Takeshi Hayashi

Reduction of Ischemic Damage by Application of Vascular Endothelial Growth Factor in Rat Brain After Transient Ischemia

Vascular endothelial growth factor (VEGF) is a secreted polypeptide and plays a pivotal role in angiogenesis in vivo. However, it also increases vascular permeability, and might exacerbate ischemic brain edema. The effect of this factor on the brain after transient ischemia was investigated in terms of infarct volume and edema formation, as well as cellular injury. After 90 minutes of transient middle cerebral artery occlusion, VEGF (1.0 ng/μL, 9 μL) was topically applied on the surface of the reperfused rat brain. A significant reduction of infarct volume was found in animals with VEGF application (P < 0.001) at 24 hours of reperfusion as compared with cases with vehicle treatment. Brain edema was significantly reduced in VEGF-treated animals (P = 0.01), and furthermore, extravasation of Evans blue was also decreased in those animals (P < 0.01). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling and immunohistochemical analysis for 70-kDa heat shock protein showed an amelioration of the stainings at 24 and 48 hours after reperfusion with VEGF treatment, which indicated reduction of neuronal damage. These results indicate that treatment with topical VEGF application significantly reduces ischemic brain damage, such as infarct volume, edema formation, and extravasation of Evans blue, and that the reductions were associated with that of neuronal injury.

Rapid Induction of Vascular Endothelial Growth Factor Gene Expression After Transient Middle Cerebral Artery Occlusion in Rats

BACKGROUND AND PURPOSEnVascular endothelial growth factor (VEGF) is a mitogen for endothelial cells and also has the potential to increase vascular permeability. Therefore, it may contribute to the recovery of brain cells from ischemic insult through potentiating neovascularization or may exacerbate brain damage by forming brain edema. However, the exact role of this protein in cerebral ischemia is not fully understood. We investigated temporal, spatial, and cellular profiles of the induction of VEGF gene expression after transient focal cerebral ischemia at both mRNA and protein levels.nnnMETHODSnWe used a transient middle cerebral artery (MCA) occlusion model. Northern blot analysis was performed to assess the chronological pattern of induction and the impact of length of ischemia on mRNA expression. Western blot analysis was performed to ensure the selective detection of immunoreactive VEGF with an antibody. Temporal, spatial, and cellular changes of immunohistochemical VEGF expression were compared with different periods of reperfusion from 1 hour to 7 days after transient MCA occlusion.nnnRESULTSn(1) Northern blot analysis revealed no detectable VEGF mRNA in the control brains. The mRNA became evident at 1 hour after reperfusion, peaked at 3 hours, and then decreased. The length of ischemia from 1 to 3 hours made no differences in the degree and temporal profile of the subsequent induction of VEGF mRNA. (2) Western blot analysis showed no band in the control brain, but two bands with molecular weights of 38 and 45 kD, corresponding to VEGF121 and VEGF165, were induced at 1 hour of reperfusion, peaked at 3 hours of reperfusion, and then decayed. (3) Neurons in the cerebral cortex of the MCA territory expressed VEGF at 1 hour after reperfusion with a peak at 3 hours and then diminished by 1 day. Pial cells of the MCA territory also expressed immunoreactive VEGF from 1 hour of reperfusion that was sustained until 3 to 7 days after reperfusion.nnnCONCLUSIONSnRapid induction of VEGF gene expression after transient MCA occlusion was demonstrated at both mRNA and protein levels. Cortical neurons and pial cells were the source of VEGF production in this model, but the temporal profiles of the induction between these cells were different. The early but dissociative induction of VEGF between neuronal and pial cells suggests different roles of the protein in their cells after transient MCA occlusion.

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.

Expression of the glial cell line-derived neurotrophic factor gene in rat brain after transient MCA occlusion

Change of the glial cell line-derived neurotrophic factor (GDNF) gene expression in rat brain was examined after transient middle cerebral artery (MCA) occlusion of adult rats. Northern blot analysis showed that the mRNA began to be induced in the occluded MCA from 1 h of reperfusion with a peak at 3 h, and almost diminished by 1 day of reperfusion. Immunohistochemical analysis with brain sections showed an expression of GDNF-like immunoreactivity in neurons of the cerebral cortex and caudate after 90 min of ischemia in a similar way to the mRNA, but the staining was more disseminated and stronger in the cerebral cortex than the caudate. No glial cell was stained in the brain sections. The present results indicate that the GDNF gene was expressed in an early stage of reperfusion in neuronal cells of the MCA territory, but that the staining property was different between in the cerebral cortex and caudate.

