Current Neurovascular Research - Volume 3, Issue 1, 2006

Early reports in the lay literature describe individuals that suffer severe brain injury, but later are able to resume either previously lost cognitive function or motor function of paralyzed limbs. For example, in The Count of Monte- Cristo, the character Monsieur Noirtier de Villefort was reported to have suffered an incomplete injury to the brain, but subsequently was able to communicate by blinking with vertical eye movements. Reports of partial recovery from incomplete lesions to the brain later began to surface in the medical literature. In 1947, the first published case described a patient with an infarction in the territory of the vertebral-basilar artery system who became tetraplegic, was without speech, but regained consciousness. Reports such as this are consistent with a "de-efferented" lesion occurring in the brainstem, usually at the level of the pons that allows cortical function to remain unimpaired, but leads to quadriplegia. In the purest sense, individuals with these presentations are considered to suffer from a "locked-in" syndrome. Yet, other reports that include the present literature describe patients with a variety of nervous system disabilities that may be exclusive of the brainstem, but these patients achieve partial or full recovery from their brain insults raising the question whether the brain is able to repair itself. As a result, it becomes essential to understand the underlying pathology that may determine an individual patient's prognosis. The etiology of the initial insult will more often than not determine the overall prognosis of the patient. For example, transient ischemic insults in the vertebral-basilar artery distribution have been reported to lead to a complete resolution of all deficits within twenty-four hours after the initial onset of vascular occlusion. In contrast, aggressive disease processes, such as malignant neoplasms that involve the posterior fossa and the pons, ultimately may lead to the demise of the patient. Yet, evaluation of a patient's ability to recover from a nervous system injury should also be considered at the cellular level. If one examines cells that are able to regenerate and proliferate, they must be able to enter and execute a normal cell cycle. However, as we acquire further knowledge of each cell's ability to enter the cell cycle during different times in its development or maturation, it becomes evident that not all cells, at least in the final stages of development, should attempt cellular pathways leading to cell cycle induction. Aberrant cell cycle induction, such as with post-mitotic neurons, can ultimately lead to the death of the cell. In this issue of Current Neurovascular Research, we present several investigations that provide insight into common cellular pathways that can serve to foster opposing outcomes in a cell during either normal physiological processes or during acute toxic insults. In particular, some of the studies address the necessary role of new cell growth and cell cycle induction for tissue repair while other studies provide an alternative perspective on cell cycle induction describing cellular pathways that utilize a "dysfunctional" cell cycle to lead to apoptotic cell destruction. In their original article, Popa-Wagner et al. employed experimental focal cerebral ischemia models in both young and aged rats and assessed cell cycle induction, cell growth, and proliferation in the brains of these animals. They report that aged rats have nestin-positive cells in the region of the brain infarct as well as an early incorporation of nestin-positive cells into the glial scar region. Moreover, the capillaries of the corpus callosum were the principal source of proliferating nestin-positive cells, but surprisingly, these cells did not significantly contribute to new neuronal cell formation in the infracted cortex of the aged rats. They hypothesize that blocked migration of these cells may produce the limited neurogenesis response, but postulate that further understanding of the active capillary proliferation response in these aged animals may be applicable to neurodegenerative diseases in the elderly. Interestingly in our next article, Ding et al. note a significant vascular response also in aged rats with new cell proliferation in the brain, but with a novel twist. Angiogenesis in the cerebral cortex was documented during a monitored exercise protocol with a significant upregulation of vascular endothelial growth factor protein, providing.........

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 neural recovery and repair. Despite the detrimental impact of infarct scar formation, the brain regions and cell types that supply the components of the scar are not well characterized. We hypothesized that premature cerebral scar formation in aged animals is associated with an altered cellular response to cerebral ischemia. Focal cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery in 3 month- and 20 month-old male Sprague Dawley rats. After 3, 7, 14, and 28 days, brain tissue was subjected to real-time reverse-transcriptase-PCR (RT-PCR) and immunostaining for 1) a cellular proliferation marker (BrdU); 2) a neuroepithelial marker (nestin); 3) an astrocytic marker (glial fibrillary acidic protein [GFAP]); 4) a neuronal marker, doublecortin; and 5) a basal lamina marker (laminin), and analyzed using 3D-reconstruction of confocal images. In this model the infarct was localized primarily in the parietal cortex. By RT-PCR there was a robust increase in nestin mRNA transcripts shortly after stroke, and this increase was particularly intense in aged rats. Accordantly, we found in aged rats a rapid delimitation of the infarct area by nestin-positive cells and an early incorporation of these cells into the glial scar. The capillaries of the corpus callosum were the major source of proliferating, nestin-positive cells, many of which were also immunoreactive for doublecortin, although a smaller population of nestin cells were associated with the ventricular walls. Despite the proliferation of nestin cells, they did not make a significant contribution to neurogenesis in the infarcted cortex, possibly because the corpus callosum impedes the migration of subventricular zone-derived nestin-positive cells into the lesioned area. We conclude that: (i) the aged brain has the capability to mount a cytoproliferative response to injury, but the timing of the cellular and genetic reaction to cerebral insult is accelerated in aged animals; (ii) the proliferating cells contribute to the formation of the glial scar, but few of the cells appear to become neurons; and (iii) the vasculature plays a hitherto unrecognized role as a source of proliferating cells after stroke. Because capillary-derived cells help to form the glial scar, elucidating the molecular basis of this phenomenon and its acceleration in the aging brain could yield novel approaches to enhancing neurorestoration in the elderly.

