Polymorphonuclear leukocyte infiltration into cerebral focal ischemic tissue: myeloperoxidase activity assay and histologic verification.

Two different techniques were utilized to identify the infiltration of polymorphonuclear leukocytes (PMN) into cerebral tissue following focal ischemia: histologic analysis and a modified myeloperoxidase (MPO) activity assay. Twenty-four hours after producing permanent cortical ischemia by occluding and severing the middle cerebral artery of male spontaneously hypertensive rats, contralateral hemiparalysis and sensory-motor deficits were observed due to cerebral infarction of the frontal and parietal cortex. In hematoxylin-and-eosin-stained histologic sections, PMN, predominantly neutrophils, were identified at various stages of diapedesis from deep cerebral and meningeal vessels at the periphery of the infarct, into brain parenchyma. When MPO activity in normal brain tissue was studied initially, it could not be demonstrated in normal tissues extracted from non-washed homogenates. However, if tissue was homogenized in phosphate buffer (i.e., washed), MPO activity was expressed upon extraction. Utilizing this modified assay, MPO activity was significantly increased only in the infarcted cortex compared to other normal areas of the brain. This was observed in non-perfused animals and after perfusion with isotonic saline to remove blood constituents from the vasculature prior to brain removal. The increased PMN infiltration and MPO activity were not observed in forebrain tissue of sham-operated control rats. Also, MPO activity was not increased in the ischemic cortex of MCAO rats perfused immediately after middle cerebral artery occlusion, indicating that blood was not trapped in the ischemic area. By using a leukocyte histochemical staining assay, activity of peroxidases was identified within vascular-adhering/infiltrating PMN in the infarcted cortex 24 hr after focal ischemia. An evaluation of several blood components indicated that increased MPO activity was selective for PMN. The observed increase of approximately 0.3 U MPO/g wet weight ischemic tissue vs. nonischemic cerebral tissues probably reflects the increased vascular adherance/infiltration of approximately 600,000 PMN/g wet weight infarcted cortex 24 hr after focal ischemia. This combined biochemical and histological study strongly suggests that PMN adhere within blood vessels and infiltrate into brain tissue injured by focal ischemia and that the associated inflammatory response might contribute to delayed progressive tissue damage in focal stroke. This modified MPO assay is a useful, quantitative index of PMN that can be utilized to elucidate the potential deleterious consequences of neutrophils infiltrating into the central nervous system after cerebral ischemia, trauma, or other pro-inflammatory stimuli.     

Increased susceptibility to ischemic brain injury in cyclooxygenase-1-deficient mice.

Cyclooxygenase-1 (COX-1), a rate-limiting enzyme in the synthesis of prostanoids, is involved in selected vasodilatatory responses of the cerebral circulation. Cyclooxygenase-1-null mice were used to determine whether COX-1 influences cerebral ischemic damage. The middle cerebral artery was occluded in COX-1 -/- and +/+ mice (n = 9/group), and lesion volume was determined in thionin-stained sections 24 or 96 hours later. Middle cerebral artery occlusion produced larger infarcts in COX-1 -/- mice, both at 24 (35 +/- 17%; P < 0.05) and 96 hours (41 +/- 16%; P < 0.05) after ischemia. The enlargement was not due to increased susceptibility to glutamate excitotoxicity, because microinjection of N-methyl-D-aspartate or kainate in the parietal cortex produced comparable lesions in COX-1 +/+ and -/- mice ( P > 0.05; n = 8/group). To examine the contribution of hemodynamic factors to the enlargement of the infarct, cerebral blood flow was monitored by laser-Doppler flowmetry in the ischemic territory (n = 6/group). Although the reduction in cerebral blood flow was comparable in the ischemic core ( P > 0.05), at the periphery of the ischemic territory the reduction was greater in COX-1 -/- mice (-58 +/- 4%) than in COX-1 +/+ mice (-34 +/- 5%; P < 0.05). It is concluded that mice lacking COX-1 are more susceptible to focal cerebral ischemia, an effect that can be attributed to a more severe cerebral blood flow reduction in vulnerable regions at the periphery of the ischemic territory. Thus, the vascular effects of COX-1 may contribute to maintain cerebral blood flow in the postischemic brain and, as such, play a protective role in ischemic brain injury.     