Delayed and selective motor neuron death after transient spinal cord ischemia: A role of apoptosis?

OBJECTIVEnThe mechanism of spinal cord injury has been thought to be related to tissue ischemia, and spinal motor neuron cells are suggested to be vulnerable to ischemia. We hypothesized that delayed and selective motor neuron death is apoptosis.nnnMETHODSnThirty-seven Japanese domesticated white rabbits weighing 2 to 3 kg were used in this study and were divided into two subgroups: a 15-minute ischemia group and a sham control group. Animals were allowed to recover at ambient temperature and were killed at 8 hours, and 1, 2, 4, and 7 days after reperfusion (n = 3 at each time point). By means of this model, cell damage was histologically analyzed. Detection of ladders of oligonucleosomal DNA fragment was investigated with gel electrophoresis up to 7 days of the reperfusion. Immunocytochemistry, in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining was also performed.nnnRESULTSnAfter 15 minutes of ischemia, most of the motor neurons showed selective cell death at 7 days of reperfusion. Typical ladders of oligonucleosomal DNA fragments were detected at 2 days of reperfusion. Immunocytochemistry showed in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end staining was detected at 2 days of reperfusion selectively in the nuclei of motor neurons.nnnCONCLUSIONnThese results suggest that delayed and selective death of the motor neuron cells after transient ischemia may not be necrotic but rather predominantly apoptotic.

Anti-P-selectin antibody attenuates rat brain ischemic injury

We examined a protective effect of anti-P-selectin monoclonal antibody against rat ischemic brain injury with 24 h of middle cerebral artery occlusion (MCAO). Anti-rat P-selectin monoclonal antibody, was intravenously injected at a dose of 1 mg/kg at 5 min before MCAO. Control animals received the same volume of vehicle. MCAO was accomplished by an insertion of nylon thread with silicone coating for 24 h. Application of anti-P-selectin antibody significantly reduced infarct size and brain water content at 24 h of MCAO. Although leukocyte infiltration was not normally detected, it became remarkably evident at 1 day of MCAO. However, treatment with ARP 2-4 significantly reduced the number of leukocytes. These results demonstrated that administration of monoclonal antibody against P-selectin attenuated infarct size and brain edema, which was associated with reduction of leukocyte infiltration.

Delayed selective motor neuron death and fas antigen induction after spinal cord ischemia in rabbits.

The mechanism of spinal cord injury has been thought to be related with tissue ischemia, and spinal motor neuron cells are suggested to be vulnerable to ischemia. To evaluate the mechanism of such vulnerability of motor neurons, we attempted to make a reproducible model for spinal cord ischemia. Using this model, cell damage was histologically analyzed. Detection of ladders of oligonucleosomal DNA fragment was investigated with gel electrophoresis up to 7 days of the reperfusion. Time course expression of Fas antigen, identified as a apoptosis-regulating molecules, was also assessed in rabbit spinal cord following transient ischemia. Spinal cord sections from animals sacrificed at 8 h, 1 day, 2 days, and 7 days following 15-min ischemia were immunohistochemically evaluated using monoclonal antibodies for Fas antigen. Following 15-min ischemia, the majority of motor neuron showed selective cell death at 7 days of reperfusion. Typical ladders of oligonucleosomal DNA fragments were detected at 2 days of reperfusion. Immunoreactivity of Fas antigen were induced at 8 h to 1 day of reperfusion selectively in motor neuron cells. The expression of Fas antigen may be related to the activation of apoptosis signal in motor neuron cells after spinal cord ischemia in rabbits.

Cyclin D1 and Cdk4 protein induction in motor neurons after transient spinal cord ischemia in rabbits.

