Current Neurovascular Research - Volume 7, Issue 3, 2010

Stroke represents one of the leading causes of death and disability in humans, but despite intense research, only a few options exist for the treatment of stroke-related infarction of brain tissue. Thus far, in experimental strokes, cell therapy appears to partly reverse some behavioral deficits. However, the mechanisms of action remain uncertain as most studies reveal only little, if any, evidence for neuronal replacement and observed behavioral improvements. This present study was performed to test rodent fetus forebrain derived neural stem cells (NSCs) implantation into rats subjected to suture-induced middle cerebral artery occlusion (MCAO). Efficacy of cell therapy was studied regarding behavior recovery, infarct volume, and protection possibility of related molecular mechanisms. Here, we show that grafted cells can home in on damaged regions by MCAO and significantly improve behavior of ischemic rats. Infarct volumes and brain atrophy were diminished after grafted NSCs treatment. Furthermore, we detected inflammation related molecules such as COX-2 and IL-1β and found that grafted NSCs treatment after ischemic stroke could repress expression of inflammation molecular protein levels. We also detected protein levels of heat shock protein 27 (HSP27) as a protective protein against apoptosis. The results showed that grafted NSCs treatment induced the protein level of HSP27 and down-regulated activity of caspase-3 compared with the vehicle control. Our results demonstrate that transplanted NSCs provide benefits in behavioral function recovery after MCAO and increase neuroprotection whilst repressing inflammatory destruction. These data reveal another essential explanation of cellular transplantation therapy in damage recovery from ischemic stroke and offer new therapeutic possibilities.

Diabetes has been found to increase the probability of vascular dementia in humans. We have investigated the effect of 4'-hydroxy-3'-methoxyacetophenone (HMAP), a NADPH oxidase inhibitor and Pitavastatin, a HMG Co-A reductase inhibitor, on Streptozotocin (STZ) diabetes induced vascular dementia in rats. Donepezil served as a positive control. The rats were administered with single dose of STZ for the induction of diabetes. Drug treatment was started after one month of STZ administration and treatment was continued till the end of the study (i.e. 56th day). On 52nd day onwards, the animals were exposed to Morris water-maze (MWM) for testing learning & memory. Serum glucose, body weight, vascular endothelial function, serum nitrite / nitrate levels, aortic & brain oxidative stress levels and brain acetylcholinesterase activity were also tested. STZ treated animals performed poorly on MWM hence reflecting impairment of learning & memory. Further STZ treatment also produced a reduction in body weight, impairment of vascular endothelial function, decrease in serum nitrite / nitrate levels, along with increase in serum glucose, aortic & brain oxidative stress levels and brain acetylcholinesterase activity. Treatment of HMAP, Pitavastatin and Donepezil significantly reversed diabetes induced impairment of learning and memory, endothelial dysfunction, and changes in various biochemical levels. It may be concluded that STZ induces vascular dementia. 4'hydroxy-3'-methoxy acetophenone and Pitavastatin may be considered as potential pharmacological agents for the management of diabetes induced vascular dementia.

Increased homocysteine (Hcy) levels contribute to a variety of cardiovascular and cerebrovascular diseases including stroke and Alzheimer's disease. Recent data has shown that elevated levels of Hcy can lead to blood-brain barrier (BBB) dysfunction and activation. However, the mechanism for Hcy-mediated dysfunction remains unclear. The aim of this study is to characterize the effects of moderate Hcy administration in rat brain capillary endothelial cells (BCECs), which serve as a simple model to study blood-brain barrier functions. This present study shows that addition of 20 μM Hcy for 6 days does not significantly affect BCEC survival, as measured by acridine orange staining, propidium iodide staining, and nitrite content. However, addition of 20 μM Hcy for 6 days does elevate lactate dehydrogenase (LDH) activity released into the supernatant of BCECs, as well as significantly enhances the transmigration of monocytes across the BCEC in a time-dependent manner. In addition, TNFα levels in BCEC are also elevated by Hcy, whereas inflammatory markers MIP3α and RANTES are significantly reduced. Finally, this study shows that intercellular adhesion molecule-1 (ICAM-1) expression is significantly enhanced by 20 μM Hcy treatment compared to control conditions. These results suggest that moderate levels of homocysteine can affect proinflammatory patterns expressed by BCECs, ultimately leading to BBB activation and dysfunction through enhanced monocyte transmigration and ICAM-1 expression.

