Yoshiyuki Matsuo

Correlation between myeloperoxidase-quantified neutrophil accumulation and ischemic brain injury in the rat. Effects of neutrophil depletion.

Background and Purpose Neutrophils have been implicated in the pathogenesis of ischemia‐reperfusion injury. The aim of the present study was to evaluate the correlation between neutrophil infiltration into ischemic tissues and brain injury after transient focal ischemia. Methods We evaluated the effects of depletion of circulating neutrophils by administration of an antineutrophil monoclonal antibody (RP3) on brain edema formation, infarct size, and neutrophil infiltration (myeloperoxidase [MPO]‐quantified) in rats with 1 hour of middle cerebral artery (MCA) occlusion. Results In the cerebral cortex perfused by the anterior cerebral artery (ACA area), there was a significant increase in MPO activity only 24 hours (P<.05) after reperfusion. In the cerebral cortex perfused by the middle cerebral artery (MCA area) and caudate putamen, MPO activity was significantly increased at 12 (MCA area, P<.01; caudate putamen, P<.05), 24 (MCA area, P<.05; caudate putamen, P<.01), and 72 hours (MCA area, P<.01; caudate putamen, P<.05) and returned to near‐normal level by 168 hours after reperfusion. Brain MPO activity after transient MCA occlusion correlated well with the appearance of neutrophils. Depletion of neutrophils by RP3 treatment completely inhibited the increase in MPO activity in the ischemic brain after 24 hours of reperfusion. In addition, treatment with RP3 significantly attenuated the postischemic increase in brain water content at 24 hours after reperfusion. RP3 also significantly reduced the size of infarct area. Conclusions These results indicate that the increase in brain MPO activity after transient focal ischemia virtually reflects the neutrophil infiltration and that neutrophil infiltration into the ischemic brain is implicated in postischemic brain injury. (Stroke. 1994;25:1469‐1475.)

Role of Neutrophils in Radical Production During Ischemia and Reperfusion of the Rat Brain: Effect of Neutrophil Depletion on Extracellular Ascorbyl Radical Formation

A growing body of experimental data indicate that oxygen radicals may mediate the brain injury during ischemia–reperfusion. One potential source of oxygen radicals is activated neutrophils. To study the role of neutrophils in radical production during cerebral ischemia–reperfusion, we evaluated the effects of depletion of circulating neutrophils by administration of an anti-neutrophil monoclonal antibody (RP3) on radical formation in rats with 1-h middle cerebral artery (MCA) occlusion. In the present study, we employed a new electron spin resonance method coupled with brain microdialysis. The method uses the endogenous ascorbyl radical (AR) concentration as a marker of oxygen radicals and requires no spin-trapping agents. In the vehicle controls, extracellular AR decreased during MCA occlusion. After reperfusion, AR significantly increased at 30 min and 1 h, returned to near basal level until 2 h, and increased again at 24 h after reperfusion. In the rats treated with RP3, AR decreased during MCA occlusion to the same extent as in the vehicle control. However, RP3 treatment completely inhibited the increase in extracellular AR after reperfusion. RP3 treatment exerted no effect on the changes in extracellular ascorbate or tissue Po2 throughout the experimental period. In conclusion, neutrophils are a major source of oxygen radicals during reperfusion after focal cerebral ischemia.

Role of cell adhesion molecules in brain injury after transient middle cerebral artery occlusion in the rat

Activated neutrophils appear to be directly involved in tissue injury after focal cerebral ischemia and reperfusion. Intercellular adhesion molecules-1 (ICAM-1) and CD11/CD18 integrins have been implicated in ischemia-reperfusion induced neutrophil endothelial adhesion and transmigration. We therefore investigated the roles of CD11a/CD18 (LFA-1) and ICAM-1 in cerebral ischemia-reperfusion injury by using monoclonal antibodies, WT1 (anti-CD11a), WT3 (anti-CD18), and 1A29 (anti-ICAM-1). Rats were subjected to 1 h of middle cerebral artery occlusion (MCAO). Individual antibodies were administered at a dose of 5 mg/kg intraperitoneally at 15 min before ischemia and immediately after reperfusion. Rats were killed at 24 h after reperfusion, and brain edema, neutrophil infiltration and infarct size were measured. Sustained enhancement of ICAM-1 expression on capillaries was observed up to 24 h (beginning between 1 and 3 h after reperfusion). While, leukocytes began to infiltrate into the ischemic hemisphere between 6 and 12 h after reperfusion. Treatment with individual antibodies against cell adhesion molecules reduced edema formation and infarct size in addition to neutrophil accumulation 24 h after reperfusion. These results strongly implicate the invasion of neutrophils in the development of post-ischemic brain injury, and suggest that interactions between CD11a/CD18 and ICAM-1 contribute to neutrophil infiltration into the ischemic brain.

