New therapeutic possibility of blocking cytokine-induced neutrophil chemoattractant on transient ischemic brain damage in rats

Brain Research 759 Ž1997. 103–111

Research report

New therapeutic possibility of blocking cytokine-induced neutrophil chemoattractant on transient ischemic brain damage in rats Yasundo Yamasaki a, ) , Yoshiyuki Matsuo b , John Zagorski c , Naosuke Matsuura a , Hiroshi Onodera d , Yasuto Itoyama d , Kyuya Kogure e a

c

Hanno Research Center, Taiho Pharmaceutical Co. Ltd., 1-27 Misugidai, Hanno City, Saitama 357, Japan b DeÕelopmental Research Center, Shionogi Pharmaceutical Co. Ltd., Osaka, Osaka 561, Japan Laboratory of Immunology, National Institute of Dental Research, National Institute of Health, Bethesda, MD 20892, USA d Department of Neurology, Tohoku UniÕersity School of Medicine, Sendai, Miyagi 980, Japan e Institute of Neuropathology, Kanto Neurosurgical Hospital, Kumagaya, Saitama 366, Japan Accepted 11 February 1997

Abstract Earlier we indicated that neutrophilic invasion into cerebral parenchyma is an important step in rat cerebral ischemia-reperfusion injury and the production of chemotactic factors, cytokine-induced neutrophil chemoattractant ŽCINC. precede the neutrophilic invasion. The aim of the present study was to evaluate the role of CINC production and the therapeutic possibility of blocking CINC activity in the transient ischemic brain damage in rats. Focal transient ischemia was produced by intraluminal occlusion of the right middle cerebral artery for 60 min. An enzyme immunoassay was used to measure the brain concentration of CINC and myeloperoxidase activity in ischemic areas was measured as a marker of neutrophilic accumulation. An immunohistochemical staining technique was used to detect the immunopositive cells for anti-CINC antibody. Further, application of anti-CINC antibody or anti-neutrophil antibody to rats was used to evaluate the role of CINC production. In ischemic areas, CINC production was detected and peaked 12 h after reperfusion, which followed 60 min of ischemia. Intraperitoneal injection of anti-neutrophil antibody 24 h before and immediately after reperfusion significantly reduced the brain water content and partially reduced the CINC production in ischemic areas. Further, immunohistochemical staining showed that anti-CINC antibody was found on the endothelial surface of venules and on parts of neutrophils that had invaded the ischemic area 6 to 24 h after reperfusion. Also, treatment with anti-CINC antibody reduced ischemic edema formation 24 h after reperfusion and the size of infarction areas 7 days after reperfusion. It thus appears that CINC, mainly produced by endothelium activated by factors released from neutrophils, plays an important role in ischemic brain damage. Furthermore, the blocking of CINC activity with antibody suggests an immuno-therapeutic approach to the treatment of stroke patients. Keywords: Cytokine-induced neutrophil chemoattractant; Focal cerebral ischemia; Ischemic neuronal damage; Neutrophil; Rat

1. Introduction It is now well established that the brain is subjected to inflammatory reactions in response to ischemic damage. The hallmark of the inflammatory reaction in the brain is leukocyte infiltration and accumulation, primarily by neutrophils w40x. Neutrophils hypothetically augment pathological processes by: Ž1. secreting lysosomal enzymes, Ž2. releasing free radicals, Ž3. decreasing blood flow by microvascular plugging, and Ž4. increasing vascular permeability w17,20,23x. Neutrophil migration into tissues is me-

)

Corresponding author. Fax: q81 Ž429. 72-8913.

diated by specific adhesion molecules on the surface of both endothelial cells and leukocytes w15,50x, and interfering with the endothelium–neutrophil adhesion interaction is an effective way in which to study the role of neutrophils in ischemic brain injury w4,25,49x. These neutrophilic functions indicate the therapeutic possibility of regulating the activity of these cells, such as by reducing their number, inhibiting adhesion molecules, and halting free radical production. Neutrophilic infiltration is thought to be directed by a transendothelial gradient of soluble attractants. C5a, leukotriene B4, and chemotactic cytokine Žchemokines. have been identified as neutrophil chemotactic factors w12,33x.