Earlier investigations from our laboratory demonstrated that the expression of matrix metalloproteinases (MMPs) was down-regulated by exogenously administered agmatine against ischemia-like injuries in the murine brain capillary endothelial (bEnd.3) cells. In our present study, we intended to investigate the mechanism involved in the inhibition of MMPs in bEnd.3 cells infected with retroviral containing human arginine decarboxylase (hADC) gene which can synthesize agmatine endogenously (ADCΔbEnd.3 cells). The ADCΔbEnd.3 cells were subjected to oxygen glucose deprivation (OGD, 6 hrs) with reperfusion (18 hrs). High performance liquid chromatography (HPLC) analysis revealed the high levels of agmatine in the ADCΔbEnd.3 cells compared to other experimental groups. The results demonstrated significant decrease in cell death and increase in the nitric oxide (NO) production in the ADCΔbEnd.3 cells. The increased expression of MMP-2 and MMP-9, and decreased expression of endothelial nitric oxide synthase (eNOS) by ischemic injury was attenuated in ADCΔbEnd.3 cells. Moreover, the expression of activating transcription factor 3 (ATF3) was increased significantly in ADCΔbEnd.3 cells. In addition, the suppression of the MMP-2 and MMP-9 expression in ADCΔbEnd.3 cells was prevented with ATF3 small interfering RNA (siRNA) treatment. These results suggest that the endogenous agmatine in ADCΔbEnd.3 cells inhibits the MMPs expression mediated via the regulation of eNOS, NO and ATF3.

Several nervous system injury models, such as sciatic crush and chronic cerebral hypoperfusion have been well studied in terms of neuroprotective effect of angelica injection. However, definitive experimental studies are lacking on diabetic peripheral neuropathy (DPN). This study sought to investigate the effects of angelica injection on DPN in type 1 diabetic rats. Diabetes was induced by single intraperitoneal injection of streptozotocin (STZ). To examine whether DPN model successed, tail-flick latency (TFL) and motor nerve conduction velocity (MNCV) were measured at 6 weeks after diabetes induction. Then, diabetic rats were treated with high- and low-dose angelica injection for 4 weeks. TFL, MNCV, morphology of sciatic nerve, myelinated nerve fiber density and the expressions of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) in soleus and sciatic nerve were measured at 10 weeks after diabetes induction. The results showed the TFL was significantly shortened (p< 0.001) and the MNCV was reduced (p< 0.01) in diabetic rats compared with normal control rats at 6 weeks after diabetes induction. The TFL was obviously prolonged and the MNCV was further reduced in diabetic control group at 10 weeks after diabetes induction. TFL, MNCV and morphology of sciatic nerve were remarkably ameliorated and myelinated nerve fiber density and the expressions of NGF and BDNF in soleus and sciatic nerve were increased in the angelica treatment groups. This study suggests angelica injection has potential therapeutic effects on DPN, and the mechanism might be related to direct increase in NGF expression and direct or indirect increase in BDNF expression.