Cerebrovascular hemodynamics and ischemic tolerance: lipopolysaccharide-induced resistance to focal cerebral ischemia is not due to changes in severity of the initial ischemic insult, but is associated with preservation of microvascular perfusion.

Lipopolysaccharide (LPS), administered 72 hours before middle cerebral artery (MCA) occlusion, confers significant protection against ischemic injury. For example, in the present study, LPS (0.9 mg/kg intravenously) induced a 31% reduction in infarct volume (compared with saline control) assessed 24 hours after permanent MCA occlusion. To determine whether LPS induces true tolerance to ischemia, or merely attenuates initial ischemic severity by augmenting collateral blood flow, local CBF was measured autoradiographically 15 minutes after MCA occlusion. Local CBF did not differ significantly between LPS- and saline-pretreated rats (e.g., 34 +/- 10 and 29 +/- 15 mL x 100 g(-1) x min(-1) for saline and LPS pretreatment in a representative region of ischemic cortex), indicating that the neuroprotective action of LPS is not attributable to an immediate reduction in the degree of ischemia induced by MCA occlusion, and that LPS does indeed induce a state of ischemic tolerance. In contrast to the similarity of the initial ischemic insult between tolerant (LPS-pretreated) and nontolerant (saline-pretreated) rats, microvascular perfusion assessed either 4 hours or 24 hours after MCA occlusion was preserved at significantly higher levels in the LPS-pretreated rats than in controls. Furthermore, the regions of preserved perfusion in tolerant animals were associated with regions of tissue sparing. These results suggest that LPS-induced tolerance to focal ischemia is at least partly dependent on the active maintenance of microvascular patency and hence the prevention of secondary ischemic injury.     

A phenothiazine derivative reduces rat brain damage after global or focal ischemia.

BACKGROUND AND PURPOSE: We previously reported that 2-(10H-phenothiazin-2-yloxy)-N,N-dimethylethanamine hydrochloride is a potent inhibitor of iron-dependent lipid peroxidation in vitro and can protect primary cultures of rat hippocampal neurons from hydrogen peroxide-induced toxicity. Because oxidants may play an important role in mediating postischemic tissue injury, we evaluated this agent in two rat models of transient cerebral ischemia. METHODS: In a model of global forebrain ischemia, 23 male Wistar rats were subjected to 10 minutes of four-vessel occlusion followed by 72 hours of reperfusion. The rats received three intraperitoneal injections of either vehicle (2% aqueous acacia) or test agent (40 mg/kg). In a model of focal stroke, 19 spontaneously hypertensive rats were subjected to 2 hours of tandem middle cerebral and ipsilateral common carotid artery occlusion followed by 24 hours of reperfusion. The rats received three intraperitoneal injections of either vehicle (2% aqueous acacia) or test agent (40 mg/kg). RESULTS: In the global model, the phenothiazine significantly protected the CA1 layer of the hippocampus, with a reduction in mean damage score from 2.1 +/- 0.3 for control rats to 1.0 +/- 0.4 for treated rats (p less than 0.05). In the transient focal stroke model, the compound reduced cortical infarct volume from 130.1 +/- 10.3 mm3 for control rats to 95.2 +/- 24.5 mm3 for treated rats (p less than 0.02). CONCLUSIONS: Although the primary mechanism responsible for the protective effect is unclear at the present time, our study is consistent with the hypothesis that oxidant-mediated lipid peroxidation may be involved in the pathophysiology of postischemic brain injury.     

Effect of plasma glucose on infarct size in focal cerebral ischemia-reperfusion

Although hyperglycemia has been shown to consistently exacerbate ischemia brain injury following global or diffuse cerebral ischemia, the effect of hyperglycemia in unilateral focal cerebral ischemia remains controversial. Recent advances in thrombolytic therapy have enhanced the clinical significance of postischemic reperfusion. We studied the effect of plasma glucose on ischemic brain injury in a newly developed focal cerebral ischemia-reperfusion model. Rats allowed free access to food until ischemic insult developed intra- and postischemic hyperglycemia and cortical infarction. Rats fasted for 24 hours had blunted hyperglycemic responses. Infarct volumes were correspondingly smaller. The protective effect of fasting was partially abolished by glucose loading during ischemia to induce intra-ischemic hyperglycemia. Glucose loading immediately or 3 hours after focal cerebral ischemia did not significantly alter the protective effect of fasting. Insulin treatment in fed rats before ischemia also reduced hyperglycemic responses and infarct volume. Timing of insulin treatment was also critical in the reduction of ischemic injury. These findings indicate that plasma glucose during the period of ischemia is an important determinant of brain injury in focal cerebral ischemia-reperfusion and there is a therapeutic window for normalization of plasma glucose to be efficacious.     

