Induction Of Middle Cerebral Artery Occlusion Research Articles

Efficacy of mild hypothermia (35 °C) and moderate hypothermia (33 °C) with and without magnesium when administered 30 min post-reperfusion after 90 min of middle cerebral artery occlusion in Spontaneously Hypertensive rats

In this study we compared the efficacy of mild (35°C) and moderate (33°C) hypothermia alone and when combined with magnesium in a transient focal cerebral ischaemia rat model. Spontaneously Hypertensive rats were subjected to 90min of transient intraluminal thread middle cerebral artery occlusion (MCAO). Thirty minutes after reperfusion animals were treated with mild (35°C/24h) or moderate (33°C/24h) hypothermia combined with either magnesium (intravenous MgSO4 infusion: 360μmol/kg, then 120μmol/kg/h for 24h) or a similar volume of saline. Control animals were maintained normothermic (37°C/24h) and received vehicle infusion (saline for 24h). Infarct volumes and functional assessment (bi-symmetrical adhesive tape removal) were measured 48h after MCAO induction. After transient MCAO, only moderate hypothermia and mild hypothermia combined with magnesium treatment significantly reduced infarct volumes by 32.9% (P=0.01) and by 24.8% (P=0.046), respectively. Mild hypothermia alone reduced infarct volume by 23.8%, but did not reach statistical significance (P=0.054), while moderate hypothermia combined with magnesium reduced infarct volume by 17.3% (P=0.17). No treatment improved adhesive tape removal time. In summary, moderate hypothermia and mild hypothermia with or without magnesium can reduce infarct volume, however magnesium may reduce the efficacy of moderate hypothermia. Given the potential advantages of mild hypothermia over moderate hypothermia in terms of side-effects and induction, and the potential beneficial effects of magnesium, these findings have important implications for the use of hypothermia for stroke.

Transfusion of hemoglobin-based oxygen carriers in the carboxy state is beneficial during transient focal cerebral ischemia

Exchange transfusion of large volumes of hemoglobin (Hb)-based oxygen carriers can protect the brain from middle cerebral artery occlusion (MCAO). Hb in the carboxy state (COHb) may provide protection at relatively low volumes by enhancing vasodilation. We determined whether transfusion of rats with 10 ml/kg PEGylated COHb [polyethylene glycol (PEG)-COHb] at 20 min of 2-h MCAO was more effective in reducing infarct volume compared with non-carbon monoxide (CO) PEG-Hb. After PEG-COHb transfusion, whole blood and plasma COHb was <3%, indicating rapid release of CO. PEG-COHb transfusion significantly reduced infarct volume (15 ± 5% of hemisphere; mean ± SE) compared with that in the control group (35 ± 6%), but non-CO PEG-Hb did not (24 ± 5%). Chemically dissimilar COHb polymers were also effective. Induction of MCAO initially produced 34 ± 2% dilation of pial arterioles in the border region that subsided to 10 ± 1% at 2 h. Transfusion of PEG-COHb at 20 min of MCAO maintained pial arterioles in a dilated state (40 ± 5%) at 2 h, whereas transfusion of non-CO PEG-Hb had an intermediate effect (22 ± 3%). When transfusion of PEG-COHb was delayed by 90 min, laser-Doppler flow in the border region increased from 57 ± 9 to 82 ± 13% of preischemic baseline. These data demonstrate that PEG-COHb is more effective than non-CO PEG-Hb at reducing infarct volume, sustaining cerebral vasodilation, and improving collateral perfusion in a model of transient focal cerebral ischemia when given at a relatively low dose (plasma Hb concentration < 1 g/dl). Use of acellular Hb as a CO donor that is rapidly converted to an oxygen carrier in vivo may permit potent protection at low transfusion volumes.