Molecular mechanism underlying leptin-mediated neuronal protection against glucose-oxygen-serum deprivation (GOSD) insult was investigated by focusing on the interactions among leptin, Interleukin-1β (IL-1β) and glutamate and their impacts on the growth of neurons under GOSD. The trypan blue dye exclusion assay, 4', 6-diamidino- 2-phenylindole (DAPI) assay, cytokine antibody array assay, immunocytochemical staining assay, glutamate determination kit, immunoblocking and chemical blocking strategies were applied to serve the study goal. Results showed that in response to 6 h of GOSD, cortical neurons can secrete significant amounts of leptin and IL-1β to protect neurons from GOSD-induced cell damage. Serine/threonine kinase Akt (Akt) and extracellular signal-related kinase (ERK) inhibitors significantly reversed leptin-mediated neuroprotection. GOSD-induced IL-1β was further enhanced by leptin in Akt/ERK-dependent manner. Blockade of endogenous leptin with specific antibodies significantly inhibited GOSDinduced IL-1β expression and increased glutamate release from GOSD neurons. IL-1 blockade with IL-1 receptor antagonist (IL-1ra) on the other hand, inhibited leptin-mediated neuroprotection and suppression of glutamate release from GOSD neurons. Pre-treating GOSD neurons with leptin and IL-1β in combined significantly increased their survival but decreased their releases of glutamate. The results indicate that leptin may act through Akt and ERK signaling pathways to protect neurons from GOSD insult; the protection was in part IL-1β dependent and through which the glutamate release from GOSD neurons was inhibited. Therapeutic values of leptin and IL-1β were suggested in the treatment of cerebral ischemia at early stage.

The endothelial-specific expression of plasmalemmal vesicle associated protein-1 (PV-1) is typical of fenestrated endothelium observed in pulmonary capillaries and some endocrine organs. In the central nervous system (CNS) it is expressed during development but disappears concomitant with maturation of the blood-CNS barrier [1]. Consistent with observations made in models of stroke, Alzheimer's disease, and tumorigenesis, we show PV-1 expression in the spinal cord specifically upregulated by pathologically-activated endothelial cells (ECs) in response to traumatic spinal cord injury (SCI). Adult female C57Bl/6 mice received a moderate T9/10 contusive SCI. PV-1 assessed by qRT-PCR and immunohistochemistry 3 hours to 14 days post-injury showed expression as early as 1 day post-SCI, with levels decreasing by 14 days. This expression was associated with microvessels in the injury epicenter and penumbral zone, with the time course and distribution correlated with progressing peripheral inflammatory cell infiltration. PV-1-immunoreactive ECs were angiogenic as demonstrated by intravascular binding of Griffonia simplicifolia isolectin B4 (IB4). ECs expressing high levels of PV-1 were anatomically and physiologically abnormal with altered/absent immunostaining for occludin and zonula occludens-1 (ZO-1), and decreased expression of glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQP4). Glucose transporter type I (Glut-1) expression decreased in affected, PV- 1 positive microvessels with little colocalization of PV-1 and Glut-1 apparent by 7 days post-SCI. These data suggest that upregulation of microvascular expression of PV-1 post-SCI may promote major components of secondary injury including extravasation of cellular and acellular mediators of inflammation and may accelerate loss of neuropil and decline in the functional and anatomical integrity of the neurovascular unit (NVU).

Chronic hypertension and cerebral amyloid angiopathy (CAA) are the main pathologies which can induce the rupture of cerebral vessels and intracerebral hemorrhagies, as a result of degenerative changes in the vascular wall. A lot of progress has been made in this direction since the successful creation of the first mouse model for the study of Alzheimer's disease (AD), as the spectrum of AD pathology includes a plethora of changes found in pure cerebrovascular diseases. We describe here some of these mouse models having important vascular changes that parallel human AD pathology, and more importantly, we show how these models have helped us understand more about the mechanisms that lead to CAA formation. An important cellular event associated with reduced structural and functional recovery after stroke in aged animals is the early formation of a scar in the infarcted region that impairs subsequent neural recovery and repair. We review recent evidence showing that the rapid formation of the glial scar following stroke in aged rats is associated with premature cellular proliferation that originates primarily from the walls of capillaries in the corpus callosum adjacent to the infarcted region. After stroke several vascular mechanisms are turned-on immediately to protect the brain from further damage and help subsequent neuroregeration and functional recovery. Although does occur after stroke, vasculogenesis is overshadowed in its protective/restorative role by the angiogenesis and arteriogenesis. Understanding the basic mechanisms underlying functional recovery after cerebral stroke in aging subjects is likely to yield new insights into the treatment of brain injury in the clinic.