1

INJURY AND THE BRAIN BARRIERS

Co-Chairs: Thomas P. Davis1, Gary A. Rosenberg2

Contributors: Ingolf E. Blasig3, Adam Chodobski4, Joanna Szmydynger-Chodobska5, Paula

Dore-Duffy6, Joe Fenstermacher7, Damir Janigro8, Eng Lo9, David Miller10, Sukriti Nag11, Martha

O’Donnell12, Darryl Peterson13, Jane Preston14, Norman Saunders15, Danica Stanimirovic16

1University of Arizona, Pharmacology Department, 1501 North Campbell Avenue, LSN 542,

Tucson, Arizona, USA 85724, davistp@email.arizona.edu, 520-626-7643

2University of New Mexico, Department of Neurology, HSC Neurology MSC10 5620 1 UMN,

Albuquerque, New Mexico, USA 97131, grosenberg@salud.unm.edu, 505-272-3315

3 Leibnizinstitute of Molecular Pharmacology, Berlin-Buch, Germany

4 Rhode Island Hospital and Brown University School of Medicine, Providence, Rhode Island,

USA

5 Rhode Island Hospital and Brown University School of Medicine, Providence, Rhode Island,

USA

6 Wayne State University, Detroit, Michigan, USA

7 Henry Ford Health System, Detroit, Michigan, USA

8Cleveland Clinic Foundation, Cleveland, Ohio, USA

9Massachusetts General Hospital, Charlestown, Massachusetts, USA

10National Institutes of Health/National Institute of Environmental Health Sciences, Research

Triangle Park, North Carolina, USA

11 University of Toronto, Toronto, Ontario, Canada

12 University of California Davis, California, USA

13 The Chicago Medical School, North Chicago, Illinois, USA

14 King’s College London, London, United Kingdom

15 University of Melbourne, Parkville, Australia

2

16National Research Council Canada, Ottawa, Ontario, Canada

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Introduction

Neurotrauma and related neuropathological states (stroke, head injury,

ischemia/reperfusion, hemorrhage, infarction and hypertension) share several common features,

one of which is an alteration of the blood-brain barrier in the area of injury, 1 which may extend to

surrounding tissues. These barriers play an important role in overall CNS homeostasis including

regulation of the flow of nutrients, ions, water, metabolites and xenobiotics from blood to CNS and

vice versa 2,3 as well as in preventing CNS entry of potentially neurotoxic plasma constituents.4,5

Thus, such changes can profoundly affect the progression of injury (including loss of neurons and

specific CNS functions), responses to therapy and the time course and extent of recovery.

Clearly, it is important to understand both the basic, underlying biology (at the functional and

molecular levels) of the barrier tissues and how these change following injury.2,3,6 As discussed

below, these issues are complex in that the barriers reflect the combined function of multiple

proteins, cellular structures and cell types and these can both sense and respond to their

immediate microenvironment, with a range of responses from subtle to dramatic.

The past two decades have seen the development of several fundamental concepts that

have collectively changed our view of barrier function. In some cases these concepts have been

integrated into our understanding of brain injury; in others they have not, leaving open the

possibility that this new knowledge can be of benefit in the clinic by providing additional markers

of barrier alteration and new therapeutic targets.

It is now recognized that blood-brain barrier permeability to solutes reflects the

contributions of both passive and carrier-mediated processes and that these contributions (and

thus integrated barrier function) are dynamic rather than static. The passive barrier is

dominated by paracellular tight junctions 6,7 and very low cellular passive permeability.8

Transporters add the capability to increase or decrease apparent permeability for certain solutes

(selectivity). Given this complex mix of determinants, it should not be surprising that the blood

brain barrier does not behave like a binary system, either open or closed. Rather it is able to

express a number of permeability states, depending on the nature of the solute under study, the

state of the tight junction proteins and the activity of appropriate transporters.