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 2 5 1 - 5

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Y. Yamasaki et al.r Brain Research 759 (1997) 103–111

Human interleukin-8 ŽIL-8. is a well-known chemokine for neutrophils and T-lymphocytes; human gro is highly chemotactic for human neutrophils and shares a receptor on neutrophils with IL-8 w8,27x. Recently, cytokine-induced neutrophil chemoattractant ŽCINC., an IL-8-like neutrophil chemoattractant, was purified from the culture fluid of rat kidney epithelial cells and cloned w42,43x. Further, it has reported that the IL-8, gro, and CINC gene contain an NF-KB element within their enhancer regions that has been shown to be necessary for transcriptional activation of these chemokines w28,31x. The primary structure of CINC revealed that it belongs to the family of human IL-8 but that it is closer in sequence homology to gro than to IL-8. It has been reported that CINC plays an important role in neutrophilic infiltration in immune complex glomerulonephritis in rats w45x. Recently, Liu et al. and we reported that CINC mRNA and protein were induced in ischemic brain damage w21,47x. However, the actual role of CINC in the inflammatory process in the transient cerebral ischemic model in rats remains to be elucidated. The results we obtained in the present study suggest that: Ž1. CINC is produced by activated endothelium andror neutrophils in ischemic areas during the reperfusion period and plays an important role in the inflammatory reaction in ischemia-reperfusion injury, and Ž2. blocking of CINC activity is a therapeutic possibility for limiting ischemic brain damage by suppressing neutrophilic infiltration.

samples was then measured by the wet and dry weight method w18x. 2.2. Measurement of CINC concentration For measurement of CINC concentration in the brain, the rats were decapitated under ether anesthesia, and the brains were dissected into the MCA, ACA, and CP areas according to the following schedule: immediately after reperfusion, and 3, 6, 12, and 24 h thereafter. For evaluation of the effect of the duration of ischemia on CINC production, brains were dissected 12 h after reperfusion following 15 or 60 min of ischemia, or after 12 h of ischemia without reperfusion. Further, in the case of evaluation of the effect of anti-neutrophil antibody treatment on CINC production, brains were dissected 6, 12, and 24 h after reperfusion. We then homogenized samples of the brain with 5 volumes of phosphate-buffered saline containing 0.5% bovine serum albumin and 0.05% Tween 20, using a polytetrafluoroethylene homogenizer Ž100 mg wet wtr0.5 ml.. Next, each homogenate was centrifuged Ž10 000 rpm, 48C, 15 min., and the resultant supernatant was collected and stored at y808C until assayed. A rat CINC enzyme-immunoassay ŽEIA. kit ŽIBL, Japan. was used according to the manufacturer’s recommendations to determine the CINC concentration in the brain. This EIA system does not react significantly with human IL-8 or rat gro beta Ždata not shown.. However, because of the possibility of cross-reactivity with other C–X–C family chemokines, we refer to the observed immunoreactivity as being CINC-like.

2. Materials and methods 2.3. Measurement of myeloperoxidase (MPO) actiÕity 2.1. Transient cerebral ischemia Adult male Wistar rats Žweight: 270–320 g; SLC. were anesthetized with a mixture of N2 OrO 2 gases Ž70:30. containing 2% halothane. Then, using a modification of the method of Longa et al. w22x, we carefully dissected the right external carotid artery after having made a median incision in the neck skin and inserted an 18-mm-long 4-0 nylon thread Žcoated with silicon. from the right external carotid artery to the right internal carotid artery to occlude the origin of the right middle cerebral artery ŽMCA.. Next, after 60 min of MCA occlusion, the thread was removed to allow complete reperfusion of the ischemic area via the right common carotid artery. Neurological deficits characterized by severe left-sided hemiparesis and right Horner’s syndrome were used as the criteria for evaluating the ischemic insults. To assess the amount of brain water 24 h after reperfusion, we dissected the brain into the cerebral cortex perfused by the MCA ŽMCA area., cerebral cortex perfused by the anterior cerebral artery ŽACA area., and caudate putamen perfused by the MCA ŽCP area. in a humidified glove box. The brain samples were dried in an oven at 1108C for 24 h, and the water content of these