Inflammatory leukocyte infiltration in focal cerebral ischemia: unrelated to infarct size

OBJECTIVE: An inflammatory host response in the ischemically injured brain is well documented. However, its pathophysiological relevance is uncertain. We investigated whether inflammatory leukocyte response in the ischemic brain alters infarct size. METHODS: The cellular inflammatory response to cerebral ischemia in Wistar-derived rats induced by the transient occlusion of the middle cerebral artery with a thread was pharmacologically upmodulated by lipopolysaccharide (LPS) or downmodulated by continuous infusion of carboxylated sialyl Lewis(x) (sLex). The effects of such experimental modulation of focal cerebral leukocyte recruitment on the extent of the resulting infarction were assessed. RESULTS: Compared to control treatments, LPS strongly enhanced (540.5 +/- 504.8 vs. 94.6 +/- 60.6, p < 0.01) and sLex decreased (32.8 +/- 29.1 vs. 97.0 +/- 49.7, p < 0.05) the numbers of neutrophils at the investigated sites in cerebral ischemia. Unexpectedly, despite such marked experimental modulation of leukocyte infiltration in the ischemic brain, the extent of the resulting cerebral infarction (percent of total hemisphere) remained unchanged under these different conditions (54.5 +/- 10.8 vs. 53.0 +/- 19.1, n.s. and 50.3 +/- 18.0 vs. 57.2 +/- 10.0, n.s., respectively). CONCLUSIONS: The striking dissociation between the massively altered inflammatory leukocyte infiltration in the ischemic brain and the unchanged infarct outcome indicates that intracerebral inflammatory leukocyte recruitment is not a major pathogenic factor in the development of ischemic tissue damage.     

Hypertonic saline worsens infarct volume after transient focal ischemia in rats

BACKGROUND AND PURPOSE: Hypertonic saline (HS) has been advocated as a hyperosmolar agent for the treatment of cerebral edema, especially after traumatic brain injury. We tested the hypothesis that continuous intravenous HS administered during reperfusion from transient focal cerebral ischemia attenuates infarct volume. METHODS: Halothane-anesthetized male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO) by the intraluminal occlusion technique. At the onset of reperfusion, rats received a 10-mL/kg intravenous bolus of 0.9% saline (SAL, n=8) or 7.5% SAL (chloride:acetate 50:50, n=8) followed by a continuous infusion for 22 hours. In a second series of experiments, ischemic damage was determined in cohorts treated with equivolumetric 3% saline (n=8) or 20% mannitol (n=8). In a third series, regional cerebral blood flow was measured ([(14)C]iodoantipyrine autoradiography) at 6 hours of reperfusion in 7.5%-SAL-treated (n=5) or SAL-treated (n=5) animals. RESULTS: In SAL rats, serum Na(+) was 137+/-3 and 138+/-2 mEq/L (mean+/-SEM) at baseline and 22 hours of reperfusion, respectively. In 7.5% SAL, serum Na(+) was 136+/-2 and 154+/-2 mEq/L at baseline and reperfusion, respectively. Physiological variables and reduction in laser-Doppler signal during MCAO and early reperfusion were not different between the 2 treatment groups. Cortical infarct volume was larger in 7.5%-SAL-treated rats (121+/-14 mm(3); 30+/-3% of contralateral cortex; P<0.05) than in SAL (64+/-15 mm(3); 16+/-4% of contralateral cortex). Striatal infarct volume was unchanged by HS therapy. Ipsilateral cortical tissue volume was increased relative to the contralateral side (by 26+/-5% with SAL; by 41+/-5% with 7.5% SAL). In contrast, ischemic damage was unaffected by 3%-SAL or 20%-mannitol treatment compared with SAL. Regional cerebral blood flow during reperfusion was heterogeneous in all animals, but there was no evidence of postischemic hypoperfusion or blood flow maldistribution in 7.5%-SAL-treated animals. CONCLUSIONS: These data demonstrate that hypernatremia resulting from postischemic HS infusion worsens cortical infarct volume in transient focal cerebral ischemia. The deleterious effect is not linked to exacerbation of delayed hypoperfusion during early reperfusion (6 hours); however, blood flow defects at later recovery time points remain to be excluded. These results may have implications for HS therapy in clinical ischemic stroke.     