BACKGROUND AND PURPOSEnThe mechanism of spinal cord injury has been thought to be related to the vulnerability of spinal motor neuron cells against ischemia. However, the mechanisms of such vulnerability are not fully understood. We hypothesized that spinal motor neurons might be lost by programmed cell death and investigated a possible mechanism of neuronal death by detection of double-strand breaks in genomic DNA and immunohistochemical analysis for cyclin D1 and cyclin-dependent kinases (Cdk) 4.nnnMETHODSnWe used a rabbit spinal cord ischemia model with a balloon catheter. Spinal cord was removed at 8 hours and 1, 2, and 7 days after 15 minutes of transient ischemia, and histological changes were studied with hematoxylin-eosin staining. In situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL), DNA fragment with gel electrophoresis, Western blot analysis for cyclin D1 and Cdk4, and temporal profiles of cyclin D1 and Cdk4 immunoreactivity were investigated.nnnRESULTSnMost motor neurons were preserved until 2 days but were selectively lost at 7 days of reperfusion. Immunocytochemistry showed positive TUNEL selectively at 2 days of reperfusion in spinal motor neuron nuclei. Typical ladders of oligonucleosomal DNA fragments were detected at 2 days of reperfusion. Immunoreactivity of cyclin D1 and Cdk4 proteins was induced selectively at 8 hours in motor neuron nuclei, which eventually died.nnnCONCLUSIONSnThese results indicate that induction of cyclin D1 and Cdk4 may be implicated in programmed cell death change after transient spinal cord ischemia in rabbits.

In vivo adenovirus-mediated gene transfer and the expression in ischemic and reperfused rat brain

In an attempt to study whether ischemic brain could express a foreign gene in vivo, a replication-defective adenoviral vector containing the Escherichia coli lacZ gene was directly injected into the ischemic or reperfused cerebral cortex of rat, and temporal and spatial profiles of the exogenous gene expression were compared with that of the control brain. Right middle cerebral artery (MCA) of rat was continuously occluded by an insertion of nylon thread for 2 days, or only transiently occluded for 90 min and then the blood flow was restored for 21 days. The adenoviral vector was administered just after the MCA occlusion or reperfusion in the case of continuous ischemia and reperfusion, respectively. Adenoviral vector was transferred into the continuous ischemic brain, and the lacZ gene was expressed until 2 days of the occlusion in the cerebral cortex of the occluded MCA territory with the number of expressing cells smaller and the staining just weaker than that of the control brain. In contrast, expression of the lacZ gene was not or only minimally observed in the reperfused brain until 2 days. However, the expression dramatically exploded at 7 days of reperfusion at a level similar to that of the control, and the expression diminished by 21 days. A few neurons in the ipsilateral thalamus, hypothalamus, and basal ganglia, and in the contralateral cerebral cortex expressed the lacZ gene at 7 days after reperfusion, a phenomenon similar to the case of the control. The majority of brain cells that expressed the lacZ gene were neurons, and a part (5-10%) were astroglial cells. Traumatic injury and immunological response in the brain were minimal both in the cases of control and ischemia/reperfusion. The present study shows an effective gene transfer and the expression in neural cells of ischemic and reperfused brains in vivo, and suggests a great potential of the gene therapy for ischemic stroke patients in the future.

Inductions of hepatocyte growth factor and its activator in rat brain with permanent middle cerebral artery occlusion

Hepatocyte growth factor (HGF) is a potent pleiotrophic peptide which has a trophic role for neuronal cells. As it exerts its effect only after a conversion to its heterodimeric active form, the activation step, which is catalyzed by an enzyme serine protease named HGF activator (HGFA), is of great importance. HGF activated by HGFA may act as a protecting agent in injured brain. In the present study, we investigated expression of immunoreactive HGF and HGFA in rat brain after permanent middle cerebral artery (MCA) occlusion. By immunohistochemical analysis, HGF and HGFA were normally expressed only in ependymal cells and choroid plexus. At 1 h after MCA occlusion, neurons in the ischemic penumbra region of the cerebral cortex slightly expressed immunoreactive HGFA. HGF was not induced at that time. At 3 h of ischemia, however, immunoreactive HGF as well as HGFA became detectable in neurons of the ischemic cerebral cortex and caudate. Immunoreactivity for HGF continued to increase until 24 h, while that for HGFA remained almost constant from 3 to 24 h. No glial or vascular endothelial cells expressed HGF nor HGFA. By Western blot analysis for HGF, a single band of molecular weight (MW) 34 kDa became apparent at 24 h, corresponding to the light chain of the active form HGF. The present study suggests that HGF and HGFA were induced in neurons under permanent ischemia with slightly different temporal profiles. Through activation by HGFA, the active form of HGF could serve as a neurotrophic factor in ischemic brain.