The rats were anesthetized with sodium pentobarbital and then perfused transcardially with 100 ml saline to flush all blood components from the vasculature. The brains were removed and frozen in liquid nitrogen, and stored at y808C for later biochemical analysis. The method of quantifying the MPO activity in ischemic areas was performed according Barone et al. w1,2x. The frozen tissue specimens were quickly weighed and homogenized ŽPolytron, 3 times with 5-s bursts. in 20 volumes of 5 mmolrl phosphate buffer ŽpH 6.0. at 48C, and then centrifuged at 30 000 = g for 30 min at 48C. The supernatant of each specimen was discarded, and the pellet was extracted by suspension in 10 vols. of 0.5% hexadecyltrimethylammonium bromide ŽSigma. in 50 mmolrl phosphate buffer ŽpH 6.0. at 258C. The samples were then frozen on dry ice, with 3 freezerthaw cycles, and sonicated for 10 s at 258C. After sonication, the samples were incubated for 20 min at 48C and centrifuged at 12 500 = g for 15 min at 48C. MPO activity in the supernatant was assayed as described by Bradley et al. w3x. The rate of formation of the colored product formed during the MPO-dependent reaction in 50 mmolrl phosphate buffer ŽpH 6.0. containing o-dianisi-

Y. Yamasaki et al.r Brain Research 759 (1997) 103–111

dine hydrochloride ŽSigma. and hydrogen peroxide was then measured. Changes in absorbance at 460 nm over a 100-s period were measured at 20-s intervals by means of a spectrophotometer, and MPO activity was calculated by use of a standardized solution ŽWAKO.. 2.4. Application of anti-CINC antibody, anti-neutrophil antibody We raised a neutralizing antibody against CINC in a goat. This antibody inhibits and neutralizes the activity of CINC specifically, as reported previously w44x. Our preliminary experiment and other reports showed that 2 mg of the IgG fraction of anti-CINC antiserum was needed to block the CINC activity in in-vivo experiments w45x. This antibody was administered intraperitoneally immediately after reperfusion. The anti-neutrophil antibody used was kindly donated by Dr. Sendo ŽYamagata University.. The generation of this anti-rat neutrophil monoclonal antibody, RP3, which selectively reacts with and depletes rat neutrophils upon in vivo administration, has been described elsewhere w37,38x. Rats were intraperitoneally administered RP3 24 h before and immediately after reperfusion. The dose of RP3 used in this study was sufficient to cause almost complete depletion of neutrophils in the blood from 6 to 24 h after the reperfusion, and had no effect on other blood cells, as previously reported w24x. 2.5. Histopathological examination Seven days after reperfusion, rats were anesthetized with sodium pentobarbital and then perfused transcardially with saline, followed by 4% buffered paraformaldehyde fixative. Next the brain of each rat was removed, postfixed, and embedded in paraffin. For evaluation of the area of neuronal damage, brain sections Ž5 mm thick. were stained with hematoxylin and eosin. The area of necrotic damage in the ischemic hemisphere was measured with a color image analyzer ŽOlympus SP500.. By light microscopy, necrotic neurons can be identified as neurons exhibiting pyknosis, cytoplasmic eosinophilia, or loss of hematoxylin affinity. Dark, scalloped, and swollen neurons were also taken to be damaged w10x. The edges of the area containing these necrotic and damaged neurons in the cortex and caudate–putamen in the ischemic hemisphere, estimated as an ‘area of pallor’, were outlined; and these areas were separately evaluated at 1.5, 0.5 and y0.5 mm from the bregma as necrotic areas w32x.

