Current Neurovascular Research - Volume 10, Issue 2, 2013

The pigmented ciliary epithelium (PCE) of mammalian eye harbors resident population of stem cells that lie in apposition with endothelial cells which release vascular endothelial growth factor (VEGF) that may influence the fate and function of these stem cells in ways that remain unclear. We examined the role of VEGF in proliferation of PCE stem cells and expression of Notch, Jagged, N-Cadherin and β-Catenin which are known to maintain proliferation state of neural stem cells. We cultured human PCE cells obtained from 12-20 weeks old fetal eyes. The neurospheres were analyzed for the proliferation capacity of PCE stem cells in presence of VEGF on 3,6 and 9 day. Real time PCR was used to quantitate the mRNA expression of above mentioned genes on PCE derived neurospheres on 3,6 and 9 day. We found increased number of neurospheres when PCE stem cells were stimulated with VEGF alongwith epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) than EGF and bFGF. BrdU immunostaining was done to analyze the proliferation of CE cells and presence of neural and retinal progenitor markers such as Nestin and Pax6 were also investigated. An increased Notch and Jagged mRNA was observed on 6th day in VEGF, EGF and bFGF treated PCE cells as compared to 0,3 and 9 day. A similar pattern was noticed with N-cadherin and β-catenin mRNA levels. These findings may clarify the role of VEGF on PCE stem cell proliferation with possible involvement of Notch, Jagged, N-cadherin and β-Catenin. The data may suggest importance of harvesting 6th day neurospheres for transplantation purposes in preclinical investigations pertaining to retinal degenerative diseases, however, additional studies are needed to substantiate the findings.

Angiogenesis is associated with improved neurologic recovery after cerebral ischemia. Human bone marrow mesenchymal stem cells (hMSCs) have been successfully used to treat ischemic stroke and were shown to induce the expression of a number of neurotrophic factors including VEGF, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) in a rat middle cerebral artery occlusion (MCAO) ischemia model. In this study, we aimed to understand the mechanism underlying the improvement of neurological function following hMSCs transplantation into MCAO rats. We established a rat MCAO model and used immunofluorescence to evaluate α-tubulin expression in the hippocampus. We used RT-PCR to determine the expression of Ang-1 and Ang-2 mRNAs after transplantation of hMSCs into MCAO rats. We showed a significant decrease in α-tubulin expression in rats with cerebral ischemia, suggesting that α-tubulin is a protective protein in cerebral ischemia Transplantation of hMSCs significantly upregulated α-tubulin levels in the hippocampus. Transplantation of hMSCs also resulted in a significant upregulation of Ang-1 and Ang-2 mRNAs in MCAO rats. Ang-2 expression was upregulated earlier than Ang-1, suggesting that 1) transplantation of hMSCs promotes angiogenesis and that 2) Ang-2 may be an initiator of angiogenesis. Our results provide a theoretical basis for the therapeutic use of hMSCs in cerebral ischemia.

Transient, severe global ischemia that arises in humans as a consequence of cardiac arrest or cardiac surgery or that is induced experimentally in animals, leads to selective and delayed neuronal death, particularly in the hippocampus. Especially, in this brain structure, clock genes are rhythmically expressed, for instance the inducible and archetypical clock gene is Period1 (Per1). An eventual involvement of its trans-activating protein products in the daytime-dependent severity of ischemia-induced cell damage is not excluded. Probably, neurons may exhibit endogenously a daytimedependent variation in the expression of predictive cell death proteins. We therefore compared the cell death machinery in the hippocampus between Per1-/-- and wildtype (WT) mice upon cerebral ischemia. Neuronal death in the hippocampal CA1-subfield, was observed in both types of mice, but the density of damaged cells in Per1-/--mice was increased by more than 23% as compared to wildtype mice. To explore the mechanisms underlying the excessive vulnerability of the hippocampus in Per1-/--mice and to address if hippocampal susceptibility inherits a daytime component, the expression of both, apoptotic and autophagic predictors of cell death was monitored. In Per1-/--mice, the expression of apoptotic/autophagic markers are altered and higher levels of the proapoptotic factors such as cytochrome c and Apaf-1 were observed as compared to WT mice. Moreover, the autophagy marker LC3B was dramatically reduced in Per1-/-- mice. Our data suggests that basal activities of apoptosis and autophagy seem to be modulated by PER1, and that the autophagic machinery is probably slowed down when this clock gene is absent. These alterations may be causal for the observed innate vulnerability of Per1-/--mice to cerebral ischemia.