Neuroprotective effects of argon in an in vivo model of transient middle cerebral artery occlusion in rats*

The neuroprotective effects of the noble gas xenon are well known. Argon, in contrast to xenon, is abundant, inexpensive, and therefore widely applicable. In this study, we analyzed the possible neuroprotective role of argon in an in vivo rat model of acute focal cerebral ischemia. Controlled laboratory study. Academic research laboratory. Male adult Sprague-Dawley rats. Twenty-two rats underwent 2 hrs of transient middle cerebral artery occlusion using the endoluminal thread model. One hr after transient middle cerebral artery occlusion induction, spontaneously breathing rats received either 50 vol % argon/50 vol % O2 (argon group, n = 11) or 50 vol % N2/50 vol % O2 (control group, n = 11) for 1 hr through a face mask. Twenty-four hrs after reperfusion, rats were neurologically and behaviorally tested and euthanized. Rat brains were stained with 2,3,5-triphenyltetrazolium chloride and infarct volumes determined by planimetry. After 2 hrs of transient middle cerebral artery occlusion in the rat, we found in the argon group a significant reduction in the overall (p = .004) and after subdivision in the cortical (p = .007) and the basal ganglia (p = .02) infarct volumes. Argon treatment resulted in a significant improvement of the composite adverse outcome (p = .034). However, there was no advantage in acute survival 24 hrs after transient middle cerebral artery occlusion (p = .361). We were able to demonstrate argon's neuroprotective effects in an in vivo experimental rat model of acute focal cerebral ischemia. Animals breathing spontaneously 50 vol % argon 1 hr after induction of transient middle cerebral artery occlusion for 1 hr by face mask showed significantly reduced infarct volumes and composite adverse outcomes.

국소 허혈성 뇌손상 흰쥐 모델에서 경두개직류전기자극이 앞다리 운동감각 기능 증진에 미치는 효과

본 연구는 국소 허혈성 뇌손상 흰쥐 모델에서 tDCS의 자극 적용시간을 달리하였을 때, 앞다리 운동감각 기능변화와 신경영양인자(GAP-43)발현에 미치는 영향을 알아보고자 하였다. 뇌손상 모델은 Sprague -Dawley계 흰쥐 80마리를 'Longa'방법을 이용하여 중대뇌동맥(middle cerebral artery)을 폐색하여 유발하였고, 실험군을 4개로 나누었다; 실험군I은 허혈성 뇌손상 유발군(n=20), 실험군II는 허혈성 뇌손상 유발 후 tDCS(10분) 적용군(n=20), 실험군III은 허혈성 뇌손상 유발 후 tDCS(20분) 적용군(n=20), 실험군IV는 허혈성 뇌손상 유발 후 tDCS(30분) 적용군(n=20)으로 나누었다. 앞다리운동감각 기능검사를 위해 수정된 앞다리배치 검사와 단일 팰릿 닿기 검사를 실시하였으며, 신경가소성에 대한 면역조직화학적 검사로 운동감각 영역에서의 GAP-43 단백질 발현을 관찰하였다. 앞다리운동감각 검사는 14일에서 실험군III (p<0.05)이 다른 군들에 비해 유의한 차이를 보였으며, 단일 팰릿 닿기 검사는 14일에서 실험군III(p<0.01)과 실험군IV(p<0.05)에서 유의한 차이를 보였다. 또한, 면역조직학적 검사는 14일에 실험군III이 다른 군들에 비해 현저한 면역양성반응의 증가를 보였다. 따라서, 0.1 mA의 강도로 20분간 적용했을 때가 앞다리운동감각 기능과 신경가역성 인자 GAP-43 발현에 가장 좋은 조건임을 알 수 있었다. This study was to investigate the effect of improve forelimb sensorimotor function and neurotrophic factor(GAP-43) expression when differing an application time of tDCS in ischemic brain injury rat model(pre, <TEX>$1^{st}$</TEX>, <TEX>$7^{th}$</TEX>, <TEX>$14^{th}$</TEX>). Focal ischemic brain injury was induced in 80 Sprague-Dawley rats through middle cerebral artery occlusion(MCAO) by 'Longa' method. And then experimental groups were randomly divided into four groups; GroupI: MCAO induction, GroupII: application of tDCS(10 min) after MCAO induction, GroupIII: application of tDCS(20 min) after MCAO induction, GroupIV: application of tDCS(30 min) after MCAO induction. Modified limb placing test and single pellet reaching test were performed to test forelimb sensorimotor function. And the histological examination was also observed through the immunohistochemistric response of GAP-43(growth-associated protein-43) in the cerebral cortex. In modified limb placing test, groupIII(p<0.05) showed significantly improve than the other groups on <TEX>$14^{th}$</TEX>). day. In single pellet reaching test, groupIII(p<0.01) and groupIV(p<0.05) significantly improved on <TEX>$14^{th}$</TEX>) day. And in immunohistochemistric response of GAP-43, group III showed significantly positive response than the other groups on <TEX>$14^{th}$</TEX> day. These results suggest that the intensity(0.1 mA)/time(20 min) condition of tDCS application has a significant impact on the sensorimotor functional recovery in focal ischemic brain injury rat models.