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the brain of each rat was removed, postfixed, and embedded in paraffin. Deparaffinized sections Ž5 mm thickness. were incubated in 0.3% H 2 O 2 in methanol for 30 min and in phosphate-buffered saline ŽPBS. with 10% rabbit serum for 60 min at room temperature. These sections were washed with PBS and incubated with the goat polyclonal anti-CINC IgG antibody Ž3 mgrml. in PBS containing 10% rabbit serum for approximately 24 h at 48C. An anti-goat avidin-biotinylated horseradish peroxidase kit was used according to the supplier’s recommendations ŽVector Laboratories, Burlingame, USA., after which the sections were immersed in 0.05% 3,3X-diaminobenzidine ŽDOJIN. in a 50 mM Tris-HCl solution containing 0.005% H 2 O 2 ŽpH 7.5.. 2.7. Statistical analysis For the statistical analysis, Dunnet’s multiple test or unpaired t-test was used; the results were compared with those obtained for the sham-operated group.

3. Results 3.1. CINC production 3.1.1. Effects of reperfusion The CINC concentration in the brain parenchyma in control rats and in rats that had been killed after 60 min of ischemia without reperfusion was below the level of detection Ž- 0.06 ngrg tissue wet weight.. CINC first became detectable at 6 h after reperfusion following 60 min of ischemia, and its level peaked at 12 h in the ACA area Ž4.3 " 1.1 ngrg tissue wet wt, n s 6 mean " S.E.M..,

2.6. Immunohistopathological detection of CINC For the immunohistopathological study, rats Ž6, 12, and 24 h after reperfusion. were anesthetized with sodium pentobarbital and then perfused transcardially with saline, followed by 4% buffered paraformaldehyde fixative. Next

Fig. 1. Graphs showing the levels of CINC production in the ischemic areas after 6, 12 and 24 h of reperfusion following 1 h of ischemia. ACA s frontoparietal cortex, supplied by the anterior cerebral artery; MCA s frontoparietal cortex, supplied by the middle cerebral artery; CPs caudate putamen, supplied by the middle cerebral artery. Results are shown as the mean"S.E.M. of 6 rats.

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Y. Yamasaki et al.r Brain Research 759 (1997) 103–111 Table 1 Effect of anti-neutrophil antibody on CINC production

Control Anti-neutrophil

n

ACA

MCA

CP

5 5

4.35"1.12 1.50"0.31

15.75"0.47 9.45"1.53 b

12.30"1.53 6.65"0.41 a

ACA santerior cerebral artery areas; MCA s middle cerebral artery areas; CPs caudate putamen perfused by MCA. Data are shown as mean"S.E.M. Values are ngrg wet wt. a P - 0.05, b P - 0.01 vs. Control Ž t-test..

Fig. 2. Graphs showing the effect of the ischemic period on the level of CINC in the brain. Open columns indicate 12 h of reperfusion following 60 min of ischemia; closed columns, 12 h of reperfusion after 15 min of ischemia; and dotted columns, 12 h of ischemia without reperfusion. Results are shown as the mean"S.E.M. of 5–6 rats. ) ) P - 0.01 vs. 12 h of reperfusion following 60 min of ischemia Žunpaired t-test..

