SELECTIVE INHIBITION OF PLASMA KALLIKREIN PROTECTS …jpet.aspetjournals.org/content/jpet/early/2006/05/... · known to stimulate the production and release of ecosanoids, cytokines,

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SELECTIVE INHIBITION OF PLASMA KALLIKREIN

PROTECTS BRAIN FROM REPERFUSION INJURY

Claudio Storini, Luigi Bergamaschini, Raffaella Gesuete, Emanuela Rossi, Diana

Maiocchi and Maria Grazia De Simoni.

Department of Neuroscience, Mario Negri Institute, Milan (C.S., R.G., M.G.D.S.)

Geriatric Unit, Ospedale Maggiore, University of Milan, Italy (L.B., E.R., D.M)

JPET Fast Forward. Published on May 16, 2006 as DOI:10.1124/jpet.106.105064

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 16, 2006 as DOI: 10.1124/jpet.106.105064

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Running title: kallikrein and brain ischemia-reperfusion

Corresponding author:

Maria Grazia De Simoni, PhD, Head of the Laboratory of Inflammation and Nervous

System Diseases, Mario Negri Institute, via Eritrea, 62, 20157 Milano – Italy. Tel *39-

0239014.505 fax *39-023546277 email: [email protected]

Number of text pages: 27

Number of tables: 0

Number of figures: 6

Number of references: 32

Number of words in the Abstract: 243

Number of words in the Introduction: 750

Number of words in the Discussion: 786

Non-standard abbreviations:

G-protein-coupled orphan receptor: GPR100


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ABSTRACT

We have studied the effect of DX-88, a selective recombinant inhibitor of human plasma

kallikrein in transient or permanent focal brain ischemia (with or without reperfusion,

respectively) induced in C57Bl/6 mice. Twenty-four hours after transient ischemia, DX-

88 administered at the beginning of ischemia (pre) induced a dose-dependent reduction

of ischemic volume, that, at the dose of 30 µg/mouse, reached 49% of the volume of

saline-treated mice. At the same dose, DX-88 was also able to reduce brain swelling to

32%. Mice treated with DX-88 pre had significantly lower general and focal deficit score.

Fluoro-Jade staining, a marker for neuronal degeneration, showed that DX-88-treated

mice had a reduction in the number of degenerating cells, compared to saline-treated

mice. Seven days after transient ischemia DX-88 protective effect, was still present.

When the inhibitor was injected at the end of ischemia (post), it was still able to reduce

ischemic volume, brain swelling and neurological deficits. DX-88 efficacy was lost when

the inhibitor was given 30 min after the beginning of reperfusion (1h post) or when

reperfusion was not present (permanent occlusion model). This study shows that DX-88

has a strong neuroprotective effect in the early phases of brain ischemia preventing

reperfusion injury and indicates that inhibition of plasma kallikrein may be a useful tool in

the strategy aimed at reducing the detrimental effects linked to reperfusion.


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INTRODUCTION

Kallikreins are serin proteases present in biological fluids and tissues. They are divided

in plasma and tissue kallikreins that differ in structural characteristics and function. They

enzymatically mediate the release of kinins from their precursors (kininogens). Kinins

include the vasoactive nonapeptide bradykinin (BK) and the decapeptide kallidin (or Lys-

BK) that are produced by two major biochemical cascades, one occurring within the

plasma and leading to BK generation and the other within the tissue leading to kallidin

production. Kinin act via binding to B1 or B2 receptors whose signaling through nitric

oxide (NO)-cGMP and prostacyclin-cAMP results in a wide spectrum of actions, as part

of the inflammatory response that develops after tissue injury (Raidoo and Bhoola,

1998). Recently a G-protein-coupled orphan receptor (GPR100) has been identified as a

novel kinin receptor, however no information are presently available on its functions and

roles (Boels and Schaller, 2003; Meini et al., 2004).

Kinin system is involved in ischemia/reperfusion injury in heart, intestine and kidney

(Souza et al., 2004; Veeravalli and Akula, 2004; Chao and Chao, 2005) and a few

studies indicate that it is involved also in the pathogenesis of ischemic injury in the brain

(Relton et al., 1997; Zausinger et al., 2002; Lehmberg et al., 2003; Xia et al., 2004;

Groger et al., 2005). Actually, all the components and the activation products of this

system have been demonstrated also in the brain (Raidoo and Bhoola, 1998), and its

activation in stroke patients has also been proved (Wagner et al., 2002).