Central and Peripheral Administration of Compound 21 Elicits Cerebroprotective Effects in Ischemic Stroke

Studies indicate that angiotensin type 2 receptors (AT2R) in the CNS exert beneficial actions in ischemic stroke. Here we tested the effects of a specific AT2R agonist, Compound 21 (Cp21), on the cerebral deficits produced by endothelin‐1 induced middle cerebral artery occlusion (MCAO). Adult male Sprague Dawley rats were pre‐treated with Cp21 (0.0075μg/h) or artificial cerebrospinal fluid (aCSF) via intracerebroventricular infusion for 7 days prior to ET‐1 induced MCAO. Cp21 treatment reduced neurological deficits and infarct size measured 72 h after MCAO induction. Infarct size was reduced to 28.2±4.0% of ipsilateral gray matter in Cp21‐treated rats (n=16) compared with 54.1±4.7% in aCSF controls (n=21; p=0.0006). Neurological deficits were reduced by Cp21 treatment as indicated by a lower Bederson Exam Score of 0.3±0.1 compared to 1.6±0.2 in control rats (p&lt;0.0001) and a higher Garcia Exam Score of 16.9±0.3 compared to 14.3±0.7 in control rats (p=0.0011). Similar results were obtained with the IP administration of Cp21. Rats received IP injections with Cp21 (0.03 mg) or 0.9% saline at 2 hours prior, and 4, 24, and 48 hours following MCAO induction. Infarct size was reduced to 19.2±3.8% of ipsilateral gray matter in Cp21 treated rats (n=6) compared with 41.5±6.8% in saline controls (n=6; p=0.004). These data are the first demonstration of a non‐peptide AT2R agonist exerting cerebroprotection following MCAO.

Improved intraischemic perfusion and pial arteriolar dilation by transfusion of pegylated hemoglobin in the carbon monoxide state

Transfusion of chemically‐modifed, cell‐free hemoglobin (Hb) during middle cerebral artery occlusion (MCAO) can reduce infarct volume, but large amounts are typically required. We tested the concept that transfusion of Hb in the CO state would promote vasodilation during MCAO without requiring large amounts of Hb to be transfused. Transfusion of 10 ml/kg of a 4% solution of pegylated COHb (PEG‐COHb) in anesthetized rats resulted in rapid release of CO and equilibration of CO with red cell‐based Hb (2–3% COHb in whole blood and plasma). Plasma [Hb] was ~0.5 g/dL. Induction of MCAO initially produced 37±8% (±SD) dilation of pial arterioles in the MCA border region that gradually subsided to 7±7% at 2 h in controls. Transfusion of PEG‐COHb at 20 min of MCAO maintained pial arterioles in a dilated state (40±13%) at 2 h, whereas transfusion of albumin (25±13%) or PEG‐Hb without CO (22±8%) had intermediate effects. When transfusion of PEG‐COHb was delayed by 90 min, penumbral laser‐Doppler flow increased from 58% to 80% of pre‐ischemic baseline, whereas transfusion of albumin or PEG‐Hb produced no significant change. We conclude that PEG‐COHb can serve as a CO donor that effectively prevents time‐dependent loss of pial arteriolar dilation during MCAO and improves blood flow in the penumbral region to levels above the threshold typically associated with eventual infarction. (Supported by NIH NS38684).