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Passive Permeability Markers

Progress:

The earliest markers used for determining disruption of the blood-brain barrier in a variety

of pathological states, which date back to the early 20th Century, were dyes such as trypan blue

or Evans blue.9,10 They have the advantage of being easily visualized, which is perhaps why they

continue to be used in spite of serious limitations. These dyes have been shown to bind firmly to

proteins in vitro,11 but may be exchangeable with tissues with higher affinity constituents in vivo.12

Thus it is not always clear whether the visualized marker is the small dye molecule (mw

<1000Da) or the albumin,11 or other plasma proteins to which it is bound.13 This is important

because there is recent evidence suggesting that the barriers in brain and spinal cord may show

increased permeability to small molecules for several days after injury,14,15 whereas the barrier to

large molecules in most investigations has been found to be closed by 3-6 hours post injury.

Since the 1960s the plant enzyme horseradish peroxidase16 has been used extensively to test

barrier integrity following neurotrauma.17,18 It has the practical advantages of being relatively easy

to visualize at the light microscopical and electronmicoscopical level. However, it has a number of

potential artifactual problems that were well recognized when it was first introduced into studies of

blood-brain barriers in the 1960s (outlined in 14)14, but which seem often to be overlooked in

more recent studies. This may explain some of the discrepancies in the literature. For example

the duration of barrier opening to HRP following neurotrauma has been variously reported as 6

hours to 24 hours plus.14 The mechanism of barrier disruption is also disputed, some authors

reporting leaks of HRP via tight junctions and others by transcellular mechanisms. A few studies

have used immunocytochemical staining for endogenous plasma proteins,9,14 such as albumin,19

fibronectin14 or IgG,20 to measure barrier integrity.9,14 These can show whether “breakdown” has

occurred, but are not suitable for defining its time course or extent.

Unresolved and New Questions:

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The time course of “barrier breakdown” to different sized molecules is important for two

reasons: (a) While barrier permeability is increased, its protective function is reduced and the

brain/spinal cord is vulnerable to entry of potentially neurotoxic agents occurring naturally in the

blood (i.e. albumin). (b) The period when the barrier is more permeable can represent a

therapeutic opportunity to use neuroprotectant agents that would not normally enter the CNS.

Indeed, there is reasonable evidence suggesting a period of 4-6 hours following several types of

trauma to the brain when the barrier is permeable to large protein-sized molecules. In the spinal

cord this period may be as long as 24 hours. Recent evidence also suggests that for molecules

<10 kDa there is a period of 4 days or more after injury when the both blood-brain and blood-

spinal cord barriers are more permeable at the site of injury.14,15 The spatial resolution of most

methods is such that it is unclear whether increased permeability is confined to the blood vessels

injured in the original trauma, although there are claims that blood vessels in surrounding neural

tissue are also affected. These claims remain controversial and largely unresolved.

Tight Junction Proteins

Progress:

At the molecular level, recent work has identified many new, critical proteins in endothelial tight

junctions3,6 and caveolae.21 The tight junction proteins: occludin, claudins 3, 5 and 12, junctional

adhesion molecule A and zonula occludens proteins have been localized in cerebral endothelium

by immunohistochemistry.22-24 In vitro studies of brain injury or stress, such as hypoxia25 and in

vivo studies of systemic inflammation26 show redistribution and reduced expression of tight

junctional proteins, which increases the paracellular barrier permeability.1,3,26 These data show

that the blood-brain barrier tight junctions are compromised with specific changes occurring at the

level of specific tight junction proteins. However, ultrastructural studies of the pathways for

visualisable tracers such as HRP in blood-brain barrier experiments in vivo have given conflicting

results concerning the route by which macromolecules such as HRP cross the disrupted blood-

brain barrier.1,17

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Unresolved and New Questions:

It is important to discover the structure, function and regulation of these tight junction

proteins in the maintenance of blood-brain barrier homeostasis and the extent to which they are

affected in brain injury or stress associated with diseases of the CNS. Such studies could be very

useful for the implementation and development of approaches that could protect the barrier. It is

also important to understand the fate and role that tight junction proteins play in maintaining the

integrity of the barrier during health and disease. Areas such as tight junction protein trafficking,

associated with barrier formation and maintenance, are critical to understand and study. Defining

the mechanism of tight junction formation of the barrier is also highly significant to understanding

the consequence of blood-brain barrier “leak” and the potential for repair.

