experimental procedures involving animals were approved by the University of California, San Francisco Institutional Animal Care and Use Committee, and conformed to National Institutes of Health guidelines. Twelve 8- to 12-week-old CCR2RFP/+CX3CR1GFP/+ male mice6,13 (fig. 1A) were used to identify bone marrow–derived macrophages. CCR2 and CX3CR1 are acronyms for chemokine (C-C motif) receptor 2 (whose cognate ligand is monocyte chemoattractant protein [MCP]-1) that is highly expressed in bone marrow–derived macrophages, and CX3C chemokine receptor 1 (CX3CR1, fractalkine receptor) that is highly expressed in resident microglia. Eighty-nine

Fig. 1. Study design and splenic macrophage depletion with clodrolip. (A) First experiment with 12 CCR2RFP/+ CX3CR1GFP/+ mice divided into two groups of six mice each treated with intraperitoneal (IP) injection of clodrolip versus CT-lip 1 h before the tibia fracture model and 25 h before tissue collection. (B) Second experiment with 24 C57BL/6J mice divided into four groups of six mice treated with IP injection of clodrolip versus CT-lip 1 h before the tibia fracture and sacrificed 12 or 24 h after the tibia fracture. (C) Third experiment with 70 C57BL/6J mice divided into four groups treated with IP injection of CT-lip versus clodrolip 1 h before the tibia fracture (20 mice per group) versus sham procedure (15 mice per group). The training session of the memory test was performed 30 min after the IP injection and 30 min before surgery, and the context session was performed 72 h after surgery. CT-lip = control liposome.

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PERIOPERATIVE MEDICINE

wild-type male mice (C57BL/6J, 10–12 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME): 29 for the cytokine expression (fig. 1B) and 70 for the behavior tests (fig. 1C). Mice did not experience unexpected lethality in the study and were euthanized according to our institutional animal care and use committee guidelines.

In Vivo Systemic Phagocyte Depletion with ClodrolipClodrolip is a liposomal formulation of clodronate (dichlo-romethylene bisphosphonic acid), a nontoxic bisphospho-nate. Liposomes are lipid vesicles consisting of concentric phospholipid bilayers surrounding aqueous compartments. In this case, liposomes are used as “Trojan horses” encapsu-lating clodronate, which are then ingested and digested by phagocytes, followed by an intracellular release and accumu-lation of clodronate. At a certain intracellular concentration, clodronate induces apoptosis of the phagocytes. Clodro-nate liposomes were obtained from clodronateliposomes.org** (Vrije Universiteit, Amsterdam, The Netherlands) at a concentration of 7 mg/ml and prepared as described previ-ously.14,15 Clodrolip (200 μl, approximately 100 mg/kg) was injected intraperitoneally 60 min before the bone fracture. Control animals received 200 μl of control liposomal solu-tion (CT-lip). No intraperitoneal or extraperitoneal damage was observed after clodrolip intraperitoneal administration.

Long-bone Fracture with Tibia Fracture SurgeryAnesthesia was induced and maintained with isoflurane by inhalation. We used a dedicated chamber for induction with 5% isoflurane for 3 min, and the operation was performed under 2% isoflurane for 10–12 min. Under aseptic surgical conditions, an open tibial fracture of the right hind limb with intramedullary fixation was performed as described pre-viously.4 Body temperature was maintained at 37° ± 0.5°C using a thermal blanket throughout the surgical procedure, and analgesia was provided by injection of buprenorphine (0.3 mg in 100 μl of saline). Sham mice for bone fracture (sham group) received the same anesthesia and analgesia as the bone fracture mice.

Measurement of IL-6 in SerumMouse blood was collected using cardiac puncture under general anesthesia (isoflurane, 3%) in separate cohorts 12 and 24 h after the bone fracture procedure (fig. 1B). Blood samples were centrifuged at 1300 rpm for 10 min at room temperature, and the serum was collected and frozen at −80°C. IL-6 is secreted by bone marrow–derived macro-phages in response to alarmins,16 and the IL-6 level in the serum is increased within the first 24 h after the tibia frac-ture.4,5 The IL-6 level in the serum is also associated with the postoperative memory dysfunction phenotype5 and is affected by clodrolip in response to lipopolysaccharide infu-sion.17 For these reasons, we decided to quantify IL-6 levels

in the serum of mice exposed to clodrolip or CT-lip using the IL-6 enzyme-linked immunosorbent assay kit (KMC0062; Invitrogen, Grand Island, NY). Results are expressed as fold increase compared with that measured in five control mice that did not receive any treatment or surgery.

Measurement of Cytokines in the HippocampusThe hippocampi of the mice were collected rapidly under a dissecting microscope, 12 and 24 h after the tibia fracture (fig. 1B), and placed in RNAlater solution (Qiagen, Valencia, CA). To avoid blood contamination, mice were perfused with saline for 5 minutes before sample collection. Total RNA was extracted using the RNeasy Lipid tissue Kit (Qiagen) treated with recombinant DNase I using a RNase-Free Dnase set (Qiagen), and reverse-transcribed to complementary DNA with a High Capacity RNA to Complementary DNA Kit (Applied Biosystems, Bedford, MA). TaqMan Fast Advanced Master Mix (Applied Biosystems) and gene-specific primers and probes used for quantitative polymerase chain reaction are as follows: β-actin (NM_007393.1), IL-6 (Mm00446190_m1), TNF-α (Mm00443258_m1), IL-1β (Mm01336189_m1), and MCP-1 (Mm00441242_m1). Quantitative polymerase chain reaction was performed using StepOnePlus (Applied Biosystems). Each RNA sample was run in triplicate, and relative gene expression was calculated using the comparative threshold cycle (∆CT) method and normalized to β-actin. Results are expressed as fold increase compared with that observed in five control mice that did not receive any treatment or surgery.

Quantification of Bone Marrow–derived Macrophages and MicrogliaTwenty-four hours after the tibia fracture surgery, the brain and spleen of the CCR2RFP/+CX3CR1GFP/+ mice were collected after intracardiac perfusion with paraformaldehyde 4% (fig. 1A). Spleen and brain (bregma, −1.0 to −1.4 mm, corre-sponding to interaural 2.7 to 2.3 mm in coronal orientation) were sectioned into 20-μm-thick slices and mounted with Vectashield DAPI (Vector Laboratories, Burlingame, CA). The expression of CCR2-RFP and CX3CR1-GFP cells was assessed using confocal images, performed with a Spectral Confocal microscope (Nikon Instruments, Melville, NY) using three laser lines (405, 488, and 561 nm). Z-stacks were rendered into a three-dimensional image using the NIS-Elements AR 3.0 software (Nikon), and the expression of CCR2-RFP and CX3CR1-GFP cells was quantified using ImageJ (National Institutes of Health, Bethesda, MD), with three different photographs per mouse taken with a 20× objective. Data are expressed as relative cell percentages nor-malized to the average value of the CT-lip group.

Behavioral Test for Hippocampus-dependent Memory with Trace Fear ConditioningFear conditioning is used to assess memory in rodents, which are trained to associate a conditional stimulus, such as a con-ditioning chamber, with an aversive, unconditional stimulus,

** http://www.clodronateliposomes.org/ashwindigital.asp?docid=26. Accessed January 7, 2013.

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such as a foot shock. Freezing behavior is an indicator of aver-sive memory that is measured when subjects are reexposed to the conditional stimulus. With this model, lesions of the hip-pocampus disrupt recall of fear responses to the presentation of the context, resulting in a diminution in freezing.18,19

For this study, we used a previously published para-digm.4–6,20 Briefly, the behavioral study was conducted using a conditioning chamber (Med Associates, Inc., St. Albans, VT) and an unconditional stimulus (two periods of foot shock of 0.75 mA during 2 s). An infrared video camera, mounted in front of the chamber, captured motion speed (Video Freeze; Med Associates).

All of the animals underwent the same training session, regardless of the specific intervention, and received their training 30–40 min after the liposomal intraperitoneal injec-tions (whether clodrolipid or CT-lip) that occurred 30 min before surgery (fig. 1C). Three days after conditioning, mice were returned to the same chamber where training had occurred for a context test. During the context test, mice were exposed just to the context and no tones or foot shocks were delivered. Freezing was recognized by the software as a total lack of movement, excluding breathing and movement of vibrissae (linear detection with a minimal freeze duration of 20 frames corresponding to 0.7 s and a motion threshold of 20 arbitrary units).4–6,20 Decrease in the percentage of time spent freezing indicated impairment of memory.

Body Weight and Maximal Motion SpeedThe body weight of the animals was measured 3 days after surgery, following assessment of freezing behavior. An infra-red video camera (Video Freeze) captured and quantified motion speed during the context test, and the maximal motion speed was recorded for each mouse.

Statistical AnalysisData are presented as mean ± 95% CI. Normality was tested with the d’Agostino–Pearson omnibus normality test. Equality of variances was tested with the F test. For two-sample comparisons, Student t tests were used (using the Welch correction if necessary); Mann–Whitney U tests were used if data were not normally distributed. For comparisons of more than two groups, means were compared using one-way ANOVA followed by Student t tests with a Bonferroni-corrected alpha level.

We used the two-way ANOVA procedure to determine whether or not time and treatment were significant factors in predicting IL-6 concentration in the serum, and IL-6, IL-1β, TNF-α, and MCP-1 messenger ribonucleic acid (mRNA) expression in the hippocampi. Given the highly skewed nature of the mRNA expression, we checked the distribution of the residuals. We applied a log transforma-tion (ln[X]) to the response of the mRNA expression before performing analysis to better adhere to the ANOVA model’s assumptions of normally distributed residuals and homosce-dasticity of residuals.

For the behavior tests, animals were tagged and allocated randomly to each group before any treatment, and research-ers were blinded to the group assignment that was revealed only after the analysis phase. A repeated measures ANOVA was performed to determine whether treatment (CT-lip and clodrolip) and the three time periods (baseline, first shock, and second shock) were significant predictors of percentage freezing time during the training session.

For this study, our primary outcome was percentage of freezing time during the context session. Based on previous freezing time data,4 we estimated that a sample of 18 C57BL/6J surgical mice per group was necessary to demonstrate a 20% increase in percentage freezing time, with 80% power at the 0.017 alpha level (after adjusting for three comparisons) to find a significant difference between clodrolip and CT-lip.

A two-tailed value of P < 0.05 was considered statistically significant for two-group comparisons, and the significance threshold was adjusted for multiple comparisons with a Bonferroni correction. Prism 5 (GraphPad Software, Inc., La Jolla, CA) was used to conduct the statistical analyses.

Results

Clodrolip Depletes Splenic Bone Marrow–derived Macrophages and Prevents Hippocampal Bone Marrow–derived Macrophage InfiltrationUsing CCR2RFP/+CX3CR1GFP/+ mice (fig. 1A), in which RFP+ bone marrow–derived macrophages and GFP+ resi-dent microglia can be tracked,6,13 we found that clodrolip depleted splenic macrophages and surgery-induced bone marrow–derived macrophage infiltration into the hippo-campus. The CCR2+ cells, which are mainly present in the splenic red pulp (fig. 2A), decreased by 96% in the clodro-lip-exposed mice (fig. 2B) (95% CI, 95–97%, P < 0.001). As shown in figure 3, the number of CCR2+ cells was also significantly reduced in the hippocampi of clodrolip-treated mice compared with CT-lip–treated mice 24 h after surgery (decrease of 76% for the dentate gyrus and 87% in the cornu ammonis 3). However, clodrolip treatment did not change the number of CX3CR1+ cells in the dentate gyrus and cornu ammonis 3 hippocampal regions (fig. 3).

Clodrolip Reduces Systemic and Hippocampal Proinflammatory CytokinesWe previously showed that proinflammatory cytokines in the blood and hippocampus increased within the first day after surgery.4 To test whether clodrolip treatment would reduce the proinflammatory cytokines, we studied serum and hip-pocampal expression 12 and 24 h after surgery (fig. 1B). Twelve hours after surgery, the rise in IL-6 in the serum was significantly attenuated in mice exposed to clodrolip (two-way ANOVA, P = 0.004 for the treatment, P = 0.003 for the time effect, and P = 0.19 for interaction) (fig. 4).

Between 12 and 24 h after surgery, the increase in mRNA hippocampal expression of IL-6, TNF-α, and IL-1 induced by

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Fig. 2. Effects of systemic macrophage depletion with clodrolip on the CCR2+ splenic cells. (A) Representative photographs of spleen section showing CCR2+ cell repartition mainly in the red pulp (RP) and less in the white pulp (WP), 24 h after tibia fracture. Top photographs are of low magnification (scale bar = 100 μm) and bottom photographs are highly magnified images (scale bar = 50 μm) in the CT-lip and the clodrolip mice. (B) Quantification of the relative percentage of CCR2+ cells in the spleen after clodrolip (n = 6, ***P < 0.001 with unpaired Student t test). CCR2 = chemokine (C-C motif) receptor 2; CT-lip = control liposome; DAPI = 4’,6’-diamidino-2-phenylindole; IP = intraperitoneal (bars = mean ± 95% CI).

Fig. 3. Effects of systemic macrophage depletion with clodrolip on the hippocampal CCR2+ and CX3CR1+ cells. (A) Representa-tive photographs of the section of interest corresponding to bregma, −1.2 mm (scale bar = 500 μm), showing the dentate gyrus (D.Gyrus) and the cornu ammonis subdivision 3 (CA.3). (B) Representative highly magnified photograph (scale bar = 20 μm) of a ramified CX3CR1+ green cell and an amoeboid CCR2+ red cell in the hippocampus. (C) Bar graph shows quantification of the relative percentage of CCR2+ cells in the dentate gyrus and the CA.3 regions after clodrolip treatment (n = 6, significant F test for both comparisons, *P = 0.02; **P = 0.002 with unpaired Student t tests with Welch’s correction). (D) Representative photo-graphs of the dentate gyrus hippocampal sections in the CT-lip (D1) and the clodrolip (D2) mice (scale bar = 100 μm) showing the decrease of CCR2+ cells after clodrolip treatment. (E) Representative photographs of the dentate gyrus hippocampal section in the CT-lip (E1) and the clodrolip (E2) mice (scale bar = 100 μm) showing the absence of CX3CR1+ cell depletion after clodrolip treatment. (F) Bar graph shows quantification of the relative percentage of CCR2+ cells in the dentate gyrus and the CA.3 regions after clodrolip (n = 6, P = 0.51 for dentate gyrus and P = 0.51 for CA.3). CT-lip = control-liposome; CCR2 = chemokine (C-C motif) receptor 2; DAPI = 4’,6’-diamidino-2-phenylindole (bars = mean ± 95% CI).

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surgery returned to almost baseline values at 24 h (fig. 5). Clo-drolip exposure significantly inhibited the surgery-induced increased expression of mRNA IL-6 (two-way ANOVA, P < 0.001 for the treatment, P = 0.002 for the time effect, and P = 0.51 for interaction), and interacted with the time-dependent decrease for TNF-α (two-way ANOVA, P = 0.03 for inter-action). Clodrolip treatment did not change IL-1β mRNA expression (two-way ANOVA, P = 0.42 for the treatment, P < 0.001 for the time effect, and P = 0.66 for interaction) (fig. 5).

Systemic macrophages are recruited into tissues by the chemoattractant MCP-1 that binds to CCR2, which is expressed on the surface of bone marrow–derived macro-phages.21,22 Following surgery, MCP-1 mRNA expression increases and is unaffected by prior exposure to clodrolip (two-way ANOVA, P = 0.64 for the treatment, P < 0.001 for the time effect, and P = 0.67 for interaction) (fig. 5D).

Clodrolip Prevents Surgery-induced Memory ImpairmentDuring the preoperative training period, learning was simi-lar in the clodrolip-exposed and the control (nonexposed) groups, with the percentage of freezing being highly associ-ated with time (fig. 6). During the context session, surgery significantly decreased percentage of freezing time in compar-ison with the sham group (52% [95% CI, 41–63%] vs. 29% [95% CI, 21 to 37%], P = 0.0012); preoperative exposure to clodrolip resulted in significantly greater freezing time than in the nonexposed surgical cohort (29% [95% CI, 21–38%] vs. 48% [95% CI: 38–58%], P = 0.004), reaching a level simi-lar to that observed in the sham-operated clodrolip-exposed mice (49% [95% CI, 36–63%], P = 0.86) (fig. 7, A and B).

Clodrolip did not affect the body weight of the mice 3 days after the injection (fig. 7C). As for maximal motion speed, the clodrolip-treated groups were no different from the CT-lip groups, even though the maximal motion speed of the surgical groups was significantly slower than the sham groups (fig. 7D).

DiscussionIn this study, we report for the first time that bone marrow–derived macrophages are required in the pathogenesis of the neuroinflammatory and memory dysfunction induced by surgery. Also, we report that a possible hippocampal signal through MCP-1 is involved in the recruitment of bone marrow–derived macrophages to this brain region. Data from rodent surgical models have provided insight into the neuroinflammatory basis for postoperative cognitive decline. This usually transient process appears to be part of a motivational system that reorganizes the organism’s priorities to facilitate recovery. To date, we have established a pivotal early role for the proinflammatory cytokine TNF-α,6 and our study demonstrated that hippocampal infiltration of bone marrow–derived macrophages also plays a role in the initiation of neuroinflammation.

Hippocampal Infiltration of Bone Marrow–derived Macrophages after SurgeryMonocyte infiltration into the brain is mainly described in acute brain injuries such as stroke23 and traumatic brain inju-ries,24 as well as chronic inflammatory brain injuries such as multiple sclerosis.13 Using long-bone fracture as a surrogate for a peripheral orthopedic surgical insult, we previously reported that CCR2+ cells were present in the hippocam-pus.6 Because microglia can also express CCR2 under certain conditions,25,26 we could not ascertain whether these CCR2-expressing cells arose from the resident macrophage popula-tion (microglia) or through an infiltration from outside of the central nervous system. Using clodrolip to specifically deplete the systemic pool of phagocytes, including bone marrow–derived macrophages, we were able to demonstrate that the CCR2+ cells in the hippocampus are a result of the recruit-ment of bone marrow–derived macrophages into the brain.

For passage into the brain, monocytes are required to overcome the blood–brain and/or blood–cerebral spinal fluid barrier27,28; these barriers can be disrupted by direct acute brain injury.29,30 Interestingly, after peripheral surgery, the blood–brain barrier is disrupted, although there is no discernible brain lesion.6 Now we show that after surgery, the hippocampus expresses MCP-1, which is capable of attracting CCR2+-expressing cells migrating through the disrupted blood–brain barrier. This increased expression of MCP-1 is unaffected by clodrolip treatment, indicating that bone marrow–derived macrophages are not a self-perpetuating source of this chemoattractant for its own recruitment. Future understanding of the source and the triggers for hippocampal MCP-1 following peripheral surgery may result

Fig. 4. Effects of systemic macrophage depletion with clodro-lip on the IL-6 serum concentration after the tibia fracture 12 and 24 h after tibia fracture in the clodrolip and CT-lip groups. Data are normalized to the average value of the control mice group that did not receive any treatment or any surgery and expressed as fold time increase (n = 6 per group and per tim-ing, two-way ANOVA, P = 0.004 for the treatment effect, P = 0.003 for the time effect, and P = 0.19 for interaction). CCR2 = chemokine (C-C motif) receptor 2; CT-lip = control liposome; DAPI = 4’,6’-diamidino-2-phenylindole; IL = interleukin-6; IP = intraperitoneal (boxes and circles = mean ± 95% CI).

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in interventional strategies designed to prevent recruitment of bone marrow–derived macrophages into the brain.

Hippocampal Bone Marrow–derived Macrophage Infiltration and Memory DysfunctionOur recent data suggest that transient hippocampal inflam-mation is the key element in postoperative memory dys-function because (1) hippocampal areas are known to be involved in memory tasks7; (2) hippocampal neuroin-flammation profile correlates with the level of memory dysfunction4,5; and (3) hippocampal neuroinflammation leads to long-term potentiation disruption.31,32 Now we report here that the absence of bone marrow–derived mac-rophage infiltration, produced by systemic depletion by clodrolip, decreases surgery-induced hippocampal inflam-mation and memory dysfunction. Therefore, postoperative bone marrow–derived macrophage recruitment into the

hippocampus plays a key role in the initiation of postopera-tive memory dysfunction.

In the context of postoperative cognitive decline, determining which cells are involved in the initiation of the inflammation response is important because, when exaggerated, this could overwhelm resolving responses and produce persistent postoperative cognitive decline. Earlier, we described that surgical trauma induces systemic release of alarmins (i.e., high-mobility group protein 1) and proinflammatory cytokines (i.e., TNF-α and IL-6).4,5 Improving our knowledge of cellular and molecular initiation mechanisms will allow insight into an ex vivo bioassay to prospectively determine whether patients are at risk.

Limitations of the StudyWe used experimental tibial fracture to generate animal postoperative memory acute dysfunction. With this model, we traumatized the bone marrow directly, which could play

Fig. 5. Clodrolip effect and early kinetic of the mRNA hippocampal expression of IL-1β, TNF-α, IL-6, and MCP-1 after tibia fracture. (A) Hippocampal mRNA expression of IL-6 relatively expressed as fold increase compared with control brain expression 12 and 24 h after tibia fracture in the CT-lip and the clodrolip-treated mice (n = 6 per group and per timing, two-way ANOVA, P = 0.0003 for the treatment effect, P = 0.0023 for the time effect, and P = 0.51 for interaction). (B) Hippocampal mRNA expression of TNF-α relatively expressed as fold increase compared with control brain expression 12 and 24 h after tibia fracture in the CT-lip and the clodrolip-treated mice (n = 6 per group and per timing, two-way ANOVA, P = 0.06 for the treatment, P < 0.0001 for the time effect, and P = 0.035 for interaction). (C) Hippocampal mRNA expression of IL-1β relatively expressed as fold increase compared with control brain expression 12 and 24 h after tibia fracture in the CT-lip and the clodrolip-treated mice (n = 6 per group and per timing, two-way ANOVA, P = 0.42 for the treatment effect, P < 0.0001 for the time effect, and P = 0.66 for interaction). (D) Hippocampal mRNA expression of MCP-1 relatively expressed as fold increase compared with control brain expression 12 and 24 h after tibia fracture in the CT-lip and the clodrolip-treated mice (two-way ANOVA, P = 0.64 for the treatment, P = 0.0005 for the time effect, and P = 0.67 for interaction). CT-lip = control liposome; IL = interleukin; MCP-1 = monocyte chemoattractant protein-1; mRNA = messenger ribonucleic acid; TNF = tumor necrosis factor (boxes = mean ± 95% CI and data are presented with a logarithmic scale).

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a key role. However, other models that did not damage bone marrow with a splenectomy33 also showed that surgery gen-erated postoperative cognitive dysfunction.

Fibrin is deposited in the hippocampus after tibia frac-ture,6 suggesting that the blood–brain barrier becomes dis-rupted and may allow the passage of clodrolip to act directly on the microglia population.34 However, we found that the systemic administration of clodrolip acts only on the num-ber of CCR2+ cells without significantly affecting the num-ber of CX3CR1+ cells (fig. 3); if clodrolip has an effect on

microglia, it may be to functionally modify them. For this reason, we cannot exclude the possibility that clodrolip does not affect the function of microglia, and that microglia do not play a key role in postoperative cognitive dysfunction.

For this study, we used a pharmacologic strategy to quickly deplete the pool of systemic macrophages. However, because clodrolip is highly toxic for monocytes and macrophages,35 it can increase the risk of postsurgical infections, generating a phenotype of its own. With a single dose, we did not observe loss of weight or other signs of sickness within the

Fig. 6. Clodrolip effect on the training session. (A) Representative record of a training session showing the motion (motion index expressed in arbitrary units) of the mouse according to the time. The two green bars represent the two shocks and the three red rectangles represent the 40-s periods used to quantify the baseline, first, and second shock freezing responses. Bar graph (B) quantifies the percentage of freezing time (n = 35), and two-way ANOVA shows a significant effect of time (P < 0.0001), no significant effect of treatment (P = 0.68), and no interaction (P = 0.61).

Fig. 7. Depletion of systemic macrophages reduces surgery-induced memory dysfunction. (A) Representative records of con-text sessions of tibia fracture mice treated with CT-lip (A1) and with clodrolip (A2) showing the motion (motion index expressed in arbitrary units) of the mice according to the time. (B) Quantification of the freezing time percentage according to the four groups (n = 15–20, *P = 0.0012 and §P = 0.004, respectively, with one-way ANOVA and Bonferroni post hoc analysis). (C) Quantifica-tion of the body weight in the four groups 3 days after surgery (n = 15–20, P = 0.02 and P = 0.03, respectively, not significant after adjustment for multiple comparisons with one-way ANOVA and Bonferroni post hoc analysis and no significant effect of the clodrolip treatment. (D) Quantification of the maximal motion speed in the four groups 3 days after surgery (n = 15–20, **P = 0.012 and ###P = 0.0003, respectively, with one-way ANOVA and Bonferroni post hoc analysis and no significant effect of the clodrolip treatment; bars = mean ± 95% CI). AU = arbitrary units; CT-lip = control-liposome.

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3 days. We performed a very short-term study, focusing on the acute exaggeration phase of neuroinflammation, and did not perform any long-term study with clodrolip. Clodrolip should be considered as a tool for mechanistic studies but cannot be proposed for clinical therapy.

To distinguish whether these recruited cells were resident or recruited systemic macrophages, we used CCR2RFP/+CX3CR1GFP/+ mice. CCR2 is receptor for MCP-1 and is mainly expressed in bone marrow–derived monocytes–macrophages. We previously showed that CD11b+ macrophages–microglia cells were recruited in the hippocampus after tibial fracture.4 In this study, however, we did not determine that CCR2+ cells were only bone marrow–derived macrophages. Further study on the role of other systemic phagocytes, including neutrophils, in our phenotype will be of most interest.

