Yasundo Yamasaki

Interleukin-1 as a Pathogenetic Mediator of Ischemic Brain Damage in Rats

BACKGROUND AND PURPOSE It has been suggested that interleukin-1 (IL-1) is a potent inflammatory mediator and that it is synthesized and secreted into the brain parenchyma. The aim of the present study is to evaluate the contribution of IL-1 to brain edema formation after focal brain ischemia. METHODS The brain water content was measured to evaluate postischemic brain injury in rats after 60 minutes of middle cerebral artery occlusion and reperfusion. The effects of exogenous application of recombinant human interleukin-1 beta (rhIL-1 beta), anti-interleukin-1 beta neutralizing antibodies (anti-IL-1 beta), and the IL-1 blocker zinc protoporphyrin (ZnPP) on brain water content were observed, and histological technique was used to measure the infarction size and number of inflammatory cells infiltrated into the brain. RESULTS Transient ischemia induced marked increase of brain water content, necrosis, and neutrophilic infiltration in the cortex perfused by the middle cerebral artery and the dorsal and ventral areas of the caudate putamen. Injection of rhIL-1 beta into the left lateral ventricle immediately after reperfusion markedly enhanced ischemic brain edema formation in these three areas in a dose-dependent manner (88.4 +/- 0.7% and 86.6 +/- 0.4% in the dorsal and ventral parts of the caudate putamen, respectively, in rats treated with 10 ng rhIL-1 beta; P < .01). rhIL-1 beta also increased the size of the brain infarction, and it tended to increase the number of infiltrating neutrophils in ischemic areas and the number of neutrophils adherent to the endothelium. In contrast, administration of anti-IL-1 beta and ZnPP into the left cerebral ventricle attenuated the postischemic increase of brain water content and decreased the size of brain infarction (83.5 +/- 2.0% and 79.9 +/- 2.0% in the dorsal and ventral parts of the caudate putamen, respectively, in rats treated with 10 micrograms anti-IL-1 beta; P < .01). The number of neutrophils that infiltrated into ischemic areas also tended to decrease with anti-IL-1 beta or ZnPP treatment. CONCLUSIONS Application of rhIL-1 beta augmented the increase of brain water content, and application of anti-IL-1 beta depressed the increase of water content. These results tended to correlate with the neutrophilic infiltration into the parenchyma. It thus appears that IL-1 beta may play an important role in ischemic brain damage after reperfusion.

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.

Neutrophil as a mediator of ischemic edema formation in the brain

We investigated the contribution of neutrophils to brain edema formation using a transient focal ischemia model in rats. Rats were given anti-neutrophil monoclonal antibody (RP3) intraperitoneally to deplete circulating neutrophils. In RP3-treated rats, ischemic brain edema formation 1 day after reperfusion was significantly decreased compared to that of saline-treated control rats. We speculate that chemical mediators released by infiltrating neutrophils alter vascular permeability and play an important role in post-ischemic brain edema formation.

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.

Possible involvement of interleukin-1 in ischemic brain edema formation

To determine the contribution of interleukin 1 (IL-1) on ischemic brain edema formation, the effect of recombinant human interleukin 1 beta (rhIL-1 beta), or zinc protoporphyrin (ZnPP) as an IL-1 blocker, on brain edema was studied in rats. The animals were subjected to 60 min of ischemia in a middle cerebral artery occlusion model. Immediately after reperfusion, rhIL-1 beta at a dose of 10 ng/2 microliters, or ZnPP at doses of 1 and 10 micrograms/2 microliters were topically applied into lateral cerebroventricle. In rhIL-1 beta-treated rats, ischemic brain edema formation was significantly increased in the dorsal and ventral areas of the caudate putamen 24 h after reperfusion, compared to that of vehicle-treated control rats. Furthermore, in ZnPP-treated rats, brain edema was decreased in both caudate-putamen areas. This suggests that IL-1 plays an important role in pathogenesis for post-ischemic brain edema.

The possible involvement of tetrodotoxin-sensitive ion channels in ischemic neuronal damage in the rat hippocampus.