Levosimendan is a novel calcium sensitizer that is an established treatment for congestive heart failure. In coronary vessels, levosimendan has a vasorelaxant, endothelium-independent effect and an antagonistic effect on endothelin-1 (ET-1). There is also some data for a neuroprotective effect in a traumatic brain injury model, and levosimendan can prevent the reduction of the luminal area of the basilar artery. We considered that patients who suffer heart attack after subarachnoid hemorrhage (SAH) might respond well to levosimendan, which might also be useful to induce hypertension in patients with cerebral vasospasm. However, the functional effects of levosimendan in the cerebrovasculature are unknown. Here, we investigated the functional role of levosimendan on rat basilar artery by assessing vasocontractile reactivity in response to ET-1, sarafotoxin S6c, acetylcholine, sodium nitroprusside, cGMP, and prostaglandin F2α (PGF). Contrary to observations in coronary vessels, levosimendan did not affect the ET-1 system in cerebral arteries; neither ET(A)-receptor-induced contraction nor ET(B)-receptor-dependent relaxation were changed. For the nitric oxide (NO) pathway, only a slight increase was detected. Rather, levosimendan caused significant and dose-dependent relaxation after PGF precontraction. To our knowledge, this is the first report that describes levosimendan-induced functional changes of cerebrovascular contractility and relaxation. Under physiological conditions, levosimendan did not influence ET(A)/ET(B)-receptor signaling or the NO pathway. Interestingly, levosimendan seemed to affect the prostaglandin system and dosedependently reversed PGF- induced contraction. We did not detect a vasospastic potential for levosimedan in cerebral arteries, suggesting that it would be safe for use in SAH patients.

Therapeutic hypothermia is a robust therapeutic tool in experimental stroke models but its clinical applications are limited. Furthermore, optimal conditions for therapeutic hypothermia, such as, temperature and the initiation and duration of cooling must be individualized. Here, we evaluated the therapeutic effects of delayed local hypothermia, administered for 44 hr after 4 hr of reperfusion in a rat model of transient middle cerebral artery occlusion (tMCAo), using a cooling device that allowed controlled local hypothermia (31°C) in brain. Histological data revealed that local hypothermia significantly reduced infarct volumes and glial hypertrophic activation. Brain water contents, IgG leakage, and Evans Blue extravasation were notably reduced by local hypothermia. Furthermore, local hypothermia had strong vasculoprotective effects, as determined by immunohistochemistry and Western blot analyses for endothelial barrier antigen (EBA), laminin, aquaporin-4, and tight junction proteins in brain. Our data indicate that delayed/prolonged local hypothermia confers neurovascular protection, reduces brain edema, and inhibits inflammatory glial activation, and suggest that hypothermic conservation of vascular structures and functions account for the therapeutic effects of local hypothermia observed in this model of experimental stroke.

The neuroprotective properties of soy isoflavone (SIF) have been demonstrated by our previous studies and others, but its potential mechanism is not clear. Because of the key role of neurovascular dysfunction in the pathogenesis of Alzheimer's disease (AD), we hypothesized neurovascular tissue might be one neuroprotective target of SIF. In the present study, learning and memory ability, β-amyloid (Aβ) expressions both in neurovascular tissue and plasma, the receptor for advanced glycation end products (RAGE), low-density lipoprotein receptor-related protein (LRP)-1, nuclear factor-κB p65 (NF-κB p65), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) expressions in neurovascular tissue were measured in Wistar rats following lateral cerebral ventricle administration of Aβ1-42 by miniosmotic pump with or without intragastric administration of SIF from 14 days before surgery to the end of experiment. The results showed that SIF could improve the impairment of learning and memory of rats induced by Aβ1-42, maintain Aβ homeostasis in brain, regulate the disordered expressions of RAGE/LRP-1 and restrain RAGE related NF-κB and inflammatory cytokines activation in neurovascular structure. These results suggested that SIF could protect Aβ-impaired learning and memory in rats, and its mechanism might be associated with the regulation of vascular Aβ transportation and vascular inflammatory reaction.

