Transient Focal Brain Ischemia Research Articles

Protein aggregation after focal brain ischemia and reperfusion.

Two hours of transient focal brain ischemia causes acute neuronal death in the striatal core region and a somewhat more delayed type of neuronal death in neocortex. The objective of the current study was to investigate protein aggregation and neuronal death after focal brain ischemia in rats. Brain ischemia was induced by 2 hours of middle cerebral artery occlusion. Protein aggregation was analyzed by electron microscopy, laser-scanning confocal microscopy, and Western blotting. Two hours of focal brain ischemia induced protein aggregation in ischemic neocortical neurons at 1 hour of reperfusion, and protein aggregation persisted until neuronal death at 24 hours of reperfusion. Protein aggregates were found in the neuronal soma, dendrites, and axons, and they were associated with intracellular membranous structures during the postischemic phase. High-resolution confocal microscopy showed that clumped protein aggregates surrounding nuclei and along dendrites were formed after brain ischemia. On Western blots, ubiquitinated proteins (ubi-proteins) were dramatically increased in neocortical tissues in the postischemic phase. The ubi-proteins were Triton-insoluble, indicating that they might be irreversibly aggregated. The formation of ubi-protein aggregates after ischemia correlated well with the observed decrease in free ubiquitin and neuronal death. The authors concluded that proteins are severely damaged and aggregated in neurons after focal ischemia. The authors propose that protein damage or aggregation may contribute to ischemic neuronal death.

Neuroprotection in transient focal brain ischemia after delayed intravenous administration of brain-derived neurotrophic factor conjugated to a blood-brain barrier drug targeting system.

Neuroprotection with brain-derived neurotrophic factor (BDNF) requires direct injection into the brain owing to poor transport of the neurotrophin through the blood-brain barrier (BBB) in vivo. The present studies investigate whether BDNF alone or conjugated to a BBB drug targeting system is neuroprotective in focal, reversible brain ischemia after delayed intravenous administration at 60 or 120 minutes after middle cerebral arterial occlusion. BDNF was conjugated to the OX26 murine monoclonal antibody to the rat transferrin receptor, which undergoes transport into brain from blood via the BBB transferrin receptor transcytosis system. After a 1-hour occlusion of the middle cerebral artery in nitrous oxide- ventilated animals with normal blood sugar, the brain was reperfused, and either BDNF or the BDNF/OX26 conjugate was administered as a single intravenous injection at a dose of 50 microg per rat. After the intravenous administration of unconjugated BDNF, there was no neuroprotection on the basis of analysis of brain at either 24 hours or 7 days after a 1-hour middle cerebral arterial occlusion. In contrast, there was a 68% and 70% reduction in cortical stroke volume at 24 hours and 7 days, respectively, after intravenous administration of 50 microg per rat of the BDNF conjugate (P<0.01). No effects on subcortical stroke volume were observed. These studies demonstrate marked neuroprotection in focal, transient brain ischemia with a single, delayed intravenous injection of BDNF if the neurotrophin is conjugated to a BBB drug targeting system. The neuroprotection is long lasting and persists for at least 7 days after a 1-hour middle cerebral artery occlusion.

Aggravation of brain injury after transient focal ischemia in p53-deficient mice

The transcriptional factor p53 is a regulatory protein which contributes to the preservation of tissue integrity by promoting either DNA repair or apoptosis. To establish the pathophysiological role of this protein in ischemia, we produced 1 h transient middle cerebral artery (MCA) occlusion in normal and in p53-deficient mice and investigated the resulting tissue damage by multiparametric imaging. Possible genetic influences on the angioarchitecture of the MCA territory and blood flow were examined by intravascular latex infusion and laser-Doppler flowmetry. Wild-type (p53 +/+), heterozygous (p53 +/−) and homozygous (p53 −/−) mice deficient for the p53 gene did not differ in respect to angioarchitecture or the effect of vascular occlusion on blood flow and general physiological parameters. Twenty-four hours after 1 h MCA occlusion, mice revealed a gene dose-dependent decline in the size of metabolic disturbances (ATP depletion and inhibition of protein synthesis) and histological injury (Cresyl Violet staining). DNA fragmentations detected by terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) did not differ in the three groups and were only present in ATP-depleted tissue. Our findings suggest that after transient focal brain ischemia p53 prevents rather than aggravates brain injury, and that this effect is brought about by mechanisms that are unrelated to the pro-apoptotic properties of this gene.

Reduction of ischemic damage by application of insulin-like growth factor-1 in rat brain after transient ischemia.

In order to investigate a possible effect of insulin-like growth factor-1 (IGF-1) on ischemic brain injury, IGF-1 was applied topically on the brain surface of reperfused rat brain after 60 min of transient middle cerebral artery occlusion. In contrast to the cases treated with vehicle, the infarct volume was greatly reduced at 24 h of reperfusion by the treatment with IGF-1. Immunohistochemical analysis in the middle cerebral artery territory showed that Caspase-3 staining was markedly reduced in the cases with IGF-1 treatment, but 72-kDa heat shock protein staining remained almost unchanged. The present results suggest that treatment with IGF-1 exerts a significant effect on ameliorating brain injury after transient focal brain ischemia. Moreover, this effect is greatly associated with the reduction of Caspase-3 staining, but is only minimally associated with a decreasd stress response at the cellular level.

