Current Neurovascular Research - Volume 2, Issue 5, 2005

Although it was initially Antoine Lavoisier who determined that oxygen is the only gas in air that sustains pulmonary respiration to prevent death and that almost 200 years later Barcroft introduced the terms "anoxic", "anemic", "histotoxic", and "stagnant" to designate the various forms of anoxia, our comprehension of human anoxic brain injury is far from complete. The term cerebral anoxia indicates any form of inadequate oxygen delivery to the brain, including hypoxemia and ischemia. Anoxic brain injury is extremely complex in nature and consists of a variety of insults to cells that involve decreased oxygen availability, systemic acidosis, hypercapnia, and sometimes superimposed ischemia. Other organs, such as the kidney and heart, can tolerate ischemic periods of up to thirty minutes, but the brain can tolerate no more than a few minutes of anoxia. Neurons survive only for minutes after the oxygen supply is reduced below critical levels. Pyramidal cells in the hippocampus, Purkinje cells of the cerebellum, and pyramidal cells of the third and fifth layers of the cerebral cortex are vulnerable to even moderate degrees of anoxia. Widespread necrosis of the cortex with the brainstem intact produces a vegetative state. More profound anoxia affecting the cortex, basal ganglia, and brainstem results in coma and subsequent death. Brief episodes of cerebral anoxia are usually well tolerated with patients escaping any irreversible deficits. Yet, an amnestic syndrome may follow transient periods of global ischemia with patients experiencing a severe antegrade amnesia and variable retrograde memory loss. Given the range of neurological disabilities that can ensue during ischemic brain injury, studies that determine the cellular mechanisms responsible for preserving both neuronal and vascular survival are essential for the development of viable therapeutics for this field. With this goal in mind, this issue of Current Neurovascular Research offers a unique perspective into not only some of the potential cellular mechanisms responsible for injury to the brain, but also the temporal parameters that appear to be intimately linked to a cell's fate. The ability to identify the temporal cellular determinants of clinical deterioration in the nervous system could bring new insight into unexplained clinical deterioration. For example, individuals with anoxic-ischemic coma of approximately six hours duration, but with unremarkable insults on brain imaging, can sometimes suffer from permanent cognitive deficits. In addition, delayed neurologic deterioration following only a brief injury to the nervous system also can ensue that includes neuropsychological impairment or unconsciousness. In their original article, He et al. illustrate that aged animals may be less susceptible to ischemic cerebral injury, but these aged animals unfortunately lose their innate ability to respond to protective measures such as ischemic preconditioning which can reduce cerebral infarct size in young animals. This significantly reduced protection normally afforded by ischemic preconditioning in young animals was markedly reduced in aged counterparts and may be associated with a reduction in expression of the N-methyl-D-aspartic acid receptor 1 as well as modified tolerance to caspase mediated cell death mechanisms. Overall, the study sheds new light on some of the temporal parameters that can determine clinical disability following cerebral ischemia and the multiple variables that need to be addressed to achieve broad clinical efficacy for both young and more senior individuals. In our next original article, Okouchi et al. follow suit with the lead article in regards to the temporal parameters that can influence cell survival by showing that chronic hyperglycemia as well as acute glucose reduction from a chronic hyperglycemic state can contribute to cellular oxidative stress. Their work has implications for a number of disease states, including diabetes and Alzheimer's disease. The authors demonstrate that chronic hyperglycemia was able to intensify methylglyoxal apoptotic cell injury and was associated with several intracellular processes that included mitochondrial redox balance, impaired glucose 6-phosphate dehydrogenase activity, and enhanced basal expression of apoptosis protease activator factor-1. Chong et al. in their original work provide important evidence that novel neuroprotective strategies, such as the administration of erythropoietin, are also critically related to the temporal modulation of intracellular apoptotic pathways. These investigators show that erythropoietin prevents neuronal β-amyloid toxicity, but that protection requires the early translocation of nuclear factor-κB from the cytosol to the nucleus to initiate an anti-apoptotic program. Without this intracellular translocation of nuclear factor-κB within a tight six hour period following β- amyloid toxicity, such as during experiments that employ the gene silencing of nuclear factor-κB, neuroprotection by erythropoietin is lost. Although the original work by Laidley et al. does not focus upon specific cellular mechanisms of ischemic cell injury, the study by these investigators presents an important analysis of the use of experimental animal models to yield scientifically sound data that can approximate clinical disease. The authors examine the use of a popular animal model for forebrain ischemia, namely the Mongolian gerbil (Meriones unguiculatus). Their work provides us with a refreshing perspective on both the benefits of this model for cerebral ischemia, but also the limitations of current commercially available strains and the considerations that should come into play for robust data analysis with this model. Our three review articles for this issue of Current Neurovascular Research complement the original articles by providing a broader overview of several of the cellular mechanisms that can contribute to the temporal determinants of cellular protection and plasticity. Han and Suk provide a thorough discussion of the neurovascular unit and the crosstalk that can occur between endothelial cells and microglia during inflammatory disorders of the nervous system. In particular, their review addresses the timely modulation required of the blood brain barrier, chemokines, and microglia for effective therapeutic strategies against neurodegenerative disease. Maiese et al. lead us into the intricate world of specific class of G-protein-linked receptors known as metabotropic glutamate receptors and their interesting role during a variety of disorders that can include amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, epilepsy, trauma, and stroke. The authors highlight the complexity of the metabotropic glutamate receptors in the nervous system. These receptors can control several cellular systems that involve neuronal, vascular, and inflammatory pathways, but function at times as a double edge sword that can either promote or prevent cellular function. Our final article by Dhanasekaran and Ren focuses upon the unique role of coenzyme Q, a ubiquitous protein in both plants and animals, that can play a vital role during neurodegenerative disease, cardiovascular disorders, and oxidative stress, such as during diabetes. In humans, coenzyme Q-10 is the predominant form and offers the advantages of being a lipid-soluble antioxidant that can rapidly alter cellular redox mechanisms, energy reserves, and stabilize mitochondrial membrane potential to control "time sensitive" pathways that may precipitate cellular injury. As our knowledge of basic cellular injury mechanisms continues to grow from the original work of Lavoisier and Barcroft, this issue of Current Neurovascular Research allows us to become increasingly more cognizant with the notion that "timing is everything" at both the cellular and clinical levels to effectively treat a broad spectrum of individuals afflicted by any disease entity.