3.1.2. Effects of anti-neutrophil antibody (RP3) Treatment with monoclonal anti-neutrophil antibody ŽRP3. 24 h before and immediately after reperfusion significantly decreased ischemic edema formation, as previously reported w24x. It also suppressed the CINC concentration in every ischemic area, especially in the MCA area, when examined 12 h after reperfusion ŽTable 1.. This suppressive effect of RP3 on CINC production was actually observed from 6 to 24 h after reperfusion Ždata not shown.. 3.2. Ischemic brain edema formation

MCA area Ž15.7 " 0.5 ngrg tissue wet wt., and in the CP area Ž12.3 " 1.5 ngrg tissue wet wt.. Thereafter, as shown in Fig. 1, the brain CINC concentration began to decrease at 24 h after reperfusion had begun, but remained at a high level in comparison with that for the sham-operated group. To ascertain the effect of the duration of ischemia on the production of CINC in the ischemic areas, we checked CINC production in rats that had been subjected either to 15 min of ischemia followed by 12 h of reperfusion or to 12 h of ischemia without reperfusion. Fifteen minutes of ischemia did not induce neuronal necrotic damage, as previously reported w29x. CINC production after 12 h of reperfusion with 15 min of ischemia was 0.07 " 0.07, 0.29 " 0.07, and 0.17 " 0.06 ngrg tissue wet wt in ACA, MCA, and CP area, respectively Ž n s 6, mean " S.E.M., Fig. 2.. These values were much less than those for the same time points after 60 min of ischemia Ž ) ) P - 0.01 unpaired t-test.. Furthermore, 12 h of ischemia without reperfusion strongly induced edema formation Žincrease in brain water content., the degree of which tended to be the same as in the case of 60 min of ischemia and 12 h of reperfusion. The brain water content in the rats subjected to 12 h of reperfusion with 60 min of ischemia were 79.4 " 1.41%, 83.6 " 0.73%, and 82.8 " 0.91%, and those in the rats subjected to 12 h of ischemia without reperfusion were 79.7 " 0.36%, 84.9 " 0.35%, and 83.6 " 0.22% in the ACA, MCA, and CP area, respectively Ž n s 6, mean " S.E.M... CINC production was observed in every area Ž4.66 " 0.41, 4.97 " 0.62, and 3.32 " 0.69 ngrg tissue wet wt in ACA, MCA, and CP area, respectively, n s 6, mean " S.E.M.., but was lower than in the case of 12 h after reperfusion with 60 min of ischemia ŽFig. 2..

To ascertain the contribution and therapeutic possibility of inhibition of CINC activity, we first observed the effect of anti-CINC antibody on ischemic brain edema formation. The antibody did not affect the number of neutrophils in the peripheral blood ŽTable 2. or the rectal temperature. Brain water content, which was measured as a marker of edema formation by the wet and dry weight method, increased from 6 to 12 h after reperfusion following 60 min of ischemia, and reached a maximum level after 24 h of reperfusion, as previously reported w47x. Brain water content of the ACA area increased from 77.5 " 0.7% Žsham operation. to 80.9 " 0.3%, that of the MCA area increased from 77.8 " 0.8% Žsham operation. to 85.1 " 1.1%, and that of the CP increased from 76.7 " 0.4% Žsham operation. to 86.5 " 0.5% after 24 h of reperfusion Ž n s 6, mean " S.E.M... Although control goat IgG treatment had little effect on brain water content in the ACA, MCA, and CP Ž80.4 " 1.4%, 85.0 " 0.7%, and 85.5 " 0.9%, respectively, n s 5, mean " S.E.M.., treatment with the antibody against CINC significantly decreased it in these areas after 24 h of reperfusion Ž78.0 " 0.5%, 81.8 "

Table 2 Effect of anti-CINC antibody on the number of circulatory neutrophil

Goat IgG Anti-CINC

n

Before ischemia

12 h after reperfusion

24 h after reperfusion

5 5

1735"87 1813"89

1856"93 1887"85

1732"98 1786"102

Data are shown as mean"S.E.M. Values are counts per mm3. Note the lack of effect on the number of circulating neutrophils upon treatment with anti-CINC antibody.