Influx and Efflux Transporters

Progress:

A literature search reveals that this aspect of barrier trauma is largely unstudied, although

recent work with animal models suggests important implications. One study on morphine

pharmacokinetics in patients with severe brain injury found that morphine permeability was

increased in “worse” affected parts of the injured brains.27 14 C-� – Aminoisobutyric acid has been

used in a few studies but at saturating concentrations,15 so that what was being measured was

non-specific transfer of a small molecule. These transporter-based influx and efflux mechanisms

are essential for control of the normal internal environment of the brain and for the supply of

essential nutrients. Disruption of these transport mechanisms is likely to contribute to secondary

consequences of neurotrauma and their repair will contribute to the damage control and to

recovery.

Striking examples of the importance of transporters as potential targets of therapy comes

from animal models of cerebral ischemia and epilepsy. First, edema following middle cerebral

artery occlusion is substantially attenuated when rats are treated with an inhibitor of the blood-

brain barrier Na-K-Cl cotransporter, a major contributor to the movement of Na+ and water into

the brain that is upregulated following ischemia.28 Second, temporary cerebral ischemia was

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found to upregulate a blood-brain barrier drug efflux pump, p-glycoprotein. Pump inhibition

enhanced both the brain uptake and the effects of two neuroprotective drugs.29 Third, epileptic

seizures appear to increase brain capillary expression of multiple drug efflux pumps and to

reduce brain uptake of antiepileptic drugs (AEDs).30 Preliminary data in animal models and

patients show that inhibition of efflux transporter activity increases both the CNS accumulation

and the effectiveness of AEDs.31

Unresolved and New Questions:

Following brain injury, there are many unresolved and new questions such as: What is

the time course of barrier disruption and repair for different size molecules? Is this similar in

different regions of the brain and spinal cord?

Which elements of the barrier (tight junction proteins, transcytosis receptors, specific

transporters) are altered and when are they altered in the process? What are the molecular

targets that underlie barrier disruption? How does this change in a time course manner?

Can information on time course, brain regions and mechanisms of barrier injury be used

to facilitate delivery of neuroprotective drugs? Would it be important to develop barrier-protective

drugs with barrier tissues as therapeutic targets? Last of all, how do patterns of barrier disruption

depend upon the nature of the injury, (i.e., ischemic stroke versus hemorrhagic stroke versus

traumatic brain injury?

Each of these unresolved questions is critical to address and when done so will cause

many more new questions which need to be answered.

Signals Disrupting Barrier Function

Progress:

Complexity dominates here. Brain injury releases a cocktail of chemical signals, including

hormones, cytokines, chemokines, enzymes, free radicals, neurotransmitters, and ions. In

addition, injury activates the brain‘s innate immune response and a variety of peripheral

neuroinflammatory responses, both chemical and cellular (macrophage infiltration; see Report on

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Inflammation and Brain Barriers). These can target all elements of the CNS including barrier

tissues. The latter are unique in that they are directly exposed to signals from both the CNS and

the periphery and can act as a conduit for exchange of signaling molecules, e.g. cytokines,

between the two compartments.

Moreover, although the major focus of previous studies has been the cerebral

endothelium, it has been clear for some time that other elements of the neurovascular unit, such

as neurons, pericytes and astrocytes, communicate directly with the endothelium and can

profoundly affect barrier function. Thus, with respect to brain injury, barrier research requires a

broader focus to define the changes occurring in other components of the neurovascular unit and

the relationship of these changes to those in and around the endothelium. Consider, for example,

the role of the extracellular matrix in barrier injury. It has been recognized for many years that

there is a biphasic opening of the blood-brain barrier in reperfusion injury.32 Correlating structure

with function has shown that the early reversible barrier opening is caused by the activation of the

constitutive matrix metalloproteinase-2 (MMP-2) by the hypoxia/ischemia. MMP-2 degrades

claudin-5 and occludin, and the breakdown of the tight junctions can be blocked by synthetic

MMP inhibitors.33 A delayed disruption of the blood-brain barrier that results from multiple factors

involved in the inflammatory response occurs after one to two days and is only slowly reversed.

This phase is associated with cytokines, free radicals and MMP-3 and MMP-9. Control of this

phase of injury has been a challenge and should be a major target of novel therapies.