The present study examines the hypothesis that aging defined by the 50% survival age compromises neuroprotection afforded by ischemic preconditioning (IPC). Sixty-four male F344 rats aged 4- and 24-months, respectively, were subjected to IPC, (3-min ischemia) or sham-surgery followed by 10-min (full) ischemia or sham-surgery 2 days later. There were 4 groups at each age: sham-surgery-sham-surgery (SS), preconditioning-sham-surgery (PS), preconditioningischemia (PI) and sham-surgery-ischemia (SI) groups. Assessments of histology and immunoreactivities of N-methyl-Daspartic acid receptor 1 (NMDAr1) and caspase-3 active peptide (C3AP) in the hippocampal CA1 region were performed 8 days after full ischemia. The CA1 "living cell ratio" was greater in the aged SI group than in the young SI group (32±6% vs. 17±5%, p<0.05), whereas the degree of protection against full ischemia afforded by IPC was reduced in the aged compared with the young (53±17% vs. 241±25%, P<0.0001). The basal level of NMDAr1 immunofluorescence was significantly higher in young animals, while the numbers of C3AP-positive cells were greater in all three aged ischemic groups as compared to respective young groups (p<0.01, p=0.055 and p<0.05). A fourth method of assessing cell damage using Fluoro Jade C labeled degenerating neurons that were also intensively eosinophilic. Counts of Fluoro Jade Cpositive cells were higher in the young SI group than in the aged SI group (P<0.05), suggesting that mechanisms of ischemic cell death may change with aging. In conclusion, aging alters mechanisms of ischemic cell death in CA1 neurons and ischemic tolerance mechanisms are blunted by aging.