Differential changes of bax, caspase-3 and p21 mRNA expression after transient focal brain ischemia in the rat

Recent studies of transient focal ischemia have focused interest on apoptotic mechanisms of neuronal cell death involving constitutive pro-apoptotic proteins. The finding of specific patterns of novel gene expression might indicate the activation of pro-apoptotic genes in previously ischemic areas. Thus, we investigated gene expression for the pro-apoptotic regulators, Bax and caspase-3, after transient focal brain ischemia, together with the p53-regulated cell cycle inhibitor, p21/WAF1/CIP1. Reversible occlusion of the middle cerebral artery for 2 h was carried out in halothane-anesthetized rats using the poly- l-lysine coated filament method. In situ hybridization was performed at 0, 1, 3, 6 h and 1, 3 and 7 d of recirculation and in sham controls. Radioactive antisense probes served for detection of bax, p21 and caspase-3 mRNAs on brain sections, and quantitative film autoradiography was combined with image-averaging techniques. Bax mRNA tended to decline after focal brain ischemia within 1 d. p21 mRNA was upregulated with a perifocal pattern at 3 h and 1 d after ischemia whereas the ischemic regions themselves failed to show significant upregulation. Caspase-3 mRNA was elevated in the resistant dorsomedial cortex at 1 d. A pro-apoptotic pattern of novel gene expression, involving Bax and caspase-3, was not observed after transient focal brain ischemia. Rather, the perifocal expression of p21 and caspase-3 mRNAs observed at 1 d after ischemia points to reactive changes in resistant brain areas.

Reduction of ischemic brain injury by topical application of insulin-like growth factor-I after transient middle cerebral artery occlusion in rats

In order to examine the effect of insulin-like growth factor-1 (IGF-1) on ischemic brain injury, IGF-1 was applied topically on the surface of reperfused rat brain after 60 min of transient middle cerebral artery occlusion (MCAO). In contrast to the cases treated with vehicle, the infarct area was greatly reduced at 24 h of reperfusion by treatment with IGF-1. Terminal deoxynucleotidyl transferase mediated dUTP-biotin in situ nick labeling (TUNEL) staining and immunoreactivity for glycogen synthase kinase 3β (GSK3β) were also markedly reduced in the brains with IGF-1 treatment. The present results suggest that the treatment with IGF-1 significantly ameliorates brain injury after transient focal brain ischemia associated with the reduction of TUNEL and GSK3β stainings.

Insulin-like growth factor-1 affects expressions of cyclin-dependent kinase 5 and its activator p35 in reperfused rat brain

Insulin-like growth factor-1 (IGF-1) was applied topically on the brain surface of reperfused rat brain after 60 min of transient middle cerebral artery (MCA) occlusion. In contrast to the cases treated with vehicle, the infarct volume was greatly reduced at 24 h of reperfusion by the treatment with IGF-1. Immunohistochemical analysis in the MCA territory showed that the increase of cyclin-dependent kinase 5 (cdk5) was greatly reduced, and that the decrease of the critical regulatory subunit of cdk5, p35, was preserved with treatment of IGF-1. The present results suggest that IGF-1 has a significant effect on ameliorating brain injury after transient focal brain ischemia with affecting the expressions of cdk5 and its activator p35.

Effects of blood sugar level on rat transient focal brain ischemia consecutively observed by diffusion-weighted EPI and (1)H echo planar spectroscopic imaging.

The effects of blood sugar level on transient focal brain ischemia were examined by consecutive diffusion-weighted EPI and (1)H echo planar spectroscopic imaging. A remote-controlled rat intraluminal suture middle cerebral artery occlusion (MCAO) model was prepared. Animals were divided into three experimental groups: control, 1 g/kg, and 2 g/kg glucose groups (n = 6 for each). Saline or glucose was infused intraperitoneally 30 min prior to MCAO. The glucose-loaded groups showed increased lactate accumulation and marked decreases in average diffusion coefficient in the ischemic region during 40-min MCAO. These changes were correlated with blood sugar levels at the onset of MCAO. After reperfusion, all rats in the control and 1 g/kg groups recovered from the ischemic changes, but three rats with marked hyperglycemia in the 2 g/kg group showed irreversible changes. The adverse effects of hyperglycemia on transient focal brain ischemia were clearly demonstrated by sequential 2D images. Magn Reson Med 42:895-902, 1999.

Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rat

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of α-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.

Expression and distribution of redox regulatory protein, thioredoxin during transient focal brain ischemia in the rat

We assayed redox regulatory protein, thioredoxin (TRX) and TRX mRNA in the rat brain after transient and permanent middle cerebral artery (MCA) occlusion. The immunoreactivity for TRX and TRX mRNA disappeared after MCA occlusion in the ischemic core regions. On the other hand, in the perifocal ischemic regions, TRX immunoreactivity and TRX mRNA was enhanced. In addition, in transient MCA occlusion, TRX induction was stronger in the hippocampus and more widespread in the contralateral cortex than in permanent occlusion. Moreover, the induced TRX was translocated into the cellular nucleus after ischemia and ischemia-reperfusion. These results suggest that TRX induction was accompanied with ROI overproduction and may play an important role not only in scavenging ROI but also in signal transduction during ischemia.

Amelioration of brain edema by topical application of glial cell line-derived neurotrophic factor in reperfused rat brain

Glial cell line-derived neurotrophic factor (GDNF) was applied topically on the brain surface of reperfused rat brain after 90 min of transient middle cerebral artery occlusion. In contrast to the cases treated with vehicle, a formation of brain edema was greatly reduced at 2 days by the treatment with GDNF. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) staining was also markedly reduced in the cases with GDNF treatment both at 1 and 2 days of reperfusion. However, amelioration of the induction of immunoreactive 70 kDa heat shock protein was only a minimum by the GDNF treatment. The present results suggest that the treatment with GDNF has a significant effect on ameliorating brain edema formation after transient focal brain ischemia, and the effect is greatly associated with the reduction of TUNEL staining, but minimally with that of stress response of cells.