Current Neurovascular Research - Volume 6, Issue 4, 2009

Oxidative stress is deeply involved in various brain diseases, including neurodegenerative diseases, stroke, and ischemia/reperfusion injury. Mitochondria are thought to be the target and source of oxidative stress. We investigated the role of mitochondria in oxidative stress-induced necrotic neuronal cell death in a neuroblastoma cell line and a mouse model of middle cerebral artery occlusion. The exogenous administration of hydrogen peroxide was used to study the role of oxidative stress on neuronal cell survival and mitochondrial function in vitro. Hydrogen peroxide induced nonapoptotic neuronal cell death in a c-Jun N-terminal kinase- and poly(ADP-ribosyl) polymerase-dependent manner. Unexpectedly, hydrogen peroxide treatment induced transient hyperpolarization of the mitochondrial membrane potential and a subsequent delayed burst of endogenous reactive oxygen species (ROS). The inhibition of mitochondrial hyperpolarization by diphenylene iodonium or rotenone, potent inhibitors of mitochondrial respiratory chain complex I, resulted in reduced ROS production and subsequent neuronal cell death in vitro and in vivo. The inhibition of mitochondrial hyperpolarization can protect neuronal cells from oxidative stress-induced necrotic cell death, suggesting a novel method of therapeutic intervention in oxidative stress-induced neurological disease.

Microglia of the central nervous system have a dual role in the ability to influence the survival of neighboring cells. During inflammatory cell activation, microglia can lead to the disposal of toxic cellular products and permit tissue regeneration, but microglia also may lead to cellular destruction with phagocytic removal. For these reasons, it is essential to elucidate not only the underlying pathways that control microglial activation and proliferation, but also the factors that determine microglial survival. In this regard, we investigated in the EOC 2 microglial cell line with an oxygen-glucose deprivation (OGD) injury model of oxidative stress the role of the “O” class forkhead transcription factor FoxO3a that in some scenarios is closely linked to immune system function. We demonstrate that FoxO3a is a necessary element in the control of early and late apoptotic injury programs that involve membrane phosphatidylserine externalization and nuclear DNA degradation, since transient knockdown of FoxO3a in microglia preserves cellular survival 24 hours following OGD exposure. However, prior to the onset of apoptotic injury, FoxO3a facilitates the activation and proliferation of microglia as early as 3 hours following OGD exposure that occurs in conjunction with the trafficking of the unphosphorylated and active post-translational form of FoxO3a from the cytoplasm to the cell nucleus. FoxO3a also can modulate apoptotic mitochondrial signal transduction pathways in microglia, since transient knockdown of FoxO3a prevents mitochondrial membrane depolarization as well as the release of cytochrome c during OGD. Control of this apoptotic cascade also extends to progressive caspase activation as early as 1 hour following OGD exposure. The presence of FoxO3a is necessary for the expression of cleaved (active) caspase 3, 8, and 9, since loss of FoxO3a abrogates the induction of caspase activity. Interestingly, elimination of FoxO3a reduced caspase 9 activity to a lesser extent than that noted with caspase 3 and 8 activities, suggesting that FoxO3a in relation to caspase 9 may be more reliant upon other signal transduction pathways potentially independent from caspase 3 and 8.

In the variant form of Creutzfeldt-Jakob disease (vCJD), ‘florid’ deposits of the protease resistant form of prion protein (PrPsc) were aggregated around the cerebral blood vessels suggesting the possibility that prions may spread into the brain via the cerebral micro circulation. The objective of the present study was to determine whether the pathology was spatially related to blood vessels in cases of sporadic CJD (sCJD), a disease without an iatrogenic etiology, and therefore, less likely to be caused by hematogenous spread. Hence, the spatial correlations between the vacuolation (‘spongiform change’), PrPsc deposits, and the blood vessels were studied in immunolabeled sections of the cerebral cortex and cerebellum in eleven cases of the common M/M1 subtype of sCJD. Both the vacuolation and the PrPsc deposits were spatially correlated with the blood vessels; the PrPsc deposits being more focally distributed around the vessels than the vacuoles. The frequency of positive spatial correlations was similar in the different gyri of the cerebral cortex, in the upper and lower cortical laminae, and in the molecular layer of the cerebellum. It is hypothesised that the spatial correlation is attributable to factors associated with the blood vessels which promote the aggregation of PrPsc to form deposits rather than representing the hematogenous spread of the disease. The aggregated form of PrPsc then enhances cell death and may encourages the development of vacuolation in the vicinity of the blood vessels.

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is a proinflammatory cytokine with neuroprotective and angiogenic properties demonstrated in animal models of cerebral ischemia but its role in human ischemic stroke is still unknown. Thus, our aim is to determine human GM-CSF plasma level in control subjects and stroke patients and its relationship to clinical outcome. Forty-three patients with middle cerebral artery occlusion who received thrombolytic therapy within the first three hours of stroke onset and nineteen healthy controls were included. Blood samples were drawn before tissue plasminogen activator (t-PA) treatment. In a group of thirteen strokes blood samples were also obtained one hour after t-PA treatment, at 24 hours of symptoms onset, at discharge and at three months. GM-CSF levels were determined by enzyme-linked immunosorbent assay (ELISA). Stroke severity and neurological outcome were assessed by National Institute of Health Stroke Scale (NIHSS) and functional outcome was scored by modified Rankin Scale (mRS) at 3 months. Baseline GM-CSF level was significantly higher in stroke patients than in healthy controls (17.8 pg/ml vs. 12.8 pg/ml); p<0.0001 and was positively correlated with NIHSS score at 12 hours (R=0.3, p=0.03). No association was detected with functional status at three months measured by mRS. Temporary profile of GM-CSF level in stroke patients gradually decreases from admission to three months. Higher plasma endogenous GMCSF level is found in stroke patients compared to controls. However, no relation was found with a better outcome. Further research is necessary for elucidating the role of GM-CSF in ischemic stroke.