The mechanism(s) of central nervous system complication associated with neurodegenerative disorders such as diabetes is unknown. Previous studies demonstrated that carbonyl stress induced by methylglyoxal (MG) mediates differential apoptosis of rat pheochromocytoma (PC12) cells in the naïve or differentiated transition states. Since chronic hyperglycemia is central to diabetic complications, and poorly differentiated cells are oxidatively more vulnerable, we currently investigated the effect of glycemic status on MG-induced apoptosis in naïve (nPC12) cells focusing on glutathione- to-glutathione disulfide (GSH/GSSG) redox signaling. nPC12 cells were exposed to 25 mM glucose acutely for 24h or chronically for 1 week. A role for glycemic fluctuation was tested in chronic high glucose-adapted cells subjected to acute reduction in glucose availability. Acute hyperglycemia potentiated MG-induced nPC12 apoptosis in accordance with cellular redox (GSH-to-Disulfide (GSSG plus protein-bound SSG)) imbalance. Chronic hyperglycemia exacerbated baseline and MG-induced apoptosis that corresponded to exaggerated loss of cytosolic and mitochondrial redox balance, impaired glucose 6-phosphate dehydrogenase (G6PD) activity, and enhanced basal expression of apoptosis protease activator factor-1 (Apaf-1). Reduced glucose availability in hyperglycemia-adapted nPC12 cells induced by acute lowering of glucose or by dehydroepiandrosterone (DHEA, G6PD inhibitor) further enhanced MG-induced apoptosis in association with greater cytosolic and mitochondrial redox and G6PD impairment and elevated basal Apaf-1 expression. These findings demonstrate that chronic hyperglycemia or acute glucose reduction from the chronic hyperglycemic state potentiates carbonyl stress, which collectively contribute to oxidative susceptibility of poorly differentiated cells such as that which occurs in brain neurons of neurodegenerative disorders like diabetes and Alzheimer's disease.

No longer considered exclusive for the function of the hematopoietic system, erythropoietin (EPO) is now considered as a viable agent to address central nervous system injury in a variety of cellular systems that involve neuronal, vascular, and inflammatory cells. Yet, it remains unclear whether the protective capacity of EPO may be effective for chronic neurodegenerative disorders such as Alzheimer's disease (AD) that involve b-amyloid (Aβ) apoptotic injury to hippocampal neurons. We therefore investigated whether EPO could prevent both early and late apoptotic injury during Aβ exposure in primary hippocampal neurons and assessed potential cellular pathways responsible for this protection. Primary hippocampal neuronal injury was evaluated by trypan blue dye exclusion, DNA fragmentation, membrane phosphatidylserine (PS) exposure, and nuclear factor-κB (NF-κB) expression with subcellular translocation. We show that EPO, in a concentration specific manner, is able to prevent the loss of both apoptotic genomic DNA integrity and cellular membrane asymmetry during Aβ exposure. This blockade of Aβ generated neuronal apoptosis by EPO is both necessary and sufficient, since protection by EPO is completely abolished by co-treatment with an anti-EPO neutralizing antibody. Furthermore, neuroprotection by EPO is closely linked to the expression of NF-κB p65 by preventing the degradation of this protein by Aβ and fostering the subcellular translocation of NF-κB p65 from the cytoplasm to the nucleus to allow the initiation of an anti-apoptotic program. In addition, EPO intimately relies upon NF-κB p65 to promote neuronal survival, since gene silencing of NF-κB p65 by RNA interference removes the protective capacity of EPO during Aβ exposure. Our work illustrates that EPO is an effective entity at the neuronal cellular level against Aβ toxicity and requires the close modulation of the NF-κB p65 pathway, suggesting that either EPO or NF-κB may be used as future potential therapeutic strategies for the management of chronic neurodegenerative disorders, such as AD.

The Mongolian gerbil (Meriones unguiculatus) has been used extensively as a model of forebrain ischemia. Its unique susceptibility to ischemia was suggested to be due to an incomplete circle of Willis. The relative ease to which ischemia can be induced combined with highly reproducible delayed CA1 cell death following a 5 min occlusion made the model popular in neuroprotection studies. Presently, this assumption was tested that complete forebrain ischemia occurs in all gerbils because increased variability was noticed in neuronal injury and behavioral outcome using this model in the last several years. Here it is reported that gerbils obtained from Charles River, the largest supplier in North America, show a high incidence (22.7% with bilateral and 38.6% with unilateral anastomoses) of posterior communicating arteries compared to another supplier of gerbils (High Oak Farms, 2.6% with bilateral and 13.2% with unilateral anastomoses, P<0.0001). This increased incidence of complete or partial circle of Willis led to less severe CA1 cell loss in Charles River gerbils (P<0.0001) compared to High Oak gerbils, with an unacceptably high level of inter-animal variability. Similarly, behavioral indices of CA1 ischemic injury (increased locomotion, habituation deficits) were also significantly attenuated in the Charles River animals. High Oak gerbils also displayed increased histological and behavioral variability relative to the pattern obtained several years ago. Thus, the gerbil model of forebrain ischemia, at least using Charles River animals, no longer produces consistent injury and behavioral alterations. Investigators are urged to consider adopting other models in future neuroprotection studies or ensure that their gerbil population lacks communicating arteries.