On the whole, the role of kinin system in the pathogenesis of brain ischemia/reperfusion

injury is not clear yet. Kallikrein action and/or BK release, during brain ischemia, are

known to stimulate the production and release of ecosanoids, cytokines, nitric oxide,


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free radicals, and excitatory amino acid neurotransmitters. On one hand release of such

mediators may induce cerebral arterial dilatation, loss of cerebrovascular auto-

regulation, endothelial cell lesions, increased capillary permeability and breakdown of

blood-brain barrier, causing vasogenic edema formation and possibly neuronal injury

and death (Relton et al., 1997; Groger et al., 2005). On the other hand, they may also

result in vasoactive, anticoagulant and pro-angiogenetic properties thus inducing

protection (Shariat-Madar et al., 2002; Xia et al., 2006).

In experimental brain stroke models, attempts to interfere with this system to obtain

protection from brain damage have yielded controversial data. To date, the role of B1

and B2 bradykinin receptors is not clearly understood. Relton (1997), Zausinger (2002;

Lumenta et al., 2006) and Ding-Zhou (2003) found a tissue protective effect and an

improvement of neurological outcome via selective inhibition of B2 receptors on brain

tissue. Accordingly B2 knockout mice are significantly protected from the ischemic insult

(Groger et al., 2005). On the other hand Lehmberg (2003) did not find any improvement

of neurological deficits or increase of vital neurons when either B1 or B2 receptors were

inhibited by specific antagonists, and when both receptors were inhibited, an increase in

mortality rate was observed. As a further degree of complexity, tissue kallikrein gene-

transfer has been reported to be protective against cerebral ischemia, an action

mediated by B2 receptors and NO production (Xia et al., 2004; Xia et al., 2006).

We choose to explore the effects of plasma kallikrein inhibition on brain ischemic injury

on the basis of the following considerations: i) kallikrein, being the precursor of BK,

results in a wide activation of the whole system irrespective of the receptors involved; ii)

although the selective involvement in ischemia/reperfusion pathogenesis of the plasma

vs. tissue cascade has not been elucidated yet, plasma compartment and the activation


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of the plasmatic cascade occurring on brain endothelial cells should be the earliest step

in this pathological condition.

We took advantage of the potent and selective plasma kallikrein inhibitor, DX-88 (Dyax

Corp., Cambridge, MA), a 60 amino acids Kunits domain (mw: 7,054 D) with high

specificity and affinity for human kallikrein (DX-88 Ki: 44 pM, C1-INH Ki: 1.3x104 pM,

aprotininin Ki: 3.0x104 pM) (Williams and Baird, 2003; Zuraw, 2005). This inhibitor is

presently used in humans for hereditary angioedema, a condition due to deficiency of

C1-inhibitor and characterized by recurrent episodes of angioedema that are though to

be due to activation of plasma kallikrein. (Williams and Baird, 2003; Zuraw, 2005;

Agostoni et al., 2004).

We studied DX-88 effects on infarct volume, neurodegeneration, brain swelling and

neurological deficits in a mouse model of cerebral transient ischemia. To specifically

address the involvement of reperfusion in DX-88 action we compared these results with

those obtained in a model of permanent ischemia (without reperfusion).


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METHODS

Animals

Male C57Bl/6 mice (26-28 g, Charles River, Calco, Italy) were housed 5 per cage and

kept at constant temperature (21 ± 1 °C) and relative humidity (60 %) with regular

light/dark schedule (7am - 7pm). Food (Altromin pellets for mice) and water were

available ad libitum.

Procedures involving animals and their care were conducted in conformity with

institutional guidelines that are in compliance with national (D.L. n.116, G.U. suppl. 40,

18 February 1992) and international laws and policies (EEC Council Directive 86/609,

OJ L 358,1; Dec.12, 1987; NIH Guide for the Care and Use of Laboratory Animals, U.S.