Hyperglycemia suppresses c-fos mRNA expression following transient cerebral ischemia in gerbils.

The c-fos proto-oncogene is activated by transient cerebral ischemia. This activation may signify a specific genetic response to ischemia affecting tolerance to ischemia and ultimate cell survival. Hyperglycemia, which enhances brain injury from transient ischemia, was studied for its effects on this gene system in gerbils by measuring c-fos mRNA 2 h after 20 min of bilateral carotid artery occlusion. Brain c-fos mRNA was increased by ischemia (11.7 +/- 5.0, p less than or equal to 0.05, fold increase) compared to nonischemic controls (1.0 +/- 1.3). Pretreatment with 1 g/kg of glucose partially reduced postischemic c-fos mRNA (6.3 +/- 1.6, p less than or equal to 0.05) while 4 g/kg of glucose completely suppressed postischemic c-fos expression (0.7 +/- 0.3, p less than or equal to 0.05). These data indicate that hyperglycemia suppresses normal postischemic gene expression and suggest the possibility that such suppression is a predictor or even a contributor to hyperglycemia-enhanced ischemic brain damage.     

Polymorphonuclear neutrophils contribute to infarction and oxidative stress in the cortex but not in the striatum after ischemia-reperfusion in rats

The present work examined whether polymorphonuclear neutrophil (PMN) infiltration contributes to cortical and striatal brain damage and oxidative stress in a model of transient focal cerebral ischemia. A 2-h occlusion of the left middle cerebral artery and ipsilateral common carotid artery was performed in rats. Administration of the neutropenic agent vinblastine (0.5 mg/kg, i.v.) resulted in a profound decrease in circulating PMNs which was associated with a 80% decrease in myeloperoxidase activity, a marker of PMN infiltration, in both the cortex and the striatum. In the cortex, vinblastine-treated animals exhibited a 44% decrease in the infarct volume and also reduced the oxidative stress (evaluated by the decrease in glutathione concentrations). By contrast, in the striatum, neutropenia modified neither the lesion size nor the oxidative stress. These results indicate that PMN contribution to postischemic injury and oxidative stress is dependent on the brain structure.     

Effect of indomethacin on edema following single and repetitive cerebral ischemia in the gerbil

BACKGROUND AND PURPOSE: Repetitive periods of cerebral ischemia result in more severe injury than a single period of ischemia of similar total duration. We investigated the possibility of prostaglandin mediation of this increased injury by attempting to modify brain edema formation with indomethacin pretreatment. METHODS: Under halothane/N2O anesthesia, groups of gerbils underwent bilateral carotid occlusion to induce forebrain ischemia. Group I underwent a single 15-minute period of carotid occlusion. Group II underwent three 5-minute periods of occlusion at hourly intervals. Groups III and IV were similar to groups I and II, respectively, but received 0.2 mg/kg indomethacin before carotid occlusion. Cortical and cerebellar water and sodium contents were determined in control animals (n = 6) at time zero and in experimental animals 24, 48, and 72 hours after ischemia (n = 6-10 gerbils/group at each time point). RESULTS: Cortical water and sodium contents in group II peaked 48 hours after insult (82.15 +/- 0.31% and 420 +/- 14 meq/kg dry wt, respectively) and were significantly higher than control and group I values at both 24 and 48 hours. Cortical water did not change from control in group I animals. Indomethacin pretreatment significantly attenuated increases in water and sodium content seen at 48 hours in gerbils undergoing repetitive ischemia (peak 80.02 +/- 0.45% and 300 +/- 39 meq/kg dry wt), but did not affect mortality. CONCLUSIONS: Indomethacin lessens edema after repetitive cerebral ischemia, suggesting that elevations of cyclooxygenase products are responsible, at least in part, for severe brain edema following repetitive ischemia.     