Y. Yamasaki et al.r Brain Research 759 (1997) 103–111

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Fig. 3. Effect of anti-CINC antibody on increased brain water content after 24 h of reperfusion following 60 min of ischemia. ACA s frontoparietal cortex, supplied by the anterior cerebral artery; MCA s frontoparietal cortex, supplied by the middle cerebral artery; CP s caudate putamen, supplied by the middle cerebral artery. Results are shown as the mean"S.E.M. of 5–6 rats. ) P - 0.05, ) ) P - 0.01 vs. goat IgG treatment ŽDunnett’s multiple test.. Note the significant reduction in brain water content after treatment with anti-CINC antibody.

1.3%, and 83.2 " 1.1%, respectively, n s 6, mean " S.E.M.. ŽFig. 3.. 3.3. MPO actiÕity We sequentially measured the myeloperoxidase ŽMPO. activity in the brain as a marker of neutrophilic infiltration in parenchyma and of neutrophilic adherence to endothelium after reperfusion following 60 min of ischemia. The MPO activity of the ischemic brain tissue without reperfusion and that with 6 h of reperfusion were below the level of detection. The MPO activity began to be detectable 12 h after reperfusion and thereafter gradually increased, as previously reported w24,47x. After 24 h of reperfusion, MPO activity was high in the ACA, MCA, and CP areas ŽTable 3.. Treatment with the antibody against CINC significantly suppressed MPO activity in the ischemic areas we examined, in comparison with the control goat IgG treatment, especially in the ACA and MCA areas 24 h after reperfusion ŽTable 3..

Fig. 4. Effect of anti-CINC antibody on the area of the necrotic neuronal damage after 7 days of reperfusion following 60 min of ischemia. The area of the necrotic neuronal damage was estimated as an ‘area of pallor’ by H.E. staining. Open circles refer to the vehicle Žgoat IgG.-treated group; and closed circles, to the anti-CINC antibody-treated group. Each point indicates the mean"S.E.M. of 5 rats. ) P - 0.05, ) ) P - 0.01 vs. goat IgG treatment Žunpaired t-test.. Note the marked reduction in the area of infarction after treatment with anti-CINC antibody.

3.4. Ischemic neuronal damage Reproducible ischemic neuronal damage was observed after 7 days of reperfusion following 60 min of ischemia.

Table 3 Effect of anti-CINC antibody on MPO activity

Sham-oper. Goat IgG Anti-CINC

n

ACA

MCA

9 9 6

0.0182 " 0.0031 b 0.0866 " 0.0140 0.0312 " 0.0048 b

0.0151 " 0.0033 0.2507 " 0.0540 0.1004 " 0.0362

CP b

a

0.0061 " 0.0016 0.0576 " 0.0318 0.0249 " 0.0054

a

ACA s anterior cerebral artery areas; MCAs middle cerebral artery areas; CP s caudate putamen perfused by MCA. Data are shown as mean " S.E.M. Values are unitsrg wet wt. Note elevation of myeloperoxidase activity in ischemia-reperfusion injury, and the significant suppression of myeloperoxidase activity by treatment of anti-CINC antibody. a P - 0.05, b P - 0.01 vs. goat IgG ŽWilliam’s multiple test..

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We observed the effect of the antibody against CINC on the area and nature of the neuronal damage 7 days after the ischemia. At this time, necrotic neurons, such as red neurons Žpyknosisreosinophilia. or ghost neurons Žcomplete loss of hematoxylinophilia., were visible in increasing numbers in the cortical and CP areas. No intact neurons were observed in these areas, as previously reported w29x. Cavitation Žareas of reabsorption of the necrotic debris by macrophages andror microglia. was seen in the preoptic area in all rats subjected to ischemia. Therefore, it was much easier to outline an ‘area of pallor’ in the cortex and CP areas. Treatment with the antibody also partially, but significantly, reduced the necrotic areas in the cortex and the CP ŽFig. 4.. The damaged area Ž%. in the cortex was 55.3 " 2.4%, 59.4 " 2.4%, and 61.5 " 2.5% at 1.5, 0.5, and y0.5 mm from the bregma, respectively, in normal goat IgG-treated rats, and 40.7 " 4.4%, 43.1 " 5.0%, and 53.3 " 4.5%, respectively, in the antibodytreated rats Ž n s 5, mean " S.E.M... 3.5. Immunohistochemical localization of CINC To localize CINC production, we employed an immunohistochemical approach. There was no positive stain-