National Research Council 1996).

Physiological parameters

Selected physiological parameters (pH, pCO2, pO2 and oxygen saturation (sO2)) were

measured in naif mice receiving saline (150µl) or 30µg of DX-88 in the same volume iv.

Blood was withdrawn from the retro-orbital plexus, five minutes after the drug injection

and loaded on G3+ cartridges (i-STAT Corporation, East Windsor, NJ, USA). The

haemogas analysis were then performed by the i-STAT 1 analyzer (i-STAT Corporation,

East Windsor, NJ, USA).

Transient ischemia

Transient focal cerebral ischemia was achieved by middle cerebral artery occlusion

(MCAO) as previously described (De Simoni et al., 2003). Anesthesia was induced by

5% isoflurane in N2O/O2 (70/30%) mixture and maintained by 1.5-2% isoflurane in the

same mixture. To confirm the adequacy of the vascular occlusion in each animal, blood


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flow was measured continuously before, during and for 10 min after occlusion by laser

doppler flowmetry (Transonic BLF-21) using a flexible 0.5 mm fiberoptic probe

(Transonic, Type M, 0.5 mm diameter) positioned on the brain surface and secured with

impression material on the skull at the following coordinates: AP =-1 mm; L= -3,5 mm

(Yang et al., 1999). Briefly, the right common carotid artery was exposed and the

external carotid artery and its branches, including the occipital artery and the superior

thyroid artery were isolated and cauterized. The pterytopalatine artery was ligated and

the external carotid artery cauterized. A 6-0 monofilament nylon suture, blunted at the tip

by heat and coated in poly-l-lysine, was introduced into the internal carotid artery

through the external carotid artery stump and advanced to the anterior cerebral artery so

as to block its bifurcation into the anterior cerebral artery and the middle cerebral artery

(MCA). The filament was advanced until a >70% reduction of blood flow, compared to

pre-ischemic baseline, was observed. At the end of 30 min ischemic period, blood flow

was restored by carefully removing the nylon filament. Intra-operative rectal temperature

was kept at 37.0±0.5°C using a heating pad (LSI Letica, Barcelona, Spain). Sham

operated mice received a midline neck incision, and the subsequent exposure of the

carotid sheath. The external carotid artery and its branches were isolated without being

ligated or cauterized. Similar to ischemic mice, sham operated mice were maintained at

37°C during surgery.

Permanent ischemia

For the permanent MCAO, a vertical midline incision was made between the left orbit

and tragus. The temporal muscle was excised and the right MCA was exposed through

a small burr hole in the left temporal bone. The dura mater was cut with a fine needle

and the MCA was permanently occluded by electrocoagulation just proximal to the origin


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of the olfactory branch. Intra-operative rectal temperature was kept at 37.0±0.5°C using

a heating pad (LSI Letica, Barcelona, Spain). Sham operated mice received an incision

between the left orbit and tragus, and the subsequent exposure of the temporal bone.

The bone was drilled, but the dura mater and the middle cerebral artery were left intact.

Similar to ischemic mice, sham operated mice were maintained at 37°C during surgery.

Drug treatment

Mice received an iv injection of saline (150 µl) or the same volume of DX-88 (discovered

by Dyax Corp. using its proprietary phage display technology) at different doses (10, 30

or 90 µg/mouse). The dose of 30µg/mouse (1.2mg/kg) of DX-88 corresponds to the

maximal effective dose used in humans to treat acute attacks in patients with hereditary

and acquired angioedema (Williams and Baird, 2003; Zuraw, 2005; Agostoni et al.,

2004). The dose of 30 µg/mouse was given at different times from ischemia, ie at the

beginning of the ischemic period (pre), at reperfusion (post) and 1h after the beginning

of ischemia (1h post)

Neurological deficits

Twenty-four hours after ischemia, each mouse was rated on two neurological function

scales unique to the mouse (Clark et al., 1997; De Simoni et al., 2003). For both scales

mice were scored from 0 (healthy mouse) to 28 (the worst performance in all

categories). The score given represents the sum of the results of all categories for each

scale. General deficit scale evaluates: hair, ears, eyes, posture, spontaneous activity,

epileptic behavior. Focal deficit scale evaluates: body symmetry, gait, climbing on a

surface held at 45°, circling behavior, front limb symmetry, compulsory circling, whisker

response to a light touch. All the experiments were run by a trained investigator blinded


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to the experimental conditions.