Fluoxetine affords robust neuroprotection in the postischemic brain via its anti-inflammatory effect

Fluoxetine is a selective serotonin reuptake inhibitor that is widely used in the treatment of major depression including after stroke. In this study, we tested whether fluoxetine protects neuronal death in a rat cerebral ischemia model of middle cerebral artery occlusion (MCAO). The administration of fluoxetine intravenously (10 mg/kg) at 30 min, 3 hr, or 6 hr after MCAO reduced infarct volumes to 21.2+/-6.7%, 14.5+/-3.0%, and 22.8+/-2.9%, respectively, of that of the untreated control. Moreover, the neuroprotective effect of fluoxetine was evident when it was administered as late as 9 hr after MCAO/reperfusion. These neuroprotective effects were accompanied by improvement of motor impairment and neurological deficits. The fluoxetine-treated brain was found to show marked repressions of microglia activation, neutrophil infiltration, and proinflammatory marker expressions. Moreover, fluoxetine suppressed NF-kappaB activity dose-dependently in the postischemic brain and also in lipopolysaccharide-treated primary microglia and neutrophil cultures, suggesting that NF-kappaB activity inhibition explains in part its anti-inflammatory effect. These results demonstrate that curative treatment of fluoxetine affords strong protection against delayed cerebral ischemic injury, and that these neuroprotective effects might be associated with its anti-inflammatory effects.     

21-Aminosteroid lipid peroxidation inhibitor U74006F protects against cerebral ischemia in gerbils

U74006F (21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-16 alpha-methylpregna-1,4,9(11)-triene-3,20-dione, monomethane sulfonate) is a novel and potent inhibitor of central nervous system tissue lipid peroxidation that is devoid of classical steroid hormonal activities. Its possible efficacy in attenuating postischemic mortality and neuronal necrosis was examined in gerbils following 3-hour unilateral carotid artery occlusion. Male Mongolian gerbils received two intraperitoneal injections of either vehicle or U74006F (3 or 10 mg/kg), the first injection 10 minutes before and the second injection at the end of the 3-hour ischemic episode. In an initial series of experiments, vehicle-treated gerbils displayed 60.9% (14 of 23) survival 24 hours after ischemia, which decreased to 34.8% (8 of 23) at 48 hours. In contrast, the 10 mg/kg U74006F-treated group showed 86.7% (13 of 15) survival at 24 hours (p less than 0.15 vs. vehicle) and 80.0% (12 of 15) survival at 48 hours (p less than 0.02). In a second series, neurons in the hippocampal CA1 subfield and the medial and lateral cerebral cortex were counted in gerbils surviving 24 hours after unilateral carotid artery occlusion. Comparison of neuronal densities in the ischemic hemisphere with those in the contralateral nonischemic hemisphere revealed significant neuronal preservation in all three brain regions of 10 mg/kg i.p. x 2 U74006F-treated gerbils. Our results show that U74006F can improve survival and attenuate neuronal necrosis in a severe brain ischemia model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cerebral mast cells regulate early ischemic brain swelling and neutrophil accumulation.

We previously observed degranulated mast cells (MC) in association with perivascular brain edema formation during focal cerebral ischemia. Brain MC are typically located perivascularly and contain potent fast-acting vasoactive and proteolytic substances. We examined in a rat model of transient middle cerebral artery occlusion (MCAO) whether, in the early phase of ischemia, MC regulate microcirculation, the blood-brain barrier (BBB) permeability, and edema formation. First, animals received MC inhibitor (cromoglycate), MC-degranulating drug (compound 48/80), or saline. Thereafter, we performed transient MCAO in gene-manipulated MC-deficient rats and their wild-type (WT) littermates, calculating brain swelling, visualizing BBB leakage by intravenously administered Evans blue albumin, and determining neutrophil infiltration with light microscopy. Cerebral blood flow, monitored by laser-Doppler flowmetry in separate experiments, was similar among pharmacological treatments. Ischemic swelling resulted in increased hemispheric volume of 13.4%+/-1.0% in controls, 8.1%+/-0.4% (39% reduction) after cromoglycate, and 25.2%+/-2.0% (89% increase) after compound 48/80 (P<0.05). Early ischemic BBB leakage was reduced by 51% after cromoglycate, and 50% enhanced by compound 48/80 (P<0.05). The cromoglycate group showed 37% less postischemic neutrophil infiltration than did controls (P<0.05). Furthermore, MC-deficient rats responded to focal ischemia with 58% less brain swelling (6.7%+/-1.2%) than did their WT littermates (15.8%+/-1.4%, P<0.05). Blood-brain barrier damage was 47% lower in MC-deficient rats than in the WT (P<0.05). Neutrophil infiltration after MCAO was decreased 47% in MC-deficient rats in comparison to WT (P<0.05). Pharmacological MC inhibition thus appears to deserve further investigation regarding reduction of brain swelling and inflammation early after stroke.     