Several kinds of injury models, such as crush, transection and graft repair have been well studied in terms of neuroprotective effect of FK506. However, definitive experimental studies are lacking on focal degeneration or ischemia. In the present study, our goal was to investigate the effect of FK506 on functional recovery of the sciatic nerve after focal ischemia, produced by stripping of the epineurial vessels. A total number of 48 Wistar rats were used for this purpose and divided into four groups (control, sham-operated, FK506-treated, and Vehicle-treated). Sciatic nerves were approached by femoral and gluteal muscle splitting. Then, epineurial vessels around the sciatic nerve were stripped in the FK506-treated and Vehicle-treated groups. After operation, 5mg/kg/day FK506 administration was initiated by subcutaneous injection until animal sacrifice. The same volume of saline was administrated to the vehicle-treated group. The functional and sensory recoveries were tested by walking pattern analysis and pinch test in every postoperative week. The animals were sacrificed in the end of the fourth postoperative week and sciatic nerve samples were harvested and processed for electron microscopic evaluation. Our data revealed that FK506 administration showed beneficial effect on subperineurial degeneration/demyelinization from functional, sensorial, and ultrastructural points of view. The sciatic nerve samples in the FK506-treated group had several remyelinated fibers compared to the vehicle-treated group. Our literature searches revealed that FK506 administration has not, to our knowledge, been studied in focal ischemic degeneration produced by stripping of the epineurial vessels.

In this study age-, circadian rhythm- and methylphenidate administration- effect on open field habituation and object recognition were analyzed. Young and adult male Wistar rats were treated with saline or methylphenidate 2.0mg/kg for 28 days. Experiments were performed during the light and the dark cycle. Locomotor activity was significantly altered by circadian cycle and methylphenidate treatment during the training session and by drug treatment during the testing session. Exploratory activity was significantly modulated by age during the training session and by age and drug treatment during the testing session. Object recognition memory was altered by cycle at the training session; by age 1.5 h later and by cycle and age 24 h after the training session. These results show that methylphenidate treatment was the major modulator factor on open-field test while cycle and age had an important effect on object recognition experiment.

Oxidative stress-induced cerebral endothelial cell dysfunction is associated with cerebral microvascular complication of primary diabetic encephaolopathy, a neurodegenerative disorder of long-standing diabetes, but the injury mechanisms are poorly understood. This study sought to determine the contribution of carbonyl (methylglyoxal, MG) stress to human brain endothelial cell (IHEC) apoptosis, the relationship to cellular redox status and mitochondrial membrane potential, and the protection by thiol antioxidant and insulin sensitizers. MG exposure induced IHEC apoptosis in association with perturbed cellular glutathione (GSH) redox status, decreased mitochondrial membrane potential (Δψm), activation of caspase-9 and -3, and cleavage of polyADP-ribose polymerase. Insulin sensitizers such as biguanides or AMP-activated protein kinase activator, but not glitazones, afforded cytoprotection through preventing Δψm collapse and activation of caspase-9 that was independent of cellular GSH. Similarly, cyclosporine A prevented Δψm collapse, while Nacetylcysteine (NAC) mediated the recovery of cellular GSH redox balance that secondarily preserved Δψm. Collectively, these results provide mechanistic insights into the role of GSH redox status and mitochondrial potential in carbonyl stressinduced apoptosis of brain endothelial cells, with implications for cerebral microvascular complications associated with primary diabetic encephalopathy. The findings that thiol antioxidant and insulin sensitizers afforded cytoprotection suggest potential therapeutic approaches.

Cerebral small vessel disease (SVD) is a major contributor to dementia in the elderly, and hypertension represents a major cause for developing the disease. However, little is known about its development and progression. Modifications of large cerebral arteries due hypertension are thought to participate to the development of small ischemic infarcts, but the status of the small vessels before the establishment of hypertension is not well defined. Using spontaneously hypertensive rats (SHR) and stroke-prone SHR (SP-SHR) as a models for SVD, we analysed the effect of hypertension on the microvasculature in the cortex and putamen, and on its relationship with astrocytes in animals aged 2 to 9 months. Compared with the normotensive Wistar-Kyoto rats (WKY), the densities of the collagen type IV-positive capillaries were significantly higher in both brain areas of young SHR and SP-SHR. In contrast, the expression of the astrocytic marker GFAP was significantly lower in these animals, whereas astrogliosis was observed after 6 months in their cortex only. To investigate if chronic hypoxia occurs due to the lower number of astrocytes in young SHR and SPSHR, we evaluated the levels of HIF-1.. in both brain regions. The accumulation of HIF-1.. was not observed at the youngest ages, but was apparent in neurons of 9-month-old SHR and SP-SHR. Our results indicate that the brains of young SHR and SP-SHR rats show evidence of cellular imbalance between microvessels and astrocytes at the neurovascular unit that may lead to their higher vulnerability to hypoxic events at older ages.