Hippocampal cytokine expression was performed with mRNA and not with protein. This is a limitation, but we considered that the potential extravasation from blood may affect protein levels. Indeed, in this model we found blood–brain barrier leakage after the tibia fracture,6 and the increase of circulating IL-6 protein in the serum could con-taminate the hippocampal samples with passive movement in the parenchyma (this phenomenon could be amplified by the perfusion itself ). By analyzing mRNA expression in the brain collected after purging blood from the vessels, we ensured that hippocampal cells were the source of proinflam-matory cytokines.

In conclusion, we showed in this study that bone mar-row–derived macrophage activation after experimental tibial fracture is directly involved in the tibia fracture–induced hippocampal bone marrow–derived macrophage infiltration and animal memory dysfunction. Understanding the cellular and biologic pathways involved in postoperative cognitive decline is a key element in designing interventions to pre-vent this disease. Reducing activation and/or migration of innate immune cells, such as systemic macrophages, into the brain represents a viable preemptive strategy.The authors thank members of the University of California, San Francisco Center for Cerebrovascular Research and the Maze Labo-ratory for their support; Niccolo Terrando Ph.D., Assistant Profes-sor, Karolinska Institute, Stockholm, Sweden, for assistance with setting up memory tests; and Israel F. Charo, M.D., Associate Direc-tor, University of California, San Francisco, Gladstone Institute, San Francisco, California, for providing the CCR2RFP/+CX3CR1GFP/+ mice.

References 1. Saczynski JS, Marcantonio ER, Quach L, Fong TG, Gross A,

Heilman KM, Gravenstein JS: Predictors of cognitive dysfunc 2008;

Resolving postoperative neuroinflammation and cognitive

in vitro.

procedures performed in rheumatic patients receiving tumor

of macrophages: Mechanism of action, preparation of lipo

pulmonary intravascular macrophages using liposomal clo

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tor alpha signaling during peripheral organ inflammation.

Stöcklein V, Bazarian JJ: Validation of serum markers for

α

their effects in the dentate gyrus. Prog Brain Res 2007;

A

of macrophages from organs and tissues. Methods Mol Biol

Liebig Explodes Heppenheim’s Apothecary Shop … or Did He?

After his chemical tinkerings exploded, schoolboy Justus Liebig (1803–1873) was expelled from his hometown’s “gymnasium” in Darmstadt, Germany. Relocating 19 miles south from 1819–20, Justus apprenticed in an apothecary shop (left) run by Gottfried Pirsch of Hep-penheim. Growing bored with his apothecary mentor, Justus resumed his previous experi-ments with unstable chemicals. His pharmacy apprenticeship terminated abruptly, after only 10 months, due to a violent explosion (right) and/or his family’s inability to keep paying Pirsch. The mischievous young Liebig would grow up to become a founder of organic chemistry and a codiscoverer of chloroform. (Copyright © the American Society of Anesthesiologists, Inc.)

George S. Bause, M.D., M.P.H., Honorary Curator, ASA’s Wood Library-Museum of Anesthesiology, Park Ridge, Illinois, and Clinical Associate Professor, Case Western Reserve University, C leveland, Ohio. [email protected].

ANESTHESIOLOGY REFLECTIONS FROM THE WOOD LIBRARY-MUSEUM

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HMGB1 Initiates Postoperative Cognitive Decline by Engaging Bone Marrow-derived Macrophages

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1 XXX 2014

ABSTRACT

Background: Aseptic trauma engages the innate immune response to trigger a neuroinflammatory reaction that results in postoperative cognitive decline. The authors sought to determine whether high-mobility group box 1 protein (HMGB1), an ubiquitous nucleosomal protein, initiates this process through activation and trafficking of circulating bone marrow–derived macrophages to the brain.Methods: The effects of HMGB1 on memory (using trace fear conditioning) were tested in adult C57BL/6J male mice; separate cohorts were tested after bone mar-row–derived macrophages were depleted by clodrolip. The effect of anti-HMGB1 neutralizing antibody on the inflammatory and behavioral responses to tibial surgery were investigated.Results: A single injection of HMGB1 caused memory decline, as evidenced by a decrease in freezing time (52 ± 11% vs. 39 ± 5%; n = 16–17); memory decline was prevented when bone marrow–derived macrophages were depleted (39 ± 5% vs. 50 ± 9%; n = 17). Disabling HMGB1 with a blocking

monoclonal antibody, before surgery, reduced postoperative memory decline (52 ± 11% vs. 29 ± 5%; n = 15–16); also, hippocampal expression of monocyte chemotactic protein-1 was prevented by the neutralizing antibody (n = 6). Neither the systemic nor the hippocampal inflammatory responses to surgery occurred in mice pretreated with anti-HMGB1 neutralizing antibody (n = 6).Conclusion: Postoperative neuroinflammation and cogni-tive decline can be prevented by abrogating the effects of HMGB1. Following the earlier characterization of the reso-lution of surgery-induced memory decline, the mechanisms of its initiation are now described. Together, these data may be used to preoperatively test the risk to surgical patients for the development of exaggerated and prolonged postopera-tive memory decline that is reflected in delirium and postop-erative cognitive dysfunction, respectively.

A SEPTIC surgical trauma provokes a neuroinflamma-tory response, presumably, to defend the organism

from further injury.1,2 When this homeostatic response is dysregulated, detrimental consequences can follow, includ-ing postoperative cognitive decline that can persist in up to 10% of surgical patients over the age of 65 yr.3,4 Although it is possible that the cognitive response to surgery may also include enhancement (if the surgery “cures” a process that

What We Already Know about This Topic

What This Article Tells Us That Is New

Copyright © 2013, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins. Anesthesiology 2014; XX:00-00

From the Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, California (S.V., V.D., M.M.), the Programme for Advanced Medical Education, Calouste Gulbenkian Foundation, Lisbon, Portugal (S.V.), INSERM, U676, Hôpital Robert Debré, Paris, France (V.D.), and the Labora-tory of Biomedical Science, Feinstein Institute for Medical Research, Manhasset, New York (K.J.T.).

Submitted for publication July 3, 2013. Accepted for publication September 24, 2013. Supported by National Institutes of Health, grant No. R01GM104194, Bethesda, Maryland. The authors declare no competing interests.

Address correspondence to Dr. Maze: Department of Anesthesia and Perioperative Care, University of California, San Francisco, 521 Parnassus Avenue, Box 0648, San Francisco, California 94143-0648. [email protected]. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. ANESTHESIOLOGY’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.

High-mobility Group Box 1 Protein Initiates Postoperative Cognitive Decline by Engaging Bone Marrow–derived Macrophages


Copyright © by the American Society of Anesthesiologists. Unauthorized reproduction of this article is prohibited.

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HMGB1 Role on Postoperative Cognitive Dysfunction

interferes with cognition) or no change (short-lived initia-tion and resolution of aseptic trauma-induced inflamma-tion), we have explored, in rodent models, the process that mediates persistent postoperative cognitive decline.1,2,5 After tissue injury the innate immune response is engaged, result-ing in penetration of bone marrow–derived macrophages (BM-DM) into the brain through a disrupted blood–brain barrier.2 Within the hippocampus these activated macro-phages release proinflammatory cytokines that are capable of attenuating long-term potentiation that is the neurobio-logic correlate of learning and memory.6,7 These processes are reversed within days through inflammation-resolving mechanisms involving both neural and humoral path-ways.2 Failure to resolve the neuroinflammatory response results in exaggerated and persistent postoperative cognitive decline.1,8,9 In an attempt to devise strategies that can detect and mitigate this risk, the most vulnerable patients need to be identified; in pursuit of this goal we sought to precisely define the initiating processes in order to devise a preopera-tive functional assay that is predictive of the patient’s likely immune response to aseptic trauma.

Alarmins, a family of damage-associated molecular pat-terns, are capable of activating the innate immune response through their interaction with pattern recognition receptors on circulating monocytes.10 In particular, high-mobility group box 1 protein (HMGB1) is an alarmin that is pas-sively released into the circulation from traumatized necrotic cells; also, HMGB1 can be rapidly secreted by stimulated

leukocytes and epithelial cells.10,11 We previously demon-strated that circulating HMGB1 increases after surgery in humans and also in a murine aseptic trauma model12,13; fur-thermore, we reported that this species of alarmin is required for trauma-induced exacerbation of the morphological and functional consequences of stroke.12

Now we describe data from experiments designed to test the hypothesis that the early release of HMGB1 triggers the neuroinflammatory and behavioral responses to trauma. These data set the stage for the development of a functional assay that assesses the initiation and resolution of inflam-matory processes that are pivotal in postoperative cognitive decline.

Materials and MethodsAnimalsAll experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco, San Francisco, Cali-fornia, and conformed to the National Institutes of Health Guidelines. All animals were fed standard rodent food and water ad libitum, and were housed (five mice per cage) in sawdust-lined cages in an air-conditioned environment with 12-h light/dark cycles. Wild-type male mice (C57BL/6J, 12–14 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME) for the behavior tests (fig. 1, A and B) and for the cytokine expression (fig. 1C).

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Tibia fracture Anti-HMGB1 vs. saline

B

Fig. 1. Study design. (A) First experiment: mice were divided in five groups treated with intraperitoneal injection of control lipo-some versus clodrolip 1 h before high-mobility group box 1 protein (HMGB1) Ag versus saline injection. Control animals received saline injections. The training session of the memory test was performed 30 min after the clodrolip/control liposome injection and 30 min before HMGB1 Ag/saline injection; and the context session was performed 72 h later. (B) Second experiment: mice were divided in four groups treated with anti-HMGB1 versus saline 1 h before tibia fracture. The training session of the memory test was performed 30 min after the intraperitoneal injection and 30 min before tibia fracture. (C) Third experiment: mice were divided in four groups treated with anti-HMGB1 versus saline 1 h before tibia fracture and sacrificed 1 and 24 h after the tibia fracture. Anti-HMGB1 = neutralizing HMGB1 monoclonal antibody; CT-lip = control liposome; HMGB1 Ag = HMGB1 antigen.


31



Animals were tagged and randomly allocated to each group before any treatment or procedure. Researchers were blinded to the group assignment, which was revealed only after the analysis phase.

Body weight was measured before any procedure or treat-ment and 3 days later, after assessment of freezing behavior.

Surgical TraumaUnder aseptic conditions, groups of mice were subjected to an open tibia fracture of the left hind paw with an intramed-ullary fixation as previously described.2,12,14,15 Briefly, mice received general anesthesia with 2% isoflurane and analgesia was achieved with buprenorphine 0.1 mg/kg administered subcutaneously, immediately after anesthetic induction and before surgical insult. Warming pads and temperature-controlled lights were used to maintain body temperature at 37° ± 0.5°C. The entire procedure from induction of anes-thesia to end of surgery lasted 12 ± 5 min.

Trace-fear ConditioningFear conditioning is used to assess learning and memory in rodents, which are trained to associate a conditional stimu-lus, such as a tone, with an aversive, unconditional stimulus, such as a foot-shock.16 Freezing behavior is an indicator of aversive memory that is measured when subjects are reex-posed to the conditional stimulus.

For this study we used a previously published para-digm.1,5,12,15,17 In brief, the behavioral study was conducted by using a conditioning chamber (Med. Associates Inc., St. Albans, VT) and an unconditional stimulus (two periods of 2-s foot-shock of 0.75 mAmp). Behavior was captured with an infrared video camera (Video Freeze; Med. Asso-ciates Inc.). Thirty minutes after a particular intervention, animals underwent the training session after which they were returned to their housing cage. Three days after train-ing, mice underwent a context test, during which no tones or foot-shocks were delivered. Freezing behavior, recognized as lack of movement, was recorded by video and analyzed by software. A decrease in the percentage of time spent freezing indicated impairment of memory.

Systemic Inflammatory Response to SurgeryOne hour and then 24 h after aseptic surgical trauma, blood was collected transcardially after thoracotomy under ter-minal isoflurane anesthesia and placed into heparin-coated syringes. Samples were centrifuged at 3,400 rotations per minute for 10 min and plasma was collected and stored at −80°C until these were assayed. Blood samples taken from animals without intervention served as controls. Plasma interleukin (IL)-6 and HMGB1 were measured using com-mercially available enzyme-linked immunosorbent kits, according to the manufacturer’s instructions (Invitrogen, Grand Island, NY, and IBL International, Toronto, Ontario, Canada, respectively).

Neuroinflammatory Response to SurgeryTwenty-four hours after surgery, mice were perfused with saline and the hippocampus was then rapidly extracted, placed in RNAlater™ solution (Qiagen, Valencia, CA) and stored at 4°C overnight. Total RNA was extracted using RNeasy Lipid tissue Kit (Qiagen). Extracted RNA was treated with recom-binant DNase I by using a RNase-Free Dnase set™ (Qiagen). Messenger RNA (mRNA) concentrations were determined with a ND-1000 Spectrophotometer (NanoDrop®; Thermo Fisher Scientific, Wilmington, DE) and mRNA was reverse transcribed to complementary DNA with a High Capacity RNA to-cDNA Kit (Applied Biosystems, Carlsbad, CA).

TaqMan Fast Advanced Master Mix (Applied Biosys-tems) and specific gene-expression assays were use for quan-titative polymerase chain reaction as follows: actin beta (NM_007393.1), IL-6 (Mm00446190_m1), tumor necro-sis factor-α (Mm00443258_m1), IL-1β (Mm01336189_m1), and monocyte chemotactic protein-1 (MCP-1) (Mm00441242_m1). Quantitative polymerase chain reaction was performed using StepOnePlus™ (Applied Biosystems). Each sample was run in triplicate, and relative gene expres-sion was calculated using the comparative threshold cycle ∆∆Ct and normalized to β-actin. Results are expressed as fold increases relative to controls.

InterventionsDepletion of BM-DM. Clodrolip is a liposomal formulation of clodronate (dichloromethylene bisphosphonic acid), a non-toxic bisphosphonate. Liposomes (lipid vesicles consisting of concentric phospholipid bilayers surrounding aqueous com-partments) encapsulate clodronate, which are then ingested and digested by phagocytes, followed by an intracellular release and accumulation of clodronate. At a certain intracellular concentration, clodronate induces apoptosis of the phago-cytes. Clodrolip was obtained from clodronateliposomes.org (Vrije Universiteit, Amsterdam, The Netherlands) at 7 mg/ml concentration and prepared as previously described.18,19 Clo-drolip (200 μl, about 100 mg/kg) was injected intraperitone-ally 60 min before aseptic surgical trauma. Control animals received 200 μl of control liposomal solution.Administration of Reagents to Simulate or Block the HMGB1 Response to Trauma. Fifty microgram per kilogram (100 μl) of recombinant HMGB1 (R&D System, Minneapolis, MN) was administered intraperitoneally. To neutralize trauma-released HMGB1, 50 μg of anti-HMGB1 neutralizing monoclonal antibody (2G7, mouse IgG2b supplied by Dr. Tracey’s Labo-ratory, Manhasset, NY) in 100 μl of saline was administered intraperitoneally, 60 min before bone fracture. Control animals received the same volume (100 μl) of the vehicle (saline).

Statistical AnalysisData are presented as mean ± SD. Normality was tested with the Kolmogorov–Smirnov normality test. Equality of vari-ances was tested with the F test. We applied a log transfor-mation (ln(X)) to the response of HMGB1 and IL-6 blood


32



concentrations and mRNA expression before performing analyses to better adhere to ANOVA model’s assumptions of normally distributed residuals and homogeneity of variance.

For comparisons of more than two groups, means were compared using one-way ANOVA followed by t tests with a Bonferroni-corrected α level. We used the two-way ANOVA procedure to determine whether or not time and antibody treatment were significant factors in predicting HMGB1 and IL-6 concentrations in the serum; this was followed by Bonferroni post hoc analyses.

For our study, the primary outcome was the percent-age of freezing time during the context session observed in anti-HMGB1 neutralizing monoclonal antibody and con-trol groups. On the basis of previous freezing time data,15 we estimated that a sample of 13 C57BL/6J surgical mice per group was necessary to demonstrate a 20% increase in percentage freezing time, with 80% power at the 0.0125 α level (after adjusting for four comparisons) to reach a significant difference.

A two-tailed P value less than 0.05 was considered statis-tically significant for two-group comparisons and the signifi-cance threshold was adjusted for multiple comparisons with a Bonferroni correction. Prism 6 (GraphPad Software Inc, La Jolla, CA) was used to conduct the statistical analyses.

ResultsHMGB1 Antigen Is Sufficient to Cause Cognitive Decline through the Participation of BM-DMA single administration of HMGB1 produced cognitive decline as evidenced by a significant reduction in freezing time (52 ± 11% vs. 39 ± 5%, n = 16 in control group, n = 17 in HMGB1 antigen group; P = 0.012; fig. 2).

Recently, we showed that depletion of BM-DM by clo-drolip exposure blocks surgery-induced neuroinflammation and cognitive decline.15 To determine whether HMGB1-induced cognitive decline requires the participation of BM-DM, mice were exposed to clodrolip or its vehicle before administration of HMGB1. Training was unaffected by the clodrolip exposure (data not shown). We lost one ani-mal in the clodrolip group and another in the control lipo-some group on day 2. According to the manufacturer this may happen if there is transference of microorganisms from the skin or by injection of a nonhomogeneous suspension of liposome. We did not experience any death in the surgical groups. The HMGB1-induced decline in contextual freezing time was prevented by previous clodrolip exposure (39 ± 5% vs. 50 ± 9%, n = 17; P = 0.039; fig. 2).

HMGB1 Antibody Prevented Postsurgical Cognitive DeclineDuring the preoperative training period, learning was simi-lar in the anti-HMGB1–exposed group and the control (nonexposed) groups (data not shown). Surgery significantly decreased the percentage of freezing time when compared with the control group (52 ± 11% vs. 29 ± 5%, n = 16 in con-trol group, n = 15 in surgical group; P < 0.001); preoperative

exposure to anti-HMGB1 attenuated the surgery-induced freezing behavior, rendering the response to be no different from that of the nonsurgical control group (52 ± 11% vs. 47 ± 11%, n = 16 in control group, n = 15 in anti-HMGB1 + surgery group, ns; fig. 3).

HMGB1 Antibody Reduces Systemic and Neuroinflammatory Response to SurgeryAs expected,12 we observed a significant decrease of the early increase of HMGB1 blood level between the first hour and 24 h after surgery (two-way ANOVA; P = 0.002 for the time effect; fig. 4). However, the neutralizing anti-HMGB1 reduced the systemic levels of HMGB1 (two-way ANOVA; P = 0.005 for the treatment effect and P = 0.010 for interac-tion between time and treatment), and this reduction was sig-nificant 1 h after the trauma (P = 0.04 with Bonferroni’s post hoc analysis; fig. 4A). With regard to IL-6 increase after sur-gery, we observed a significant decreased within the first day (two-way ANOVA; P = 0.015 for the time effect) HMGB1 neutralizing antibody was also a significant predicting fac-tor for IL-6 concentration (two-way ANOVA; P < 0.001 for treatment effect). Using HMGB1 neutralizing antibody, we observed a reduction in IL-6 concentration 1 h after trauma (P = 0.006 with Bonferroni’s post hoc analysis; fig. 4B).

Twenty-four hours after surgery, the increase in hippo-campal mRNA expression of IL-6 (n = 6; P = 0.009) and

Control

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Fig. 2. Effects of high-mobility group box 1 protein (HMGB1) on cognitive decline. Contextual fear response reveals hip-pocampal-dependent memory impairment at postoperative day 3 (Arm A). Quantification of the freezing time percentage according to the five groups (n = 15–17; *P = 0.012 control vs. HMGB1 Ag, and #P = 0.039 HMGB1 Ag vs. clodrolip + HMGB1 Ag, with one-way ANOVA and Bonferroni post hoc analysis). HMGB1 Ag = HMGB1 antigen.


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tumor necrosis factor-α (n = 6; P < 0.001) was blocked by exposure to the neutralizing anti-HMGB1 antibody. Treat-ment did not significantly change mRNA transcription of IL-1β (n = 6; P = 0.085; fig. 5, A–C).

Circulating chemokine C-C motif receptor 2-expressing-expressing BM-DM are recruited into the brain by the che-moattractant, MCP-1.12 After surgery there was an increase of hippocampal mRNA transcription of MCP-1 (n = 6; P = 0.003); treatment with the anti-HMGB1 neutralizing anti-body prevented surgery-induced expression of MCP-1 (n = 6; P = 0.005; fig. 5D).

ConclusionThis study posits that HMGB1, when released from a sterile traumatic injury, plays a pivotal role in postoperative mem-ory dysfunction. Together with the detection of the cell type involved in the initiation of the surgery-induced inflamma-tory cascade these findings establish both the precise ele-ments of the immune response that need to be interrogated for establishing risk of dysregulated trauma-induced inflam-mation as well as putative targets for interventions designed to limit or reverse persistent postoperative cognitive decline.

The independent role of the alarmin HMGB1 in disrupt-ing cognitive processing was established by the fact that a single injection of HMGB1 was capable of reproducing a deficit in an hippocampal memory test similar to the surgi-cal phenotype that we previously described.1 Furthermore, its dependence on bone marrow–derived monocytes was evidenced by the attenuation of HMGB1-induced cognitive decline if clodrolip was first administered to induce apopto-sis of these circulating phagocytes (fig. 2); this is similar to findings of a previous report in the setting of surgery.1,5

After the aseptic trauma of elective surgery, either experi-mentally (fig. 4A) or clinically, HMGB1 is released into the circulation where it can interact with pattern recognition

0

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151516 15

Fig. 3. Effects of preoperative administration of high-mobility group box 1 protein (HMGB1) neutralizing monoclonal anti-body on postsurgical memory impairment. Quantification of the freezing time percentage on contextual testing according to the four groups at postoperative day 3 (Arm B) (n = 15–16; ****P < 0.001 control vs. surgery, and **P = 0.001 surgery vs. anti-HMGB1 + surgery and with one-way ANOVA and Bonfer-roni post hoc analysis). Anti-HMGB1 = neutralizing HMGB1 monoclonal antibody.

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Fig. 4. Effects of preoperative administration of high-mobility group box 1 protein (HMGB1) neutralizing monoclonal anti-body on the HMGB1 and interleukin (IL)-6 serum concentration at 1 and 24 h after tibia fracture (Arm C). The dotted lines represent the average values for respectively for HMGB1 and IL-6 concentration of six control mice without any treatment or surgery (n = 6). (A) Levels of HMGB1 serum concentration 1 and 24 h after surgery. After log transformation of the raw data, we observed with two-way ANOVA significant time and treatment effects (P = 0.002 for the time effect; P = 0.005 for the treatment effect, and P = 0.010 for interaction between time and treatment) and a significant difference between the two groups at 1 h (22.00 ± 16.54 vs. 5.29 ± 4.13 surgical control vs. anti-HMGB1 + surgery; P = 0.040 with two-way ANOVA with Bonferroni’s post hoc analysis). (B) Levels of IL-6 serum concentration 1 and 24 h after surgery. After log transforma-tion of the raw data, we observed with two-way ANOVA significant time and treatment effects (P = 0.015 for the time effect; P < 0.001 for the treatment effect; and P = 0.015 for interaction between time and treatment) and a significant difference between the two groups at 1 h (24.24 ± 12.87 vs. 4.90 ± 4.47 surgical control vs. anti-HMGB1 + surgery; P = 0.006 with two-way ANOVA with Bonferroni’s post hoc analysis). Anti-HMGB1 = neutralizing HMGB1 monoclonal antibody.


34



receptors (toll-like receptors 2 and 4 as well as receptor for advanced glycation end products) on immunocytes.15 By neutralizing the early release of alarmins, HMGB1 antibody both decreased surgery-induced inflammation (figs. 4 and 5) as well as cognitive decline (fig. 3), further establishing the importance of hippocampal inflammation for the develop-ment of postoperative memory dysfunction.1,5

After peripheral surgery, chemokine C-C motif receptor 2-expressing cells migrate to the brain, attracted by signal-ing from hippocampal MCP-1, a chemokine that regulates migration and infiltration of monocytes/macrophages.15,20 Interestingly, by depleting BM-DM, the expression of MCP-1 in the surgical model remained unaffected, indicat-ing that BM-DM are not the self-perpetuating source of this chemoattractant for its own recruitment.15 We now show that treatment with HMGB1 antibody was able to prevent the synthesis of MCP-1 in the hippocampus, establishing the dependence of its increased expression on the release of HMGB1. Taking our previous report15 and our current findings together, it follows that HMGB1 is signaling to the hippocampus to produce the chemoattractant, MCP-1, through a mechanism that is independent of BM-DM; this HMGB1-dependent hippocampal expression of MCP-1 could involve either a neural or humoral pathway.

Accumulating evidence indicates that HMGB1 can stimulate migration of not only monocytes, but also vari-ous types of cells including neurite,21 smooth muscle cells,22 tumor cells,23 mesoangioblast stem cells,24 dendritic cells,25,26 and neutrophils.27,28 This could explain how blocking the effect of the release of HMGB1 may have blocked signaling of other HMGB1-derived factors. Although BM-DM play a necessary role, they may not be sufficient to completely explain the cognitive decline seen after surgery; there may be other cells and factors involved in the genesis of postopera-tive cognitive dysfunction.

The following caveats apply when interpreting our find-ings. Our surgery model involves disruption of the bone marrow with an intramedullary pin for internal fixation of the broken bone. The bone marrow itself produces soluble factors that affect immune cells, such as a prolif-eration-inducing ligand A (APRIL), B-cell activating fac-tor (BAFF), both belonging to the tumor necrosis factor family, CXCL12, IL-6, IL-7, and macrophage inhibitory factor.29 The possibility exists that surgical trauma that does not involve damage to the bone marrow may not use the same panoply of alarmins. In addition to its role as a pri-mary lymphoid organ, the bone marrow can act as a host for various mature lymphoid cell types. Several subsets of

0

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Fig. 5. Effects of high-mobility group box 1 protein (HMGB1) neutralizing monoclonal antibody on hippocampal transcription of interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, and monocyte chemotactic protein (MCP)-1 24 h after tibia surgery (Arm C). (A) IL-6 messenger RNA (mRNA), (B) TNF-α mRNA, (C) IL-1β and (D) MCP-1 (n = 6; *P = 0.023; **P = 0.009; ***P < 0.001; #P = 0.003; ##P = 0.005 with one-way ANOVA and Bonferroni post hoc analysis). Anti-HMGB1 = neutralizing HMGB1 monoclonal antibody.