The neurovascular unit is composed of a microvascular endothelium, neuron, and glial cell elements that are in physical proximity to the endothelium. The vascular system provides oxygen, glucose, and hormones for brain cells and guides the cells to appropriately respond to the local environment. Conversely, the brain cells, especially glial cells, can regulate the function of blood vessels in response to local requirements. The disruption of the neurovascular coordination was observed in a variety of inflammation-related diseases in brain, such as infectious diseases, stroke, vascular dementia, and multiple sclerosis. Inflammatory responses resulting from infections or injury of the brain activate the endothelium and glial cells to various degrees depending on the type, titer, or strength and duration of exposure to the agents or insults. The activation of endothelial and microglial cells may be modulated by the action of cytokines or other substances secreted from these cells. In an effort to understand the pathogenesis and find rational treatments against inflammatory disorders in brain, studies have been separately carried out using either endothelial cells or microglia. Increasing evidence, however, indicates that a crosstalk between these two cell types is important for the brain inflammation. Here, we review recent advances that provide insights into the coordinated interaction between the vascular and microglial systems, including the role of the specialized endothelium in regulating the immune response that occurs within CNS, the influence of microglial cells on the properties of endothelial cells, and the effects of endothelium on the state of microglial activation.

Metabotropic glutamate receptors (mGluRs) share a common molecular morphology with other G protein- linked receptors, but there expression throughout the mammalian nervous system places these receptors as essential mediators not only for the initial development of an organism, but also for the vital determination of a cell's fate during many disorders in the nervous system that include amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, Multiple Sclerosis, epilepsy, trauma, and stroke. Given the ubiquitous distribution of these receptors, the mGluR system impacts upon neuronal, vascular, and glial cell function and is activated by a wide variety of stimuli that includes neurotransmitters, peptides, hormones, growth factors, ions, lipids, and light. Employing signal transduction pathways that can modulate both excitatory and inhibitory responses, the mGluR system drives a spectrum of cellular pathways that involve protein kinases, endonucleases, cellular acidity, energy metabolism, mitochondrial membrane potential, caspases, and specific mitogen-activated protein kinases. Ultimately these pathways can converge to regulate genomic DNA degradation, membrane phosphatidylserine (PS) residue exposure, and inflammatory microglial activation. As we continue to push the envelope for our understanding of this complex and critical family of metabotropic receptors, we should be able to reap enormous benefits for both clinical disease as well as our understanding of basic biology in the nervous system.

Coenzyme Q (ubiquinone, 2-methyl-5,6-dimethoxy-1,4-benzoquinone), soluble natural fat quinine, is crucial to optimal biological function. The coenzyme Q molecule has amphipathic (biphasic) properties due to the hydrophilic benzoquinone ring and the lipophilic poly isoprenoid side-chain. The nomenclature of coenzyme Q-n is based on the amount of isoprenoid units attached to 6-position on the benzoquinone ring. It was demonstrated that coenzyme Q, in addition to its role in electron transport and proton transfer in mitochondrial and bacterial respiration, acts in its reduced form (ubiquinol) as an antioxidant. Coenzyme Q-10 functions as a lipid antioxidant regulating membrane fluidity, recycling radical forms of vitamin C and E, and protecting membrane phospholipids against peroxidation. The antioxidant property, high degree of hydrophobicity and universal occurrence in biological system, suggest an important role for ubiquinone and ubiquinol in cellular defense against oxidative damage. Coenzyme Q-10 is a ubiquitous and endogenous lipid-soluble antioxidant found in all organisms. Neurodegenerative disorders, cancer, cardiovascular diseases and diabetes mellitus and especially aging and Alzheimer's disease exhibit altered levels of ubiquinone or ubiquinol, indicating their likely crucial role in the pathogenesis and cellular mechanisms of these ailments. This review is geared to discuss the biological effect of coenzyme Q with an emphasis on its impact in initiation, progression, treatment and prevention of neurodegenerative, cardiovascular and carcinogenic diseases.