To determine the role of tetrodotoxin-sensitive ion channels in post-ischemic selective neuronal death, the effect of tetrodotoxin on ischemia-induced brain cell injury was studied in rats. The animals were subjected to 20 min of cerebral ischemia in a four vessels occlusion model. Thirty min before ischemia, tetrodotoxin at a dose of 10(-7) or 10(-6) M was topically applied into the hippocampal CA1 subfield. Morphological changes in the CA1 subfield were evaluated 7 days after ischemia and compared with those of a vehicle-injected group. The average cell density of CA1 pyramidal neurons ipsilateral to the injection (cells/mm, mean +/- S.E.M.) was 27 +/- 7 (n = 6) in the vehicle-treated group, and 56 +/- 13 (n = 6) and 83 +/- 17 (n = 6) in the group treated with tetrodotoxin at doses of 10(-7) and 10(-6) M, respectively. Tetrodotoxin mitigated the ischemic hippocampal neuronal damage in a limited but dose-dependent manner. This suggests that activation of tetrodotoxin-sensitive ion channels might contribute to the process of the ischemic neuronal damage.

Weekly Dosing With the Platelet-Derived Growth Factor Receptor Tyrosine Kinase Inhibitor SU9518 Significantly Inhibits Arterial Stenosis

Abstract— The platelet-derived growth factor (PDGF) ligands and their receptors have been implicated as critical regulators of the formation of arterial lesions after tissue injury. SU9518 (3[5-{5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl}-2,4-dimethyl-1 H-pyrrol-3-yl]propionic acid) is a novel synthetic indolinone that potently and selectively inhibits the cellular PDGF receptor kinase and PDGF receptor-induced cell proliferation. Inhibition of PDGF receptor phosphorylation in cell-based assays occurs within 5 minutes after drug exposure and persists for >6 hours after drug removal. The pharmacokinetics indicate plasma levels that exceeded the effective concentration required to inhibit the PDGF receptor in cells for up to 8 hours or 7 days after a single oral or subcutaneous administration, respectively. In the rat balloon arterial injury-induced stenosis model, once-daily oral or once-weekly subcutaneous administration of SU9518 reduced intimal thickening of the carotid artery (ratio of neointimal to medial area, 1.94±0.38 versus 1.03±0.29 [P <0.01] 2.21±0.32 versus 1.34±0.45 [P <0.01], respectively). These studies provide the rationale to evaluate PDGF receptor tyrosine kinase inhibitors, including inhibitors related to the indolinone, SU9518, for the treatment of arterial restenosis.

Temporal Profiles of Nerve Growth Factor β‐Subunit Level in Rat Brain Regions After Transient Ischemia

To determine the role of nerve growth factor (NGF) in ischemic brain damage, we measured the temporal and regional changes in the level of NGF in the hippo‐campal subfields, the cerebral cortex, the striatum, and the septum at 1, 2, 7, and 30 days after transient forebrain ischemia using a highly sensitive sandwich‐type enzyme im‐munoassay system for the β‐subunit of mouse 7S NGF (β‐NGF). We also analyzed glial fibrillary acidic protein immunoreactivity in the hippocampus to ascertain the contribution of reactive astrocytes to NGF production after an ischemic insult. In the CA1 subfield of the hippocampus, the level of β‐NGF decreased slightly 2 days after ischemia (not significant), at which time CA1 pyramidal cell loss began to occur, and increased by 40% 30 days after ischemia (p < 0.05). A marked increase in glial fibrillary acidic pro‐tein‐positive astrocytes in the CA1 subfield 2–30 days after ischemia suggests that the reactive astrocytes participated in a gradual increase in the level of β‐NGF after recirculation. The level of β‐NGF in the dentate gyrus decreased transiently 2 days (p < 0.05) and 7 days (p < 0.01) after ischemia, followed by recovery to the level of control animals 30 days after ischemia. The level of β‐NGF in the septum gradually decreased 7 days (−27%, p < 0.05) and 30 days (−43%, p < 0.01) after ischemia. The levels of β‐NGF in the cerebral cortex and striatum remained unaltered throughout the observation period. These results suggest that the alteration of NGF content occurs not only in the CA1 subfield, where pyramidal cells are depleted, but also in areas with minimal histological damage. Septal decrease in NGF may reflect the reduction of retrograde NGF transport from hippocampus to septal cholinergic neurons.

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.

Decreased expression of neurotrophin-3 mRNA in the rat hippocampus following transient forebrain ischemia

The expression profile of neurotrophin-3 (NT-3) mRNA in the rat hippocampus after forebrain ischemia was investigated by Northern blot and S1 nuclease protection analyses. The NT-3 transcripts in the hippocampus immediately decreased after ischemic insult, became undetectable within 3 h and remained at undetectable levels for at least 7 days. In contrast, the expression of c-fos and c-jun mRNA transiently increased both in the cerebral cortex and in the hippocampus. These results suggest that brain ischemia triggers dynamic changes in gene expression including a neurotrophic factor, which may cause functional and/or morphological changes of the neuronal network.