The effect that exercise has on angiogenesis in the aging rat is unknown. We initiated this study with the intent to determine if exercise could induce angiogenesis in aging rats, as well as in adult rats reported previously. The markers we used to determine our endpoint were vascular endothelial growth factor (VEGF) and angiopoietin 1 and 2, as well as vascular density. Aged (22 month old) female Fisher 344 rats (n=16) were exercised on a treadmill 30 minutes each day for 3 weeks, or housed as non-exercised controls for the same duration. At the end of the exercise protocol, a significant (p<0.01) increase in the density of microvessels was found within the cerebral vasculature of the rats. Exercise was also associated with a significantly (p<0.01) increased mRNA expression of angiopoietin 1 and 2 in the aged cohort of rats. A mild but significant (p<0.01) increase in the four isoforms of VEGF mRNA (120, 144, 164, 188) were observed, with VEGF120 and VEGF144 being more markedly up-regulated than the other two. VEGF protein expression was also significantly (p<0.01) increased. This study demonstrates that angiogenesis can be induced in aging rats via exercise. The induced angiogenesis was associated with overexpression of angiogenic factors. These results support the hypothesis that an angiogenic response to chronic physical exercise is maintained with aging.

Either the absence or dysfunction of a number of critical pathways, such as those that involve the nuclear retinoblastoma protein (Rb) and the transcription factor E2F1, may account for the aberrant induction of the cell cycle in post-mitotic neurons that can be responsible for oxidative stress-induced apoptotic cellular destruction. Yet, it is unclear whether early programs of apoptotic injury that involve membrane phosphatidylserine (PS) exposure and calreticulin expression as well as later phases of apoptotic injury with nuclear DNA injury require the critical modulation of Rb and E2F1. We demonstrate that both the post-translational of phosphorylation of Rb to prevent E2F1 transcription as well as the protein integrity of Rb are closely aligned with the modulation of cell cycle induction in post mitotic neurons during oxidative stress. More importantly, we illustrate that both the initial onset of apoptosis with either membrane PS exposure or calreticulin analysis as well as the more terminal phases of apoptosis that involve nuclear DNA degradation proceed concurrently in the same neuronal cells with cell cycle induction. Progression of attempted cell cycle induction is closely associated with the phosphorylation of Rb, its inability to bind to E2F1, and the degradation of the Rb protein. Inhibition of Rb phosphorylation using cyclin dependent kinase inhibitors maintains the integrity of the E2F1/Rb complex and is neuroprotective during free radical exposure. Furthermore, maintenance of the integrity of the Rb protein is specifically dependent upon caspase 3-like activity, since caspase 3 can cleave Rb during free radical activity and this degradation of Rb can be blocked during the inhibition of caspase 3 activity. Our studies not only highlight the critical role of attempted cell cycle induction during oxidative stress-induced neuronal apoptotic injury, but also bring to light the significant impact of the Rb and E2F1 pathways upon early apoptotic programs that can directly influence both intrinsic cell survival as well as extrinsic inflammatory cell activation.

Inflammation may be one of the independent risk factors contributing to many neurological diseases. Moreover, there is an emerging body of data indicating that statins may have neuroprotective action. Recent studies suggest that CD40-CD40 ligand (CD40L) system is proven to be an important mediator of several auto-immune and chronic inflammation diseases. To address whether lovastatin produces neuroprotection as a potential novel anti-inflammatory pathway through the inhibition of CD40 expression, we examined the possible effects of lovastatin on expression of CD40, apoptosis, level of nitric oxide (NO) and nitric oxide synthase (NOS) activity induced by tumor necrosis factor α (TNF-α) in the cerebral vascular endothelial cells (CVECs) involved in cerebrovascular diseases. Preincubation with lovastatin (10-7, 10-6 and 10-5 mol/l) for 24 hours (h) protected CVECs from TNF-α-induced decrease of cellular viability. Further, lovastatin inhibited the TNF-α-induced increases of NO level, NOS activity, apoptotic cells and CD40 expression in a dosedependent manner, and anti-CD40 antibody also inhibited the cellular apoptosis induced by TNF-α. In conclusion, our data provide evidence to support a direct pro-inflammatory effect of CD40-CD40L signaling pathway in CVECs, and lovastatin possesses an anti-inflammatory effect independent of its lipid-lowering action involved in the cerebrovascular diseases.