Given the plethora of signals generated and the capability of barrier tissues to respond to

so many factors, one looks for a rational way to decide which signals/receptors, etc, should be

considered as priority targets in therapy and when. This point brings up an important and

troubling issue. So many stroke treatments that were promising in extensive animal studies

proved to be disappointing when brought to clinical trials. Indeed, we still have only one effective

treatment to limit stroke damage, tissue plasminogen activator, administered within 3 hours of an

ischemic stroke. From this one could easily conclude that available animal models are inadequate

to mimic all the critical aspects of stroke in patients. Could this be a result of species differences,

the age of the animals, or a lack of appreciation of the complexity of events, signals and targets?

9

Unresolved and New Questions:

Following injury, what is the time course of release of signaling molecules (CNS and

systemic) and how does this correlate with barrier disruption (tight junctions and transporters) and

repair? What is the role of the barrier tissues in hemorrhagic transformation?

Another important unresolved question is: can synergy be obtained by targeting more

than one signaling event, such as free radical damage, MMPs and proinflammatory cytokines,

and at the optimal times.

In the area of model development, can we develop better models for defined CNS

injuries, models that more closely mimic the human condition?

Stroke and Head Injury

Progress:

Stroke and head injury are two major areas of clinical blood-brain barrier (BBB) research.

The insights from in vitro studies of the molecular aspects of the BBB have been translated into in

vivo studies where disruption of the tight junction proteins is seen in experimental models of

injury, including reperfusion after stroke. Greater understanding of the extracellular matrix has

come from recent studies on the proteolytic disruption of basal lamina proteins, such as laminin,

fibronectin, and type IV collagen, and the tight junctions.34

In the past five years, the role of magnetic resonance imaging (MRI) has emerged as an

important method to visualize and quantify BBB opening in vivo. Fenstermacher and colleagues

adapted an autoradiographic graphical method to the MRI.35,36 Using Gadolinium-DTPD and fast

T1-weighted scanning sequences, they were able to measure the permeability coefficient, ki, in

rats with stroke. This is an important advance over the invasive methods that need to have tissue

samples because it provides visual information on the sites of BBB disruption, which is otherwise

not available with quantitative methods, and it is noninvasive. In addition, MRI can be used to

measure infarct size and cerebral perfusion in the intact animal so that long-term behavioral

measurements can be made. This combination of measurements on the same animal provides a

powerful means to assess therapies, which may have beneficial early effects, but later

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detrimental ones. More importantly, the MRI graphical method that has been developed for

animals (Fig. 1) can be applied to studies in humans.

Unresolved and New Questions:

There are unresolved and new questions that need to be answered to achieve the goals

associated with advancing the area of stroke and head injury, one major obstacle is the expense

of the imaging equipment and the need for teams of scientists and engineers to carry out these

studies. This will necessitate designation of specialized centers for human studies and other

centers for molecular studies. A major problem is the lack of effective treatments for either head

trauma or stroke. Collaboration will be needed between academia and by big pharmaceutical

companies to speed discovery of therapies in these areas. This may require studies of multiple

agents to block several aspects of BBB damage; such studies provide unique challenges. These

obstacles can be overcome by the joining together of multiple centers for consolidation of studies,

sharing of information, and training of the next generation of experts on the BBB. The need to

train this next generation of BBB experts is critical to advance the field but remains unresolved.

Overall Concepts that need to be Developed

The prevailing medical textbook vision of brain barrier tissues as static structures whose

properties reflect solely tight junctional permeabilities is misleading and detrimental to research.

To fully understand the role that barrier tissues play in brain injury, this vision must be changed to

incorporate the following major concepts:

1) Barrier properties are a function of both paracellular and cellular elements, including tight

junctions, transcytotic mechanisms and specific influx and efflux transporters and

channels.

2) Barrier properties are dynamic, responding to signals received from both the CNS and the

periphery.

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3) The brain capillary endothelium is itself one element of a neurovascular unit (along with

pericytes, astrocytes and neurons) that integrates environmental signals and together

determines blood-brain barrier function.

4) Brain barrier properties (functional and molecular), susceptibility to injury (response to signals)

and inherent neuroprotective mechanisms likely vary with region.

5) Brain barrier disruption is an important contributor to secondary injury following neurotrauma

and thus barrier protective therapeutics could provide an opportunity for neuroprotection.

Summary Points

What needs to be done?