Uptake of radiolabeled ions in normal and ischemia-damaged brain

The regional concentrations of nine radiochemicals were measured in rat brain after induction of cerebral ischemia to identify tracers concentrated by brain undergoing selective neuronal necrosis. Transient (30 minute) forebrain ischemia was produced in the rat; 24 hours after cerebral recirculation the radiochemicals were injected intravenously and allowed to circulate for 5 hours. The brain concentrations of the radiochemicals in dissected regions were determined by scintillation counting. Forebrain ischemia of this nature will produce extensive injury to striatal neurons but will spare the great majority of neocortical neurons at 24 hours. The regional concentrations of these radiochemicals varied considerably in both control and ischemic animals. In postischemic animals, 4 radionuclides (63Ni, 99TcO4, 22Na, and [3H]tetracycline) were concentrated in the irreversibly damaged striatum in amounts ranging from 1.4 to 2.4 times greater than in normal tissue. The concentrations of 65Zn, 59Fe, 32PO4, and 147Pm in postischemic brain were similar to or less than those in normal brain. The concentration of [14C]EDTA was increased in injured and uninjured brain of postischemic rats. Autoradiographic analysis of the distribution patterns of some of these ions in normal animals showed that 99TcO4, 22Na, 65Zn, and 59Fe were distributed more uniformly throughout the brain than were 32PO4, 63Ni, and 147Pm. At 24 or 48 hours after ischemia, 63Ni, 99TcO4, and 22Na were preferentially concentrated in the damaged striatum and hippocampus, whereas 65Zn, 59Fe, 32PO4, and 147Pm did not accumulate in irreversibly injured tissue. Of the radiochemicals tested to date, Ni, TcO4, and tetracycline may be useful for diagnosing ischemic brain injury in humans, using positron emission tomography.     

Functional deterioration of endothelial nitric oxide synthase after focal cerebral ischemia

Endothelial nitric oxide synthase (eNOS) dysfunction is related to secondary injury and lesion expansion after cerebral ischemia. To date, there are few reports about postischemic alterations in the eNOS regulatory system. The purpose of the present study was to clarify eNOS expression, Ser1177 phosphorylation, and monomer formation after cerebral ischemia. Male Wistar rats were subjected to transient focal cerebral ischemia. Endothelial nitric oxide synthase messenger RNA (mRNA) and protein expression increased ∼8-fold in the ischemic lesion. In the middle cerebral artery core, eNOS-Ser1177 phosphorylation increased 6 hours after ischemia; however, there was an approximately 90% decrease in eNOS-Ser1177 phosphorylation observed 24 hours after ischemia that continued until at least 7 days after ischemia. Endothelial nitric oxide synthase monomer formation also increased 24 and 48 hours after ischemia (P<0.05), and protein nitration progressed in parallel with monomerization. To assess the effect of a neuroprotective agent on eNOS dysfunction, we evaluated the effect of fasudil, a Rho-kinase inhibitor, on eNOS phosphorylation and dimerization. Postischemic treatment with fasudil suppressed lesion expansion and dephosphorylation and monomer formation of eNOS. In conclusion, functional deterioration of eNOS progressed after cerebral ischemia. Rho-kinase inhibitors can reduce ischemic lesion expansion as well as eNOS dysfunction in the ischemic brain.     