35



bone marrow cells have been shown to support immune cell function.29

Given that HMGB1 resides in the nucleus and functions as an essential nonhistone chromatin-binding protein, there are no HMGB1 knockout animals. For this study we then decided to use a pharmacologic strategy to quickly deplete the pool of systemic macrophages. However, because clodrolip is highly toxic to all phagocytes, it can increase the risk of post-surgical infections, generating a phenotype of its own.30 With a single dose, we did not observe loss of weight or other signs of sickness within 3 days. As this was a short-term study, focus-ing on the acute exaggeration phase of neuroinflammation, we are unable to extrapolate from these data the long-term effects and did not perform any long-term study with clodrolip. Clo-drolip should be considered a tool for mechanistic studies.

In previous reports using this model of surgical trauma, we documented the effectiveness of preoperative administra-tion of interventions such as IL-1 receptor antagonist, anti-tumor necrosis factor monoclonal antibody and activation of the α7 subtype of nicotinic acetylcholine receptor in pre-venting postoperative memory dysfunction.1,2,5 Each of these affect important host defense mediators, and risk of infec-tion needs to be evaluated before considering these agents.

These studies on the initiation of trauma-induced cogni-tive decline, coupled with our previous reports on the reso-lution of postoperative cognitive decline,1,2,5 sets the stage for the development of an ex vivo bioassay that can test the function of the innate immune response to trauma. Such an assay may be capable of prospectively identifying surgical patients at increased risk for the development of exaggerated and persistent cognitive decline; stratification of a surgical cohort, enriched for the development of cognitive decline, can result in a randomized trial to test efficacy of interven-tions using fewer surgical patients.

The authors thank members of the Hellman Laboratory, University of California, San Francisco, San Francisco, California, and Pedro Gambus, M.D., Staff Anesthesiologist, Department of Anesthesia, Hospital Clinic de Barcelona, Spain, for their support, and Mitchell Marubayashi, M.Sc., and Stuart Dilg, B.A., volunteers in the Maze Laboratory, University of California, San Francisco, San Francisco, California, for their assistance in the behavior tests.

References

Role of interleukin-1beta in postoperative cognitive dysfunc-

-

-

α in

their effects in the dentate gyrus. Prog Brain Res 2007;

-

-

-perfusion. Crit Care 2009; 13:R174

-

A

--

-


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H: Receptor for advanced glycation end products-binding -

-

Poll T: Role of toll-like receptors 2 and 4, and the recep-

in vivo

-


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The Neuroinflammatory Response of Postoperative Cognitive Decline

38

The neuroinflammatory responseof postoperative cognitive decline

Susana Vacas†‡, Vincent Degos†§, Xiaomei Feng†, and Mervyn Maze†*

†Department of Anesthesia and Perioperative Care, University of California San Francisco, 521Parnassus Avenue, San Francisco, CA 94143-0648, USA; ‡Programme for Advanced MedicalEducation, Lisbon, Portugal, and §Departments of Anesthesiology and Critical Care, GroupeHospitalier Pitie-Salpetriere, Assistance Publique-Hopitaux de Paris, Universite Pierre et MarieCurie, Paris, France

Background: Aseptic surgical trauma provokes a homeostatic neuroinflammatory

response to promote healing and protect the organism from further injury.

When this response is dysregulated, harmful consequences can follow, including

postoperative cognitive decline.

Sources of data: We performed a comprehensive search on PubMed related to

postoperative cognitive dysfunction (POCD).

Areas of agreement: Although the precise pathogenic mechanisms for POCD

remain unclear, certain risk factors are known.

Areas of controversy: The mechanisms that lead to exaggerated and persistent

neuroinflammation and the best way to counteract it are still unknown.

Areas for developing research: It is imperative that we identify the underlying

processes that increase the risk of cognitive decline in elderly surgical patients.

In this review we explore non-resolution of inflammation as an underlying cause

of developing exaggerated and persistent POCD. If interventions can be

developed to promote resolution of neuroinflammation, the patient’s

postoperative recovery will be enhanced and long-term consequences can be

prevented.

Keywords: neuroinflammation/cognitive decline/neurodegeneration/surgery,sleep

Accepted: February 1, 2013

British Medical Bulletin 2013; 106: 161–178

DOI:10.1093/bmb/ldt006& The Author 2013. Published by Oxford University Press. All rights reserved.

For permissions, please e-mail: [email protected]

*Correspondence address.

Department of

Anesthesia and

Perioperative Care,

University of California

San Francisco, 521

Parnassus Avenue,

San Francisco, CA 94143-

0648, USA.

E-mail: mazem@

anesthesia.ucsf.edu

Published Online April 4, 2013

at University of California, San Francisco on January 22, 2014

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Introduction

Impairment of cognition after surgery is a disturbing reality.Postoperative delirium (POD) is characterized by inattention, disorga-nized thinking and altered level of consciousness with acute onset andfluctuating course. While some patients develop POD, others develop alater onset form of postoperative cognitive decline known as post-operative cognitive dysfunction (POCD).

It is estimated that POCD occurs in .10% of non-cardiac surgicalpatients1 over 60 years old,2 and is independently associated with poorshort-term and long-term outcomes, including an increased risk ofmortality.3,4 Although several risk factors have been identified, theexact pathophysiology that underlies POCD remains undefined.

The thesis that neuroinflammation is a possible cause of POCD hasrecently been tested. Data from preclinical studies support the conceptthat inflammation is a possible pathogenic mechanism for post-operative cognitive dysfunction.5–9 Increased expression of interleukinsin mouse hippocampus following minor surgery was associated withcognitive decline,5,10 corroborating the view that surgery-induced neu-roinflammation can result in cognitive impairment. Surgical patientsexhibit elevations of pro-inflammatory cytokines in both the centralnervous system and the systemic circulation, the extent of which mayrelate to the degree of cognitive decline.11,12 Assuming that neuroin-flammatory changes noted postoperatively in rodents also occur inhumans, reasons must be sought why POCD is a relatively infrequentclinical event (+10%) whereas neuroinflammation always occurs.Among the possibilities include the fact that the neuroinflammatorychanges are usually evanescent and do not normally cause a long-lasting consequence in animal models. Several clinical conditions cantransform the self-limiting postsurgical neuroinflammatory responseinto one that is persistent. The persistence in neuroinflammation maybe due to dysfunction in the inflammation-resolving mechanism.Alternatively, a normal neuroinflammatory response to surgery mayhave long-lasting detrimental effects in settings of neurological path-ology, whether clinically evident or not. In a recently completed pro-spective study, patients who had previously suffered a stroke were moreat risk of POCD even though they had no neurological sequelae fromthe remote stroke event.13,14 Epidemiologic studies have suggested thatneurodegenerative disorders, such as Alzheimer’s disease (AD), may beaccelerated by surgery15–18 and that exacerbates dementia in ADpatients19 while increasing the occurrence of dementia20. However, thisrelationship has recently been challenged21.

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This review considers the possible role of inflammation in the devel-opment of POCD in the setting of underlying systemic and neurologicdiseases; it will also discuss future research possibilities that might helpidentify vulnerable patients with whom interventional strategies couldbe invoked.

Clinical condition

Clinical studies distinguish POD from POCD. The Diagnostic andStatistical Manual of Mental Disorders (DSM-IV-TR)22 defines thestandards necessary for a diagnosis of delirium. These include disturb-ance of consciousness, change in cognition, inattentiveness and a fluc-tuating time course. Clinically, this diagnosis is often made using theconfusion assessment method (CAM), a simple four-question screeningtool that has a sensitivity of 94% and a specificity of 89%. A variationknown as the CAM-ICU is often used in the intensive care unit (ICU)setting and is useful in sedated or intubated patients. Diagnosis ofdelirium in the cases of dementia can also be accomplished with theappropriate tools.23

There are three subtypes of delirium: hyperactive (25%), hypoactive(50%) and mixed (25%). The hypoactive subtype is the most frequent-ly missed and may actually be associated with greater mortality thanthe hyperactive subtype.24 The incidence of POD is between 10 and55% in postoperative patients, depending on the type of procedure thepatient underwent, with a higher percentage in orthopedics comparedwith general surgery patients.25 Additionally, the incidence of POD issignificantly higher in elderly patients. It is estimated that up to 50%of elderly patients suffer from delirium after surgery.26 Furthermore,over 40% of hospitalized patients with delirium suffer from psychoticfeatures, including visual hallucinations. It typically manifests itselfwithin 24–48 h postoperatively, with exacerbation of symptoms atnight, perhaps due to circadian disturbances. The implications of PODare significant. It is associated with increased morbidity and a 1-yearmortality that approaches 40%.27 The estimated healthcare-associatedcosts related to delirium are astronomic. They made up nearly$7 billion of Medicare expenditures in 2004.28

Persistent cognitive decline is predominantly seen in the elderly29 andis termed POCD. POCD is diagnosed by the International Society ofPostoperative Cognitive Dysfunction as subtle deficits in one or morediscrete domains of cognition, e.g. attention, concentration, executivefunction, verbal memory, visuospatial abstraction and psychomotorspeed.30 This condition typically develops over weeks to months,and is long-lasting. The diagnosis requires sensitive presurgical and

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postsurgical neuropsychiatric testing.31 As a consequence of this com-plication, patients can lose their employment or independence, whichcan seriously reduce their quality of life.4,14,32

An international multicenter study of POCD (ISPOCD) reportedmemory impairments in more than a quarter of the patients 1 week afternon-cardiac surgery and in 10% after 3 months in patients older than60 years. Follow-up studies have shown similar incidences with somereports describing cognitive decline persisting for up to 1 year aftersurgery. Both because the number of major surgical interventions(requiring anesthesia) exceeds 230 million worldwide33 and because ofthe increasing prevalence of surgical interventions in patients .65 yearsold, this age group will become the largest segment of surgical patientsby 2020.34 If current rates hold steady, we can expect that millions ofelderly patients will run the risk of developing POCD. This possibilityraises the stakes considerably: not only on an individual level, but alsoon a societal scale.

Risk factors

Studies have sought to identify factors that may contribute to POCD,some of which include surgery, anesthesia and patient-related factors.

Non-modifiable patient factors

Patient-related risk factors include: advanced age,1,2,14,35–42 educa-tion,1,2,14,38,40,42 genetic polymorphism (apolipoprotein E4)43–45 andseveral other comorbidities.

Advanced age is associated with infirmities, many of which can besuccessfully treated with surgery. Unfortunately, persistent cognitiveimpairments can develop as a side effect of these surgical procedures.29

An increase in the aging population and improvements in anesthesiaand surgery have led to increases in the number of elderly patientsundergoing surgery. Therefore, it is likely that postoperative centralnervous system dysfunction will become increasingly common.

Systemic disease: metabolic syndrome

Roughly 25% of the 45 million surgical patients in the US have meta-bolic syndrome (MetaS),46 though its precise definition and diagnosticcriteria continue to evolve.47 MetaS, comprising insulin resistance,visceral obesity, hypertension and dyslipidemia, increases the risk of

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postoperative complications contributing to a significant higher mortal-ity rate.48–51 While each of the subphenotypes that define MetaS has astrong genetic component, lifestyle factors that contribute to thiscluster of conditions include sedentary behavior and a diet with a highcaloric content from saturated fats and/or simple carbohydrates.52

Many complications of MetaS (including atherosclerosis) are inflamma-tory in nature and the pathologic metabolism in adipose stores may bethe source of pro-inflammatory adipokines.53 Conversely, with littleadiponectin to attenuate activation of the transcription factor NF-kB inmacrophages, expression of genes for pro-inflammatory cytokines isincreased;54 up-regulated NF-kB activity in morbid obesity can be recti-fied with adiponectin.55 Roughly a quarter of the American adultpopulation have MetaS; 50% of cardiac surgery patients are affectedwith MetaS.56 Recent evidence indicates that patients suffering fromMetaS may be particularly susceptible to POCD.50,57

Neurologic disease

The two most common causes of dementia are vascular dementia andAD, although most cases of dementia have both types of pathology.Pre-operative cognitive impairment, such as mild cognitive impairment(possible prodrome for AD), may already exist in many elderly patientswho incidentally present at surgery. Although perioperative cognitivedecline and AD may share certain neuropathologic and biochemicalmechanisms, there is no direct evidence linking the involvement ofAD-type pathogenic mechanisms and POCD in humans and only weakepidemiological evidence associating surgery with onset of AD.58

Epidemiological studies have suggested that neurodegenerative disor-ders, including AD, may be accelerated by surgery.15 However, largeretrospective studies have thus far not associated surgery or anesthesiawith further dementia and AD.21 Evidence from animal models sug-gests that inhaled anesthetic exposure increases pathology normallyassociated with AD, including increase in b-amyloid peptide andb-acting cleavage enzyme;59 anesthesia-induced hypothermia increasedtau hyperphosphorylation by decreasing phosphatase 2A activity.60

Symptomatic pre-operative neurologic diseases, including dementiaand any disease of the central nervous system, are often considered ex-clusion criteria for POCD studies.14 Interestingly, cerebral vascularaccidents (without residual deficit) were associated with risk factors forPOCD, suggesting for the first time that a pre-operative ischemic braininsult could influence the possibility of POCD. However, no causallink between pre-operative cerebral vascular accident and POCD hasbeen established yet in experimental or human investigations.





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Postoperative neurodegeneration, akin to AD, is observed in agedrats;61 while inflammation-resolution mechanisms have not been inves-tigated, it is known that these mechanisms decline with advancingage.62,63 AD-type neurodegeneration is accelerated by neuroinflamma-tion,64 raising the possibility that failure to resolve neuroinflammationmay provoke neurodegenerative changes that cause persistent cognitivedecline. It is noteworthy that anesthesia alone, at higher concentrationsand for more prolonged periods, has been reported to produce AD-likeneurodegeneration although this has been challenged.65–68

The anesthetized state

There are several risk factors directly related to anesthesia that may beinvolved in the pathogenesis of POCD. Intra-operative hypotension,hypoxia, embolism, medications and postoperative infections have allbeen described as risk factors for POCD. Although general anestheticsare capable of producing long-lasting cognitive dysfunction undercertain circumstances,69,70 the incidence of POCD is similar after re-gional and general anesthesia,71 the reason why attention has beenfocused on the role of the surgical intervention itself in the genesis ofthis condition. Postoperative pain is a possible etiologic factor inPOCD.72 Epidural analgesia with local anesthetics and/or opioids maybe better than parenteral analgesics for the control of postoperativepain and the prevention of early POCD.73 Furthermore, patients whoare prescribed postoperative oral analgesics experience less POCD com-pared with those receiving parenteral medication.74 Even thoughstudies have shown the potential benefit of intra-operative monitoringof anesthetic depth and cerebral oxygenation as a pragmatic interven-tion to reduce postoperative cognitive impairment,75 this factor stillremains a controversial issue as some studies have shown no relationbetween deeper states of anesthesia and the emergence of POCD.76 Insupport of the latter position, a number of recent studies show thatanimals exposed to short-duration isoflurane do not develop memoryimpairment.5

Sleep

Sleep is crucial for the repair of many types of injury and disease,especially with regard to the central nervous and immune systems; italso has anabolic, restorative properties that improve both neurocog-nitive and immune function. During non-rapid eye movement(NREM) sleep, slow-wave activity performs a homeostatic function

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to reduce the strength of synapses that has been acquired duringwakeful activity.77 This synaptic homeostasis improves subsequentcognitive function by allowing new changes in synaptic strength. Forexample, both NREM and REM sleep are necessary for the consoli-dation of learning and memory while sleep deprivation results incognitive dysfunction.78

Polysomnographic studies revealed extreme sleep disruption in ICUpatients with decreases in total sleep-time, altered sleep architecture(predominance of stage 1 and 2 sleep, decreased or absent stage 3NREM and REM sleep) and sleep fragmentation;79,80 also, up to 50%of the total sleep-time occurred during daytime. Lack of sleep hygieneresults in cognitive dysfunction,81,82 contributes to delirium,83 adverse-ly affects immunity84,85 and independently increases both morbidityand mortality.86 Sleep disruption during hospital care has the potentialto adversely impact on patients’ outcome and also provides a direct fi-nancial cost with respect to the length of hospital stay and depletion ofhealthcare resources.

Preclinical studies have shown the detrimental effect of lack of sleepon cognition.87 In addition, perioperative sleep deprivation induced sig-nificant neuroinflammatory changes.88–90 The exact mechanism forthe deleterious consequences of a ‘double hit’ (aseptic surgical traumaand sleep deprivation) is still poorly understood, though it has beenshown to increase the expression of inflammatory cytokines in thebrain.88,89 Sedative practices have also shown to be a main causativefactor for this disruption.91,92

Anesthetics have different action targets and ultimately different con-sequences. The pivotal work of the MENDs trial92–94 indicated thebenefits of a specific anesthetic agent, dexmedetomidine, in theoutcome of ICU populations. a2 adrenergic agonists converge on sleeppathways within the brainstem, while those that act by modulating theGABAA receptor converge at the level of the hypothalamus. Severalstudies have now demonstrated the association between the use ofbenzodiazepine (BZD) and increased incidence93 and duration95 of de-lirium in ICU patients, although the relationship of the developmentand duration of delirium to sleep disruption has not yet been thorough-ly ascertained. Acute withdrawal from long-term sedation with BZDsand opiate narcotics results in profound sleep disruption.96 Thus,thoughtful attention must be paid in selecting an anesthetic agent thatbest mimics natural sleep in order to decrease the decline of cognitivefunction. These efforts have to be maintained for the entireperioperative period.





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Neuroinflammatory response to surgery

Activation of the immune system after surgery is associated with cognitivedecline.5,7 Tissue trauma releases damage-associated molecular patterns(DAMPs) that are recognized by pattern recognition receptors (PRRs),which then trigger an immune response in a manner remarkably similar tothat of microbial-derived pathogen-associated molecular patterns(PAMPs).97–99 Among PRRs, Toll-like receptors (TLRs) are of critical im-portance, recognizing various ligands (including PAMPs and DAMPs) andactivating TLR signals along different pathways, thereby increasing the syn-thesis and release of pro-inflammatory mediators. Although the function ofTLR4 during lipopolysaccharide (LPS) endotoxemia100 has been deeplyexplored, the pathways of infection-mediated neuroinflammation and cog-nitive decline seem to be distinct from that of aseptic surgical trauma.7 Oneof the most important DAMPs (released from dead or dying cells throughnon-apoptotic processes101) is high-mobility group box 1 (HMGB1).HMGB1 can bind and signal through a family of PRRs that are evolution-arily conserved.102 Clinical conditions such as sepsis, arthritis and stroke allrelease massive amounts of HMGB1.103 Both DAMPs and PAMPs con-verge on NF-kB to increase synthesis and release of pro-inflammatory cyto-kines,104 including TNF-a, which disrupt blood brain barrier (BBB)integrity.5,7,8,105 Early activation of the innate immunity through DAMPs(HMGB1 and cytokines) will introduce the initial response to surgeryresulting in neuroinflammation and concomitant cognitive decline.7

Following injury, this ‘transient’ inflammation is a necessary tissuerepair process that promotes healing. Macrophages are highly heteroge-neous hematopoietic cells found in nearly every tissue in the body andhave been defined as the sentinels of the innate immune system. Theyare also key players in the resolution of inflammation and are criticalto tissue repair, wound healing and restoration of homeostasis.106 Inaddition, macrophages are responsible for sensing, integrating and ap-propriately responding to a bewildering array of stimuli from its localmicroenvironment. Macrophage responses are mediated through twodistinct and mutually exclusive activation programs, termed classical(or M1) and alternative (or M2).107 These activation programs wereinitially defined by their antimicrobial activities; classical activationoccurs in response to bacterial infections and results in highly inflam-matory macrophages with high phagocytic and bactericidal poten-tial.107 In contrast, alternative activation occurs in response to parasiticinfections and promotes antiparasitic functionalities as well as thoseinvolved in tissue repair and remodeling.108 Both programs promotedifferentiation of neighboring macrophages to their same activationstate and potently inhibit maturation of the other.

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Neuroinflammation after surgery is likely to include a pro-inflamma-tory phase and an anti-inflammatory phase (neural and humoral path-ways mediate the switch between these two phases).109,110 With respectto the humoral factors, resolvins, lipoxins and maresins (macrophagemediators in resolving inflammation), derived from polyunsaturatedfatty acids (PUFAs), are novel lipid mediators that promote the reso-lution of inflammation. Protective actions of D-series resolvins havebeen observed in both acute and chronic inflammatory diseases, such asperitonitis, ischemia/reperfusion injury and sepsis. Resolvins both limitPMN infiltration and enhance macrophage phagocytosis by transducingsignaling mechanisms that originate at specific receptors on humanPMN, monocytes and macrophages. Lipoxins were the first mediatorsrecognized to have both anti-inflammatory and pro-resolving actions.111

Maresins are newly identified macrophage mediators with the sameproperties.112 They are capable of dampening the pro-inflammatory re-sponse by inhibiting macrophage NF-kB activity and polarizing macro-phages into an M2 phenotype.113–115 Dietary supplementation withPUFAs in patients with MetaS corrects many of the metabolic derange-ments116 as well as the pro-inflammatory markers.117 Regarding the re-solving inflammatory state mediated by neural factors, DAMPs activatethe efferent arc of the inflammatory reflex via NF-kB, termed the cholin-ergic anti-inflammatory pathway. At its splenic nerve terminus, vagaloutflow releases adrenergic agonists (rather than the usual cholinergicneurotransmitter); these catecholamines activate b2 adrenergic receptorson CD3 T lymphocytes that are capable of synthesizing and releasingacetylcholine needed to mediate inhibition of macrophage NF-kB activ-ity by signaling through the a7 subtype of nicotinic acetylcholine recep-tors (a7 nAChR). Ultimately, it inhibits synthesis and release ofpro-inflammatory cytokines from circulating immunocompetentcells.104,118,119

The neural cholinergic reflex is very important in resolving theinflammatory pathogenesis of several diseases including sepsis,120

rheumatoid arthritis121 and colitis.122

Furthermore, the cholinergic anti-inflammatory pathway also modu-lates the function of T regulatory cells,123 which influences the produc-tion of anti-inflammatory cytokines (IL-10 and IL-4)124 and alternativemacrophage activation that promotes the resolution of inflamma-tion.125 IL-4 is the cytokine responsible for polarizing macrophagefrom the pro-inflammatory classically activating (M1) to the reparativealternatively activating (M2) phenotype. In the mouse models of type IIdiabetes, there is a relative lack of T regulatory cells and an imbalanceof M1/M2 macrophages, which might contribute to persistent low-grade inflammation.126 Abnormalities of the switching mechanism maycause a non-resolving chronic inflammatory state that could create the





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circumstances for persistent cognitive decline. Recently, it has beenshown that MetaS will contribute to exaggerated and persistent PODin a rat model of tibial fracture.127 In addition, dysfunctional resolutionof inflammation was found to be associated with behavioral deficitsafter surgery in metabolic syndrome rats.128 However, the detailedmechanisms are still unclear and more research is warranted.

Studies have shown the importance of this reflex for resolvingDAMP-induced neuroinflammation, pro-inflammatory cytokine release,neuroinflammation and cognitive decline; stimulating the a7 nAChR inmacrophages, inhibited NF-kB activity which in the quiescent state pre-cludes postoperative memory impairment by preventing monocyte mi-gration into the hippocampus.129 Drugs used clinically in theperioperative period, including anesthetics, exert anti-cholinergic activ-ity that may translate into non-resolution of inflammation and PCD inthe form of delirium.130 Advanced age is associated with decline in cho-linergic function, which may be relevant in explaining the high preva-lence of POCD in elderly patients.

When inflammation does not subside, it can contribute to the patho-genesis of diseases.106 Through a permeable BBB, CCR2-expressingbone marrow-derived macrophages (BM-DM) are attracted, by thenewly expressed chemokine, MCP-1, into the brain parenchyma. Themacrophages synthesize and release a variety of pro-inflammatory cyto-kines that interfere with processes required for memory. Macrophage-specific Ikappa B kinase (IKK)b coordinates activation of NF-kB; whenit is deleted, it prevents BBB disruption and BM-DM infiltration into thehippocampus following surgery.129 Transgenic mice that overexpressHsp72 and inhibit NF-kB activity have attenuation of postoperativeneuroinflammation and cognitive decline.131,132

Learning and memory processes rely on the hippocampus, a region ofthe brain that contains a large number of pro-inflammatory cytokinereceptors.133,134 The hippocampus has the highest density of IL-1receptors, and although IL-1b is required for normal learning andmemory processes, higher levels can also produce diminished cognitivefunction.135,136 Recent studies have suggested a role for cytokines suchas interleukins-1 and -6 in the genesis of POCD. The relative preva-lence of the TNF-a receptor, as well as other PRR, on the endotheliumof this brain region may account for its vulnerability to systemicpro-inflammatory cytokines.137

Surgical trauma in animal models is associated with the persistent ac-tivation of macrophages in the CNS that are capable of maintaning ele-vated levels of IL-1b, and other pro-inflammatory cytokines, such asTNF-a and IL-65. These changes are correlated with cognitive dysfunc-tion seen in animal models (contextual fear memory,5,7,9 spatial learn-ing99,138 or reversal learning98). IL-1 is released in response to a wide

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range of infectious, inflammatory or toxic insults, IL-1.99,139

Sub-clinical inflammation following administration of LPS substantiallyincreases IL1-b levels and cognitive deterioration after surgery.8 In add-ition, several studies suggest that the marked and sustained expressionof inflammation-related enzymes, such as cyclooxygenase- 2, plays animportant role in secondary events that amplify cerebral injury after is-chemia.140 Patients also exhibit a robust neuroinflammatory responseto peripheral surgery with an initial rise in pro-inflammatory cytokinesin the CSF12,141 as well as release of reactive oxygen species andendothelins.102,142,143

Can we identify vulnerable patients pre-operatively?