Clinical features and therapeutic strategies of cervicocranial arterial dissection (CCAD) are still unclear. A retrospective review was conducted on 71 CCAD patients. Diagnosed by DSA and outcome evaluation was through mRS scores follow-up 12 months. All patients were allocated into three groups according to clinical situation: 1) subarachnoid hemorrhage (SAH), 2) ischemic symptoms and 3) mass effect. CCAD with anterior circulation arterial dissection (ACAD) had higher ischemia than that with posterior circulation arterial dissection (PCAD) (p=0.023). The non-aneurysmal dissection (NAD) patients were susceptible to ischemia (p=0.00) and patients with aneurismal dissection (AD) were more susceptible to SAH (p=0.001); The outcome of patients with SAH was significantly worse than patients with other manifestations (p=0.012). Following up one year, the outcome of CCAD involving posterior inferior cerebellar artery (PICA) was significantly worse than the other area (p=0.035). There was no statistically significant difference in mRS scores between endovascular treatment and conservative treatment (p=0.052) at one year follow-up. Patients suffering from SAH that received endovascular treatment experienced improved outcomes than patients with conservative treatment (p=0.033). The patients in the ACAD, NAD and extracranial CCAD groups were more likely to suffer from ischemia, while patients in the AD group were susceptible to SAH. CCAD with SAH or involving PICA had poor prognoses. The therapeutic efficacy of conservative treatment is nearly equal to endovascular treatment in CCAD patients follow up 12 months; however, endovascular treatment may decrease the risk of mortality for the patient with SAH.

In the present study, three bi-3-azaoxoisoaporphine derivatives were synthesized and intracerebroventricularly administrated to BALB/c mice. The antidepressant actions in stroke-induced depressive like behavior in mice were examined using despair swimming test and tail suspension test. The results reported that bilateral common carotid arteries occlusion caused a significant abnormality of the normal behaviors. Behavioral models demonstrated that synthesized compounds showed antidepressant action. The most antidepressant active compound was DIME2 (4,4'-dimethyl-7H,7'H- [6,6'-bibenzo[e]perimidine]-7,7'-dione), which decreased the immobility time and increased the swimming and climbing times in despair swimming model. DIME2 also showed similar results in decreasing the immobility time in the tail suspension model. In open field tests, DIME2 at 0.1μg/μl showed a significant activity in the modification of the distance movement and the number and duration of rearing versus bilateral common carotid arteries occlusion (P<0.001). Furthermore, bilateral common carotid arteries occlusion caused a significant increase in the water consumption and significant decreasing in the sucrose consumption which are indicated as a state of anhedonia, a well known common symptom of transient ischemic stroke-induced depressive like behavior, versus normal group (P<0.001). In conclusion, bi- 3-azaoxoisoaporphine derivatives can be considered as antidepressant agents for post stroke-induced depressive like behavior therapy.

Sympathetic nerve activity (SNA) is increased in both patients and experimental animals with renal failure. The kidney is a richly innervated organ and has both efferent and afferent nerves. Renal denervation shows protective effects against renal failure in both animals and humans. The underlying mechanisms include a decrease in blood pressure, a decrease in renal efferent SNA, a decrease in central SNA and sympathetic outflow, and downregulation of the reninangiotensin system. It has been demonstrated that re-innervation occurs within weeks after renal denervation in animals but that no functional re-innervation occurs in humans for over two years after denervation. Renal denervation might not be renal protective in some situations including bile duct ligation-induced renal failure and ischemia/reperfusion-induced acute kidney injury. Catheter-based renal denervation has been applied to patients with both early and end stage renal failure and the published results so far suggest that this procedure is safe and effective at decreasing blood pressure. The effectiveness of renal denervation in improving renal function in patients with renal failure needs to be further investigated.

Excess reactive oxygen species (ROS) generation and oxidative stress in vascular tissue is associated with many diseases. Glutathione (GSH), one of the most abundant low molecular weight non-protein thiols, modulates physiological levels of ROS and is involved in the cell's oxidative stress response. The GSH/GSSG redox couple is commonly used in measuring oxidative stress status. The imbalance of GSH is reported in many disease states including atherosclerosis, cancer, neurodegenerative disease, and aging. The importance of GSH in modulation of intracellular ROS involves both its protective defense against the damaging effects of oxidative stress and its role in facilitating ROS cell signaling. In this paper, we review significant results obtained from mass balance and kinetic reactions based computational and mathematical models of GSH participation in oxidative stress. The focus is on the mediation of ROS and oxidative stress with respect to the antioxidant capacity of the cell. We discuss the role of GSH in the redox state of the cell, maintaining homeostasis through GSH synthesis, scavenging of free radicals, modulating hydrogen peroxide level and interacting with nitric oxide pathways.