Leukocyte Recruitment and Ischemic Brain Injury

Leukocytes are recruited into the cerebral microcirculation following an ischemic insult. The leukocyte–endothelial cell adhesion manifested within a few hours after ischemia (followed by reperfusion, I/R) largely reflects an infiltration of neutrophils, while other leukocyte populations appear to dominate the adhesive interactions with the vessel wall at 24 h of reperfusion. The influx of rolling and adherent leukocytes is accompanied by the recruitment of adherent platelets, which likely enhances the cytotoxic potential of the leukocytes to which they are attached. The recruitment of leukocytes and platelets in the postischemic brain is mediated by specific adhesion glycoproteins expressed by the activated blood cells and on cerebral microvascular endothelial cells. This process is also modulated by different signaling pathways (e.g., CD40/CD40L, Notch) and cytokines (e.g., RANTES) that are activated/released following I/R. Some of the known risk factors for cardiovascular disease, including hypercholesterolemia and obesity appear to exacerbate the leukocyte and platelet recruitment elicited by brain I/R. Although lymphocyte–endothelial cell and –platelet interactions in the postischemic cerebral microcirculation have not been evaluated to date, recent evidence in experimental animals implicate both CD4+ and CD8+ T-lymphocytes in the cerebral microvascular dysfunction, inflammation, and tissue injury associated with brain I/R. Evidence implicating regulatory T-cells as cerebroprotective modulators of the inflammatory and tissue injury responses to brain I/R support a continued focus on leukocytes as a target for therapeutic intervention in ischemic stroke.     

Tissue plasminogen activator does not increase neuronal damage in rat models of global and focal ischemia

BACKGROUND: Intravenous tissue plasminogen activator (tPA) is the only approved treatment for acute ischemic stroke. Recent results from both in vitro and in vivo animal model experiments suggest the possibility that tPA is neurotoxic. METHODS: The authors evaluated the putative neurotoxicity of tPA in both global and focal animal models of ischemic stroke. Global ischemia was induced in male Wistar rats using a modified four-vessel occlusion technique, with percentage neuronal injury assessed at 7 days through necrotic and normal cell count in the CA1 region. Transient focal ischemia was induced in male spontaneously hypertensive rats subjected to middle cerebral artery clipping, with measurement of cortical infarct volume at 24 hours. tPA was administered in 1, 5, or 10 mg/kg doses given intravenously as a 10% bolus, 90% over the following hour, analogous to current human treatment protocols. RESULTS: In the global model, percent hippocampal injury was 60%+/-23%, 66%+/-26%, 55%+/-26%, and 52%+/-12% in the saline control, 1, 5, and 10 mg/kg tPA groups, respectively. In the focal model, after 120 minutes of ischemia, the control infarct size was 151+/-39 mm3, and for the group given 10 mg/kg of tPA, it was 158+/-28 mm3. CONCLUSIONS: Despite sublethal insults, with moderate injury induced by ischemia, there was no evidence that increasing doses of tPA exacerbated ischemic injury.     

Increase in endogenous brain superoxide dismutase as a potential mechanism of lipopolysaccharide-induced brain ischemic tolerance.

A low dose (0.5 mg/kg) of lipopolysaccharide (LPS), administered 72 hours before 60-minute middle cerebral artery occlusion, induced a delayed neuroprotection proven by the significant decrease (-35%) of brain infarct volume in comparison with control, whereas infarct volumes remained unchanged in rats treated 12, 24, or 168 hours before ischemia. This delayed neuroprotective effect of LPS was induced only with low doses (0.25 to 1 mg/kg), whereas this effect disappeared with a higher dose (2 mg/kg). The delayed neuroprotection of LPS was induced in the cortical part of the infarcted zone, not in the subcortical part. The beneficial effect of LPS on consequences of middle cerebral artery occlusion was suppressed by dexamethasone (3 mg/kg) and indomethacin (3 mg/ kg) administered 1 hour before LPS, whereas both drugs had no direct effect on infarct volume by themselves, suggesting that activation of inflammatory pathway is involved in the development of LPS-induced brain ischemic tolerance. Preadministration of cycloheximide, an inhibitor of protein synthesis, also blocked LPS-induced brain ischemic tolerance suggesting that a protein synthesis is also necessary as a mediating mechanism. Superoxide dismutase (SOD) could be one of the synthesized proteins because lipopolysaccharide increased SOD brain activity 72 hours, but not 12 hours, after its administration, which paralleled the development of brain ischemic tolerance. In contrast, catalase brain activity remained unchanged after LPS administration. The LPS-induced delayed increase in SOD brain content was suppressed by a previous administration of indomethacin. These data suggest that the delayed neuroprotective effect of low doses of LPS is mediated by an increased synthesis of brain SOD that could be triggered by activation of inflammatory pathway.     