We believe that non-resolution of inflammation is a factor that contri-butes to the pathogenesis of POCD, which in turn significantlyincreases morbidity and mortality in surgical patients. We might bewitnessing a perfect and unfortunate storm of factors with regard toPOCD: to put it another way, given the rise in surgeries and increasingnumber of elderly patients worldwide, the stakes could not be higher.

Vulnerable patients need to be identified and risk/benefit should beconsidered before contemplating the efficacy of surgical intervention.Advanced age, MetaS, patients prone to AD and poor selection ofsedative agents may each result in an exaggerated and persistent neu-roinflammatory response to surgery. Further studies are needed tounderstand which patients will suffer from exacerbated inflammationwith the aim of developing a biomarker that is quick to assay for clini-cians and easy to comprehend for patients and their families.Concurrently, clinical interventions need to be further developed topromote the resolution of neuroinflammation in postoperative patientpopulations. Following both tracks, we anticipate that postoperativerecovery for vulnerable patients will be greatly enhanced and possiblelong-term consequences, such as postoperative neurodegeneration, canbe significantly reduced.

Conclusions

In the majority of patients, postoperative neuroinflammation is part ofthe normal protective mechanism to peripheral trauma and resolvesproperly with no residual cognitive consequences. Indeed, it is alsopossible that surgery for a chronic inflammatory disease may result incognitive improvement by eliminating disease-inducing cognitive impair-ment that may be associated with chronic inflammatory disease. That





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said, some risk factors, such as MetaS, patients prone to AD and poorselection of sedative agents, may each promote the intractable persist-ence of neuroinflammatory response to surgery. For an increasingnumber of patients with advanced age, POCD is alarmingly common,making postoperative central nervous system dysfunction a loomingpublic health crisis, given the world’s rising elderly population.

Additional study is essential to elucidate the risk factors, preventativestrategies and the underlying pathophysiology of this disorder. If thesestudies can succeed in identifying patients prospectively, or earlyenough in the advent of persistent inflammation, interventions can bejudiciously and appropriately launched.

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Sleep

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Consequences of Sleep Fragmentation in the Perioperative Setting

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Consequences of Sleep Fragmentation in the Perioperative Setting Susana Vacas, Vincent Degos, Mervyn Maze

1. Author: Susana Vacas (MD)

• Title: MD • Affiliation: Department of Anesthesia and Perioperative Care, University of California San

Francisco, San Francisco, CA, USA • Email: [email protected] • Contribution: This author helped design the study, conduct the study, collect the data, analyze

the data and prepare the manuscript • Attestation: Susana Vacas approved the final manuscript. Susana Vacas attests to the integrity of

the original data and the analysis reported in the manuscript. Susana Vacas is the archival author. • Conflicts of interest: None.

2. Author: Vincent Degos (MD, PhD)

• Title: MD, PhD • Affiliation: INSERM, Paris, France • Email: [email protected] • Contribution: This author helped analyze the data and prepare the manuscript • Attestation: Vincent Degos approved the final manuscript. Vincent Degos attests to the integrity

of the original data and the analysis reported in the manuscript. • Conflicts of interest: None.

3. Author: Mervyn Maze (MB, ChB)

• Title: MB, ChB • Affiliation: Department of Anesthesia and Perioperative Care, University of California San

Francisco, San Francisco, CA, USA • Email: [email protected] • Contribution: This author helped design the study, analyze the data and prepare the manuscript • Attestation: Mervyn Maze approved the final manuscript. Mervyn Maze attests to the integrity of

the original data and the analysis reported in the manuscript. • Conflicts of interest: None.

Name of Department and Institution: This work is presented by the Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, CA, USA

Short Title: Sleep fragmentation and surgery

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ABSTRACT (400 words) Background: Sleep is integral to biological function and sleep disruption can result in both physiological and psychological dysfunction. The acute cognitive consequences of sleep loss has been an active field of recent investigation, evidence suggests that sleep disruption in critically ill older adults can result in acute decrements in cognitive functioning. Surgery activates the innate immune system, inducing neuroinflammatory changes that interfere with cognition. The fact that patients with sleep disorders have an increased likelihood of exhibiting postoperative delirium encourages us to investigate the contribution of perioperative SF to the neuroinflammatory and cognitive responses of surgery. Methods: The effects of 24h sleep fragmentation (SF) and surgery were explored on adult C57BL/6J male mice. SF procedure started at 7 am with the home-cages being placed on a large platform orbital shaker cycled every 120 seconds (30 sec on/90 sec off). This procedure lasted for 24h. Stabilized tibia fracture was performed either before or after the 24h SF procedure. Separate cohorts of mice were tested for systemic and hippocampal inflammation and cognition. Results: Twenty-four hours of SF induced non-hippocampal memory dysfunction and increase in systemic IL-6. SF and surgery caused hippocampal-dependent memory impairment, although memory impairment was not exacerbated by combining SF with surgery. One day after either SF or surgery there was a significant increase in IL6 mRNA and TNF-alpha mRNA. These increments were more pronounced when either pre or post operative SF was combined with surgery. Conclusions: We show that while SF and surgery can independently produce significant memory impairment, perioperative SF significantly increased hippocampal inflammation without further cognitive impairment. The dissociation between neuroinflammation and cognitive decline may relate to our use of a sole memory paradigm that does not capture other aspects of cognition, especially learning.

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INTRODUCTION Importance of sleep for memory Sleep restores and repairs several mechanisms related to learning and memory.1 Slow wave activity weakens the synaptic strengthening that occurs during wakefulness and restores the brain to a state that is capable of appropriately processing sensory input in succeeding periods of wakefulness.2,3 Importance of continuous sleep Unlike sleep deprivation, sleep fragmentation (SF) does not necessarily affect total sleep time, but reduces the total amount of time spent in the deeper levels of sleep. Further, the brain’s capacity to successfully respond to cognitive challenges through compensatory recruitment becomes overwhelmed if the patient is not presented with appropriate and continual sleep. Optogenetic activation of orexinergic neurons, which play a key role in arousal processes, demonstrate that a minimal unit of uninterrupted sleep is crucial for memory consolidation.4 Interrupted, fragmented or restricted sleep is a consequence of many diseases, including the respiratory disorder Obstructive Sleep Apnea (OSA). OSA consequences OSA is not just highly disruptive to steady breathing but also to constant and continuous sleep. It is also the most pervasive sleep disorder, increasing the risk of heart failure, stroke, coronary heart disease, and cognitive impairment.5-8 Studies also suggest that the cognitive and structural deficits in OSA may be secondary to SF and repetitive nocturnal intermittent hypoxemia.9 Further, it is an independent risk factor for the development of postoperative delirium.10 Surgery and sleep Cognitive dysfunction, either alone or as part of delirium, is an adverse outcome for surgical/ICU patients that result in serious consequences, including higher mortality rates. Surgery activates the innate immune system, inducing neuroinflammatory changes that cause subsequent decline in cognitive function.11 These changes are necessary in order to defend the organism from further injury, but when this response is dysregulated, they can lead to cognitive decline.11,12 SF by itself can also induce activation of inflammatory mechanisms, more specifically TNF-α-dependent pathways, which can itself impair the immune system.13,14 Polysomnographic studies have revealed profound sleep disruption in intensive care unit patients, with decreases in total sleep-time, altered sleep architecture, and SF.15

The fact that patients with sleep disorders have an increased likelihood of exhibiting postoperative delirium encourages us to investigate the contribution of perioperative SF to the neuroinflammatory and cognitive responses of surgery.

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METHODS Animals All the experiments were conducted under approval of the Institutional Animal Care and Use Committee of the University of California, San Francisco, and conformed to the National Institutes of Health Guidelines. Experiments were performed using 12-14 weeks old male C57BL/6J mice (Jackson Laboratories, Bay Harbor, ME), under a 12:12 hours light-dark cycle, in a constant temperature and humidity controlled environment, with free access to food and water. Animals were tagged and randomly allocated to each group before any treatment or procedure (fig. 1 Study design). Researchers blinded to the group assignment performed all neurobehavioral tests. For those animals in which behavior was assessed, no blood harvesting or tissue sampling was performed, in order to obviate possible confounding effects of fear conditioning testing on inflammatory markers.16 Body weight was measured before any procedure or treatment and daily after that. Sleep fragmentation Sleep was interrupted for 30 sec every 2 min as previously described.17 Briefly, animals were kept in their home cage and placed on an analog orbital shaker (OS-500, VWR, Champaign, IL). Repetitive on/off cycling of the shaker (30 sec on/90 sec off), set at 100 rpm, was controlled by a timer (traceable controller, Fisher Scientific, Pittsburg, PA). A metal cage cardholder was suspended from the top of the cage, creating an additional audible stimulus when the holder knocked against the side of the cage. Standard laboratory chow was supplemented with aqueous gel. Surgical trauma Under aseptic conditions, groups of mice were subjected to a tibial fracture as previously described.18 Mice were anesthetized with 2% isoflurane, and analgesia was achieved with buprenorphine 0.1 mg/kg subcutaneously immediately after anesthetic induction and before skin incision. Warming pads and temperature-controlled lights were used to maintain body temperature at 37±0.5ºC. Aseptic conditions were maintained throughout the procedure. The entire procedure from induction of anesthesia to end of surgery lasted 10 ± 3 min. Novel object recognition (NOR) NOR is based on the spontaneous tendency of rodents to spend more time exploring a novel object than a familiar one. The choice to explore the novel object reflects the use of non-hippocampal learning and recognition memory. For our study we used a previously described protocol.19 Briefly, animals were familiarized for two days to the testing environment that differed from the home cage. In a first phase, animals were presented with the to-be familiarized object in the testing environment (10 min). Following the sample-object exposure, animals were returned to the home-cage for a retention period (1 hour). In the second phase, animals were returned to the environment and presented with two-objects: the previously experienced sample object and a novel object (3-5 min). Object recognition was distinguished by the animal spending more time exploring the novel object. To score time of object interaction from video, XNote Stopwatch® was used. Trace-fear Conditioning (TFC) Fear conditioning is used to assess learning and memory in rodents, which are trained to associate a conditional stimulus, such as a tone, with an aversive, unconditional stimulus, such as a foot-shock20. Freezing behavior is an indicator of aversive memory that is measured when subjects are re-exposed to the conditional stimulus. For this study we used a previously published paradigm.11,21,22 Briefly, the behavioral study was conducted using a conditioning chamber (Med. Associates Inc., St. Albans, VT) and an unconditional stimulus (two periods of 2-second foot-shock of 0.75 mAmp). Three days after the training session, mice were returned into the same chamber where training had occurred for a context test, during which no tones or foot-shocks were delivered. Freezing behavior in response to the context was

62

recognized by the software as a total lack of movement excluding breathing but including movement of fur, vibrissae and skeleton. The percentage of time spent freezing over the total time spent in the chamber to accomplish the test was used to score memory and learning abilities. A decrease in the percentage of time spent freezing indicated impairment of these abilities. Systemic Inflammatory response After termination of both SF procedure and surgery (7 am on day one), blood was collected transcardially under terminal isoflurane anesthesia into heparin-coated syringes, Samples were centrifuged at 3400 rpm for 10 minutes and plasma was collected and stored at -80˚C until assaying. Blood samples taken from animals without intervention served as controls. Plasma IL-6 was measured using a commercially available ELISA kit (Invitrogen, Grand Island, NY). Neuroinflammatory response to surgery After termination of both SF procedure and surgery (7 am on day one), the hippocampus was rapidly extracted under a dissecting microscope and placed in RNAlater™ solution (Qiagen, Valencia, CA, USA). Total RNA was extracted using RNeasy Lipid tissue Kit (Qiagen). Extracted RNA was treated with recombinant DNase I using a RNase-Free Dnase set™ (Qiagen). mRNA concentrations were determined with a ND-1000 spectrophotometer (NanoDrop® Thermo Fisher Scientific, Wilmington, DE) and mRNA was reverse transcribed to cDNA with a High Capacity RNA to-cDNA Kit (Applied Biosystems, Carlsbad, CA). TaqMan Fast Advanced Master Mix (Applied Biosystems) and specific gene expression assays were used for qPCR as follows: beta-actin (ACTB) (NM_007393.1), IL-6 (Mm00446190_m1), TNF-a (Mm00443258_m1) and IL-1b (Mm01336189_m1). qPCR was performed using StepOnePlus™ (Applied Biosystems). Each sample was run in triplicate, and relative gene expression was calculated using the comparative threshold cycle ΔΔCt and normalized to ACTB. Results are expressed as fold-increases relative to controls. Statistical analysis Data are presented as mean ± standard deviation (SD). For comparisons of more than two groups, means were compared using one-way analysis of variance (ANOVA) followed by t-tests with a Bonferroni-corrected alpha level. Based on previous freezing time data23, we estimated that a sample of 10 C57BL/6J surgical mice per group was necessary to demonstrate a 20% increase in percentage freezing time, with 80% power at the 0.0125 alpha level (after adjusting for 4 comparisons) to reach a significant difference. A two-tailed p value < 0.05 was considered statistically significant for 2-group comparisons and the significance threshold was adjusted for multiple comparisons with a Bonferroni correction. Prism 6 (GraphPad Software Inc, La Jolla, CA) was used to conduct the statistical analyses.

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RESULTS Twenty-four hours of sleep fragmentation induces non-hippocampal memory dysfunction and increase in systemic IL-6 NOR test was assessed after 24h of SF. Sleep fragmented animals were unable to differentiate between the familiar and novel object (time of object interaction 6.02±3.43 sec versus 5.56±3.08 sec, ns) as opposed to the control group spending significantly more time at the novel object relative to the familiar (7.23±2.61 sec versus 11.93 ±5.88 sec, p<0.01) (fig.2 NOR control vs SF). This dysfunction was associated with an increase in systemic IL-6 (p<0.01). (Fig.3 ELISA IL6 control vs SF). Pre and postoperative SF enhanced neuroinflammatory response to surgery Twenty-four hours after surgery or initiation of SF we observed a significant increase in hippocampal mRNA expression of IL-6 (n=5, p<0.05 and p<0.01) and TNF-α (n=5; p<0.01 and p<0.001, respectively). These changes were even more pronounced when pre or post operative SF was combined with surgery (fig. 4). Perioperative SF did not affect surgery-induced memory impairment Surgery significantly decreased the percentage of freezing time when compared to the control group (49.30±5.77% versus 30.26±3.27% n=10, p<0.001). SF insult also induced cognitive decline (49.30±5.77% versus 32.90±5.80, n=10, p<0.01). Memory impairment was not further exacerbated when SF is applied either pre (43.91±13.12%) or postoperatively (38.47±12.32%) (fig. 5).

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CONCLUSIONS Summary of results: dissociation between neuroinflammation and cognitive test This study posits that SF induces significant changes that underlie memory, indicating a loss of cognitive function in non-hippocampal and hippocampal dependent domains. We show that while SF and surgery can independently produce significant memory impairment, perioperative SF significantly increased hippocampal inflammation (fig. 4) without further cognitive impairment (fig. 5). This peculiar dissociation between neuroinflammation and cognitive decline may relate to our use of memory paradigms that do not capture all aspects of cognition. Sleep rebound and preconditioning effect We chose a SF model that is characterized by a decrease in REM and NREM sleep.17 This type of sleep disturbance closely resembles both patients with OSA and also the type of sleep disorder reported in acute care facilities.24,25 During recovery, mice exhibit a rebound in REM sleep time and an increase in the depth of NREM sleep as measured by delta (1-4Hz) power in the EEG.17 Although SF and surgery separately produced memory dysfunction, when combined, cognitive function reverted back to the control state. It may be possible that by day three the rebound REM and NREM sleep is enough to restore the brain to a state that is capable of appropriately processing sensory input. It has been shown that rebound sleep shortly after stroke onset may be causally associated with neuroprotection through an ischemic preconditioning mechanism.26 The neuroprotective effects of acute sleep deprivation are in agreement with previous reports of neuroprotection and attenuation of microglial responses in rats subjected to global ischemia27. Caveats: SF method may induce stress One of the caveats of our study is that sleep deprivation/SF methods can induce stress responses, which may affect memory,28-31 while some studies have shown that SF methods only modestly elevate plasma corticosterone.32 It could be argued that stress is a confounding factor, producing immobility and thus influencing the amount and extent of freezing in fear conditioning. Our study’s design attempts to address this concern by delaying the context session in the trace fear paradigm test by three days after surgery and/or SF; we also chose a SF method where animals are kept in their original environmentally enriched home cages, thus mitigating the amount of stress. From our previous work we have demonstrated that the surgical memory dysfunction phenotype is still present at day three while inflammatory markers are back to normal levels. Stress corticosteroids may underlie some of the deficits identified in both sleep-fragmented humans as well as the animals in our model. Even if our model does have a stress response associated, it has been shown that this is also a normal response to sleep deprivation in humans.33 Cortisol levels are high early in the morning and low at the time of sleep onset and partial acute sleep loss, which delays the recovery of the hypothalamus-pituitary-adrenal from early morning circadian stimulation, is thus likely to involve an alteration in negative glucocorticoid feedback regulation.33 These data support the need to adopt interventions that can provide patients with appropriate rest in order to mitigate neuroinflammation. While we realize that the etiology of cognitive dysfunction in surgical/ICU patients is multi-factorial, if the restorative and reparative benefits of sleep mitigate the development of inflammation, this may result in shorter ICU or postoperative lengths of stay. Subsequent studies are warranted in order to both understand the mechanisms involved in the reversion of memory function after SF but also to improve our knowledge of POCD and ameliorate its adverse effects.

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Figures

Figure 1

Study design. (A) First experiment; mice were divided in 2 groups: control versus 24h SF. The training session of the NOR memory test was performed immediately after cessation of SF procedure. (B) Second experiment; mice were divided in 5 groups and tested according to trace fear conditioning paradigm: control; 24h SF; surgery; preoperative SF; postoperative SF. The training session of the memory test was performed right before any intervention and the context session was performed 3 days later. (C) Third experiment; Mice were divided in 5 groups and sacrificed 24h after the start of the intervention: control; 24h SF; surgery; preoperative SF; postoperative SF. Downwards blue arrow represents tibia surgery. SF = sleep fragmentation, NOR = Novel Object Recognition.

Figure 2

Effect of 24h SF on non-hippocampal memory function assessed by using novel object recognition test. Figure 3

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Effect of 24h SF on systemic IL-6 serum concentration (n=5; ** p<0.01, with two tailed t-test). SF = sleep fragmentation.

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Effects of perioperative SF on hippocampal transcription of IL-6, TNF-α, IL-1β and MCP-1 24h after tibia surgery. (n=5; * p<0.05; ** p<0.01; *** p>0.001 with one-way ANOVA and Bonferroni post hoc analysis). SF = sleep fragmentation.

Figure 5

Effects of Perioperative SF on Cognitive Decline. Contextual fear response reveals hippocampal-dependent memory impairment at day 3. Quantification of the freezing time percentage according to the five groups (n=10; ** p<0.01, *** p<0.001, with one-way ANOVA and Bonferroni post hoc analysis).

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8 Ayalon, L., Ancoli-Israel, S. & Drummond, S. P. A. Obstructive Sleep Apnea and Age A Double Insult to Brain Function? American Journal of Respiratory and Critical Care Medicine 182, 413-419, doi:10.1164/rccm.200912-1805OC (2010).

9 Ayalon, L., Ancoli-Israel, S., Aka, A. A., McKenna, B. S. & Drummond, S. P. A. Relationship Between Obstructive Sleep Apnea Severity and Brain Activation During a Sustained Attention Task. Sleep 32, 373-381 (2009).

10 Flink, B. J. et al. Obstructive Sleep Apnea and Incidence of Postoperative Delirium after Elective Knee Replacement in the Nondemented Elderly. Anesthesiology 116, 788-796 (2012).

11 Cibelli, M. et al. Role of interleukin-1beta in postoperative cognitive dysfunction. Ann Neurol 68, 360-368, doi:10.1002/ana.22082 (2010).

12 Terrando, N. et al. Resolving postoperative neuroinflammation and cognitive decline. Ann Neurol 70, 986-995, doi:10.1002/ana.22664 (2011).

13 Tang, Y., Preuss, F., Turek, F. W., Jakate, S. & Keshavarzian, A. Sleep deprivation worsens inflammation and delays recovery in a mouse model of colitis. Sleep Med 10, 597-603, doi:S1389-9457(09)00052-5 [pii]

10.1016/j.sleep.2008.12.009 [doi] (2009). 14 Mullington, J. M., Haack, M., Toth, M., Serrador, J. M. & Meier-Ewert, H. K. Cardiovascular,

inflammatory, and metabolic consequences of sleep deprivation. Prog Cardiovasc Dis 51, 294-302, doi:S0033-0620(08)00091-1 [pii]

10.1016/j.pcad.2008.10.003 [doi] (2009). 15 Drouot, X., Cabello, B., d'Ortho, M.-P. & Brochard, L. Sleep in the intensive care unit. Sleep

medicine reviews 12, 391-403, doi:10.1016/j.smrv.2007.11.004 (2008). 16 Nguyen, K. T. et al. Exposure to acute stress induces brain interleukin-1beta protein in the rat. J

Neurosci 18, 2239-2246 (1998). 17 Sinton, C. M., Kovakkattu, D. & Friese, R. S. Validation of a novel method to interrupt sleep in

the mouse. J Neurosci Methods 184, 71-78, doi:10.1016/j.jneumeth.2009.07.026 (2009). 18 Harry, L. E. et al. Comparison of the healing of open tibial fractures covered with either muscle

or fasciocutaneous tissue in a murine model. J Orthop Res 26, 1238-1244, doi:10.1002/jor.20649 [doi] (2008).

19 Bevins, R. A. & Besheer, J. Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study 'recognition memory'. Nat. Protocols 1, 1306-1311, doi:http://www.nature.com/nprot/journal/v1/n3/suppinfo/nprot.2006.205_S1.html (2006).

20 Kim, J. J. & Fanselow, M. S. Modality-specific retrograde amnesia of fear. Science 256, 675-677 (1992).

21 Terrando, N. et al. Tumor necrosis factor-alpha triggers a cytokine cascade yielding postoperative cognitive decline. Proc Natl Acad Sci U S A 107, 20518-20522 (2010).

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22 Terrando, N. et al. The impact of IL-1 modulation on the development of lipopolysaccharide-induced cognitive dysfunction. Crit Care 14, R88, doi:10.1186/cc9019 (2010).

23 Degos, V. et al. Depletion of Bone Marrow-derived Macrophages Perturbs the Innate Immune Response to Surgery and Reduces Postoperative Memory Dysfunction. Anesthesiology 118, 527-536, doi:10.1097/ALN.0b013e3182834d94 (2013).

24 Friese, R. S., Diaz-Arrastia, R., McBride, D., Frankel, H. & Gentilello, L. M. Quantity and quality of sleep in the surgical intensive care unit: are our patients sleeping? J Trauma 63, 1210-1214, doi:10.1097/TA.0b013e31815b83d7

00005373-200712000-00003 [pii] (2007). 25 Aurell, J. & Elmqvist, D. Sleep in the surgical intensive care unit: continuous polygraphic

recording of sleep in nine patients receiving postoperative care. Br Med J (Clin Res Ed) 290, 1029-1032 (1985).

26 Weil, Z. M. et al. Sleep deprivation attenuates inflammatory responses and ischemic cell death. Exp Neurol 218, 129-136, doi:10.1016/j.expneurol.2009.04.018 (2009).

27 Hsu, J. C., Lee, Y. S., Chang, C. N., Ling, E. A. & Lan, C. T. Sleep deprivation prior to transient global cerebral ischemia attenuates glial reaction in the rat hippocampal formation. Brain Res 984, 170-181 (2003).

28 Hairston, I. S. et al. Sleep deprivation elevates plasma corticosterone levels in neonatal rats. Neuroscience letters 315, 29-32, doi:10.1016/s0304-3940(01)02309-6 (2001).

29 McEwen, B. S. Sleep deprivation as a neurobiologic and physiologic stressor: allostasis and allostatic load. Metabolism-Clinical and Experimental 55, S20-S23, doi:10.1016/j.metabol.2006.07.008 (2006).

30 Payne, J. D. et al. The role of sleep in false memory formation. Neurobiology of Learning and Memory 92, 327-334, doi:10.1016/j.nlm.2009.03.007 (2009).

31 Tartar, J. L. et al. Experimental sleep fragmentation and sleep deprivation in rats increases exploration in an open field test of anxiety while increasing plasma corticosterone levels. Behavioural Brain Research 197, 450-453, doi:10.1016/j.bbr.2008.08.035 (2009).

32 Ringgold, K. M., Barf, R. P., George, A., Sutton, B. C. & Opp, M. R. Prolonged sleep fragmentation of mice exacerbates febrile responses to lipopolysaccharide. J Neurosci Methods 219, 104-112, doi:10.1016/j.jneumeth.2013.07.008 (2013).

33 Leproult, R., Copinschi, G., Buxton, O. & Van Cauter, E. Sleep loss results in an elevation of cortisol levels the next evening. Sleep 20, 865-870 (1997).

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Sleep and Anesthesia: Common Mechanisms of Action

71

Sleep and AnesthesiaCommon Mechanisms of Action

Susana Vacas, MDa,b, Philip Kurien, MDa,Mervyn Maze, MB ChB, FRCA, FRCP, FMedScia,*

KEYWORDS

� Anesthesia � Sleep � Circadian rhythm � Dexmedetomidine

KEY POINTS

� Anesthetic drugs induce unconsciousness by altering neurotransmission at multiple sites.