Cyclosporin A protects against ischemia-reperfusion injury in the brain

We investigated the protective effect of Cyclosporin A (CsA) against ischemia-reperfusion injury in the brain using a transient focal ischemia model in rats. In CsA-treated rats, ischemic brain edema formation 1 day after reperfusion in the cerebral cortex perfused by the middle cerebral artery (MCA) and infarct size were decreased compared with those in olive oil treated control rats. These results suggest that CsA is beneficial in reducing ischemia-reperfusion injury, possibly by the suppression of immunological reactions.

Transient Increase of Cytokine-Induced Neutrophil Chemoattractant, a Member of the Interleukin-8 Family, in Ischemic Brain Areas After Focal Ischemia in Rats

BACKGROUND AND PURPOSE We have indicated that neutrophils play an important role in cerebral ischemia-reperfusion injury. Neutrophils are also known to adhere to the endothelial wall through adhesion molecules and to infiltrate into the tissue, and this neutrophilic invasion correlates with the concentration gradient of chemotactic factors. The aim of the present study was to evaluate the role of cytokine-induced neutrophil chemoattractant (CINC) on brain damage in rats from transient ischemia. METHODS The brain water content was measured to evaluate postischemic brain injury in rats with 60 minutes of middle cerebral artery occlusion with perfusion. An enzyme-linked immunosorbent assay was used to evaluate the blood and brain concentrations of CINC, and enzymatic and histological techniques were used to measure the neutrophilic infiltration into the brain. RESULTS The increase of water content was first observed at 6 hours after reperfusion, after which this increase was gradual, with brain edema peaking from 24 to 48 hours after reperfusion. Neutrophilic infiltration into the parenchyma and myeloperoxidase activity were first noted 12 hours after reperfusion, after which a marked increase occurred from 24 to 48 hours after reperfusion. In the ischemic brain areas, CINC was first detected at 3 hours after reperfusion. The CINC level peaked at 12 hours after reperfusion (9.15 +/- 0.45 ng/g wet wt, n = 5 and then gradually reduced from 24 to 48 hours after reperfusion (5.35 +/- 0.95 ng/g wet wt, n = 5, and 1.25 +/- 0.10 ng/g wet wt, n = 5, respectively). Interestingly, the serum CINC concentration was transiently elevated from 3 to 6 hours after reperfusion. No CINC production was detected in the brain of rats subjected to 60 minutes of ischemia without reperfusion. CONCLUSIONS A marked increase in CINC concentration was detected in brain and serum during early reperfusion. This suggests that the time course of CINC production precedes brain edema formation and neutrophilic infiltration. It thus appears that CINC may play an important role in neutrophilic infiltration in ischemic lesion and in brain edema formation after ischemia-reperfusion injury.

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

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 (CINC) 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.

Role of platelet-activating factor and thromboxane A2 in radical production during ischemia and reperfusion of the rat brain

Oxygen radicals produced by activated neutrophils have been involved in brain injury during ischemia-reperfusion. Platelet-activating factor (PAF) is a candidate as one of the mediators of neutrophil activation during cerebral ischemia-reperfusion. Recent evidence indicates that PAF-induced neutrophil activation is mediated by thromboxane A2 (TXA2). To study the role of PAF and TXA2 in radical production during cerebral ischemia-reperfusion, we evaluated the effects of a PAF antagonist, Y-24180, and a TXA2 antagonist, S-1452, on radical formation in rats with 1 h middle cerebral artery (MCA) occlusion. In the present study, we employed a new electron spin resonance (ESR) method coupled with brain microdialysis. The method uses the endogenous ascorbyl radical (AR) concentration as a marker of oxygen radicals and requires no spin-trapping agents. In the vehicle controls, extracellular AR from the ischemic brain cortex decreased during MCA occlusion. Following reperfusion, AR significantly increased at 30 mm and 1 h, returned to near the basal levels at 2 h, and increased again at 24 h after reperfusion. In the rats treated with S-1452 or Y-24180, AR decreased during MCA occlusion to the same extent as in the vehicle control. However, pretreatment with Y-24180 or S-1452 significantly attenuated the increase in extracellular AR after reperfusion, while it exerted no effect on the changes in extracellular ascorbate or tissue pO2 throughout the experimental period. In conclusion, PAF and TXA2 might contribute to cerebral ischemia-reperfusion injury by increasing the generation of oxygen radicals.