Neuroprotective FK506 does not alter in vivo nitric oxide production during ischemia and early reperfusion in rats.

BACKGROUND AND PURPOSE: Previous studies have demonstrated that the immunosuppressant FK506 provides neuroprotection in experimental brain injury and suggest that this action may be mediated by suppression of neuronal nitric oxide synthase activation that occurs after ischemic depolarization. We sought to determine whether FK506 reduces histological injury after middle cerebral artery occlusion (MCAO) in the rat and whether the neuroprotective effect is mediated via suppression of in vivo nitric oxide (NO) production during ischemia or early reperfusion. METHODS: Under controlled conditions of normoxia, normocarbia, and normothermia, halothane-anesthetized male Wistar rats were subjected to 2 hours of MCAO by the intraluminal occlusion technique in a blinded, randomized experimental trial. Ipsilateral parietal cortical laser-Doppler flowmetry was monitored throughout ischemia. Animals were randomly assigned to 4 pretreatment groups: intravenous FK506 0.3 mg/kg or 1. 0 mg/kg, vehicle (cremaphor), or an equivalent volume of saline administered 30 minutes before MCAO. Infarction volume was assessed by a triphenyltetrazolium chloride staining at 22 hours of reperfusion. In separate experiments, microdialysis probes were placed bilaterally into the striatum. Rats were perfused with artificial cerebrospinal fluid containing 3 micromol/L [14C]- L-arginine for 3 hours and then subjected to 2 hours of right MCAO. Intravenous 0.3 mg/kg FK506 or cremaphor was given 30 minutes before right MCAO. Right-left differences between [14C]-L-citrulline in the effluent were assumed to reflect differences in NO production. RESULTS: All values are mean+/-SE. FK506 at 0.3 mg/kg reduced infarction volume in cortex: 40+/-12 mm3 compared with saline (109+/-15 mm3) and cremaphor vehicle (148+/-23) (P<0.05). Striatal infarction was also reduced by low-dose FK506: 16+/-4 mm3 versus 36+/-4 mm3 and 34+/-4 mm3 in saline and vehicle groups, respectively (P<0.05). High-dose treatment reduced infarction volume in cortex (61+/-14 mm3, P<0.05 from saline and vehicle groups) and in striatum (22+/-5 mm3, P<0.05 from saline and vehicle groups). [14C]-L-citrulline recovery via microdialysis was markedly enhanced in ischemic compared with nonischemic striatum. However, ischemia-evoked [14C]-L-citrulline recovery was not different in FK506-treated rats compared with vehicle-treated animals. CONCLUSIONS: These data demonstrate that FK506 provides robust neuroprotection against transient focal cerebral ischemia in the rat. The mechanism of protection in vivo is not through attenuation of ischemia-evoked NO production during MCAO and early reperfusion.     

Neutrophils do not contribute to infarction,oxidative stress,and NO synthase activity in severe brain ischemia

Polymorphonuclear leukocytes (PMNs) were reported to contribute to ischemia-reperfusion-induced brain damage. The present work examined whether PMN infiltration is deleterious in a severe model of transient focal cerebral ischemia and in which part PMNs contribute to oxidative stress and nitric oxide (NO) production. A 20-min occlusion of the left middle cerebral artery and both common carotid arteries was performed in rats. Infarction was maximal 24 h after reperfusion, while accumulation of PMNs in infarcted tissue was not significant before 48 h. Moreover, neutropenia induced by vinblastine (0.5 mg/kg iv) significantly decreased by 60-80% PMN infiltration 48 h after reperfusion but did not reduce the infarct volume. Thus PMNs do not contribute to cerebral injury in our model. Furthermore, decreased PMN infiltration modified neither oxidative stress evaluated by glutathione concentrations nor NO synthase activities 48 h after reperfusion. In conclusion, our results suggest that PMNs are not involved in severe cerebral ischemia and that anti-PMN strategies may be inefficient in some pathological conditions.