Cerebroprotective action of angiotensin 1‐7 in a rat model of ischemic stroke

Recent progress in cardiovascular therapy suggests that stimulation of Angiotensin Converting Enzyme 2 (ACE2), production of Angiotensin 1‐7 (Ang 1‐7), and activation of the Ang 1‐7 receptor, Mas, are viable targets for disease prevention and treatment. We proposed that central administration of Ang 1‐7 via lateral ventricular cannula will provide cerebroprotection in a rat model of Endothelin‐1 (ET‐1)‐induced middle cerebral artery occlusion (MCAO). Prior to ET‐1 induced MCAO, rats were treated with intracerebroventricular Ang 1‐7 (1µg/h) or artificial Cerebrospinal Fluid (aCSF) for 5 days. Ang 1‐7 treatment reduced neurological deficits and infarct size measured 72 h after MCAO induction. Infarct size was reduced to 15.23 ± 5.77% (n = 9) of ipsilateral gray matter in the Ang 1‐7 treated rats compared with 36.94 ± 7.56% (n = 12) in artificial Cerebrospinal Fluid (aCSF) controls. Neurological deficits were also reduced in Ang 1‐7 treated rats as indicated by a lower Benderson Exam Score of 0.22 ± 0.15 (n = 9) compared to 1.08 ± 0.31 (n = 12) in control rats, a higher Garcia Exam Score of 17.56 ± 0.18 (n = 9) compared to 14.00 ± 1.10 (n = 12) in control rats, and improved sunflower seed eating from 146 ± 23 s and 14 ± 1 (n = 9) shell pieces compared to 293 ± 7 s and 29 ± 1 shell pieces (n = 12) in control rats. This provides the first demonstration of the cerebroprotective properties of Ang 1‐7 during ischemic stroke.

Combined magnesium and mild hypothermia (35 °C) treatment reduces infarct volumes after permanent middle cerebral artery occlusion in the rat at 2 and 4, but not 6 h

Background and purpose Using transient focal and global cerebral ischemia models in the rat, we have previously shown that MgSO 4 is not neuroprotective unless it is combined with mild hypothermia. This study establishes a therapeutic time window for combined MgSO 4 and mild hypothermia treatment after permanent middle cerebral artery occlusion (MCAO). Methods Rats were subjected to permanent intraluminal thread MCAO and animals were treated 2, 4 or 6 h after ischemia with a MgSO 4 infusion (360 μmol/kg, then 120 μmol/kg/h) and mild hypothermia (35 °C) or with vehicle for 24 h. At the 2 h time point, treatment with hypothermia alone and MgSO 4 alone were also assessed. Infarct volumes were measured 48 h after MCAO induction. Results After permanent MCAO, combined MgSO 4 and hypothermia treatment reduced infarct volumes by 54% at 2 h ( P = 0.048) and by 39% at 4 h ( P = 0.012), but there was no treatment effect detected at 6 h or in the hypothermia alone or MgSO 4 alone groups. Conclusions These findings support our earlier work highlighting the neuroprotective effect of MgSO 4 when combined with mild hypothermia, even when treatment is delayed by several hours.

ANTI‐OXIDANT ACTIVITY MEDIATED NEUROPROTECTIVE POTENTIAL OF TRIMETAZIDINE ON FOCAL CEREBRAL ISCHAEMIA–REPERFUSION INJURY IN RATS

1. The present study was designed to investigate the neuroprotective effect of trimetazidine (TMZ) following focal cerebral ischaemia-reperfusion (I/R) injury in rat forebrain. 2. Cerebral I/R injury was induced in rats by middle cerebral artery occlusion (MCAO) for 2 h, followed by reperfusion for 22 h. Trimetazidine (5 and 25 mg/kg, i.p.) was administered 1 h after induction of MCAO. The effects of TMZ were investigated by measuring neurological deficit, volume of infarct and brain swelling after 22 h reperfusion. Oxidative stress and inflammatory reactivity were assessed by estimating anti-oxidant markers and myeloperoxidase (MPO) activity in brain homogenates. Rectal temperature was measured during the study. The effects of TMZ on blood-brain barrier (BBB) permeability and apoptosis were also investigated in rat brain. Apoptosis was observed by DNA fragmentation studies using agarose gel electrophoresis. 3. Treatment with TMZ significantly (P < 0.01) reduced infarct volume and brain swelling. Superoxide dismutase (SOD) activity was reduced in ipsilateral hemispheres of vehicle (saline)-treated reperfused (RI) animals. Treatment with TMZ significantly restored SOD activity (P < 0.01) and glutathione levels (P < 0.05) after reperfusion compared with RI animals. Lipid peroxidation, MPO activity, BBB permeability and rectal temperature were all significantly (P < 0.01, P < 0.05 and P < 0.001, respectively) reduced in TMZ-treated animals compared with RI animals. 4. These results suggest that TMZ protects the brain against cerebral I/R injury and that this neuroprotective activity could be mediated by its anti-oxidant properties. The reduction in rectal temperature by TMZ treatment may be responsible for maintaining the delicate energy balance during I/R injury in rat brain and could have contributed to the neuroprotective activity of TMZ.