Quantification of infarct size and brain swelling

Mice were deeply anesthetized with Equitensin (120 µl/mouse ip) and brains were

rapidly frozen by immersion in isopentane at –45 °C for 3 min before being sealed into

vials and stored at -70°C until use. For lesion size determination, forty-micron coronal

brain cryosections were cut serially at 320 µm intervals and stained with neutral red

(Neutral Red Gurr Certistain, BDH, England) (De Simoni et al., 2003). On each slice,

infarcted areas were assessed blindly and delineated by the relative paleness of

histological staining tracing the area on a video screen. The infarcted area and the

percentage of brain swelling was determined by subtracting the area of the healthy

tissue in the ipsilateral hemisphere from the area of the contralateral hemisphere on

each section (Swanson et al., 1990; De Simoni et al., 2003). Infarct volumes were

calculated by the integration of infarcted areas on each brain slice as quantified with

computer-assisted image analyzer and calculated by Analytical Image System (Imaging

Research Inc., Brock University, St. Catharines, Ontario, Canada).

Brain transcardial perfusion

Mice were deeply anesthetized with Equitensin (120 µl/mouse ip) and transcardially

perfused with 20 ml PBS 0.1 mol/L pH 7.4, followed by 50 ml of chilled

paraformaldehyde (4%) in PBS. After carefully removing the brains from the skull, they

were transferred to 30% sucrose in PBS at 4°C overnight for cryoprotection. The brains

were then rapidly frozen by immersion in isopentane at –45°C for 3 min before being

sealed into vials and stored at –70°C until use.

Assessment of neurodegeneration

Fluoro-Jade labelling was carried out on perfused brains (Schmued et al., 1997). Briefly,


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20 µm mounted sections were dried and rehydrated in ethanol (100%-75%) and distilled

water. Then, they were incubated in 0.06% potassium permanganate, washed in

distilled water and transferred to 0.001% Fluoro-Jade staining solution. After staining,

the sections were rinsed in distilled water, dried, immersed in xylene and coverslipped.

Neuronal count

At 7 days after ischemia the lesioned area is not evaluable by the paleness of the tissue

following neutral red staining because of the large microglial activation and cell

recruitment to the lesion site. Therefore, at this time point, the ischemic lesion was

assessed by neuronal count. After cresyl violet staining, three 20 µm sections at 640 µm

intervals from ipsi- and contralateral hemispheres were selected for neuronal count. The

first section was at stereotaxic coordinates anteroposterior +0.86 from bregma. Thus the

selected slices include the striatum, the brain area corresponding to the ischemic core.

The amount of neuronal loss was calculated by pooling the number of stained neurons

in the three sections of both hemispheres and expressed as percentage of contralateral

hemisphere. An Olympus BX61 microscope, interfaced with Soft Imaging System

Colorview video camera and AnalySIS software was used. Segmentation was used in

order to discriminate neurons from glia on the basis of cell size. The quantitative

analysis was performed at 40X magnification by an investigator blinded to the treatment.

Experimental groups

Eighteen experimental groups were prepared: a) two groups receiving 30 µg of DX-88 or

saline (n=4) for physiological parameter analysis; b) eight groups receiving 10, 30, 90 µg

of DX-88 or saline pre, 30 µg of DX-88 or saline post and 1h post (n= 8-10) for

evaluation of neurological deficits, ischemic volume and brain swelling at 24 hours after


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ischemia/reperfusion; c) two groups receiving 30 µg of DX-88 or saline pre (n= 6-8) for

determination of neurodegeneration by Fluoro Jade staining 24 hours after

ischemia/reperfusion; d) two groups receiving 30 µg of DX-88 or saline pre (n= 6-8) for

evaluation of neuronal loss seven days after ischemia/reperfusion; e) two groups

receiving 30 µg of DX-88 or saline pre (n= 5-7) for evaluation of ischemic volume seven

days after permanent ischemia; f) one group of transient MCAO sham-operated mice for

neurological deficits and neurodegeneration evaluation 24 hours after surgery (n=3); g)

one group of permanent MCAO sham-operated mice for ischemic volume seven days

after surgery (n=3).