P/Q-type Ca2+ channel blocker ω-agatoxin IVA protects against brain injury after focal ischemia in rats

Recently, P/Q-type Ca2+ channels have been shown to be involved in neurotransmission in the central nervous system in mammals. We evaluated the effects of the P/Q-type Ca2+ channel blocker omega-agatoxin IVA (omega-Aga-IVA) on brain edema formation and infarct size determined after 24 h of reperfusion following 1 h of middle cerebral artery (MCA) occlusion in rats. Intracerebroventricular (i.c.v.) treatment with omega-Aga-IVA significantly attenuated the postischemic increase of brain water content. omega-Aga-IVA also significantly reduced the size of the infarct area determined by triphenyltetrazolium chloride staining after 24 h of reperfusion. omega-Aga-IVA (30 pmol, i.c.v.), which exhibited a neuroprotective effect, had no significant effect on the magnitude of intra- and postischemic brain temperature when compared with vehicle-treated rats. This indicates that the postischemic neuroprotective effect of omega-Aga-IVA is produced by a direct and not an indirect effect via hypothermia. These results suggest that P/Q-type Ca2+ channels may be involved in the development of focal ischemic brain injury and that blockers of these channels may be therapeutically useful against ischemic injury.

Inflammation of the Brain after Ischemia

Cytokines which promote emigration of leukocytes from the vascular lumen into the injured brain tissue are produced at the site of incipient cerebral infarction. The blood-borne invaders then accelerate the decomposition of brain cells by their toxic by-products, phagocytic action, and by the immune reaction. Recently accumulated data in our laboratories and other research facilities show that depleting the amount of circulating leukocytes or administering anti-inflammatory chemicals such as cytokine blocking agents, anti-adhesion molecule antibodies, and immunosuppressants effectively minimize the size of ischemia induced cerebral infarction. Based on the fact the leukocyte invasion of the affected brain tissue occurs 6 to 24 hours after onset of ischemia, administration of an anti-inflammatory therapy may widen the therapeutic window against stroke.

Effect of a novel thromboxane A2 receptor antagonist, S-1452, on postischemic brain injury in rats.

Background and Purpose Arachidonate metabolites have been implicated in the development of cerebral injury after ischemia. Particular importance has been placed on the balance of thromboxane A2 and prostaglandin I2 because of its regulative activity on platelet functions and arterial tone. The purpose of the present study was to shed light on the role of thromboxane A2 in postischemic brain injury. Methods We evaluated the effects of S-1452, a novel thromboxane A2 receptor antagonist, on brain edema, infarct areas, and survival rate in rats with middle cerebral artery occlusion. A transient middle cerebral artery occlusion model was produced by inserting a piece of silicon-coated nylon thread into the internal carotid artery. Results The ratio of plasma thromboxane B2 to 6-keto-prostaglandin Flα significantly rose at 0 hour (P<.05), 1 hour (P<.01), 3 hours (P<.05), and 12 hours (P<.05) and then nearly returned to the normal level at 24 hours after reperfusion following 1-hour occlusion. Pretreatment with S-1452 (5, 10, or 50 mg/kg PO) significantly attenuated the increase in postischemic water content in the cerebral cortex perfused by the anterior cerebral artery and the cerebral cortex perfused by the middle cerebral artery in a dose-dependent manner but slightly attenuated it in the caudate putamen 24 hours after reperfusion following 1-hour occlusion. Pretreatment with S-1452 (10 mg/kg PO) also significantly decreased the areas of infarction in the front parts of the cerebrum. The survival rate of animals after 2 hours of occlusion tended to be improved by treatment with S-1452 (10 mg kg−1 d−1 PO), although there was no statistical significance. Conclusions Our results suggest that thromboxane A2 is closely related to postischemic brain injury in the early phase of recirculation and that S-1452 may have a protective effect on postischemic brain injury.