The mechanisms involved in endothelial dysfunction are multifactorial. A correlation between oxidative stress and derangements of nitric oxide synthase (NOS) pathways in altered endothelial homeostasis has been most studied and demonstrated in different pathophysiological conditions. NOS activities are regulated by endogenous inhibitors such as asymmetric dimethyl-L-arginine (ADMA) that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Since recent data demonstrated that some endothelial dysfunction may be related to reduced expression and/or activity of DDAH, the aim of the present research was to investigate the expression of DDAH-2 and NOS isoforms in high glucosemediated oxidative stress. Endothelial cells were incubated with normal (7 mM) and high concentrations (33 mM) of Dglucose for 5 days; mannose (26 mM) plus D-glucose (7 mM) was used as osmotic control. Data obtained in the present study show that the exposure for 5 days to high glucose increases oxidative stress, reduces DDAH-2 and eNOS expression and increases iNOS expression. These results indicate that DDAH-2 and iNOS/eNOS dysregulation may play a key role in high glucose-mediated oxidative stress, suggesting that selective modulation of DDAH isoforms may result in selective inhibition/activation of NOS isoforms, thereby providing a novel strategy of approach in vascular complications of several pathologies.

The rabbit eye presents a valuable model to study the effects of vascular occlusion on the function and structure of myelinated nerve fibers. The rabbit eye has a band of myelinated nerve fibers within the intraocular compartment that are supplied by a narrow band of retinal vasculature. These vessels were transiently occluded (∼8 hours) using laser photocoagulation and the transmission of electrical signals along the nerve fibers was assessed by recording the visual evoked response (VER). Morphological damage was assessed by histological techniques. The ischemic insult produced no permanent change in retinal function as assessed by electroretinography, but the VER was suppressed, indicating failure of nerve fiber transmission. Histologically, the visible damage to the region supported by the retinal vasculature worsened following reperfusion, showing evidence of demyelination and necrosis followed by macrophage responses and gliosis. This rabbit model of ischemia/reperfusion of the retinal vasculature offers a rare opportunity to reliably study the response of myelinated nerve fibers to ischemia/reperfusion insults and has demonstrated the susceptibility of myelinated nerve fibers to such insults.

Long-term disabilities are the main outcome of cerebral strokes, though some of the deficits show receding signs in the weeks and months following the "brain attack". Studies show that neurogenesis is induced in the hippocampus and subventricular zone (SVZ) in animal models of ischemia, and that new neurons are generated at the sites of degeneration, where they replace some of the lost nerve cells. The enhanced neurogenesis suggests the involvement of the hippocampus and SVZ in the physiopathology of cerebral strokes, and the generation of new neuronal cells at the sites of degeneration suggests that the central nervous system (CNS) may attempt to repair itself. In this manuscript, we will review the studies on adult neurogenesis in cerebral strokes, discuss the contribution of adult neurogenesis to the physiopathology of strokes, and its underlying mechanisms.

Accelerated atherosclerosis and microvascular complications are perhaps the leading cause of coronary heart disease, blindness and renal failure, which could account for disabilities and high mortality rates in patients with diabetes. Several thrombogenic abnormalities have been shown to play a central role in the pathogenesis of these devastating complications. However, the molecular mechanism for thrombogenic diathesis in diabetes is not fully elucidated. A recent clinical study, the Diabetes Control and Complications Trial-Epidemiology of Diabetes Interventions and Complications (DCCT-EDIC) Research, has revealed that the reduction in the risk of progressive retinopathy and nephropathy resulting from intensive therapy in patients with type 1 diabetes persist for at least several years, despite increasing hyperglycemia. Further, intensive therapy during the DCCT resulted in decreased progression of carotid intima-media thickness six years after the end of the trial as well. These clinical studies strongly suggest that so-called 'hyperglycemic memory' causes chronic abnormalities in diabetic vessels that are not easily reversed, even by subsequent, relatively good control of blood glucose. Among various biochemical pathways activated under diabetes, the process of formation and accumulation of advanced glycation end products (AGEs) and their mode of action are most compatible with the theory 'hyperglycemic memory'. In this review, we discuss the role of AGEs in thrombogenic abnormalities in diabetes.