� a2-Agonists, such as dexmedetomidine, are associated with changes in neuronal activity similar tothose seen in deeper stages of non–rapid eye movement sleep.

� The effects of anesthetics on circadian rhythm possibly lead to immune deregulation.

� Thoughtful attention must be paid to selecting an anesthetic agent that best mimics natural sleep.

INTRODUCTION

“You are going to go to sleep now” is an oft-repeated colloquialism in every anesthesiologist’sdaily practice. The phrase might be useful in allay-ing the fears of nervous patients, but does generalanesthesia actually mimic sleep? Do they travel onthe same neural pathways? To what degree doesthe comparison accurately reflect the underlyingmechanisms involved?

Sleep, especially non–rapid eye movement(NREM) sleep, and anesthesia may use commonneuronal and genetic substrates. Anesthetics actthrough sleep neural circuits but not necessarilyin the same way.

AROUSAL PATHWAYS

To promote and sustain cortical arousal, neuronalpathways have developed two parallel ascendingneuronal pathways. The first branch activates thethalamic relay neurons that are crucial for trans-mission of information to the cortex. Cholinergicsignaling originating from the laterodorsaltegmental (LDT) and pedunculopontine tegmental(PPT) nuclei and the basal forebrain promote the

a Department of Anesthesia and Perioperative Care, UAvenue, San Francisco, CA 94143-0648, USA; b ProgrGulbenkian Foundation Av, 45-A Berna, Lisbon 1067-001* Corresponding author.E-mail address: [email protected]

Sleep Med Clin 8 (2013) 1–9http://dx.doi.org/10.1016/j.jsmc.2012.11.0091556-407X/13/$ – see front matter � 2013 Elsevier Inc. Al

cortically activated states of wakefulness andrapid eye movement (REM) sleep. The secondbranch bypasses the thalamus, activating neuronsin the lateral hypothalamic area and basal fore-brain (BF), and throughout the cortex. Thispathway originates from monoaminergic neuronsin the upper brainstem and caudal hypothalamus.The locus coeruleus (LC) provides norepinephrine-mediated inhibition of the ventrolateral preoptic(VLPO) nucleus in the hypothalamus.1,2 Therefore,g-aminobutyric acid (GABAA)-mediated andgalanin-mediated inhibition of the ascendingarousal circuits by the VLPO nucleus is inhibitedand the awake state is promoted.

SLEEP PATHWAYS

Sleep is under thecontrol of twoprocesses, a circa-dian clock that regulates the appropriate timing ofsleep and wakefulness across the 24-hour dayand a homeostatic process (sleep homeostasis)that regulates sleep need and intensity accordingto the time spent awake or asleep.3 Sleep isa nonhomogeneous state that can be divided intoNREM sleep and REM (paradoxic) sleep. The brainareas identified as important in sleep fall into 2

niversity of California San Francisco, 521 Parnassusamme for Advanced Medical Education, Calouste, Portugal

l rights reserved. sleep.theclinics.com

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mailto:[email protected]

http://dx.doi.org/10.1016/j.jsmc.2012.11.009

http://sleep.theclinics.com

Vacas et al2

general groups: those with an arousing influenceand active during wakefulness, namely the LC,dorsal raphe (DR), tuberomammillary (TMN), andBF; and those active primarily during sleep, namelythe VLPO. The median preoptic area contains bothwake-active and sleep-active neurons.Discrete neurochemical changes accompany the

different types of sleep with cholinergic (in brain-stem and forebrain), noradrenergic (LC), and sero-tonergic (DR) all becoming less active in NREMsleep, whereas cholinergic activity increases inREM sleep.4 Activity in the VLPO is increased inNREM sleep and the GABAergic/galanin inputfrom VLPO inhibits the histaminergic TMN nucleus.Orexinergic pathways from the perifornical nucleusare inactive during NREM sleep (Fig. 1).

The relatively quiescent LC facilitates a series ofchanges that includes activation of the galanin/GABA-containing neurons of the VLPO nucleusthat terminate on and inhibit aminergic neuronswithin the tuberomammillary nucleus.5

Anesthetic-induced unconsciousness resultsfrom specific interactions of anesthetics, with theneural circuits regulating sleep and wakefulness.

Fig. 1. Simplified NREM sleep-promoting pathway. An inhibpanies endogenous NREM sleep, releases a tonic noradrebelieved to release GABA into the TMN which inhibits its rand thus induces loss of consciousness. A number of pathwprojects to all the ascending monoaminergic, cholinergic anPeF), which project to the cortex where they release arouness. (From Nelson LE, Guo TZ, Lu J, et al. The sedative compin an endogenous sleep pathway. Nat Neurosci 2002;5(10):

WHERE DO ANESTHETICS COLLIDE IN SLEEPPATHWAYS?

Thecurrently available imaging techniquescanonlyindirectly measure neuronal activity, for examplethrough changes in blood flow, glucose metabo-lism, or oxygen concentration. One can then under-stand the difficulty in fully comprehending themechanisms by which anesthesia induces sleep/unconsciousness. A common finding betweenNREM sleep and anesthesia in imaging studies isthe deactivation of the thalamus leading to corticalinhibition. Anesthetic drugs induce unconscious-ness by altering neurotransmission at multiple sitesin the cerebral cortex, brainstem, and thalamus.The different actions of the different available anes-thetics have made the understanding of the exactmechanism even more difficult. Effects of modernanesthetics on subsequent sleep behavior areknown for some, but not all, anesthetics. No oneelectroencephalogram (EEG) pattern characterizesthe anesthetized state. Different anesthetics anddoses have distinct profiles with respect to theEEG activity.

ition of noradrenergic neurons in the LC, which accom-nergic inhibition of the VLPO. The activated VLPO iselease of arousal-promoting histamine into the cortex,ays are involved in NREM sleep; the sleep-active VLPOd orexinergic arousal nuclei (TMN, LC, DR, PPTg, LDTg,

sal-promoting neurotransmitters to promote wakeful-onent of anesthesia is mediated by GABA(A) receptors979–84; with permission.)

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Sleep and Anesthesia 3

Although some agents act on excitatory syn-apses, others act through potentiation of inhibi-tory synaptic receptors. The GABAA receptors areneurotransmitter-gated chloride channels that existon cells that may also contain nicotinic acetylcho-line receptors, glycine receptors, and serotonintype 3 receptors. Anesthetics such aspropofol, eto-midate, and barbiturates exert their effect throughenhancement of GABA-mediated channel activa-tion and prolong postsynaptic inhibitory currents,suppressing neuronal excitability. In brain regionscontaining neurons that promote wakefulness,GABAergic inhibition has been shown to cause anincrease in sleep. These brain regions include theDRnucleus, TMN,medial preoptic area, andventro-lateral periaqueductal gray.6,7 In a series of studiesinvolving GABAergic agents, it was reported thatunlike in NREM sleep, these hypnotic agents didnot alter noradrenergic activity in the LC (seeFig. 1).1 Instead these agents converged on theNREM sleep pathway at the level of the hypothal-amus.8 However, short-term administration of theGABAergic agent propofol permitted normalrecovery after a period of sleep deprivation.9,10

At clinical concentrations, drugs such as N2O,xenon, and ketamine have little or no effect onGABAA receptors. Instead, these anestheticspotently inhibit N-methyl-D-aspartate (NMDA)receptors, which are excitatory cation channelsactivated by glutamate. These agents reduceexcitatory signals in critical neuronal circuits,causing unconsciousness. Glutamate levels inthe PPT are greater during wakefulness asopposed to NREM and REM sleep.11,12 The disso-ciative state that is produced by ketamine anes-thesia can be in part attributed to the differentregions to which ketamine promotes glutamaterelease (nucleus accumbens,13 prefrontal cortex,14

and anterior cingulate15). It has been shown thatisoflurane and sevoflurane reduce glutamaterelease16–18 and inhibit its uptake,19 but fewin vivo studies elaborated on understanding theexact mechanism by which isoflurane modulatesglutamatergic transmission.

REM sleep rebound after exposure to volatileanesthetics suggests that these volatile anestheticsdonot fully substitute for natural sleep.20 Inhumans,isoflurane anesthesia alone (without surgery) resultsin no change in subsequent REM or NREM sleep,but a shift in NREM sleep from slow-wave sleep tolighter (I and II) stages.21 On the other hand, it hasbeen shown that wake-active orexinergic neuronsare inhibited by isoflurane and sevoflurane, andthat waking up from anesthesia uses neural circuitsdistinct from those necessary to become anes-thetized but similar to the neural circuitry that pro-motes arousal.22 Furthermore, isoflurane depresses

serotonin levels on hypoglossal motoneurons indogs23 and on mice hippocampus.24

The molecular targets for dexmedetomidine arecentral a2-adrenergic receptors. It has been shownthat a2-agonists transduce its hypnotic responseafter binding to the a2A-receptor subtype

25 in theLC.26 Through signaling processes that involveboth pertussis toxin–sensitive G proteins27 andeffector mechanisms that include inhibition of ad-enylyl cyclase27 and ligand-gated calciumchannelsaswell asactivationof inwardly rectifyingpotassiumchannels,28 the noradrenergic neurons becomehyperpolarized and are less likely to achieve anaction potential. a2-Agonists such as dexmedeto-midine are associated with similar changes inneuronal activity, as is seen in deeper stages ofNREMsleep2,29 apart from the absenceof inhibitoryeffect on the orexinergic neurons in the perifornicalnucleus.8 A functional magnetic resonance studyshowed that a thalamic nucleus, which receivesafferent input from orexinergic neurons, is activatedduring an arousal stimulus in a2-agonist–sedatedsubjects.30 Children sedated with dexmedetomi-dine showed an EEG pattern that was identical tothat seen in stage 2 NREM sleep.31

Although acetylcholine (ACh) plays a primary rolein generating the brain-activated states of wakeful-ness and REM sleep, cholinergic receptors are nota main target of common anesthetics. Nonethe-less, ACh interacts with other transmitter systemsthat are targets of sleep pharmacology, forexample theGABAergic agents. The clinical findingthat physostigmine (acetylcholinesterase inhibitor)reverses propofol sedation, causing arousal,suggests that propofol produces unconscious-ness, in part, by disrupting cholinergic neurotrans-mission.32 In vitro studies showed that propofol,isoflurane, sevoflurane, and ketamine inhibitmuscarinic and nicotinic ACh receptors,33–37

providing support that these agents cause seda-tion, in part, by inhibiting cholinergic neurotrans-mission in brain regions that regulate arousal.

CIRCADIAN RHYTHM

The 2-process model of sleep homeostasis asdescribed by Borbely andWirz-Justice38 integratessleep debt (“process-s”) and circadian rhythm(“process-c”). This model implicates circadianrhythmand sleep as intertwined, codependent pro-cesses. Experimentally, distinguishing process-cfrom process-s presents a challenge in decon-structing causative factors in sleep disorders.Nevertheless, there is a growing body of evidencethat suggests circadian rhythmcan be altered inde-pendent of sleep deprivation and that anestheticscan specifically change circadian rhythm.

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Circadian rhythmicity is thought to be controlledby the suprachiasmatic nucleus and is establishedby processing external cues (zeitgebers), such aslight, into systemicmediators, such as temperature,adrenergic signaling, and circulating hormones (eg,cortisol and melatonin). This process serves tomaintain a diurnal pattern, presumably to coordi-nate intracellular or intersystem function by re-synchronizing intrinsic cellular molecular clocks. Inbrief, the molecular core of the circadian clockinvolves the heterodimerization of CLOCK andBMAL1,whichact as thecanonical armof the clock,and the heterodimerization of PER1/2 and CRY1/2,which are critical components of the negative feed-back arm18; stabilizing proteins RORa, REV-ERB,DEC, DBP, and E4BP4 act as additional repressorsor activators of the canonical arm,39 and theseproteins oscillate through the day and translocatefrom the cytoplasm to the nucleus in a highly coor-dinated fashion to provide a reliable rhythm ofapproximately 24 hours. Disruption of circadianprocesses is being studied as a relevant contrib-uting factor to multiple human conditions altering,among others, immunity.

CIRCADIAN RHYTHM AND ANESTHESIA

An initial indication that circadian rhythm is impor-tant in anesthetic delivery is the time-of-day vari-ance in susceptibility to general anesthetics.40,41

Indeed, the greatest therapeutic effect of generalanesthetics in animal models occurs during theanimals’ rest phase for propofol42 and ketamine.43

A volatile anesthetic effectmay also vary accordingto a diurnal pattern; halothane administration in ratsvariedwith a lowerminimumalveolar concentrationrequirement during the rest phase in comparisonwith the active phase.44 Recently, using bees asa model system, 6-hour isoflurane administrationduring the rest phase failed to alter the circadianactivity patterns of the hive, whereas isofluraneadministered in the active phase significantlyaltered the circadian activity of the hive.45 Takentogether, the time-of-day administration of anes-thesia is likely important in both the dose-dependent effect and themaintenance of circadianrhythm.As parameters for outlining circadian rhythm,

cortisol and melatonin levels can be used to makeassumptions about the effect of general anesthesiaon daily cycling in human subjects. Most humanstudies following these variables involve generalanesthesia with the confounding aspect of surgery,and do not incorporate the natural underlyingcycling of these hormones adequately. Given theseand other significant caveats, a propofol-basedanesthetic appears to decrease the amount of

plasma cortisol during surgery in comparison withsevoflurane.46 Postoperatively, following a thio-pental/sevoflurane-based or thiopental/isoflurane-based anesthetic, cortisol levels are elevated inmen who underwent long-duration surgery forlarynx or pharynx cancer.47 Conversely, in womenundergoing laparoscopic procedures for pelvissurgery, anesthesia with thiopental/sevofluranereduced amounts of cortisol 2 to 4 hours aftersurgery compared with thiopental/isoflurane anes-thesia.48 Given that these studies were designedto investigate the anesthetic effects on stressresponses and not on circadian rhythm directly,there remains only a suggestion that altered cortisollevels may interfere with circadian rhythm afteranesthesia and surgery.Investigation into the effect of anesthesia on

the cycling of melatonin points more directly toa circadian rhythm effect. A comparison of generalanesthesia (thiopental/isoflurane) with spinal anes-thesia for orthopedic procedures showed a signifi-cant reduction in melatonin levels in the firstpostsurgical night compared with baseline levels;interestingly, there was no significant difference inthe reductionofmelatoninbetween theexperimentalgroups, indicating that theeffectwas independentofthe anesthesia, and pointing to the possibility ofa significant surgical influence of postoperativeopiate use on melatonin secretion.49 In patientsundergoing general anesthesia (thiopental/isoflur-ane/fentanyl) for laparoscopy, a modest reductionin 13-hour average melatonin secretion was notedin the evening after surgery compared with the pre-surgical night, with a large increase in melatoninsecretion the second night after surgery.50 Corrobo-rating these data, in patients undergoing majorabdominal surgery with general anesthesia andconcomitant use of a thoracic epidural, there wasa similarmodest reduction in basal melatonin secre-tion on the first postoperative day, followed bya significant increase on the second postoperativeday.51 Following urine metabolites of melatonin(aMT6s), general anesthesia (thiopental/propofol)decreased the maximal concentration and delayedthe peak of aMT6s.52 Short mask-inhalation anes-thetics (21 minutes average duration) for dilationand curettage showed no difference in melatoninsecretion compared with nonsurgical controls.53

Experimental models allow for more preciseexamination of melatonin secretion and generalanesthesia apart from surgical effect. Rats anesthe-tized with propofol for 25 to 30 minutes around thepeak of serum melatonin secretion showed a sig-nificant reduction in melatonin secretion for the 3hours following anesthesia, a subsequent increase20 hours after the anesthetic, and a phase advanceof cyclical melatonin secretion of 40 minutes

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consistent with the approximate duration of anes-thesia.40 Whether in humans or rats, it seems con-sistent that melatonin levels are reduced in theimmediate postoperative/anesthetic period witha rebound phenomenon observed thereafter, al-though it remains unclear in human subjects whichcomponent of the observed effect can be attribut-able to either surgery or anesthesia.

The effect of anesthetics on intrinsic molecularcircadian clocks is beginning to be explored inexperimental models. In rats, 6 hours of sevoflur-ane anesthesia changed the expression patternof approximately 1.5% of 10,000 genes surveyed.Of interest, the expression of Per2 was the onlycircadian protein in the brain that was significantlyreduced after the anesthetic.54 The effect ofreduced expression of Per2 and an auxiliary clockgene Dbp persists for 24 hours.55 Infusions ofboth propofol and dexmedetomidine likewisereduced the expression of Per2 in rat brain 6 hoursafter anesthetic delivery, but the effect persisted foronly 24 hours in the case of dexmedetomidine.56

Further investigation demonstrated that a 4-hoursevoflurane anesthetic blunted Per2 mRNAproduction in the suprachiasmatic nucleus inresponse to a light stimulus, and created a delayedactivity rhythm in anesthetized mice.57 Repressionof Per2 expression by sevoflurane anesthesia wasmost significant when administered between thehours of 8 AM and 12 PM in comparison with othertime points, but activity patterns of anesthetizedanimals were delayed for all time points of anes-thetic administration.58 Bees anesthetized witha 6-hour course of isoflurane during the day hada reduction in the amplitude of Cry expressionand phase delay of both Cry and Per2 expressionin whole bee brains in comparison with bees anes-thetized during the evening, leading to alterations incircadian governed homing patterns and foragingtimes.45 With respect to the molecular clock,general anesthesia appears to significantly affectcritical clock proteins in a time-of-day–dependentfashion, and corresponds to changes in activitypatterns consistent with circadian disruption.

CIRCADIAN CONTROL OF IMMUNEFUNCTION

A separate line of investigation has focused on theinfluence of circadian rhythm on immune function.Clinically, timing is relevant in terms of suscepti-bility to infection,59 asthma attacks,60 or flares ofrheumatic arthritis,61 all of which suggest circadianprinciples underlying these immune-mediatedprocesses.

Circulating pools of many immune cells’ cyclethroughout the day indicate the influence of

circadian timing. Natural killer (NK) cells peak inboth activity and numbers in the human circulationin the early morning, and likely marginate awayfrom circulation during other times of the day.62 Inmouse models, after lipopolysaccharide (LPS)administration, macrophages circulate cyclicallyto the spleen.63 Of importance, the circadianmolecular clock exists and functions in NKcells,64,65 macrophages, dendritic cells, and Bcells.63,66 Indeed, approximately 8% of the macro-phage genome is classified as falling under thecontrol of circadian transcription factors,63 andcritical immune transcription factors such as signaltransducer and activator of transcription family(STATs) and nuclear factor kB (NF-kB) are regu-lated by clock proteins.67 Study of the functionalconsequence of disturbing circadian molecularclock proteins in immune subsets is generatinginteresting data. Injection of LPS at specific timepoints caused significantly elevated cytokineproduction in both macrophage-restricted Bmal1knockout (KO), and systemic Rev-Erba KO mice,compared with the chronometric 12-hour oppositetime point (antiphasic control).68Cry1/2 double KOmice have elevated NF-kB activity causingincreased baseline inflammatory cytokine expres-sion in vitro, and generated greater inflammationwhen challenged with LPS in vitro and in vivo.69

Examining lymphocyte function has similarly eluci-dated at least some aspect of circadian gating.Per2KOmouse lymphocytes had a robust increasein proliferative capacity after being immunizedin vivo when compared with their antiphasiccontrol.70 Isolated T cells cultured from mouselymph nodes proliferated after stimulation ina circadian manner, an effect that was abolishedin Clock mutant mice.71 Whether by observationor direct experimentation these data showed thatcertain immune cells traffic according to apparentcircadian parameters, possess oscillating intrinsicmolecular clocks, have critical transcription factorscontrolled by clock proteins, and have altered func-tion when clock proteins are perturbed.

SLEEP DISRUPTION AND COGNITIVEDYSFUNCTION IN SEDATED PATIENTS IN THEINTENSIVE CARE UNIT

Sleep disruption in critically ill patients is acommon occurrence in the intensive care unit(ICU), with the potential to adversely affectpatients’ outcome and also to provide a directfinancial detriment with respect to the length ofhospital stay and depletion of health careresources. Early polysomnographic studies hadrevealed extreme sleep disruption in ICU patientswith decreases in total sleep time, altered sleep

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architecture (predominance of stage 1 and 2 sleep,decreased or absent stages 3 and 4 NREM andREM sleep), and sleep fragmentation72,73; also,up to 50% of the total sleep time occurred duringdaytime. Among the possible causes that con-tribute to sleep disruption in the ICU are thoserelated to the patient’s acute illness and comor-bidities, environmental factors (including noiseand inappropriate light), and iatrogenic factorsincluding frequent care-related interruptions andmedications prescribed for analgesia and seda-tion.74,75 Among those that are potentially ame-nable to modification, excessive noise does notcontribute as much as was anticipated,76 andattention has focused on sedative practices.77

Sedative-hypnotic agents are widely used to facil-itate sleep in the ICU; however, depending on thesedative agent, it may not produce appropriatesleep hygiene and instead will aggravate theproblem by producing fewer of the restorativeproperties of natural sleep.Several studies have now demonstrated the

association between the use of benzodiazepines(BZDs) and increased incidence78 and duration79

of delirium in medical ICU patients, although therelationship of the development and duration ofdelirium with sleep disruption was not ascertained.Acute withdrawal from long-term sedation withBZDs and opiate narcotics results in profoundsleep disruption.80 The pivotal work of the MENDstrial78,81,82 indicated the benefits of a specificanesthetic agent, dexmedetomidine, in theoutcome of the ICU population.

SUMMARY

Appropriate sleep hygiene is crucial for repair instates of disease and injury, and in restoring func-tion especially in the central nervous and immunesystems. Lack of sleep hygiene results in cognitivedysfunction, contributes to the delirium that isprevalent in patients within the ICU, adverselyaffects immunity, and independently increasesboth morbidity and mortality. Anesthetics used inhospital care have different action targets and, ulti-mately, different consequences. Those which actby modulating the GABAA receptor converge atthe level of the hypothalamus, whereas a2-adren-ergic agonists converge on sleep pathways withinthe brainstem. Thus, thoughtful attention must bepaid to selecting an anesthetic agent that bestmimics natural sleep. Future studies will furtherelucidate the benefits of dexmedetomidine asa good anesthetic/sedative candidate to mimicnatural sleep.While the fields of investigation into the anes-

thetic effect on circadian rhythm and the circadian

influence on immune function remain disparate,there exists the plausible concatenation that anes-thetics, by altering circadian rhythm (possiblyindependent of sleep deprivation), affect immunefunction. Given that anesthetics are often used ad-junctively to facilitate therapeutic interventions, itwould be worthwhile to elucidate whether moreprecise applications of anesthesia could improveimmunologic outcomes for patients who requirethem.Anesthesia is not the same as sleep. The actions

of anesthetics on sleep pathways and the restor-ative properties of natural sleep for the centralnervous system are undeniable and essential, yetthey also advance a concomitant advantage tothe immune system, with fewer infections anda greater likelihood of survival from sepsis.

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Bone Fracture Exacerbates Murine Ischemic Cerebral Injury

Participated in study design, conducted qPCR experiments and participated in data analysis

and interpretation and final preparation of the manuscript.

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ABSTRACT

Background: Bone fracture increases alarmins and proin-flammatory cytokines in the blood, and provokes macro-phage infiltration and proinflammatory cytokine expression in the hippocampus. We recently reported that stroke is an independent risk factor after bone surgery for adverse out-come; however, the impact of bone fracture on stroke out-come remains unknown. We tested the hypothesis that bone fracture, shortly after ischemic stroke, enhances stroke-related injuries by augmenting the neuroinflammatory response.Methods: Tibia fracture (bone fracture) was induced in mice one day after permanent occlusion of the distal middle cere-bral artery (stroke). High-mobility-group box chromosomal protein-1 (HMGB1) was tested to mimic the bone frac-ture effects. HMGB1 neutralizing antibody and clodrolip

(macrophage depletion) were tested to attenuate the bone fracture effects. Neurobehavioral function (n = 10), infarct volume, neuronal death, and macrophages/microglia infil-tration (n = 6–7) were analyzed after 3 days.Results: We found that mice with both stroke and bone frac-ture had larger infarct volumes (mean percentage of ipsilateral hemisphere ± SD: 30 ± 7% vs.12 ± 3%, n = 6, P < 0.001), more severe neurobehavioral dysfunction, and more macrophages/microglia in the periinfarct region than mice with stroke only. Intraperitoneal injection of HMGB1 mimicked, whereas neutralizing HMGB1 attenuated, the bone fracture effects and the macrophage/microglia infiltration. Depleting macro-phages with clodrolip also attenuated the aggravating effects of bone fracture on stroke lesion and behavioral dysfunction.Conclusions: These novel findings suggest that bone fracture shortly after stroke enhances stroke injury via augmented inflammation through HMGB1 and macrophage/microglia infiltration. Interventions to modulate early macrophage/microglia activation could be therapeutic goals to limit the adverse consequences of bone fracture after stroke.