Behavioral and Histologic Neuroprotection of Aqueous Garlic Extract After Reversible Focal Cerebral Ischemia

The objective of the present study was to investigate the effects of aqueous garlic extract (AGE) on neurobehavioral activities, malondialdehyde (MDA) and reduced glutathione (GSH) levels, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), and sodium-potassium ATPase (Na(+),K(+)-ATPase) activities, and glutamate and aspartate content in a middle cerebral artery (MCA) occlusion (MCAO) model of acute cerebral ischemia in rats. The right MCA of male Wistar rats was occluded for 2 hours using intraluminal 4-0 monofilament, and reperfusion was allowed for 22 hours. MCAO caused significant depletion in GSH and its dependent enzymes (GPx, GR, and GST) and significant elevation of MDA, glutamate, and aspartate. The activities of Na(+),K(+)- ATPase, SOD, and CAT were decreased significantly by MCAO. The neurobehavioral activities (grip strength, spontaneous motor activity, and motor coordination) were also decreased significantly in the MCAO group. All of the alterations induced by ischemia were significantly attenuated by pretreatment with AGE (500 mg/mL/kg of body weight, i.p.) 30 minutes before the induction of MCAO and correlated well with histopathology by decreasing the neuronal cell death following MCAO and reperfusion. These findings suggest that AGE effectively modulates neurobehavioral and neurochemical changes in focal ischemia, most probably by virtue of its antioxidant properties.

Effect of Saffron (Crocus sativus) on Neurobehavioral and Neurochemical Changes in Cerebral Ischemia in Rats

The modifying effects of Crocus sativus (CS) stigma extract on neurobehavioral activities, malondialdehyde (MDA), reduced glutathione (GSH), glutathione peroxidase, glutathione reductase, glutathione S-transferase, superoxide dismutase (SOD), catalase (CAT), and Na(+),K(+)-ATPase activities, and glutamate (Glu) and aspartate (Asp) content were examined in the middle cerebral artery (MCA) occlusion (MCAO) model of acute cerebral ischemia in rats. The right MCA of male Wistar rats was occluded for 2 hours using intraluminal 4-0 monofilament, and reperfusion was allowed for 22 hours. MCAO caused significant depletion in the contents of GSH and its dependent enzymes while significant elevation of MDA, Glu, and Asp. The activities of Na(+),K(+)-ATPase, SOD, and CAT were decreased significantly by MCAO. The neurobehavioral activities (grip strength, spontaneous motor activity, and motor coordination) were also decreased significantly in the MCAO group. All the alterations induced by ischemia were significantly attenuated by pretreatment of CS (100 mg/kg of body weight, p.o.) 7 days before the induction of MCAO and correlated well with histopathology by decreasing the neuronal cell death following MCAO and reperfusion. The present results may suggest the effectiveness of CS in focal ischemia most probably by virtue of its antioxidant property.

Therapeutic Efficacy of Basic Fibroblast Growth Factor on Experimental Focal Ischemia Studied by Magnetic Resonance Imaging

The purpose of this study was to evaluate the effect of intravenous infusion of basic fibroblast growth factor (bFGF) in a permanent ischemia model at the subacute phase (2 weeks) as well as at 24 hours and 1 week using T2-weighted magnetic resonance imaging (MRI). The middle cerebral artery (MCA) in Sprague-Dawley rats was occluded using an intraluminal suture method. The rats were randomly divided into 2 groups to receive either bFGF (45 mircrog/kg/hr) or saline solution. The infusion was started 30 minutes after MCA occlusion (MCAO) and continued for 3 hours. Regional cerebral blood flow (rCBF) was measured using laser Doppler flowmetry throughout the infusion. T2-weighted MRI was carried out before MCAO, 24 hours after MCAO, and days 7 and 14 after MCAO. Although an elevation in rCBF was seen after the infusion, no significant change between the groups was observed. A significant difference between the bFGF and saline groups in T2-derived lesion volume was observed at 24 hours (P < .05), on day 7 (P < .05), and on day 14 (P < .01). The percentage of lesion area calculated from the ipsilateral hemisphere using hematoxylin and eosin staining on day 14 showed a significant difference between the bFGF and saline groups (P < .05). No significant change in the number of bromodeoxyuridine (BrdU)-labeled cells between the groups was observed. This study demonstrates that bFGF, infused intravenously starting 30 minutes after the induction of permanent MCAO, significantly reduces region volume even at day 14, as well as at days 1 and 7, compared with the corresponding saline group.