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RESULTS

Physiological parameters

DX-88 did not modify the physiological parameters tested (pH: 7,12±0.005 and

7.13±0.015; pCO2: 43.1±0.4 and 42.35±2.35; pO2: 58.5±2.5 and 54±1; sO2: 80.5±1.5

and 77±2 for saline- and DX-88-treated mice respectively; data are expressed as

mean±SEM)

Transient MCAO: 24h outcome

C57Bl/6 mice treated with DX-88, given at the beginning of the ischemic period, had a

dose-dependent reduction of ischemic volume to 38.4% of the volume of saline-treated

mice (12.11±1.44 mm3 and 4.65±0.88 mm3, saline and DX-88 treated mice,

respectively) with the dose of 90µg/mouse (Fig.1). Also 30µg/mouse was able to

significantly reduce the ischemic lesion (5.94±1.10 mm3, reduction to 49% of saline),

while 10µg/mouse was ineffective (Fig.1).

Similarly, DX-88 was also effective in markedly reducing brain swelling at the dose of

30µg/mouse when given at the beginning of the ischemic period (8.96%±1.57% and

2.85%±0.54%, for saline and DX-88 treated mice, respectively, or a reduction to 32%,

Fig.2, lower panel).

When given at the end of 30 min ischemia, but before reperfusion, 30µg/mouse of DX-

88 were still able to significantly reduce the ischemic volume (5.07±1.83 mm3, reduction

to 42%) and brain swelling (4.16%±0.86%, reduction to 46.4%, Fig.2, post). DX-88

efficacy was lost when the inhibitor was given 30 min after the beginning of reperfusion

(Fig.2, 1h post).

To evaluate if the marked reduction of the ischemic lesion observed 24h after ischemia


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resulted in reduced functional impairment, neurological deficits were evaluated at 24h in

mice receiving saline or 30µg/mouse of DX-88 at the beginning of ischemic period (pre)

(Fig.3). While saline treated mice showed stable scores, those who received DX-88 had

significantly reduced general (11.5±1.01 and 8.87±1.13, means of saline and DX-88

treated mice, respectively, Fig.3, upper panel) and focal (18.9±2.14 and 11.62±1.87,

means of saline and DX-88 treated mice, respectively, Fig.3, lower panel) deficit scores.

The same results were obtained when the inhibitor was given at the end of ischemia, but

before reperfusion (8.17±1.01 means of general and 11.67±2.33 means of focal deficits,

Fig.3). No change in functional impairment was observed when the inhibitor was given

after 30 min of reperfusion.

We then analyzed the effect of ischemia and DX-88 treatment on neurodegeneration to

assess if neurons were actually spared by the inhibitor treatment. The staining with

Fluoro-Jade, a marker for neuronal degeneration, evaluated 24h after ischemia, showed

the presence of degenerating neurons in selected brain areas. As expected, in saline-

treated mice, positive cells were consistently observed in hippocampus and striatum

where they appeared as extended clusters of fluorescent cells (3 out of 3 mice, Fig.4A-

C). In mice treated with 30µg of DX-88 pre the number of degenerating cells was greatly

reduced and the general structure of the brain tissue, well preserved compared to

saline-treated mice. These mice showed small groups of Fluoro-Jade positive cells both

in striatum and hippocampus (Fig.4B-D). Positive cells could not be found in the other

brain regions in these mice indicating the focal nature of the lesion.

Sham-operated mice did not show any neurological deficits nor neurodegeneration (data

not shown).

Transient MCAO: 7 day outcome


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To evaluate if the protective effect of DX-88 was long lasting, we assessed the neuronal

loss 7 days after induction of ischemia/reperfusion and drug treatment. DX-88 protective

effect was still present at this time point (14%±2.18% and 3.7%±1.5%, saline- and DX-

88-treated mice, respectively, Fig.5).