STROKE is the leading cause of disability in adults1 and an important risk factor for bone fracture.2 In the United

States, ≈70,000 stroke victims suffer from bone fracture within the first year after their stroke.3,4 A small proportion of these stroke patients experience bone fracture within the first 24 h after the ischemic stroke, but the impact of bone fracture on acute stroke lesion is unknown. In a database cohort study including more than 270,000 European patients hospitalized for stroke from 1987 to 1996, the authors found that overall, 9% of the stroke patients had a subsequent fracture.4 Inter-estingly, the increase in risk was most evident immediately

Bone Fracture Exacerbates Murine Ischemic Cerebral Injury

Vincent Degos, M.D., Ph.D.,* Mervyn Maze, M.B., Ch.B.,† Susana Vacas, M.D.,‡ Jan Hirsch, M.D.,§ Yi Guo, M.D.,‖ Fanxia Shen, M.D.,# Kristine Jun, B.S.,# Nico van Rooijen, Ph.D.,** Pierre Gressens, M.D., Ph.D.,†† William L. Young, M.D.,‡‡ Hua Su, M.D.§§

* Associate Researcher, Center for Cerebrovascular Research and Department of Anesthesia and Perioperative Care, University of Cali-fornia, San Francisco, San Francisco, California, and INSERM, Hôpi-tal Robert Debré, Paris, France. † Professor and Chair, ‡ Postdoctoral Fellow, Department of Anesthesia and Perioperative Care, Univer-sity of California, San Francisco, and Program for Advanced Medi-cal Education, Lisbon, Portugal. § Assistant Professor, Department of Anesthesia and Perioperative Care, University of California, San Francisco. ‖ Postdoctoral Fellow, # Assistant Researcher, ** Professor, Vrije Universiteit Medical Center, Department of Molecular Cell Biol-ogy, Faculty of Medicine, Amsterdam, The Netherlands. †† Professor, INSERM, Hôpital Robert Debré. ‡‡ Professor and Vice-Chair, Center for Cerebrovascular Research, Departments of Anesthesia and Peri-operative Care, Neurological Surgery, and Neurology, University of California, San Francisco. §§ Associate Professor, Center for Cerebro-vascular Research and Department of Anesthesia and Perioperative Care, University of California, San Francisco.

Received from the Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, Cali-fornia. Submitted for publication June 25, 2012. Accepted for pub-lication January 29, 2013. Supported by grant nos. R01NS0027713, P01NS044155, and R21NS070153 from the National Institutes of Health, Bethesda, Maryland; AHA10GRNT3130004 from the American Heart Association, Dallas, Texas; the Department of Anesthesia and Perioperative Care, University of California, San Francisco; INSERM, Paris, France; Fondation des Gueules Cassées, Paris, France; Institut Servier, Paris, France; and Société Française d’Anesthésie Réanimation, Paris, France.

Address correspondence to Dr. Su: Department of Anesthesia and Perioperative Care, University of California, San Francisco, 1001 Potrero Avenue, Building 10, Room 1206, San Francisco, California 94110. [email protected]. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. ANESTHESIOLOGY’S articles are made freely accessible to all read-ers, for personal use only, 6 months from the cover date of the issue.

What We Already Know about This TopicAs many as 250,000 patients annually throughout the world will suffer a bone fracture within a day after stroke, but the impact of bone fracture on recovery from stroke has not been examined

What This Article Tells Us That Is NewIn mice, infarct volume from experimental stroke was 2.5-fold larger if a bone fracture was performed 1 day after strokeDepleting macrophages and neutralizing high-mobility-group box chromosomal protein-1 attenuated the aggravating ef-fects of bone fracture on stroke

Murine Bone Fracture and Ischemic Stroke

Degos ET AL.

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10.1097/ALN.0b013e31828c23f8

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Deepa Koshi

2013

Copyright © 2013, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins.

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after the stroke. Based on the data provided by Kanis et al.,4 the estimated incidence of having a bone fracture within 24 h of a stroke is 2.4–3.6/100,000, corresponding to 1–1.5% of stroke patients. Thus, about 7,000–11,000 patients in the United States and 167,000–250,000 worldwide will experi-ence a fall-fracture within the first day of the stroke. More recently, an American study confirmed that the hazard ratio of suffering from a hip fracture during the first 24 h after the stroke diagnosis was significantly increased by 3.9 (95% CI of 2.1–7.3), when compared to that of a nonstroke population.3

Our retrospective review of more than 400,000 surgical patients revealed that stroke is an independent risk factor for poor outcome after orthopedic bone surgery but not abdominal aortic surgery,5 suggesting a specific interaction between stroke and bone surgery. Understanding the impact and underlying mechanism of this interaction will enable clinicians to intervene appropriately and to design target-selective neuroprotective strategies perioperatively.

In a model of aseptic bone fracture, circulating alarmins, including high-mobility-group box chromosomal protein-1 (HMGB1) and proinflammatory cytokines,6,7 increase; in addition, aseptic bone fracture provokes macrophage infil-tration and proinflammatory cytokine expression in the hip-pocampus.8 HMGB1, a nonhistone DNA-binding protein, stabilizes nucleosome formation and DNA repair;9 addition-ally, it activates pattern recognition receptors (e.g., toll-like receptors 2 and 4, and the receptor for advanced glycation end-products) on bone marrow-derived monocytes and mac-rophages to initiate an innate immune response.10 Because sys-temic and local inflammation in the acute phase of ischemic stroke may have deleterious effects on stroke outcome,11–13 and because the risk of bone fracture in the stroke population is mainly just after the brain insult,3,4 we tested the hypothesis that bone fracture, one day after ischemic stroke, aggravates brain damage and functional consequences of stroke.

Materials and MethodsAnimalsAll experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco, and conformed to the National Institutes of Health Guidelines. All animals were fed standard rodent food and water ad libitum, and were housed (5 mice per cage) in sawdust-lined cages in an air-conditioned environment with 12-h light/dark cycles.

Wild-type male mice (C57BL/6J, 10–12 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME). C-C motif Chemokine receptor-2 red fluorescent protein (CCR2RFP/+) CX3CR1 green fluorescent protein (CX3CR1GFP/+) mice (10–12 weeks old)14 were provided by Katerina Akas-soglou, Ph.D.; Israel F. Charo, M.D., Ph.D.; and Kim Baeten, Ph.D. (Associate Investigator, Associate Director, and Postdoctoral Fellow, respectively, University of Cali-fornia, San Francisco Gladstone Institute, San Francisco,

CA). CCR2 and CXCR1 are acronyms for chemokine (C-C motif ) receptor 2 (monocyte chemoattractant protein-1), highly expressed in bone marrow-derived macrophages, and CX3C chemokine receptor 1 (fractalkine receptor), highly expressed in resident microglia, respectively.

Animals were tagged and randomly allocated to each group before any treatment. Researchers blinded to the group assign-ment performed all neurobehavioral tests, infarct volume, and cell counting. Based on preliminary data, in corner tests, there was a standard deviation of 15% in the percentage of left turns 3 days after permanent middle cerebral artery occlu-sion (pMCAO). We estimated that a sample of nine mice per group was necessary to find a significant difference between the pMCAO mice and the pMCAO+bone fracture mice with 80% of power if the difference was 20%. For this reason, we included n = 10 mice per group for each behavior test’s comparison.

Human Blood SamplesUnder an approved protocol by the University of California, San Francisco Committee on Human Research (Study number: H5636-20263-09), four individuals presenting with osteoar-thritis elective for total knee replacement under spinal anesthesia were enrolled. Blood was drawn immediately before and after the tourniquet was released using an uncoated tube. Blood sam-ples were centrifuged at 1300 rpm for 10 min at room tempera-ture and the serum samples were immediately frozen at −80°C.

pMCAO for Stroke ModelFollowing anesthesia (isoflurane, 2%), under aseptic surgical condition, animals received a left craniotomy and a dissection of the dura. The left middle cerebral artery was permanently occluded (pMCAO) using electrical coagulation just proximal to the pyriform branch. Rectal temperature was maintained at 37 ± 0.5°C using a thermal blanket throughout the surgical procedure. Surface cerebral blood flow was monitored during the procedure using a laser Doppler flowmeter (Vasamedics Inc., Little Canada, MN). Mice were excluded from further analysis when the surface cerebral blood flow in the ischemic core was more than 15% of the baseline after pMCAO, or if the artery injuries with the coagulator generated a massive bleeding. Animals were allowed to recover spontaneously from the anesthetic under warm conditions and received one intraperitoneal injection of buprenorphine (0.3 mg in 100 μl saline). Control mice were subjected to craniotomy without arterial occlusion but with the same amount and duration of anesthesia and the same amount of buprenorphine (0.3 mg in 100 μl saline) used for stroke mice. In this study, a total of 6 C57BL/6J mice were euthanized during the pMCAO procedures due to massive bleeding induced by vascular surgical injury, and were replaced by other mice from the same cage. No mouse was lost during the experiment’s 3-day duration.

Tibia Fracture Surgery for Bone Fracture ModelTwenty-four hours after the pMCAO procedure, animals were given general anesthesia with 2% isoflurane inhalation. Under aseptic surgical conditions, animals received an open tibia

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fracture of the right hind limb with an intramedullary fixa-tion, as previously described.6 Animals were allowed to recover spontaneously from the anesthetic under warm conditions and received one intraperitoneal injection of buprenorphine (0.3 mg in 100 μl saline). Rectal temperature was maintained at 37 ± 0.5°C using a thermal blanket throughout the surgical pro-cedure. Repeated measurements of arterial systolic blood pres-sure were performed using the tail-cuff method (ML125M, AD Instruments, Colorado Springs, CO) as previously described.15 The mice subjected to craniotomy and pMCAO had similar tail arterial systolic blood pressure before the bone fracture procedure (data not shown). Control mice for bone fracture received hind limb hair shaving with the same amount and duration of anesthesia and analgesia (buprenorphine, 0.3 mg in 100 μl saline) as for the bone fracture mice. Body weight of the animals was measured before pMCAO and immediately after the neurobehavioral tests. Mice with pMCAO alone presented a significant loss of weight when compared to control mice, but the groups of mice with pMCAO with and without bone fracture were not different. HMGB1 neutralized antibody and clodrolip did not influence the loss of body weight (data not shown). The tibia fracture surgery did not present any lethality.

Chemical ReagentsBased on serum levels of HMGB1 after mice tibia fracture,7 we injected 50 μg/kg (100 μl) of recombinant HMGB1 (R&D System, Minneapolis, MN) intraperitoneally 24 h after the stroke. Control animals received the same volume (100 μl) of the vehicle (saline). To neutralize HMGB1 in the blood, we injected anti-HMGB1 antibodies (chicken IgG, IBL Interna-tional, Toronto, Canada), 200 μg in 100 μl (corresponding to 10 mg/kg) intraperitoneally, 60 min before the bone frac-ture. Control animals received the same volume (100 μl) of the control chicken IgG antibodies (IBL international, 10 mg/kg). Clodronate liposomes (clodrolip) were obtained from clo-dronateliposomes.org (Vrije Universiteit, Amsterdam, Neth-erlands) at 7 mg/ml concentration and prepared as previously described elsewhere.16,17 Clodrolip (200 μl, about 100 mg/kg) was injected intraperitoneally 60 min before the bone fracture. Control animals received 200 μl of control liposomal solution.

Behavioral TestsAll tests were conducted 3 days after the pMCAO.Corner Test. As previously described,18 the corner test was used to detect sensorimotor and postural asymmetries. Mice were placed between two boards with identical dimensions (30 × 20 cm). When mice neared the corner, both sides of their vibrissae were stimulated. The mice would rear forward and upward, and then turn back to face the open end. Normal mice would turn to the left or right side with equal frequency, whereas the stroke mice would turn more often to the ispilat-eral side of the lesion (left). The percentage of left turns was recorded in three different sets of 10 trials. Turning movements not incorporated in a rearing movement were not recorded.Adhesive Removal Test. To assess the forepaw lateral sen-sitivity and point out a possible somatosensory neglect, we

performed the adhesive removal test.19 Briefly, adhesive tape (0.3 × 0.3 cm) was applied on each paw. The time that it took for the mice to remove the tape from each paw was recorded with a maximum testing time of 120 s. Mice were trained three times daily for 4 days before the surgery to obtain an optimal level of performance.

Evaluation of Infarct VolumeThree days after the pMCAO, brain samples were collected after paraformaldehyde 4% perfusion. A series of 20-μm-thick coronal sections was obtained. One in every 10 sections was stained with cresyl violet (one section per 200-μm-thick tissue) and sections were digitized. After binary imaging, the infarct and the ipsilateral hemisphere areas were out-lined using Image J software (National Institutes of Health, Bethesda, MD) and then measured.20 The infarct and ipsilat-eral hemisphere volumes were estimated as the sum of each area multiplied by 200 μm. The ratio of infarct volume ver-sus ipsilateral hemisphere volume was calculated.

Measurement of Mice HMGB1 in SerumSix hours after the bone fracture procedure, blood was collected by cardiac puncture under general anesthesia (isoflurane, 3%). Blood samples were centrifuged at 1300 rpm for 10 min at room temperature and the serum was collected and frozen at –80°C. HMGB1 levels in the serum of the mice and the human samples were quantified using the HMGB1 ELISA kit (IBL International).

Measurement of Cytokines in the Brain LesionThe left frontal-parietal cortical region of the mice was rapidly collected under a dissecting microscope 6 h after the bone fracture, and placed in RNAlater solution (Qia-gen, Valencia, CA). To avoid blood contamination, mice were perfused with saline for 5 minutes before sample col-lection. Total RNA was extracted using RNeasy lipid tis-sue kit (Qiagen) treated with recombinant DNase I using a RNase-Free DNase Set (Qiagen), and reverse-transcribed to complementary deoxyribonucleic acid with a High Capacity RNA to cDNA Kit (Applied Biosystems, Carls-bad, CA). TaqMan Fast Advanced Master Mix (Applied Biosystems, CA) and gene specific primers and probes used for real-time polymerase chain reaction are beta-actin (NM_007393.1), interleukin-6 (IL-6, Mm00446190_m1), tumor necrosis factor-α (Mm00443258_m1), and IL-1β (Mm01336189_m1). Real-time polymerase chain reaction was performed using StepOnePlus (Applied Bio-systems). Each RNA sample was run in triplicate, and relative gene expression was calculated using the compara-tive threshold cycle ∆CT and normalized to beta-actin. Results are expressed as fold increases relative to controls.

Histological Analysis 3 Days after Ischemia OnsetImmunohistochemical staining was performed using a series of 20-μm-thick coronal sections. All the quantifications were

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performed using the sections in the same anatomical region (bregma 1.2–1.4 mm). For immunostaining, sections were incubated with the following primary antibodies: CD68 (1:50, AbD Serotec, MCA1957, Raleigh, NC) and neuronal nuclei (1:500, MAB377, Millipore, Bedford, MA). Sections were then incubated with Alexa Fluor 647-conjugated, Alexa Fluor 594-conjugated, and Alexa Fluor 488-conjugated IgG (1:500, Invitrogen, Carlsbad, CA). Negative controls were performed by omitting the primary or the secondary anti-bodies in the staining procedures. Terminal deoxynucleoti-dyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay was performed using the dedicated kit (ApopTag, Millipore) per instructions in the manual. The neuronal nuclei-TUNEL double staining was verified with confocal imaging and quantified using image J at the periinfarct region inside the cortical infarct border, using three different pictures per mice taken under 40× objective (fig. 1). CD68+ cells, CCR2+ cells, CX3CR1+ cells, and CCR2+ with CX3CR1+ double-positive cells were counted separately, using three different pictures per mice taken under 40× objective (fig. 1) at the periinfarct region outside the infarct border.21 CD68 staining and expression of CCR2-RFP and CX3CR1-GFP were verified using confocal images (data not shown) and quantified using Image J with three different pictures/mice taken under 40× objective. The effectiveness of clodrolip in the depletion of macrophages was verified by CD68 staining of 20-μm-thick spleen sections.

Statistical AnalysesData are presented as mean ± SD. Gaussian distribution was tested with d’Agostino and Pearson omnibus normality test. Equalities of variances were tested with the F test. For dual comparisons, t tests (Student and Mann–Wittney for non-Gaussian distribution, and Welch correction of unequal variances) were used when appropriate. For multiple com-parisons, means were compared using one-way ANOVA fol-lowed by Bonferroni post hoc correction.

Comparison of the human and mice HMGB1 expres-sions before and after bone fracture was performed with two-way ANOVA. Correlations were analyzed using the Pearson r coefficient. A two-tailed P value < 0.05 was considered sta-tistically significant. Prism 5 (GraphPad Software Inc., La Jolla, CA) was used to conduct the statistical analyses.

ResultsBone Fracture Aggravates Functional and Morphological Consequences of StrokeMice with stroke alone exhibited neurobehavioral defi-cits, e.g., increased time to remove adhesive on the con-tralateral right paw and more turns to the lesion side (left turn) in the corner test, than sham-operated mice sub-jected to craniotomy only (fig. 1, A and B). These neu-robehavioral abnormalities of stroke were significantly

worsened by bone fracture, with longer latency times to remove the adhesive on the contralateral (P < 0.001, fig. 1A) paw and higher percentage of left turns in the corner test (P < 0.001, fig. 1B). Bone fracture alone did not affect neurobehavioral function, and the time to remove adhesive on the ipsilateral paw was not increased in the groups of mice with stroke alone and bone fracture alone (data not shown).

Compared to mice with stroke alone, mice with both stroke and bone fracture had infarct volumes almost three times larger (P < 0.001, fig. 1, C and D) and more TUNEL positive neurons in the periinfarct region right inside the border of the cortical infarct area (fig. 1, E and F).

Bone Fracture Shortly after Stroke Exacerbates Brain InflammationThirty hours after occlusion, mice with stroke alone exhibited higher transcript expression than controls in the left hemisphere (lesion side) for IL-1β and tumor necrosis factor-α, but not for IL-6 (fig. 2, A–C). Six hours after the bone fracture (corresponding to 30 h after the stroke), mice with both injuries had higher transcript levels of IL-6 in the left hemisphere than mice with stroke (P = 0.01, fig. 2A). The IL-1β level also trended higher in mice with both injuries than mice with stroke alone (fig. 2B, P = 0.09). Furthermore, mice with both injuries presented higher transcript expression for IL-6, IL-1β, and tumor necrosis factor-α than mice with bone fracture only (fig. 2, A–C).

Using CCR2RFP/+CX3CR1GFP/+ mice,14 we demonstrated that bone fracture increased both bone marrow-derived CCR2+ macrophages (P = 0.01) and resident CX3CR1+ microglia in the periinfarct region (P < 0.001), compared to mice with stroke only (fig. 2, D and E). About 20% of the CCR2+ cells and ≈10% of the CX3CR1+ cells expressed both CX3CR1 and CCR2 (data not shown). Mice with both stroke and bone fracture showed a trend toward more double-positive cells than mice with stroke only. The number of CCR2+ cells positively correlated with CX3CR1+ cell number (r = 0.40, P = 0.03). CCR2+ macrophages and active CX3CR1+ microglia in the periinfarct region expressed CD68, whereas CX3CR1+ cells in the contralateral hemisphere (inactive microglia) did not (data not shown)22; mice with both stroke and bone fracture had more CD68+ cells in the periinfarct region than mice with stroke alone (P < 0.001, fig. 3, A and B). The ratio of CD68-positive cells correlated with the ratio of neuronal nuclei TUNEL-positive cells (fig. 3C; r2 = 0.70, P < 0.001).

HMGB1 Plays a Causative Role in the Exacerbating Effects of Bone Fracture on Stroke InjuryHMGB1 increased in the serum within 6 h of bone fracture in both patients and mice (fig. 4, A and B). HMGB1 (50 μg/

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Fig. 1. Bone fracture enhances neuronal injury. A, Quantification of the time used to remove the tape from the right paw (con-tralateral) in adhesive removal test. (n = 10, # P < 0.001 vs. mice subjected to sham procedures for stroke and bone fracture, § P < 0.001 vs. bone fracture group * P < 0.001 vs. stroke group). B, Quantification of corner test. (n = 10, # P < 0.001 vs. mice subjected to sham procedures for stroke and bone fracture, § P < 0.001 vs. bone fracture group, * P < 0.001 vs. stroke group). C, Representative images of cresyl violet-stained brain sections (bregma 1.3 mm, scale bar: 1 mm) of stroke (C1) or stroke and bone fracture mice (C2). The red squares correspond to the three regions used to quantify neuronal nuclei (NeuN)-terminal de-oxynucleotidyl transferase deoxyuridine triphosphate nick end-labeling (TUNEL)–positive cells. The green squares correspond to the three regions used to quantify the CX3CR1, CCR2, and CD68 cells. D, The bar graph shows the quantification of infarct volumes (n = 7, * P < 0.001). E, Representative NeuN (red) and TUNEL (green) costained pictures of stroke (E1) or stroke and bone fracture mice (E2). Insert in E2 is a 3 dimensional reconstructed confocal image showing a nucleus stained positively for both NeuN and TUNEL (yellow). F, Bar graph shows the quantification of TUNEL+ neurons (n = 7, * P < 0.001).

kg), injected intraperitoneally 1 day after stroke, produced significant larger infarct volumes (fig. 4C) and more TUNEL-positive neurons in the infarct area than the saline-treated stroke mice (13 ± 2% vs. 43 ± 10%, P < 0.001, n = 6). HMGB1 treatment increased CD68+ cells in the periinfarct region (fig. 4D). HMGB1-treated stroke mice demonstrated more severe neurobehavioral dysfunction with increased adhesive removal time (fig. 4E) and percentage of left turns in the corner test (fig. 4F).

Intraperitoneal administration of HMGB1 neutralizing antibodies (10 mg/kg) immediately before bone fracture atten-uated fracture-enhanced infarct volume (fig. 5A). HMGB1 neutralizing antibodies also attenuated TUNEL-positive

neurons in the infarct area (47 ± 9% vs. 17 ± 4%, P < 0.001, n = 6), the number of CD68+ cells in the periinfarct region (fig. 5B), and the behavioral dysfunction (fig. 5, C and D).

Systemic Macrophages Play a Causative Role in the Exacerbating Effects of Bone Fracture on Stroke InjuryWe selectively depleted macrophages16 through intraperi-toneal injection of liposomal formulation of clodronate (100 mg/kg) (clodrolip) immediately before bone fracture. This intervention drastically reduced the number of CD68+ cells in the spleen (data not shown) and decreased by three times the number of CD68+ cells in the periinfarct region (P < 0.001, fig. 6A). Clodrolip treatment prior to the bone

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Fig. 3. Bone fracture exacerbates the recruitment of activated macrophages. A, Representative images of CD68 antibody stained section. A1, A low magnified picture showing the border zone (B.Z.) and the periinfarct region outside (P.I.) and inside the border (core). CD68+ cells formed a band just outside the infarct border. scale bar: 100 μm A2 and A3 are representative pictures taken in the periinfarct region outside the infarct border of mice with stroke (A2) or stroke plus bone fracture mice (A3). Scale bar: 50 μm. B, Bar graph shows the quantification of CD68+ cells in the periinfarct region (n = 7, * P < 0.001). C, Correlation between the num-bers of CD68+ cells and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labeling (TUNEL)+ neurons (r2 = 0.70, P < 0.001). DAPI = 4’,6-diamidino-2-phenylindole.

Fig. 2. Bone fracture exacerbates brain inflammation. Relative mRNA expression of interleukin (IL)-6 (A), IL-1β (B), and tumor necrosis factor (TNF)-α (C) 6 h after bone fracture and 30 h after the stroke in the stroke lesion (n = 5; [A] * P = 0.01 vs. stroke group without fracture, § P = 0.01 vs. bone fracture group; [B] # P < 0.001 vs. sham procedures for stroke § P < 0.001 vs. bone fracture group; [C] # P = 0.03 vs. the group subjected to sham procedures for stroke and § P = 0.002 vs. bone fracture group). The mice with stroke and bone fracture showed a trend toward higher IL-1β in the brain tissue than the mice with stroke only (P = 0.09). (D) Representative picture taken in the periinfarct region of CCR2RFP/+CX3CR1GFP/+ mice with stroke and bone fracture. D1. Low-magnified (scale bar: 100 μm) and D2, high-magnified (scale bar: 50 μm) images show that there are many CCR2+ (red) and CX3CR1+ (green) cells. Some cells are CCR2–CX3CR1 double positive (yellow). (E) The bar graph shows quantification of the percentage of CCR2+, CX3CR1+, or CCR2&CX3CR1+ cells amount total (4’,6-diamidino-2-phenylindole [DAPI]-positive nuclei) cells in the periinfarct region (n = 5, * P = 0.01 and ** P < 0.001).

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Fig. 4. High-mobility-group box chromosomal protein-1 (HMGB1) injection mimics the negative impact of bone fracture on stroke injury. A, HMGB1 levels in the human serum before, and 3, 6, 9, 12, and 24 h after total knee replacement surgery. Red dots repre-sent the levels in individual patients. The green curve shows the global shape of the time course (n = 4). B, Comparison of HMGB1 in human and mice serum before and after bone fracture (n = 4 for human and n = 5 for mice * P < 0.001 vs. before bone fracture). C, Intraperitoneal injection of HMGB1 increased the infarct volume of stroke mice (n = 6, * P = 0.003). D, Intraperitoneal injection of HMGB1 increased CD68+ cells in the periinfarct region (n = 6, * P < 0.001). E, Intraperitoneal injection of HMGB1 increased the time for stroke mice to remove the tape from the right paws (n = 10, * P < 0.001). F, Intraperitoneal injection of HMGB1 increased the percentage of left turn in the corner test (n = 10, * P < 0.001). DAPI = 4’,6-diamidino-2-phenylindole; ip = intraperitoneal.

Fig. 5. Neutralized high-mobility-group box chromosomal protein-1 (HMGB1) antibodies attenuate the negative impact of bone fracture on stroke injury. A, Representative images of cresyl violet-stained brain sections (bregma 1.3 mm, scale bar: 1 mm) of stroke mice with bone fracture procedure receiving control antibodies (A1, control antibody [CTab]) or anti-HMGB1 antibodies (A2, HMGB1 neutralized antibody [HMGB1ab]). B, The bar graph shows quantification of the infarct volumes (n = 7, * P < 0.001). C, Quantification of CD68+ cells in the periinfarct region (n = 6, * P < 0.001). D, Adhesive removal time of the right paw of mice treated with CTab or HMGB1ab (n = 10, * P < 0.001). E, Percentage of left turn in the corner test of mice treated with CTab or HMGB1ab (n = 10, * P < 0.001). DAPI = 4’,6-diamidino-2-phenylindole.