ing for CINC on the brain parenchymal venules in the sham-operated rats. After 6 h of reperfusion, weak immunoreactivity for CINC was observed in the venules, but the intensity of the immunoreactivity became higher by 12 h of reperfusion. Thereafter, the immunoreactivity in the vessels tended to decrease, as observed 24 h after the start of reperfusion ŽFig. 5.. No immunoreactivity was observed in vessels in the non-occluded hemisphere. Some infiltrating neutrophils in the ischemic areas were also immunopositive for CINC after 24 h of reperfusion ŽFig. 5..

4. Discussion The acute-phase response elicited by a transient cerebral ischemia is characterized by a pathophysiological cascade of proinflammatory reactions Že.g., cytokine production, fever, complement activation, leukocytosis, acute-phase protein synthesis, and tissue neutrophil infiltration. w1,2,13,46,48x. Neutrophilic infiltration is thought to be directed by a transendothelial gradient of soluble attractants, such as leukotriene B 4 and chemokines w27,33x. Cytokine-induced neutrophil chemoattractant ŽCINC. is

Fig. 5. Representative photographs showing the expression of CINC-like immunoreactivity in the ischemic area of the rat brain: ŽA. sham-operated rat; ŽB. after 6 h of reperfusion; ŽC. after 12 h of reperfusion; and ŽD. after 24 h of reperfusion. Sixty minutes of ischemia was produced in panels B–D. Note the positive staining for CINC on the endothelial cells of venules Žarrow. and on the neutrophils that had infiltrated into the ischemic areas Žarrowhead..

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the rat equivalent of human gro, a member of the family of human IL-8-like molecules, and is thought to be responsible for neutrophil recruitment into the extravascular space w42x. However, the actual sources and role of CINC in the inflammatory process in cerebral ischemia in the rat were not heretofore known. Thus, the aim of the present study was to define the source of CINC, and the role of CINC, in the ischemically damaged rat brain. Firstly, the concentration of CINC was measured by an enzyme immunoassay technique. CINC in the brain increased markedly by 12 h of recirculation after 60 min of ischemia. This phenomenon was associated with severe tissue damage, and marked infiltration of neutrophils into the parenchyma. As CINC production was little after 12 h of reperfusion with 15 min of ischemia, which caused no necrosis and no emigration of neutrophils, we documented that cellular events initiated by reperfusion following a significant degree of ischemia were necessary for more CINC production. Furthermore, permanent ischemia also induced the production of CINC, but its production was less, even though drastic brain edema formation was observed without marked emigration of neutrophils. These data indicate that intense brain cell damage and emigration of neutrophils into the lesion site are needed to raise the CINC concentration in the tissue. These findings also mean that energy depletion andror lack of oxygen could not induce CINC production and that reperfusion, mainly active oxygen species formation after reperfusion, may contribute to augment the production of CINC. Potential sources of oxygen-derived free radicals include mitochondria, xanthine oxidase, prostaglandin biosynthesis, and infiltration of neutrophils associated with the inflammatory response after ischemia-reperfusion injury w30,41x. Matsuo et al., using a new electron spin resonance method coupled with microdialysis, recently reported that free radicals were mainly generated by neutrophils during reperfusion in this model and that this free radical generation was depressed by anti-neutrophil antibody w26x. Therefore, in order to define the role of the neutrophil in CINC production, we administrated an anti-neutrophil antibody ŽRP3.. Depletion of the circulating neutrophils significantly, but only partially, lowered the CINC concentration from 6 to 24 h after the start of reperfusion. Thus, cells in addition to neutrophils must also participate in CINC production in the post-ischemic lesion. Utilizing a polyclonal anti-CINC antibody, we examined the CINC-producing compartment immunohistochemically. While brain slices from the sham-operated rats showed no immunopositive reaction, the venules in the ischemic and penumbra regions showed markedly immunopositive sites after 12 and 24 h of reperfusion following 60 min of ischemia. CINC expression was also detected in some of the invading neutrophils at 24 h of reperfusion. Besides that of the neutrophil, participation of the endothelial cell in CINC production during reperfusion after ischemic brain damage was evident.

