Shinsaku Nishio

The differences between high and low-dose administration of VEGF to dopaminergic neurons of in vitro and in vivo Parkinson's disease model

Vascular endothelial growth factor (VEGF) has previously been shown to display neuroprotective effects on dopaminergic (DA) neurons. In this study, we investigated whether the effects of VEGF were dose-dependent or not. First, VEGF was shown to be neuroprotective on 6-hydroxydopamine (6-OHDA)-treated murine DA neurons in vitro, although the 1 ng/ml of VEGF displayed more neuroprotective effects than 100 ng/ml. Furthermore, using 2 sizes of capsules (small/large) with different secreting quantities, 6-OHDA-treated rats receiving the small capsule filled with VEGF-secreting cells (BHK-VEGF) into the striatum showed a significant decrease in amphetamine-induced rotational behavior in number and a significant preservation of TH-positive fibers compared to those receiving the large BHK-VEGF capsule as well as those receiving BHK-Control capsule. Rats receiving the large BHK-VEGF capsule showed much more glial proliferation, angiogenesis, and brain edema around the capsule than those with the small one. High-dose administration of VEGF might cause poor circulation related to brain edema, although low-dose administration of VEGF displays neuroprotective effects on DA neurons. Our results demonstrate the importance of administration dose of VEGF, suggesting that low-dose administration of VEGF might be desirable for Parkinsons disease.

Hypothermia-Induced Ischemic Tolerance

ABSTRACT: Delayed resistance to ischemic injury can be induced by a variety of conditioning stimuli. This phenomenon, known as delayed ischemic tolerance, is initiated over several hours or a day, and can persist for up to a week or more. The present paper describes recent experiments in which transient hypothermia was used as a conditioning stimulus to induce ischemic tolerance. A brief period of hypothermia administered 6 to 48 hours prior to focal ischemia reduces subsequent cerebral infarction. Hypothermia‐induced ischemic tolerance is reversed by 7 days postconditioning, and is blocked by the protein synthesis inhibitor anisomycin. Electrophysiological studies utilizing in vitro brain slices demonstrate that hypoxic damage to synaptic responses is reduced in slices prepared from hypothermia‐preconditioned animals. Taken together, these findings indicate that transient hypothermia induces tolerance in the brain parenchyma, and that increased expression of one or more gene products contributes to this phenomenon. Inasmuch as hypothermia is already an approved clinical procedure for intraischemic and postischemic therapy, it is possible that hypothermia could provide a clinically useful conditioning stimulus for limiting injury elicited by anticipated periods of ischemia.

Inhibition of excitatory neuronal cell death by cell-permeable calcineurin autoinhibitory peptide

In glutamate‐mediated excitatory neuronal cell death, immunosuppressants (FK506, Cys‐A) are powerful agents that protect neurons from apoptosis. Immunosuppressants inhibit two types of enzyme, calcium/calmodulin‐dependent protein phosphatase (calcineurin: CaN), and peptidyl‐prolyl cis‐trans‐isomerase (PPIase) activity such as the FKBP family. In this study, we used a protein transduction approach to determine the functional role of CaN and to produce a potential therapeutic agent for glutamate‐mediated neuronal cell death. We created a novel cell‐permeable CaN autoinhibitory peptide using the 11 arginine protein transduction domain. This peptide was highly efficient at transducing into primary culture neurons, potently inhibited CaN phosphatase activities, and inhibited glutamate‐mediated neuronal cell death. These results showed that CaN plays an important role in excitatory neuronal cell death and cell‐permeable CaN autoinhibitory peptide could be a new drug to protect neurons from excitatory neuronal death.

Detection of Lipid Peroxidation and Hydroxyl Radicals in Brain Contusion of Rats

To examine the relationship between the free radicals and brain tissue damage, we investigated the intensity of brain hydroxyl (OH) radical generation and lipid peroxidation in the rat contusion injury model. A unilateral contusion was induced by a weight-drop method. All rats were decapitated six hours after the injury, and brain samples were taken from three portions (core, peripheral, and distal) to examine the specific gravity as an indicator of brain edema, generation of OH using an electron paramagnetic resonance spectrometer (EPR), and malondialdehyde (MDA) and 4-hydroxyalkenals production. Analysis of the specific gravity revealed cerebral edema on the ipsilateral side in the injured group. The signal intensity of EPR in the core and peripheral portions in the contusion group was significantly higher than that in the distal portion of the contusion group and that of all portions in the control animals. No significant difference was observed between the core and peripheral portions of the contusion group. The MDA and 4-hydroxyalkenals production was significantly higher in the core and peripheral portions than in the distal portion of the contusion group and that of all portions of the control group. The degree of posttraumatic brain edema was closely correlated with the increase of DMPO-OH adduct, MDA, and 4-hydroxyalkenals. These results support the current concept that free radical production following traumatic brain injury may induce lipid peroxidation and may be the direct cause of edema formation.

Hypothermic preconditioning induces rapid tolerance to focal ischemic injury in the rat.