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Fig. 6. Depletion of macrophage reduces the negative impact of bone fracture on stroke injury. A, Representative picture taken in the periinfarct region (bregma 1.3 mm) of mice treated with control liposome (A1, control liposome [CT-lip]) and clodrolip (A2). Scale bar: 100 μm. B, A bar graph shows the quantification of CD68+ cells in the periinfarct region (n = 7, * P < 0.001). C, Quan-tification of infarct volume (n = 7, * P < 0.001). D, Quantification of adhesive removal time of the right paw (n = 10, * P < 0.001). E, Quantification of the percentage of left turn (n = 10, * P < 0.001). F, Synthesis of possible mechanisms underlying the nega-tive impact of bone fracture on stroke injury: 1, Alarmins including high-mobility-group box chromosomal protein-1 (HMGB1) is released into blood after bone fracture. 2, HMGB1 interacts with its receptors on innate immune cells including macrophages. 3, Systemic macrophages are recruited to the stroke lesion site in the brain; together with activated microglia, they release neu-rotoxic molecules. 4, Exacerbation of neuroinflammation, neuronal cell death, and behavioral dysfunction. 5, Increased neuronal death further increases the release of alarmins and chemokines, triggering a vicious cycle through HMGB1–macrophage activa-tion. DAPI = 4’,6-diamidino-2-phenylindole.

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fracture significantly reduced infarct volume (fig. 6B), neu-ronal injury (neuronal nuclei-TUNEL–positive cells of 37 ± 6% vs. 17 ± 4%, P = 0.001, n = 6), and neurobehavioral dysfunction (fig. 6, C and D) in mice subjected to stroke and bone fracture.

DiscussionSterile tissue injury, caused by surgery (fig. 4, A and B), ischemia–reperfusion,23 or hemorrhagic shock,24 provokes release of the alarmin HMGB1 that can initiate systemic inflammation and cause remote organ injury.25 Previously, we showed that bone fracture increased CD11b-positive cells in the hippocampus.6,8 Using CCR2RFP/+CX3CR1GFP/+ mice, we found that a majority of the infiltrated CD11b-positive cells are bone marrow-derived macrophages.8 In our study, both species of monocytes increased in the periinfarct region of stroke mice with tibia fracture; additionally, some of the monocytic cells were positive for both CCR2 and CX3CR1.

We postulate that after bone fracture, HMGB1 is released into the blood and interacts with pattern recognition receptors (toll-like receptors 2 and 4 as well as the receptor for advanced glycation end-products) on immunocytes, including macrophages. Together with activated microglia, systemic macrophages are recruited to the site of the stroke lesion where they are capable of releasing proinflammatory cytokines, which then exacerbate neuroinflammation that causes neuronal cell death and worsening behavior dysfunction in a feed-forward manner. Supporting these interpretations are data from the “sufficiency/necessity” experiments involving HMGB1 (figs. 4 and 5) and the necessity experiments involving systemic macrophages (fig. 6). Thus, our data demonstrate that bone fracture aggravates the functional and morphological consequences of stroke, and further suggest that this occurs through engagement of the innate immune response by the alarmin HMGB1 that likely enhances neuroinflammation in the periinfarct region (fig. 4D).

Stroke is associated with increased risk of severe fall-related bone fracture,26,27 with 4–7% of patients suffering from bone fracture within the first year of their stroke.4 Previously, we reported that circulating levels of HMGB1 increase after aseptic tibia fracture in mice, and that this is accompanied by an influx of macrophages and expres-sion of proinflammatory cytokines in the hippocampus6,7; others have shown that inflammation is an important modulatory factor in stroke-related injuries and poststroke recovery.11–13,28,29 Circulating levels of IL-6 in humans cor-related positively with imaged brain infarct volume assessed by imaging analysis, and negatively with 1-yr survival.30 In our study, IL-6 transcript did not significantly increase in the ipsilateral hemisphere of stroke mice. However, subsequent bone fracture significantly increased messenger RNA expres-sion of IL-6 in the ipsilateral hemisphere of stroke mice, which was accompanied by increased infarct volume and more severe neurobehavioral dysfunction. Our data support enhanced inflammation for mediating the negative impact

of bone fracture on the functional and morphological con-sequences of stroke.

We acknowledge that the direct link between the number of CD68+ cells and the number of TUNEL+ neurons could not be determined by this study. However, we found that the depletion of the CD68+ cells with clodrolip reduced the ratio of TUNEL+ neurons to the total number of neurons. We have also found a significant correlation between the number of CD68+ cells and the TUNEL+ neurons (fig. 3C). More-over, in vitro studies about macrophage/microglia’s activa-tion showed that excitotoxic and/or inflammation stressors can induce neuronal toxicity.31,32 All together, the evidence suggests that the activation of macrophages/microglia can be involved in early neuronal cell death and thus, exacerbate lesions and behavioral dysfunction.

We found that both CCR2-RFP+ bone marrow-derived macrophages and CX3CR1-GFP+ microglia increased in the periinfarct region of stroke mice with bone fracture. In addition, some of the cells were positive for both CCR2 and CX3CR1, suggesting that some of the bone marrow-derived macrophages acquired microglia phenotype. This phenomenon was previously observed in an experimental autoimmune encephalomyelitis model.14 Depleting the systemic macrophages with clodrolip prior to bone fracture reduced CD68+ cells infiltration, infarct volume, and neu-robehavioral dysfunction. We do not know if intraperito-neal injection of clodrolip affects resident microglia in the brain. However, our data show that both macrophages and activated microglia are capable of playing important roles in bone fracture-induced exacerbation of stroke injury.

Limitations of the StudyWe showed that bone fracture shortly after stroke enhances stroke injury via augmented inflammation through HMGB1 and macrophage/microglia infiltration. This experimental study mimics a relevant rare scenario, in which ischemic stroke patients suffer bone fracture within the first 24 h of the ischemic insult.3,4 Kanis et al.4 showed that ≈9% of stroke patients experienced a bone fracture after stroke and that 10–15% of the fracture occurs on the first day of stroke, which means that 1–1.5% of stroke patients will have a fracture on the first day of stroke. Interestingly, more than 80% of them would be older than 60 yrs. Because the elderly patient subgroup is on the rise in Western countries, this incidence should increase in the next decades.

These results give rise to questions and issues that need to be addressed in future studies. Because we report on only one time point, is it possible that bone fracture occurring at different time points, both before or after stroke, would have a different impact on stroke outcome? Could our neutralizing strategies regarding HMGB1 and systemic macrophage have blocked the alarmin and inflammatory response of the stroke itself? Given that HMGB1 has not only a neurotoxic effect in the acute stage of ischemic stroke,33 but also a protective effect in the latter stages of stroke,11,12 could an alternative timing of the

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administration of the neutralizing antibody have produced a different response? Shichita et al.34 recently showed that block-ing the release of HMGB1 with neutralized antibody was only protective when it was given during the MCAO procedure but not 6 h after the ischemic injury. Even if these data argue against the use of neutralizing HMGB1 as a neuroprotective strategy for delayed stroke lesion, our data show that inhibit-ing HMGB1 appears to be effective in preventing a second increase of HMGB1 induced by a bone fracture surgery 24 h after the stroke lesion. Further studies are needed to evaluate the effect of bone fracture occurring at different time points (before the stroke or a longer time period after).

In this study, we found that two treatments (neutraliza-tion of HMGB1 and systemic macrophage depletion) were neuroprotective in our model of pMCAO+bone fracture, and our data suggest that this neuroprotection was induced by the reduction of the macrophage recruitment. However, these two strategies can also indirectly affect other proin-flammatory pathways, such as trauma-induced hyperther-mia, that could provide neuroprotection as well.

We measured the ratio of the immune cells (CD68, CX3CR1, and CCR2) in the periinfarct region just outside the infarct border, as shown in figures 1C and 3A. These measures were performed at the same coordinates for all the mice (bregma +1.3 mm) for consistency. It is important to note that the variation in the infarct size can affect the loca-tion of the infarct border. We cannot exclude the possibility that the quantification of the macrophages was influenced by the location of the infarct border.

Using CX3CR1-GFP; CCR2-RFP mice, we found that bone fracture increased the numbers of both CCR2+ and CX3CR1+ cells in the brain. For this reason, we considered that the increase in the two cell populations was associ-ated with the phenotype, and thus decided to focus on the CD68+ cells. CD68 antibody stains for a lysosomal protein that is mainly expressed in the activated phagocytosis cells. In the normal brain, there are few CD68+ cells, whereas a significant number of CD68+ cells is present in the periin-farct side of the border zone of pMCAO.21 Although we cannot distinguish CCR2+ and CX3CR1+ cells by CD68 antibody staining, we were able to show that the neutralized antibody and the clodrolip treatments reduced CD68+ cells in the periinfarct side of the border zone.

We selected two well-established behavior tests that exhibit reproducibility in a pMCAO model.18,19 As shown in figure 1, tibia fracture alone did not significantly influence mouse performance in these tests. However, we could not completely rule out the influence of bone fracture on this function in mice with pMCAO and bone fracture, as these mice may have more severe hind-limb dysfunction due to increased inflam-mation in the fracture. Furthermore, even if we show that the frequency of neuronal cell death increases in mice with pMCAO and bone fracture, we cannot rule out that cortical edema induced by the activation of the neuroinflammation does not play a role in increased behavioral dysfunction.

In summary, we demonstrated that bone fracture shortly after ischemic stroke increases stroke-related neuronal injury and neurobehavioral dysfunction in mice. HMGB1 and macrophage/microglia play a causal role in the negative impact of bone fracture on stroke outcomes. Our findings regarding modulation of HMGB1 level and macrophage/microglia activities pose possible intervention opportunities for patients with both stroke and bone fracture.

The authors thank members of the Center for Cerebrovascular Re-search and the Maze laboratory (both from the University of Califor-nia, San Francisco, San Francisco, California) for their support, as well as Katerina Akassoglou, Ph.D.; Israel F. Charo, M.D., Ph.D.; and Kim Baeten, Ph.D. (Associate Investigator, Associate Director, and Postdoc-toral Fellow, respectively, University of California, San Francisco; Glad-stone Institute, San Francisco, California), for providing the CCR2RFP/+

CX3CR1GFP/+ mice.

References

preoperative predictors of outcomes in orthopedic and vas

acute coronary syndrome or stroke and the operation. Ann

matory and immune responses in vascular disease. Stroke

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93

Metabolic Syndrome

94

Surgery Results in Exaggerated and Persistent Cognitive Decline in a Rat Model of the Metabolic Syndrome Participated in study design, in data analysis and interpretation and final preparation of the

manuscript.

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1098 May 2013

ABSTRACT

Background: Postoperative cognitive decline can be repro-duced in animal models. In a well-validated rat model of the Metabolic Syndrome, we sought to investigate whether sur-gery induced a more severe and persistent form of cognitive decline similar to that noted in preliminary clinical studies.Methods: In rats that had been selectively bred for low and high exercise endurance, the low capacity runners (LCR) exhibited features of Metabolic Syndrome (obesity, dyslip-idemia, insulin resistance, and hypertension). Tibial fracture surgery was performed under isoflurane anesthesia in LCR and high capacity runner (HCR) rats and cognitive function

was assessed postoperatively in a trace-fear conditioning para-digm and Morris Water Maze; non-operated rats were exposed to anesthesia and analgesia (sham). Group sizes were n = 6.Results: On postoperative D7, LCR rats had shorter freez-ing times than postoperative HCR rats. Five months postop-eratively, LCR rats had a flatter learning trajectory and took longer to locate the submerged platform than postoperative HCR rats; dwell-time in the target quadrant in a probe trial was shorter in the postoperative LCR compared to HCR rats. LCR and HCR sham rats did not differ in any test.Conclusion: Postoperatively, LCR rats diverged from HCR rats exhibiting a greater decline in memory, acutely, with persistent learning and memory decline, remotely; this could not be attributed to changes in locomotor or swim-ming performance. This Metabolic Syndrome animal model of surgery-induced cognitive decline corroborates, with high fidelity, preliminary findings of postoperative cognitive dys-function in Metabolic Syndrome patients.

M ILD transient cognitive decline after surgery may occur commonly; however, some patients develop a

more severe form that meets the Diagnostic and Statistical Manual of Mental Disorders IV classification of delirium or a longer-lasting form, referred to as postoperative cognitive dysfunction. It has been estimated that 11% of patients undergoing elective surgery develop delirium between post-operative days 1–4; surgical patients with this complication

Surgery Results in Exaggerated and Persistent Cognitive Decline in a Rat Model of the Metabolic Syndrome

Xiaomei Feng, M.D., Ph.D.,* Vincent Degos, M.D., Ph.D.,† Lauren G. Koch, Ph.D.,‡ Steven L. Britton, Ph.D.,§ Yinggang Zhu, M.D.,‖ Susana Vacas, M.D.,# Niccolò Terrando, B.Sc., Ph.D.,** Jeffrey Nelson, M.S.,†† Xiao Su, M.D., Ph.D.,† Mervyn Maze, M.B., Ch.B.‡‡

* Post-doctoral Fellow, Department of Anesthesia and Periopera-tive Care, University of California, San Francisco, San Francisco, Cali-fornia, and Department of Anesthesiology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China. † Associate Researcher, Department of Anesthesia and Perioperative Care, Uni-versity of California, San Francisco. ‡ Associate Professor, Department of Anesthesiology, University of Michigan Medical School, Ann Arbor, Michigan. § Professor, Department of Anesthesiology, University of Michigan Medical School. ‖ Visiting Assistant Professor, Department of Anesthesia and Perioperative Care, University of California, San Francisco. # Post-doctoral Fellow, Department of Anesthesia and Perioperative Care, University of California, San Francisco. ** Depart-ment of Anesthesia and Perioperative Care, University of California, San Francisco. †† Associate Specialist, Department of Anesthesia and Perioperative Care, University of California, San Francisco. ‡‡ Pro-fessor and Chair, Department of Anesthesia and Perioperative Care, University of California, San Francisco.

Received from the Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, Califor-nia. Submitted for publication August 4, 2012. Accepted for publica-tion January 2, 2013. The LCR-HCR rat model system was funded by the National Center for Research Resources grant (R24 RR017718) and is currently supported by the Office of Research Infrastructure Programs/OD grant (ROD012098A) from the National Institutes of Health, Bethesda, Maryland (Lauren G. Koch and Steven L. Britton), University of Michigan Medical School, Ann Arbor, Michigan. Addi-tional support was provided by the Department of Anesthesia and Perioperative Care, University of California, San Francisco.

Address correspondence to Dr. Maze: Department of Anesthesia and Perioperative Care, University of California, 521 Parnassus Avenue, C455, San Francisco, California 94143-0648. [email protected]. Information on purchasing reprints may be found at www.anesthesiol-ogy.org or on the masthead page at the beginning of this issue. ANESTHE-SIOLOGY’S articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.

What We Already Know about This TopicThe contributions of surgery, anesthesia and pathophysiologi-cal factors in postoperative cognitive decline are difficult to resolve in clinical studiesMetabolic syndrome might enhance inflammation-mediated postoperative complications, including cognitive dysfunction

What This Article Tells Us That Is NewUsing a rat model, metabolic syndrome produced greater memory impairment and persistent learning and memory decline following tibial fracture surgery under isoflurane an-esthesiaFurther studies are necessary to determine the mechanisms of these effects, and how metabolic syndrome exacerbates postoperative cognitive dysfunction

Persistent POCD and Rodent Metabolic Syndrome

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require more intensive care and have a higher mortality rate.1 Because postoperative cognitive dysfunction is not classified in the Diagnostic and Statistical Manual of Mental Disorders IV, it lacks firm diagnostic criteria; if a threshold of “worse than expected” postoperative cognitive decline is used, then postoperative cognitive dysfunction occurs in >10% of non-cardiac surgical patients over the age of 65 years.2

With neither standardized cognitive domain testing nor appropriate controls, some have challenged whether the fre-quency of persistent postoperative cognitive decline is beyond that expected from “natural,” age-dependent deterioration.3–5 In order to overcome the lack of appropriate controls in clini-cal studies (i.e., surgical patients who are randomized to a cohort that does not receive surgery), we have set up models of rodents in which to explore the independent effects of sur-gery and anesthesia, and to address the molecular and cellular mechanisms that contribute to this condition.6–9 For animal models to be valid, these models need to reproduce salient features of the clinical syndrome. Patient factors, including increasing age, less higher education, postoperative compli-cations (respiratory and infectious), and re-operation, had all been reported to increase the likelihood of developing post-operative cognitive decline.2,10 We, and others, have shown that more severe and persistent cognitive decline occurs in the settings of perioperative infection11 and advanced age.12

The induction of short-lived neuroinflammation, following the release of damage-associated molecular patterns from trau-matized tissue, appears to be necessary for the organism’s central nervous system-mediated “sickness behavior;” this is comprised of a fever, anorexia, somnolence, and cognitive impairment, and injured animals in this state remain sedentary promoting healing rather than risking further injury. Our recent preclinical studies have revealed that the transient cognitive decline that follows aseptic surgical trauma occurs by engaging the innate immune system through nuclear factor-κB-dependent signaling to release cytokines that disrupt blood brain barrier integrity.6–9 Through a permeable blood brain barrier, bone marrow-derived macrophages migrate into the brain parenchyma promoting neuroinflammation that is capable of interfering with processes required for learning and memory.8,13

Two recent preliminary reports from Hudetz et al. have drawn attention to an exacerbation of early postoperative cog-nitive decline, as well as a greater likelihood of the persistence of postoperative cognitive decline in patients with Metabolic Syndrome (MetaS) who undergo either cardiac or non-cardiac surgery, respectively.14,15 In order to investigate the possible mechanisms, we reverted to a validated animal model of MetaS.

Most of the mouse models of MetaS are created by the extinc-tion or modification of a single gene ± a dietary perturbation. These gene knock-out/knock-in approaches reveal the depen-dence on that gene, together with the subsequent adaptations, for the resulting loss or gain of function. These approaches may fail to model the human phenotype because they do not recon-stitute complex gene-gene interaction. Amongst the several rat models of MetaS, we have chosen one that not only has the

appropriate phenotype but also has been generated in a manner that is “lifestyle-related.” Starting in 1995, Koch and Britton applied divergent artificial selection for intrinsic low and high endurance running capacity starting with a founder population of genetically heterogeneous rats. Thirty generations of selec-tion have produced lines of low capacity runners (LCRs) and high capacity runners (HCRs) that differ by 7-fold in treadmill running capacity.16 The LCR rats contain features of the MetaS, including elevated low density lipoproteins, cholesterol, blood pressure, triglycerides, fasting glucose, insulin, C-reactive pro-tein, and visceral adiposity being 100 g heavier than the HCR rats at 12 weeks.17 The LCR rats have a low intrinsic aerobic capacity that results in easy fatigability; this may contribute to their sedentary behavior18 which is thought to be causally related to the development of the MetaS.19,20

Using this rat model of MetaS, we have explored whether cognitive decline is more severe in the early postoperative period and whether it is likely to be more persistent in the LCR rats that have the features of MetaS; establishment of this an abnormal immune response to elective surgery can set the stage for a thorough exploration of the mechanisms that can contribute to these and other inflammation-medi-ated postoperative complications.

Materials and MethodsAnimalsAll experimental procedures involving animals were approved by the University of California, San Francisco Institutional Animal Care and Use Committee, and con-formed to National Institute of Health guidelines. Animals were handled in strict accordance with good animal practice. LCRs and HCRs were developed by Koch and Britton.16 The LCR rats are maintained as genetically heterogeneous lines by using a rotational mating paradigm that minimizes inbreeding, thereby maintaining genetic complexity and allowing combinations of allelic variants at multiple inter-acting loci to be enriched by selection pressure.16,21

Only male rats aged between 4 and 5 months were used. All animals were fed standard rodent chow and water ad libitum, and were housed (2 rats per cage) in sawdust-lined cages in an air-conditioned environment with 12-h light/dark cycles. Animals were tagged and randomly allocated to the surgery or sham group before any procedure was undertaken; researchers were blinded to the group assignment during assessments, prior to the analysis phase. Because of the difference in color and weight between the LCR (brown, approximately 400 g) and HCR (white approximately 300 g) rats, it is not possible for the observer to be blinded for the phenotype, but they were blinded for the treatment group. Forty-eight rats were included with no lethality or exclusion.

SurgeryUnder general anesthesia with 2.1% isoflurane in 0.30 FiO2, rats underwent an open tibial fracture of the left hind paw

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with an intramedullary fixation under aseptic surgical condi-tions as previously described.7–9 The surgical field was main-tained as sterile throughout the procedure. Briefly, the left hind paw of surgical animals was meticulously shaved and disinfected with povidone iodine. Following a median inci-sion, a 20 G pin was inserted into the intramedullary canal, the periosteum was stripped, and osteotomy was performed. The wound was irrigated, the skin was sutured, and the ani-mals were allowed to recover spontaneously from the anes-thetic. During the procedure, temperature was monitored and maintained at 36.5–37.5°C with the aid of warming pads (Harvard Apparatus, Holliston, MA) and a temperature-con-trolled light. Buprenorphine (0.1 mg/kg) was given subcuta-neously to provide analgesia after the induction of anesthesia and before skin incision. The sham rats were exposed to anes-thesia and analgesia as above and had the paw shaved.

Twenty-four rats were used for trace-fear conditioning (TFC) assessed 7 days after surgery and an additional 24 rats were used for Morris Water Maze (MWM) assessed 5 months after surgery.

Behavioral StudiesTrace Fear Conditioning. TFC was used to assess hippocam-pal-dependent memory in rodents as previously described.7–9 The clear acrylic TFC chamber (Med Associates Inc., St Albans, VT), with dimensions of 32 cm long, 25 cm wide, and 25 cm high, included a floor constructed of stainless steel bars that was connected to a shock delivery system (Med Associates). The chamber was wiped with a pine-scented cleaner (5% Pine Scented Disinfectant; Midland, Inc., Sweetwater, TN) before and after each session, and training and assessment was performed in a room illuminated with overhead fluorescent bulbs with a ventilation fan providing background noise (65 db). During the training, rats were allowed to explore this context for 3 min, after which they were presented with an auditory cue (75–80 dB, 5 kHz, con-ditional stimulus for 20 s). The unconditional stimulus, a 2-s foot shock (0.8 mAmp), was administered 20 s after ter-mination of the auditory tone. Rats were removed from the chamber after an additional 30 s. Rats anticipate the shock by “freezing,” which is defined as the absence of all move-ment except for respirations; this defensive posture reflects learned fear. When placed in the same context on a subse-quent occasion, the learned fear is recalled, and the amount of learning and recall is measured by the amount of freezing. Surgery was performed within 30 min after training. Mem-ory of the learned fear was assessed 7 days later by returning the rat into the same chamber in which it was trained, in the absence of tone and shock. Behavior was recorded by a Polaris digital video recorder (Cohu Electronics Division, Poway, CA). Each animal’s behavior was scored every 5 s during the 5 min observation period and a percentage was calculated using the formula 100×f/n, where f is the num-ber of freezing events per rat and n is the total number of observations per rat.

Open Field. A standardized measure of general motor func-tion is spontaneous activity. At the conclusion of the con-textual fear response, rats were placed in a wooden open field apparatus (45” square; 18” high) in which the floor was subdivided into 25 blocks (9” square) with thin white stripes, and activity was recorded by the Polaris digital video recorder (Cohu Electronics Division, Poway, CA). The number of line crossings and rearings performed in a 5 min epoch was scored by an observer that was unaware of group assignment.22

Morris Water MazeFive months after surgery, separate cohorts of rats were inves-tigated in the MWM in the following manner.23

Cueing Procedure. A platform (diameter, 10.3 cm) was placed one inch above the level of warm (24°C), opaque water in a circular pool (diameter, 180 cm; depth, 50 cm). The platform was rendered more visible from the surface by mark-ing its edge with bright, yellow tape. For each of the three sessions, the rat was placed in the water and emerged from the water onto the platform within 60 s; if not, the rat was guided towards the raised platform. A different platform site was used for each session. This cueing procedure enables the rat to real-ize that they can escape the water by locating a platform.Spatial Reference Memory. The pool is surrounded by visual cues and the platform is submerged below the surface. Daily, two training sessions spaced 7 h apart, were performed; for each session, the rat was released from one of three assigned locations facing the wall of the tank, resulting in one short, one medium, and one long swim per session, in random order. Rats were given 90 s to locate the hidden platform; if the rat failed to locate the hidden platform within the allotted time, the rat was guided to the platform. In either case, the rat was removed from the platform after 15 s. To minimize any bias associated with platform location, equal numbers of rats in each group were assigned one of the four quadrant locations of the platform for the duration of training. The time to reach the platform (latency), path length, swimming speed, and time-integrated distance to the platform were analyzed using an EthoVision video tracking system (Noldus Instruments, Wageningen, Holland) that was set to analyze 10 samples per second. The mean difference of decrease in the escape latency to the submerged platform per session, as well as the escape latency for the final session (session number 10), were analyzed.Probe Trials. To assess memory retention for the hidden platform location, a probe trial, with the platform removed from the tank, was performed immediately after the last training session. During the 60-s probe trial, the proportion of time spent in the quadrant in which the platform previ-ously resided (“target quadrant”), as well as each of the other quadrants, was determined for a 60-s interval.

Statistical AnalysisData are presented as a mean ± SD. We tested for normal distribution of the data with the d’Agostino and Pearson

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omnibus test and the equality of variances with the F-test. For comparisons of the two independent variables (strain [HCR/LCR] and surgery [fracture/sham]), we performed two-way ANOVA; this was followed by four pairwise stu-dent t tests with a Bonferroni correction (Bonferroni cor-rected alpha = 0.05/4 = 0.0125).

For TFC, we performed two-way ANOVA, testing the percentage of freezing time followed by four pairwise t tests (Bonferroni corrected alpha = 0.05/4 = 0.0125).