A therapeutic window for intravenous administration of autologous bone marrow after cerebral ischemia in adult rats

The primary objective of this study was to test the hypothesis that intravenous administration of autologous bone marrow cells could improve functional recovery after middle cerebral artery occlusion (MCAO) for 45 min in the rat and to determine specific time windows for efficacy. Mononuclear cells from autologous bone marrow were transfected with the LacZ reporter gene, and injected intravenously into rats at 3–72 h after induction of MCAO. Histological analysis of the ischemic lesion at 14 days after transplantation revealed reduced ischemic lesion volume. Lesion volume was 250±45 mm 3 ( n=6) after MCAO without cell transplantation. Lesions were minimally detected by absence of 2,3,5-triphenyltetrazolium chloride (TTC) staining when bone marrow cells were infused 3 h after lesion induction. Lesions were clearly detected beginning with the 6-h postlesion group and became progressively larger at 12, 24 and 72 h (80±25, 140±18, and 180±22 mm 3, respectively; n=6 for each group). Transplanted LacZ + bone marrow cells accumulated extensively in and around the ischemic lesions, and immunohistochemistry suggests some neuronal and glial lineage differentiation. Behavioral testing (Morris water maze and Treadmill stress test) indicated greater functional recovery in the treated group. These findings suggest that early intervention with intravenous administration of autologous mononuclear cells from bone marrow can reduce lesion size in the MCAO model in the rat, and improve functional outcome.

Early effects of acid-base management during hypothermia on cerebral infarct volume, edema, and cerebral blood flow in acute focal cerebral ischemia in rats.

Although the frequency for the use of moderate hypothermia in acute ischemic stroke is increasing, the optimal acid-base management during hypothermia remains unclear. This study investigates the effect of pH- and alpha-stat acid-base management on cerebral blood flow (CBF), infarct volume, and cerebral edema in a model of transient focal cerebral ischemia in rats. Twenty Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) for 2 h during normothermic conditions followed by 5 h of reperfusion during hypothermia (33 degrees C). Animals were artificially ventilated with either alpha- (n = 10) or pH-stat management (n = 10). CBF was analyzed 7 h after induction of MCAO by iodo[(14)C]antipyrine autoradiography. Cerebral infarct volume and cerebral edema were measured by high-contrast silver infarct staining (SIS). Compared with the alpha-stat regimen, pH-stat management reduced cerebral infarct volume (98.3 +/- 33.2 mm(3) vs. 53.6 +/- 21.6 mm(3); P > or = 0.05 mean +/- SD) and cerebral edema (10.6 +/- 4.0% vs. 3.1 +/- 2.4%; P > or = 0.05). Global CBF during pH-stat management exceeded that of alpha-stat animals (69.5 +/- 12.3 ml x 100 g(-1) x min(-1) vs. 54.7 +/- 13.3 ml x 100 g(-1) x min; P > or = 0.05). The regional CBF of the ischemic hemisphere was 62.1 +/- 11.2 ml x 100 g(-1) x min(-1) in the pH-stat group versus 48.2 +/- 7.2 ml x 100 g(-1) x min(-1) in the alpha-stat group ( P> or = 0.05). In the very early reperfusion period (5 h), pH-stat management significantly decreases cerebral infarct volume and edema as compared with alpha-stat during moderate hypothermia, probably by increasing CBF.

Abstracts of Literature.

Abstracts of Literature.

Reduction of ischemic brain injury by anti-P-selectin monoclonal antibody after permanent middle cerebral artery occlusion in rat