Permanent MCAO: 7 day outcome

Lastly, the effect of plasma kallikrein inhibitor was then assessed in a model of

permanent brain ischemia, i.e. without reperfusion. Seven days after permanent

ischemia mice treated with 30µg/mouse of DX-88, at the beginning of ischemia, did not

show a reduction of the ischemic volume compared to saline-treated mice (14.71±0.98

mm3 and 18.77±3.11 mm3, saline- and DX-88-treated mice, respectively, Fig.6).

Sham-operated mice did not show ischemic cerebral lesion (data not shown).


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DISCUSSION

The results of this study show that DX-88, a selective plasma kallikrein inhibitor, reduced

the ischemic volume, both infarct size and swelling, and the functional impairment in

mouse transient ischemia, showing a remarkably high degree of neuroprotection. This

protection was persistent and related to reperfusion. DX-88 dose-dependently reduced

the ischemic volume. Thirty and ninety micrograms of the inhibitor were able to reduce

the ischemic volume. The use of Fluoro-Jade staining to evidence degenerating

neuronal cells, allowed us to investigate whether DX-88 action resulted in sparing of

these cells and showed that 24h after ischemia, Fluoro-Jade positive cells were

markedly decreased in treated mice compared to mice receiving saline.

Beside the brain ischemic lesion, oedema is a serious complication in post ischemic

injury. It results from local vasodilatation, increased microvascular permeability and

retention of fluid in injured tissue. The increase in blood flow and vascular permeability

at inflammatory sites causes plasma to leak into the brain parenchyma, provoking

swelling, and the plasma inflammatory mediators, such as free radicals, cytokines,

activated complement and kinin system factors, can directly affect neuronal survival. The

plasma kallikrein inhibitor, DX-88, was able to counteract swelling formation further

supporting the hypothesis that the release of bradykinin in brain tissue plays an

important role in brain edema after focal ischemia (Groger et al., 2005).

An additional important aspect when assessing the protective effect of a compound on

ischemia-reperfusion injury is the evaluation of functional deficits. DX-88 induced a

progressive recovery in both general and focal neurological deficits indicating an

amelioration of the appearance of the mice, their motor performance, their reactivity and

response to stimuli. In order to assess whether the favorable effect of DX-88 treatment


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was merely due to a delay in degenerating processes or to a real neuroprotection, we

analyzed the neuronal loss 7 days after ischemia/reperfusion and fully confirmed the

observation that neurodegeneration was actually inhibited by DX-88 treatment.

At variance with this finding, DX-88 did not affect the ischemic volume in the permanent

model of ischemia, indicating that the compound was effective only if reperfusion was

present, i.e. its protective effect is exerted by inhibiting events related to reperfusion.

This is further supported by the observation that the protective effect of DX-88 is not

present when the inhibitor is given 30 min after the beginning of reperfusion in the

transient ischemia model. Although reperfusion of ischemic brain induced by

thrombolythic agents is an effective therapy for stroke, this phenomenon can also

contribute to tissue damage by several mechanisms that include activation of

endothelium, increased production of oxygen radicals, inflammatory cytokine induction,

leukocyte recruitment and edema formation (Hallenbeck and Dutka, 1990; Kaur et al.,

2004; Latour et al., 2004). Thus, the lack of neuroprotection when reperfusion was not

allowed (permanent ischemia model) or when DX-88 was administered after reperfusion

(transient ischemia model) indicates that plasma kallikrein plays a pivotal role in the

mechanism of ischemic brain injury at reperfusion, and supports the idea that blockade

of kallikrein may be an effective strategy for prevention of the inflammatory injuries

occurring early during reperfusion of ischemic organs, including brain.

Previous data of ours (De Simoni et al., 2003; De Simoni et al., 2004; Storini et al.,

2005) showed a powerful neuroprotective action of C1-INH when given to C1q-/- mice

before reperfusion. C1-INH, the endogenous inhibitor of the activated first component of

complement classical pathway (C1) is a major inhibitor of plasma kallikrein, thus our

previous data, together with the present ones allow us to hypothesize that part of the


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powerful neuroprotective effect of C1-INH in ischemia/reperfusion brain injury is due to

an action on the kinin system.