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It has been reported that reoxygenation of heart cells, or treatment of them with interleukin-1b or tumor necrosis factor-a causes a rapid induction of CINC production by heart cells w36x. Further, it has been shown that hypoxia induced IL-8 gene expression in endothelial cells and oxidant stress-regulated IL-8 gene expression in leukocytes and epithelial cells w6,19x. Our present results and our hypothesis are in line with these reports. In our previous report w47x, we suggested that the concentration gradient of CINC between the blood and brain tissue may work as an important factor in neutrophilic infiltration into ischemic brain parenchyma. However, given that a haptotactic gradient of CINC molecules could exist either on the endothelial cell surface or in the extracellular matrix, it is possible that CINC-induced neutrophil emigration and migration into the brain tissues is caused by this haptotactic mechanism, rather than a chemotactic one, as was shown for IL-8 w9,35x. Further investigations are needed to evaluate other potent factors that may attract neutrophils from endothelial cells into the brain parenchyma. As a second part of our study, we evaluated the role of CINC in ischemic brain damage after reperfusion by utilizing an intraperitoneal injection of anti-CINC antibody. Administration of the IgG fraction of anti-CINC antibody did not alter the number of neutrophils in the peripheral blood, but both MPO activity and ischemic brain water content in the post-ischemic brain tissue were suppressed significantly. As a result, post-ischemic brain damage was minimized. It has been reported elsewhere that invasion of neutrophils into the ischemic area is noxiously implicated in the development of post-ischemic brain injury. Leukocyte accumulation andror activation after reperfusion can exacerbate tissue injury through rheological effects associated with microvascular occlusion w7x, as well as through the release of a variety of deleterious mediators. The latter include: leukotrienes, which induce a conformational change in CD18 on leukocytes to promote their adherence to endothelial cells and are chemotactic w5x; oxygen free radicals, which can increase vascular permeability and damage the endothelium w11x; and platelet-activating factor, which increases vascular permeability and is neurotoxic w14x. Further, granular, enzymatic constituents including several cytotoxic lysosomal proteases induce subsequent neuronal damage. Therefore, we checked the size of infarction areas, as judged by loss of pallor, and the existence of necrotic neurons, which can be identified by their pyknosis and cytoplasmic eosinophilia, after 7 days of reperfusion. Application of anti-CINC antibody immediately after reperfusion reduced the necrotic area significantly. The neutrophils that invaded the ischemic areas might have successively stimulated the endothelial cells to produce CINC. Therefore, inhibition of neutrophilic infiltration into ischemic areas in the early stages after reperfusion can minimize subsequent neutrophilic infiltration and

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neuronal damage, resulting in a reduction in the size of infarction areas. Finally, we wish to stress that the current study serves to establish the importance of CINC production in transient cerebral ischemic damage in the rat, and provides insight into the synthesis of CINC and its role in the inflammatory cascade. Inflammatory reactions, such as leukocytosis and elevated body temperature, are often observed in patients with acute stroke w16,34,39x. This study also suggests that the human C–X–C chemokine, such as IL-8 or the gro family of polypeptides, may be similarly important in the early stages of stroke in man. In addition to increasing our understanding of the role of chemokines in ischemic tissue damage, therapeutic approaches that target chemokines directly or enhance the body’s mechanisms for controlling their activity will be important in developing therapeutic measures for use against the damage consequent to stroke.

w12x

w13x

w14x

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