The selectin family of adhesion molecules is involved in adhesion of leukocyte to microcirculatory system and the transmigration into brain parenchyma. Although the role of P-selectin may be important in the pathogenesis of brain ischemia, a possible protective effect on ischemic brain injury by blocking P-selectin has not been reported. We have examined the effects of a novel anti-P-selectin antibody on ischemic brain injury after 24 h of permanent middle cerebral artery occlusion (MCAO) in rat. Male Wistar rats were subjected to MCAO by an insertion of a silicone rubber cylinder for 24 h. Anti-rat P-selectin monoclonal antibody, ARP 2-4 was injected intravenously at a dose of 1 mg kg–1 at 5 min before the induction of MCAO. Control animals received the same volume of vehicle solution. Regional cerebral blood flow (rCBF) was measured immediately after and at 8 h of MCAO. At decapitation of rats at 24 h of permanent MCAO, infarct size was compared between the antibody and vehicle treated group. In addition, immunohisto- chemistry for leukocyte infiltration and HSP72, and histochemistry for TUNEL were also compared. Pretreatment with ARP 2-4 improved rCBF at 8 h of MCAO (55.4% ± 11.7% of control, n = 5) as compared to vehicle group (24.2% ± 77.8%, n = 5, p < 0.02). Although leukocyte infiltration was not normally detected by monoclonal antibodies for CD11a and CD18, it became remarkably evident at 7 day of MCAO. Although HSP72 and TUNEL were not also detected in sham control brains, they were induced in neurons of the MCA area at 7 day of MCAO. Treatment with ARP 2-4 significantly reduced the numbers of leukocyte and neurons with positive HSP72 and TUNEL stainings. These results demonstrated that an administration of a monoclonal antibody against P-selectin improved rCBF, and attenuated infarct size that was associated with reduction of leukocyte infiltration. Furthermore, treatment with the antibody reduced both HSP72 and TUNEL stainings. These data suggest an important role of P-selectin in ischemic brain damage, and a future therapeutic potential to human stroke patients. [Neurol Res 1999; 21: 269-276]

Increased Formation of Reactive Oxygen Species after Permanent and Reversible Middle Cerebral Artery Occlusion in the Rat

In barbiturate-anesthetized rats, we induced 3 hours of permanent middle cerebral artery occlusion (MCAO) by an intraluminal thread (n = 6), or 1 hour MCAO followed by 2 hours of reperfusion (n = 6). Through a closed cranial window over the parietal cortex, the production of reactive oxygen species (ROS) was measured in the infarct border using online in vivo chemiluminescence (CL) while monitoring the appearance of peri-infarct depolarizations (PID). The borderzone localization of the ROS and direct current (DC) potential measurements was confirmed in additional experiments using laser-Doppler scanning, mapping regional CBF changes through the cranial window after permanent (n = 5) or reversible (n = 5) MCAO. CL measurements revealed a short period (10 to 30 minutes) of reduced ROS formation after vessel occlusion, followed by a significant increase (to 162 +/- 51%; baseline = 100%; P < .05) from 100 minutes of permanent MCAO onward. Reperfusion after a 1-hour period of MCAO led to a burst-like pattern of ROS production (peak: 489 +/- 330%; P < .05). When the experiments were terminated 3 hours after induction of MCAO, CL was still significantly increased above baseline after permanent and reversible MCAO (to 190 +/- 67% and 211 +/- 64%, respectively; P < .05). Simultaneous DC potential recordings detected 6.4 +/- 2.7 PID in the first, 4.7 +/- 2.3 in the second, and 2.8 +/- 2.0 in the third hour after permanent MCAO. In animals with reversible MCAO, PID were abolished from 15-minutes recirculation onward. There was no temporal relationship between ROS production and peri-infarct DC potential shifts. In conclusion, using a high temporal resolution ROS detection technique (CL), we found that permanent MCAO (after an initial decrease) was accompanied by a steady increase of ROS production during the 3-hour observation period, while reperfusion after 1 hour of MCAO produced a burst in ROS formation. Both patterns of ROS production were not related to the occurrence of PID.

Hyperglycemia delays terminal depolarization and enhances repolarization after peri-infarct spreading depression as measured by serial diffusion MR mapping.

We investigated the effect of hyperglycemia on the initiation and propagation of spreading depression-like peri-infarct ischemic depolarization (SD) induced by focal cerebral ischemia in rats. Peri-infarct SD were monitored during the initial 15 minutes after remotely induced middle cerebral artery occlusion (MCAO) using serial diffusion weighted magnetic resonance imaging. Maps of the apparent diffusion coefficient (ADC) were calculated and ADC decreases were monitored over time. Hyperglycemic rats (n = 6) had a significant prolongation of the time from induction of MCAO to the start of the ADC decrease as compared with normoglycemic control rats. The time to the maximal ADC decrease was significantly delayed and recovery of transient ADC declines in the area adjacent to the ischemic core was significantly faster in hyperglycemic rats. We conclude that hyperglycemia delays the terminal depolarization in the ischemic core and supports a faster repolarization in severely mal-perfused penumbral tissue after SD, which reflects the increased availability of energy substrates in the state of hyperglycemia.