1. Resources specifically focused on brain barrier research

• Funding for international (cross continent), translational research projects at the level of

2-3 laboratories within the same discipline but having complementary knowledge.

• Increased highly targeted funding opportunities with specified set-asides

• Generation of databases listing BBB specific genes, reagents, antibodies, primers, siRNA

etc. Development of a BBB- Gene-Map similar to the brain-map published in Nature

January 2007

2. Training/Personnel

• Too few groups investigating BBB trauma at the mechanistic level (driven traditionally by

physiologists and pharmacologists). There is a critical need for cellular and molecular

biologists, neurobiologists and immunologists investigating trauma and the BBB.

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• A critical mass of BBB researchers is lacking. This limits the intellectual advance of the

field and does not give scope for replication of key findings or even dissemination to

peripheral fields. Establish a graduate student program specifically designed to attract

high quality students to work on the BBB with the opportunity to spend time “rotating” in

other BBB laboratories in the USA or abroad.

• BBB research in neurotrauma should be developed as a distinct area – until today it is

hidden in many different disciplines (i.e. research on barrier trauma damage is mostly a

minor component of neurotrauma research using out of date methods).

• Cell biologists and protein biochemists working on tight junctions and isolation of

transporter biology need to be attracted into the BBB field. Most research by this group of

researchers is performed in epithelial cells, which are distinct from endothelial cells.

• Better behavioral methods are required for evaluating effects of barrier dysfunction during

development. This will probably require attracting behavioral scientists into the field.

Conclusion and Single Most Important Issue

Future directions of research in the BBB of trauma and stroke will involve studies of both

the natural history of brain barrier disruption in human subjects and parallel studies using

proteomics 37,38 and genomics,39 of the molecular events in animal models and in vitro.3,6,7 It is

crucial to define the natural history in humans to improve the experimental models used today,

but it is also important to use modern genomic and proteomic methods to discover new

mechanisms that could lead to successful human therapy. Pathway analysis should identify

upstream genes that will be potential therapeutic targets for amelioration of the deleterious effects

of blood-brain barrier disruption in trauma and other pathologies. In parallel, the structure,

function and regulation of the proteins tightening the blood-brain barrier have to be elucidated to

define novel target molecules and modulators for barrier protection (protein structure

factories).3,6,40 To limit brain injury and speed recovery, there is an urgent need for high

resolution, non-invasive, in vivo tools (molecular probes and detection technology) that will

facilitate monitoring of brain barrier function (tight junctions, transporters and channels) within the

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components of the neurovascular unit. These tools should include CNS-deliverable, molecular

level reporters of expression (small molecules that bind specifically) and of activity (substrates) as

well as inhibitors that can be used in therapy. To aid translational studies, these molecules should

be usable in both cell-based and animal-based models as well as in patients. In addition,

appropriate imaging technology (PET, MRI multiphoton/confocal) should permit both time-based

and spatially based monitoring over a range of resolutions. Advances in imaging are the single

most critical issue for the advancement of understanding in the field of Brain Injury and for the

development of new treatments.

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Figure Legend

Figure 1: In vivo studies of the blood-brain barrier in rat with transient ischemia and reperfusion,

using the suture method. A) Brain section of a spontaneously hypertensive rat after 90 min of

ischemic middle cerebral artery occlusion (MCAO) with a suture and 3 h of reperfusion. A

confocal image of a blood vessel in the non-ischemic piriform cortex is shown. The astrocytes are

immunolabeled with GFAP (green) and the tight junction protein, claudin-5 is immunostained red.

Note the linear bands of claudin-5 surrounded by the GFAP-positive astrocytic foot processes. B)

A similar image from the ischemic piriform cortex showing disruption of the claudin-5 positive

bands. C) An MRI of an untreated Wistar rat with a 2 h MCAO followed by 3 h of reperfusion. The

red represents regions of high permeability calculated from the Gd-DTPA contrast enhanced

image using the Patlak Plot method. D) An MRI permeability map of a rat treated with the matrix

metalloproteinase inhibitor, BB-1101. The region of increased permeability is absent. The insert

shows the molecular components of the tight junction. (Modified from Yang et al.33 and Hawkins

and Davis3; MRI courtesy of Rohit Sood, PhD, MD. University of New Mexico, U.S.A.)

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Figure 1