Regarding the MWM test, based on previous data24 (SD of the percentage of dwell time in target quadrant = 15), we estimated that a sample of 6 rats per group was neces-sary to find a significant difference between the LCR and the HCR surgery groups, with 80% of power if the differ-ence was 25%. For the spatial reference memory experi-ments, the escape latency to the submerged platform of the 4 groups in the last session was analyzed with two-way ANOVA followed by four pairwise t tests (Bonferroni cor-rected alpha = 0.05/4 = 0.0125). To analyze the mean differ-ence of decrease per session within the 10 sessions, we used a mixed-effects linear regression model25 with a three-way interaction involving session number, intervention (surgery or sham), and strain (LCR/HCR) with all lower order terms as predictors. We performed four additional post hoc models for pairwise comparisons of groups to determine whether there was a faster improvement in the latency time to plat-form amongst pairings across sessions; a pairing was deemed significantly different if the interaction term for the session and group was significant in the model (Bonferroni cor-rected alpha = 0.05/4 or 0.0125). For the probe trial, we compared the proportions of dwell time spent in each of the quadrants for the groups with a two-way ANOVA followed by four pairwise t tests (Bonferroni corrected alpha = 0.05/4 or 0.0125).

A two-tailed P value < 0.05 was considered statistically significant and data were analyzed using Stata 11.2 software (StataCorp, College Station, TX).

ResultsAnimals were assessed by open field testing after the con-textual fear response on day 7, in order to exclude possible locomotor impairments that could confound the TFC assess-ment. Spontaneous movement was not different in the LCR versus HCR rats with or without surgery (fig. 1). Acute post-operative memory, assessed by the percentage of time spent freezing when the rat was placed in the same context as the pre-operative TFC training, was impaired in both cohorts of postoperative rats on postoperative day 7. Two-way ANOVA revealed a significant interaction between the strain and intervention (P = 0.02); the degree of memory impairment, as reflected by the percentage freezing, was greater in the postoperative LCR than the postoperative HCR rats (20 ± 4, vs. 33 ± 6, P = 0.006, fig. 2).

In the MWM test, the swimming speed was similar in all groups tested at 5 months (fig. 3A). Although the strain of

the rats presented a significant effect on the weight recorded one week prior to the MWM (P < 0.001), surgery had no significant effect, suggesting that all groups continued to thrive throughout the course of the study (fig. 3B).

LCR surgery rats had significantly less overall improvement with successive tests compared to HCR surgery, as evidenced by a significantly longer time required to locate the submerged platform after the final, 10th session (P = 0.007 for two-way ANOVA strain effect). Pairwise post hoc t tests revealed that this strain effect was significant in the surgery rats (LCR surgery vs. HCR surgery, 34 ± 21 vs. 15 ± 6, P = 0.001, fig. 4A). A three-way interaction in the mixed-effects model was significant (P = 0.010), indicating that the effects of surgery and strain could not be considered in isolation when explaining variation in the rate of change in time to the submerged platform over successive trials. The mean rate of improvement (seconds/per session) was significantly less (P < 0.001) for the postoperative LCR rats (2 s/session) than for the sham-LCR rats (6 s/session) in a pairwise comparison of groups (fig. 4B). The difference in improvement per session between postoperative LCR rats and postoperative HCR rats (3 s/session, P = 0.010) was also significant after applying a Bonferroni correction.

Fig. 1. Effect of phenotype and surgery on spontaneous mo-bility in open field test. On postoperative day 7, vertical (num-ber of rearings, A) and horizontal (number of grid-crossings, B) movement was recorded for 5 min. Data were analyzed with two-way ANOVA with post hoc pairwise t test com-parisons with Bonferroni correction; results are expressed as mean and SD (n = 6). There was no statistically significant difference noted in pairwise comparisons. HCR = high capac-ity runner phenotype; LCR = low capacity runner phenotype; surg = surgery.

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The modeled curves reveal that the improvement in the escape latency per session to locate the hidden platform in the LCR surgery group was smaller than the LCR sham group (fig. 4B), whereas the improvements of HCR surgery and HCR sham rats were similar (fig. 4C).

In the probe trial, two-way ANOVA revealed a significant interaction between strain and intervention (P = 0.02) for the target quadrant (in which the platform formerly resided); post hoc analyses revealed that the percentage of time that the postoperative LCR rats spent in the target quadrant was significantly shorter than for each of the other groups (for example—LCR surgery vs. HCR surgery 24 ± 7 vs. 40 ± 6, P = 0.007, fig. 5). Apart from the postoperative LCR group, groups spent significantly more time in the target quadrant than in each of the others, while the postoperative LCR rats spent equivalent time in each of the four quadrants.

DiscussionCognitive decline after surgery, whether temporary in the form of Postoperative Delirium or persistent, possibly in the form of Postoperative Cognitive Dysfunction, appears to be associated with long-term adverse outcomes including withdrawal from the workplace, loss of independent living, and an increase in mortality rate.2,26 Therefore, it is vital to understand the risk factors and pathogenesis that may con-tribute to these postoperative complications.

In a series of preclinical studies, we had noted that postoperative cognitive decline is due to the engagement of the innate immune system in response to aseptic

trauma.6–9,27,28 The expression of cognitive decline is due to neuroinflammation that produces a constellation of symptoms, referred to as “sickness behavior,” which is thought to protect the traumatized organism from further injury and to facilitate the healing process.29 This is a short-lived process in which neuroinflammation is curtailed by feedback mechanisms that involve both neural (especially the parasympathetic nervous system), as well as humoral (including oxygenated and nitrogenated lipid) factors.30,31 Disruption of these precisely regulated processes may contribute to abnormal quantitative and qualitative responses to aseptic trauma.8 The existence of a disease that causes either exacerbation of the acute postoperative decline and/or the persistence of cognitive impairment, validates the animal model as one in which to explore pathogenic mechanisms and, thereby, possible therapeutic interventions.14,15

MetaS, comprised of insulin resistance (hyperglycemia that can progress to Type 2 diabetes mellitus), visceral obesity, hypertension, and dyslipidemia increases the risk of postoperative complications contributing to a significantly higher mortality rate.14,15,32 In a recent review of a case series of coronary artery bypass surgical patients, those suffering

Fig. 2. Acute effect of surgery and phenotype on hippo-campal-dependent memory. After pre-operative training in a trace-fear conditioning paradigm, the percent time that a rat spent freezing during a 5 min epoch was assessed on post-operative day 7 when placed in the same context as the train-ing. Data were analyzed with two-way ANOVA with post hoc pairwise t test comparisons with Bonferroni correction and results are expressed as mean and SD (n = 6). A significant interaction was noted between strain and surgery (P = 0.02). Post hoc pairwise comparison revealed that each of the surgi-cal groups exhibited significantly less freezing time than their own sham cohort HCR; furthermore, the LCR surgical group exhibited significantly less freezing time than the HCR surgi-cal group. There was no significant difference between the LCR/sham and HCR/sham groups. * P < 0.01. HCR = high capacity runner phenotype; LCR = low capacity runner phe-notype; surg = surgery.

Fig. 3. Effect of surgery and phenotype on swim speed and thriving. (A) Five months after surgery, swimming speed was measured during the spatial reference memory portion of the Morris Water Maze. Data were analyzed with two-way ANO-VA with post hoc pairwise t test comparisons with Bonferroni correction; results are expressed as mean and SD (n = 6). There was no statistically significant difference noted for ei-ther interactions or for pair-wise comparisons. (B) Weights were measured five months after surgery and data were ana-lyzed with two-way ANOVA; this showed a significant effect of the strain without any effect of the surgery (n = 6, P <0.001 for strain, P = 0.93 for surgery, and P = 0.81 for interaction by strain and surgery). HCR = high capacity runner phenotype; LCR = low capacity runner phenotype; surg = surgery.

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with MetaS had a longer postoperative stay although overall outcome was not affected.33 Many of the complications of MetaS (including atherosclerosis) are inflammatory in nature with the pathological adipose stores being the source of pro-inflammatory adipokines.34 It is estimated that more than a quarter of the American adult population has MetaS; its prevalence is probably over-represented in the surgical population, with half of all patients undergoing cardiac surgery being afflicted by this syndrome.32 Recent evidence indicates patients suffering from MetaS may be particularly susceptible to postoperative cognitive decline.14,15

Starting in 1995, Koch and Britton applied divergent artificial selection for intrinsic low and high endurance run-ning capacity starting with a founder population of geneti-cally heterogeneous rats. Thirty generations of selection have produced lines of LCRs and HCRs that differ 7-fold in treadmill running capacity16; the LCR rats contain fea-tures of the MetaS, including elevated low density lipo-proteins, cholesterol, blood pressure, triglycerides, fasting glucose, insulin, C-reactive protein, and visceral adiposity

being approximately100 g heavier than the HCR rats at 12 weeks.18 The LCR-HCR rats represent polygenic substrate that can be used for exploring medically-relevant features, such as postoperative cognitive decline, that may be asso-ciated with the MetaS.14,15 The major genetic hypothesis is that contrasting alleles, causative of the trait differences, have been enriched or fixed differentially between the lines.

In the behavioral paradigms that we have used to interro-gate cognitive domains of learning and memory, non-surgi-cal LCR and HCR rats did not differ in either TFC or in the MWM. For the MWM experiments, we applied a mixed-effects model that took into account the strain, the type of surgery and the repeated measures. This model allowed us to gain power and to capture the rate of learning of the rats. Other tests, which are perhaps more “subtle” than the ones that we employed, do show a behavioral phenotype in non-surgical rats.35 Postoperatively, the LCR rats exhibit both an early exacerbation of memory decline as well as a persis-tent abnormality in both learning and memory. Non-cog-nitive factors that could have contributed to the behavioral

Fig. 4. Effect of remote surgery and phenotype on learning. (A) Five months after surgery, rats were tested twice daily for 5 con-secutive days, for their ability to locate a submerged platform in a Morris Water Maze. For each of the ten sessions, the latency (s) to escape the water and find the platform was the average of three scored trials. Data were analyzed at session 10 with two-way ANOVA showing a significant effect of the strain (P = 0.007 for strain), but not for surgery, or for the interaction by strain and surgery. Post hoc pairwise t test comparisons with Bonferroni correction revealed that escape latency to the submerged platform in LCR surgery rats was significantly longer than for the HCR surgery rats (* P = 0.01). Mixed-effects linear regression model of the effect of surgery on learning in LCR (B) and HCR (C) rats. The average raw values for each session from A were analyzed, taking into account the three-way interaction involving session number, surgery/sham and a session-squared term. Pair-wise comparisons with Bonferroni correction revealed that the slope was significantly less in LCR surgery rats compared to the LCR sham rats (P < 0.001); additionally, there was a significant difference in slope between LCR surgery rats and HCR surgery rats (P = 0.010). The curves for sham and surgery HCR rats were similar (C). HCR = high capacity runner phenotype; LCR = low capacity runner phenotype; surg = surgery.

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assessments, such as altered spontaneous movement in TFC or the swim speed in MWM, are not different between the LCR and HCR rats.

It is noteworthy that the HCR rats may not represent “normal” rats and are more akin to organisms that are per-forming at a higher level of efficiency for energy expenditure compared to “normal” rats. As eight strains of rats formed the original cross-breeding reagents, it is not possible to ascertain which strain of rat is the normal one to be used as a con-trol. Therefore, we can only comment that postoperatively, the LCR rats diverge from the HCR rats and are not able to opine whether this difference relates to deterioration in func-tion from normal for the LCR rats and/or an improvement from normal for the HCR rats. However, it is noteworthy that the HCR rats do not appear to recover from acute post-operative memory decline faster than that noted in our earlier study involving male Sprague Dawley rats in a Y-maze test.6

Our findings set the stage for a thorough exploration of the reasons why MetaS induces differences in postop-erative cognitive function. Recently, we reported that when

the cholinergic neural feedback mechanism for surgery-induced neuroinflammation is disrupted in wild-type mice, an exacerbation of postoperative cognitive decline follows.8 It is noteworthy that patients with MetaS have a disorder in cholinergic function.36,37 Further exploration in these rat reagents may reveal the mechanism for this abnormal-ity in cholinergic function and, thereby, reveal targets for interventions.

While we have concentrated on the reasons why the brain is a target for postoperative inflammatory complications, it will be important to consider whether other postoperative complications with a putative inflammatory basis, includ-ing conversion from acute to chronic postoperative pain and thrombo-embolic complications, may have a similar propen-sity to occur in MetaS possibly on the basis of dysregulation of mechanisms involved in the initiation and/or resolution of inflammation.

The authors acknowledge the insights on linear mixed effects mod-eling provided by Pedro Gambus, M.D., Visiting Professor, Depart-ment of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, California.

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ium in the surgical patient. Psychosomatics 2001; 42:68–73 2. Monk TG, Weldon BC, Garvan CW, Dede DE, van der Aa

MT, Heilman KM, Gravenstein JS: Predictors of cognitive dys-function after major noncardiac surgery. ANESTHESIOLOGY 2008; 108:18–30

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4. Ehlenbach WJ, Hough CL, Crane PK, Haneuse SJ, Carson SS, Curtis JR, Larson EB: Association between acute care and critical illness hospitalization and cognitive function in older adults. JAMA 2010; 303:763–70

Zeger SL, McKhann GM: Cognitive and neurologic outcomes

6. Wan Y, Xu J, Ma D, Zeng Y, Cibelli M, Maze M: Postoperative impairment of cognitive function in rats: A possible role for cytokine-mediated inflammation in the hippocampus. ANESTHESIOLOGY 2007; 106:436–43

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Fig. 5. Remote effect of surgery and phenotype on memory. Five months after surgery, immediately following the last ses-sion in the spatial reference test (described in Figure 4), rats were returned to the Morris Water Maze in which the sub-merged platform had been removed. The probe trial consist-ed of measuring the % of time spent within a 60 s epoch, in the quadrant in which the platform formerly resided; this is referred to as dwell time in target quadrant. In addition, the time spent in the opposite quadrant, clockwise quadrant (CW), and counterclockwise (CCW) quadrant were also mea-sured. Each quadrant’s data were analyzed separately with two-way ANOVA followed with post hoc analyses, and results are expressed as mean and SD (n = 6). For the target quad-rant, there was a significant interaction between strain and surgery (P = 0.04) and the dwell time was significantly lower (* P < 0.01) in the LCR surgery rats than for either the HCR surgery or LCR sham groups. For the opposite quadrant, there was a significant strain and surgery effect (P = 0.003 for the surgery, P = 0.04 for the strain and P = 0.12 for the inter-action) and the percent of dwell time in the LCR surgery rats was significantly higher in LCR sham rats (* P < 0.01). The four groups spent similar time in CW and CCW quadrants. HCR = high capacity runner phenotype; LCR = low capacity runner phenotype; surg = surgery.

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DISCUSSION AND CONCLUSION

POCD has long-term consequences, including higher mortality rate and costs

associated with the care of the cognitively impaired. According to rodent

models of postoperative cognitive decline, activation of the innate immune

response following aseptic surgical trauma results in the elaboration of

hippocampal proinflammatory cytokines, which are capable of disrupting long-

term potentiation, the neurobiologic correlate of memory. We used a specific

pharmacologic strategy to acutely deplete systemic phagocytes before an

aseptic surgical trauma with an experimental tibia fracture. Depletion of BM-

DM reduced the circulating inflammatory mediators, reducing migration of

immune cells into the brain, and postoperative memory deficits. We also

showed that the recruitment of BM-DM is mediated by hippocampal signaling

through MCP-1. Following the characterization of the resolution of surgery-

induced memory decline, we sought to describe the mechanisms involved in

its initiation. We found that a single injection of HMGB1 antigen is sufficient to

cause cognitive decline through the activation and trafficking of circulating

BM-DM to the brain. HMGB1 antibody reduced the systemic and

neuroinflammatory response to surgery, preventing post-surgical cognitive

decline.

Studies have sought to identify factors that may contribute to POCD, which

include surgery, as well as in-patient care factors, and patient-related factors.

We tried to understand the causal relationship between a common occurrence

during in-hospital care, sleep disruption, and cognitive impairment. We found

that SF independently produced significant memory impairment, although

perioperative SF significantly increased hippocampal inflammation without

further cognitive impairment.

If we divide the possible risk factors into categories of modifiable/non-

modifiable and patient related/environmental, we can both disentangle the

causes of the neuroinflammatory cascade and also to focus on possible

clinical adjustments and applications to stave it off. Using a rodent model, we

demonstrated that surgery aggravates functional and morphological

104

consequences of stroke by depleting macrophages. The effects of bone

fracture on stroke were attenuated by neutralizing HMGB1.

The fact that patients with MetaS have a significantly greater likelihood of

exhibiting cognitive decline than a cohort of patients without MetaS,

encouraged us to investigate the extent and severity cognitive decline in

MetaS patients. Using a genetically heterogenous lineage of MetaS rat model,

we showed that MetaS produced greater memory impairment and persistent

learning and memory decline following tibia fracture surgery.

In the majority of patients, postoperative neuroinflammation is part of the

normal protective mechanism to peripheral trauma and resolves properly with

no residual cognitive consequences. Indeed, it is also possible that surgery for

a chronic inflammatory disease may result in cognitive improvement by

eliminating disease-inducing cognitive impairment that may be associated

with chronic inflammatory disease. That said, some risk factors, such as

MetaS, patients prone to neurological disease, and poor selection of sedative

agents, may each promote the intractable persistence of neuroinflammatory

response to surgery. For an increasing number of patients with advanced age,

POCD is alarmingly common, making postoperative central nervous system

dysfunction a looming public health crisis given world's rising elderly

population.

Understanding the cellular and biologic pathways involved in postoperative

cognitive decline is a key element in designing interventions to prevent this

disease. Reducing activation and/or migration of innate immune cells, such as

systemic macrophages into the brain, represents a viable preemptive strategy.

We demonstrated that BM-DM activation after experimental tibia fracture is

directly involved in the tibia fracture–induced hippocampal BM-DM infiltration

and animal memory dysfunction. HMGB1, when released from a sterile

traumatic injury, plays a pivotal role in postoperative memory dysfunction.

Together with the detection of the cell type involved in the initiation of the

surgery-induced inflammatory cascade, these findings establish both the

precise elements of the immune response that need to be interrogated for

establishing the risk of dysregulated trauma-induced inflammation as well as

105

putative targets for interventions designed to limit or reverse persistent

postoperative cognitive decline.

These studies on the initiation of trauma-induced cognitive decline, coupled

with previous reports on the resolution of postoperative cognitive decline12,14,44 set the stage for the development of an ex-vivo bio-assay that can test the

function of the innate immune response to trauma. Such an assay may be

capable of prospectively identifying surgical patients at increased risk for the

development of exaggerated and persistent cognitive decline; stratification of

a surgical cohort, enriched for the development of cognitive decline, can result

in a randomized trial to test with efficacy of interventions using fewer surgical

patients.

Clearly, the surgical effect of this neuroinflammatory trigger is just one

possible mechanism. Indeed, environmental culprits can also offer a window

into the postoperative cognitive decline conundrum. One great suspect in this

complex puzzle is that of sleep fragmentation: SF induces significant changes

that underlie memory, indicating a loss of cognitive function in non-

hippocampal and hippocampal dependent domains. We show that while SF

and surgery can independently produce significant memory impairment,

perioperative SF significantly increased hippocampal inflammation without

further cognitive impairment. This peculiar dissociation between

neuroinflammation and cognitive decline may relate to our use of memory

paradigms that do not capture all aspects of cognition. These data support the

need to adopt interventions that can provide patients with appropriate rest in

order to mitigate neuroinflammation. While we realize that the etiology of

cognitive dysfunction in surgical/ICU patients is multi-factorial, if the

restorative and reparative benefits of sleep mitigate the development of

inflammation, this may result in shorter ICU or postoperative lengths of stay. If

the restorative and reparative benefits of sleep mitigate the development of

cognitive dysfunction, this will result in shorter ICU and postoperative lengths

of stay for critically ill patients with a concomitant reduction in healthcare

costs. Furthermore, it is possible that the restorative properties of sleep for

106

cognition in the central nervous system can extend to the immune system with

less infection and/or greater likelihood of survival from sepsis68,86

In a recently completed prospective study, patients who had previously

suffered a stroke were more at risk for POCD even though they had no

neurological sequelae from the remote stroke event5,87. We have

demonstrated that bone fracture shortly after ischemic stroke increases

stroke-related neuronal injury and neurobehavioral dysfunction in mice.

HMGB1 and macrophage/microglia play a causal role in the negative impact

of bone fracture on stroke outcomes. By modulating the level of HMGB1 level

and macrophage/microglia activities we could be opening for possible

intervention opportunities for patients with both stroke and bone fracture.

We have also tried to explore that modifiable patient related factors also bear

a great influence on POCD development. In a rat model of MetaS we report

that these animals have greater memory impairment and persistent learning

and memory decline following tibia fracture surgery. These findings set the

stage for a thorough exploration of the reasons why MetaS induces

differences in postoperative cognitive function and if appropriate patient

optimization before surgery could mitigate the development of POCD.

We believe that non-resolution of inflammation is a factor that contributes to

the pathogenesis of POCD, which in turn significantly increases morbidity and

mortality in surgical patients. We might be witnessing a perfect and

unfortunate storm of factors with regard to POCD: to put it another way, given

the rise in surgeries and increasing number of patients with chronic conditions

worldwide, the stakes could not be higher.

Vulnerable patients need to be identified and risk/benefit should be

considered before contemplating the efficacy of surgical intervention.

Advanced age, MetaS, patients prone to neurological disease, and poor

selection of sedative agents may each result in exaggerated and persistent

neuroinflammatory response to surgery. Further studies are needed to

understand which patients will suffer from exacerbated inflammation with an

aim toward developing a biomarker that is quick to assay for clinicians and

107

easy to comprehend for patients and their families. Concurrently, clinical

interventions need to be further developed to promote the resolution of

neuroinflammation in the postoperative patient population. Following both

tracks, we anticipate that postoperative recovery for vulnerable patients will be

greatly enhanced and possible long-term consequences, such as

postoperative neurodegeneration, can be significantly reduced.

Additional study is essential to elucidate the risk factors, preventative

strategies, and underlying pathophysiology of this disorder. If these studies

can succeed in identifying patients prospectively, or early enough in the

advent of persistent inflammation, interventions can be judiciously and

appropriately launched.

108

TABLES

Table 1 – Risk Factors for Postoperative Delirium Risk factors for POD Incidence

of delirium (%)

References

Patient Related factors Advanced age 42-92 88 15 89 42.3 90 6.8 91 ? 63,92-104

Education ? 94,102,105 Male sex ? 96,106 Apolipoprotein E4 ? 104

Comorbidities Preoperative depression ? 105,107,108 Metabolic Syndrome Obstructive Sleep Apnea 24 Preoperative cognitive impairment

24 89 55.6 90 ? 88,92-95,100-104,106,108-112

Other comorbidities 18.3 99 ? 63,88,90,93,94,97,99,108,109,113

Surgery and anesthesia Duration of surgical procedure

40 90 ? 63,97

Pain management ? 98,114 Metabolic derangement 24 89

13-41 113 ? 63,88,102,110

Other Tobacco use 53.3 99 ? 97

History of alcohol or drug abuse

20 89 ? 92,96,100,102,103,105,107,110

Preoperative anxiety ? 115,116 Sleep deprivation ? 63

109

Table 2 – Risk Factors for Postoperative Cognitive Dysfunction Risk factors for POCD Assessment

in days Incidence of POCD (%)

Reference

Patient Related factors

Advanced age ≤ 1wk 41.4 5 29 8,9 11.4 117 5.8 118 ? 119-124

1wk – 3mo 12.7-14 5,8,9 ? 117,119,120,122,124,125

3mo – 6mo ? 119,122 >1yr 16 8

? 119,122,126 Education (< high school)

≤ 1wk 27 8,9 13.2 124 ? 5,120,122

1wk – 3mo 9 8 9 9 ? 5,120,122,125

>1yr 10 8 ? 122,126

Male sex ≤1wk ? 123 Menopause 1wk – 3mo ? 127

3mo – 6mo ? 127 Apolipoprotein E4 ≤ 1wk 11.7 128

21.8 129 1wk – 3mo 10.3 128

41.7 130 Comorbidities Preoperative

depression ≤1wk ? 120,121,131 1wk – 3mo ? 120,131 3mo – 6mo ? 131 6mo -1yr ? 131 >1yr ? 126

Metabolic Syndrome ≤1wk ? 22,23 1wk – 3mo ? 22,23

Preoperative cognitive impairment

≤ 1wk ? 120 1wk – 3mo ? 120

Other Comorbidities ≤1wk 3.2-6.3 118 31.7 124 ? 122,123

1wk – 3mo 11 5 ? 22,122,130

>1yr ? 122,126 Surgery and Anesthesia

Second operation ≤ 1wk 43 8 54 9

1wk – 3mo 20 8 14 9

Long duration of surgical procedure/anesthesia

≤ 1wk 33 8 29 124 ? 123

1wk – 3mo 9 8 ? 132

6mo -1yr ? 132 >1yr 11 8

110

Postoperative infection ≤ 1wk 39 8 33 9

1wk – 3mo 14-19 8,9 >1yr 19 8

Respiratory complication

≤ 1wk 59 8 40 9

1wk – 3mo 14-15 8,9 >1yr 14 8

Anesthetic type ≤ 1wk ? 124 1wk – 3mo ? 124

Pain management ≤ 1wk ? 133 Other Tobacco use ≤ 1wk ? 123

History of alcohol or drug abuse

≤ 1wk 26.8 124 1wk – 3mo ? 118,124,134

111

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Susana Rodrigues Vacas - run.unl.ptrun.unl.pt/bitstream/10362/13251/1/Vacas Susana TD 2014.pdf · BACKGROUND Impairment of cognition after surgery is a disturbing reality. Postoperative