Stressful, preconditioning stimuli can elicit rapid and delayed forms of tolerance to ischemic injury. The identification and characterization of preconditioning stimuli that are effective, but relatively benign, could enhance the clinical applicability of induced tolerance. This study examines the efficacy of brief hypothermia as a preconditioning stimulus for inducing rapid tolerance. Rats were administered hypothermic preconditioning or sham preconditioning and after an interval of 20-120 min were subjected to transient focal ischemia using a three-vessel occlusion model. The volume of cerebral infarction was measured 24 h or 7 days after ischemia. In other experiments, the depth or duration of the hypothermic stimulus was manipulated, or a protein synthesis inhibitor (anisomycin) was administered. Twenty minutes of hypothermia delivered 20 or 60 (but not 120) min prior to ischemia significantly reduces cerebral infarction. The magnitude of protection is enhanced with deeper levels of hypothermia, but is not affected by increasing the duration of the hypothermic stimulus. Treatment with a protein synthesis inhibitor does not block the induction of rapid tolerance. Hypothermic preconditioning elicits a rapid form of tolerance to focal ischemic injury. Unlike delayed tolerance induced by hypothermia, rapid tolerance is not dependent on either de novo protein synthesis or the duration of the preconditioning stimulus. These findings suggest that the mechanisms underlying rapid and delayed tolerance induced by hypothermia differ fundamentally. Brief hypothermia could provide a rapid means of inducing transient tissue protection in the context of predictable ischemic events.

Induction of Cu,Zn-superoxide dismutase after cortical contusion injury during hypothermia

To determine the effect of hypothermia on superoxide injury after cerebral contusion, the induction of Cu,Zn-superoxide dismutase was examined 6 h after contusion in rats using Northern blotting. Cu,Zn-superoxide dismutase gene expression increased at the periphery of the contusion, which may indicate the severity of the superoxide stimulus. This increase was preserved after contusion under hypothermia, which may show that superoxide injury is still severe although brain edema is decreased.

Effects of lecithinized SOD on sequential change in SOD activity after cerebral contusion in rats.

To analyze the effect of lecithinized superoxide dismutase (SOD) on superoxide accumulation after traumatic brain injury (TBI) in rats, we studied the SOD activity by NBT-reducing method and the expression of Cu,Zn-SOD mRNA by Northern blot analysis. As determined by the specific gravity method, the administration of lecithinized SOD decreased brain edema in the periphery of the lesion at 6 hr after contusion. SOD activity, without lecithinized SOD administration, increased at the peripheral portion at 30 min after contusion, but decreased to normal level at 6 hr after TBI. By administration of lecithinized SOD, the increase of SOD activity was preserved until 6 hr after TBI. The expression of Cu,Zn-SOD mRNA increased in the core lesion, peripheral portion, and contralateral hemisphere until 6 hr after TBI, then was suppressed in all three areas by lecithinized SOD. These results support the hypothesis that superoxide anions may play an important role in the development of brain edema after TBI, and that leciyhinized SOD appears to prevent brain edema through a protective effect against superoxide anions.

Effects of hypothermia and rewarming on evoked potentials during transient focal cerebral ischemia in cats

Abstract We examined the effects of mild to moderate hypothermia and the influence of rewarming on electrophysiological function using somatosensory evoked potentials (SEPs) in transient focal ischemia in the brain. Nineteen cats underwent 60 min of left middle cerebral artery occlusion under normothermic (36 ° -37 ° C, n = 6) or hypothermic (30 ° -31 ° C, n = 13) conditions followed by 300 min of reperfusion with slow (120 min, n = 6) or rapid (30 min, n = 7) rewarming. Whole-body hypothermia was induced during ischemia and the first 180 min of reperfusion. SEPs and regional cerebral blood flow were measured before and during ischemia and during reperfusion. The specific gravity of gray and white matter was examined as the indicator of edema. During rewarming, SEP amplitudes recovered gradually. After rewarming, SEPs in the normothermic and rapid rewarming groups remained depressed (20%-40% of pre-occlusion values); however, recovery of SEPs was significantly enhanced in the slow rewarming group (p < 0.05). Hypothermia followed by slow rewarming reduced edema in gray and white matter. Rapid rewarming did not reduce edema in the white matter. The recovery of SEPs correlated with the extent of brain edema in transient focal ischemia. Rapid rewarming reduced the protective effect of hypothermia. [Neurol Res 2002; 24: 621-626]

Complications of embolization for cerebral arteriovenous malformations.

Embolization is recognized as an important adjunct in the treatment of cerebral arteriovenous malformations (AVMs). We reviewed our results of embolizations for AVMs and discussed procedure-related complications. Eleven complications were recorded in 68 consecutive patients (16%). Of these, four were technical problems including a glued catheter, inability to withdraw the catheter, vessel perforation by the microcatheter, and coil migration. Other complications included three cases of ischemic symptoms due to retrograde thrombosis, two cases of asymptomatic cerebral infarction, one case of asymptomatic small haemorrhage due to venous occlusion, and one case of post-embolization haemorrhage of unknown etiology. Our morbidity rate was 7%, mortality rate was 0%, and asymptomatic complication rate was 9%, retorospectively. Further improvements to endovascular techniques and devices are required.

Effects of lecithinized SOD on contusion injury in rats.

To analyze the effect of lecithinized superoxide dismutase (SOD) on superoxide accumulation after traumatic injury, the expression of Cu,Zn-SOD mRNA was examined after contusion in rat using Northern blotting. As determined by specific gravity, lecithinized SOD decreased brain edema. The expression of Cu,Zn-SOD mRNA increased at the core, peripheral and contralateral hemisphere of injury. These increases were then suppressed by lecithinized SOD. Our results support the hypothesis that superoxide may play an important role in edema formation after contusion, and that lecithinized SOD appears to prevent brain edema through a protective effect against superoxide injury.