Recently Xia (Xia et al., 2004; Xia et al., 2006) and colleagues showed a protective

effect of delayed tissue kallikrein gene-transfer, the production of the peptide occurring

2-5 days after brain ischemia. This action was linked to promotion of angiogenesis and

neurogenesis. These data together with ours indicate that the kinin system, similarly to

other inflammatory system and molecules (McIntosh et al., 1996; Walport, 2001b;

Walport, 2001a) may have a dual role, detrimental and beneficial, mainly depending on

the degree and/or time of activation. We can hypothesize that early after the ischemic

injury, when the contact-kinin system is fully activated, its inhibition is protective, while at

later times kinin vasoactive properties result in protective effects.

In our model of brain ischemia/reperfusion, DX-88 induced a dose-dependent reduction

of the ischemic volume and swelling, it lowered the number of dying neurons and

significantly lowered functional neurological impairment. Its protective effect was still

present seven days after ischemia. The experimental data indicate that the inhibition of

plasma kallikrein protects from reperfusion injury, and support this strategy for the

treatment of ischemic stroke aimed at reducing detrimental effects linked to reperfusion.


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ACKNOWLEDGEMENTS

The DX-88 was supplied by Dyax Corp., Cambridge, MA.


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FOOTNOTES

The study was partially supported by Dyax Corp., Cambridge, MA.

This work was presented at Brain 05, Meeting of International Society of Cerebral Blood

Flow and Metabolism Amsterdam, June 2005.

Please send reprint request to: Maria Grazia De Simoni, PhD, Mario Negri Institute, via

Eritrea, 62, 20157 Milano – Italy, [email protected]


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LEGENDS FOR FIGURES

Figure 1

Infarct volume assessed 24 h after ischemia in mice receiving saline or different doses

of DX-88 at the beginning of the ischemic period. Data are expressed as mean ± SEM

(n=8-10 mice per group). A representative ischemic lesion is outlined in the brain slices

(upper panel).

** P < 0.01 versus saline, one way ANOVA and Dunnett as post-hoc test.

Figure 2

Infarct volume (upper panel) and brain swelling (lower panel) assessed 24 h after

ischemia in mice receiving saline or 30µg/mouse of DX-88 at the beginning of ischemic

period (PRE), at reperfusion (POST) and 30 min after the beginning of reperfusion (1h

POST).

Data are expressed as mean ± SEM (n=8-10 mice per group).

* P < 0.05, ** P < 0.01 versus saline, one way ANOVA and Dunnett as post-hoc test, for

infarct volume, Kruskal-Wallis as post-hoc test, for brain swelling.

Figure 3

General (upper panel) and focal (lower panel) neurological deficit score evaluation (0-

28) assessed 24h after ischemia in mice treated with 30µg/mouse of DX-88 at the

beginning of ischemic period (PRE), at the end of ischemia but before reperfusion

(POST), 30 min after the beginning of reperfusion (1h POST) or with saline. Data are

expressed as mean and SEM (n=8-10 mice per group).


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* P < 0.05, versus saline, one way ANOVA and Kruskal-Wallis as post-hoc test.

Figure 4

Fluoro-Jade labeling of degenerating neurons in different brain areas of representative

ischemic mice, 24 h after ischemia. Clusters of Fluoro-Jade positive neurons can be

observed in the hippocampus-CA2 of saline treated mice (A), but not mice receiving

30µg of DX-88 (B). Several groups of positive cells can be observed in the striatum of

saline (C), while only a few scattered fluorescent cells can be found in the same brain

area of mice treated with DX-88 (D) (n=6-8 mice per group). Scale bar: 100 µm

Figure 5

Neuronal count performed 7 days after ischemia/reperfusion in ischemic mice treated

with 30µg/mouse of DX-88 pre or saline. Data are expressed as neuronal loss

percentage (nuclei) of contralateral hemisphere (n=6-8 mice per group).

** P < 0.01 versus saline, Mann-Whitney test.

Figure 6

Infarct volume (mm3) assessed 7 days after permanent ischemia in mice receiving saline

(SAL) or 30µg/mouse of DX-88. Data are expressed as mean ± SEM (n=5-7 mice per

group). A representative ischemic lesion is outlined in the brain slices (upper panel).


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