Frank A. Welsh - US grants

The goal of this investigation is to determine whether tissue acidosis plays a critical role in the development of ischemic damage and, if so, by which biochemical mechanisms. Using a model of reversible, unilateral hypoxia-ischemia in the mouse, we will first develop a regional, quantitative method for brain pH. Second, ischemic alterations of brain pH will be correlated with the accumulation of lactate during hypoxia-ischemia and with postischemic recovery of energy metabolism. Third, the pH changes during hypoxia-ischemia will be modified (a) by administering CO2 and (b) by partially inhibiting the glycolytic pathway in order to alter the recovery of energy metabolism. Finally, the ischemic change in glycolytic intermediates other than lactate will be determined in glucose-pretreated animals to detect deleterious alterations not related to acidosis. The second major aim is to identify the biochemical processes which are affected by acidosis/glucose-pretreatment. Because NADH is acid-labile, we will measure brain levels of NADH, NAD+, and their degradation products using enzymatic methods and high performance liquid chromatography (HPLC). In addition, we will attempt to alter the size of the NAD pool by administering the precursor, nicotinamide, and a false precursor, 6-amino-nicotinamide. The effect of acidosis/glucose-pretreatment on brain levels of Adenyl, Guanyl, Uridyl, and Cytidyl Nucleotides during and after hypoxia-ischemia will be determined using HPLC. Finally, the degradative activity of acid phosphatase will be assayed in hypoxic-ischemic brain using both in vitro and in vivo methods.

Current therapies for human stroke have limited efficacy. Recent studies indicate that there are protective mechanisms residing within the brain that can be activated using preconditioning stimuli. Identification of these mechanisms may provide new therapeutic targets for stroke in humans. The central hypothesis of this proposal is that preconditioning upregulates inhibitors of inflammation that protect the brain against ischemia. Enhancing the expression of inflammatory inhibitors after stroke is a novel strategy to attenuate ischemic damage. The central hypothesis will be tested in four specific aims. First, the timecourse of and regional expression of selected inhibitorsof inflammation will be measured after preconditioning. Second, the causative relationship between these inhibitors and neuroprotection will be assessed by determining whether attenuation of their expression abrogates the induction of tolerance to ischemia. Third, we will determine whether agents that specifically activate Toll-like receptors upregulate inhibitors of inflammation and induce tolerance to ischemia. Finally, we will determine whether neurotrophic factors, known to be upregulated by preconditioning, themselves increase the expression of inhibitors of inflammation and whether antagonism of this expression attenuates the protective effects of the neurotrophins against ischemia. These studies will test whether upregulation of inhibitors of inflammation is a major mechanism contributing to ischemic tolerance. Enhancement of endogenous pathways that suppress inflammation is an innovative and promising strategy for the treatment of stroke in humans.

Trauma to the Central Nervous System (CNS) initiates a cascade of physiologic and molecular events that culminate in cellular injury and damage. Recent studies indicate that cells respond to trauma and other insults by altering the expression of specific genes. Among the genes expressed following CNS trauma are the immediate early genes (IEGs), many of which encode transcription factors. A second group of genes induced by CNS injury are those encoding heat-shock proteins (HSPs). Although the expression of these genes may be involved in the attempted recovery of function following brain trauma, little is known about the identity of the genes induced by trauma or the temporal/anatomic relationship between this induction and traumatic injury. Moreover, the consequences of post-traumatic induction of IEGs and HSPs remains poorly defined. This study will use molecular biology techniques to identify key changes in gene expression following experimental brain injury and relate these changes to histologic injury. Using in situ hybridization and immunohistochemistry, we will determine the timecourse and regional expression of several IEGs (c-fos, c-jun, junB, zif-268) in several models of brain trauma. We will then determine whether trauma-induced expression of IEGs is followed by expression of critical target genes, including proenkephalin, brain-derived neurotropic factor (BDNF), calbindin-D28K, and microtubule-associated protein (MAP2). We will also determine the timecourse and regional expression of hsp72, hsp90, and hsp32 (heme-oxygenase) following brain injury. Finally, we will correlate changes in gene expression with indicators of cell injury, including cellular calcium staining, blood-brain-barrier function, histologic damage and immunocytochemistry for glutamic acid decarboxylase (GAD). These studies will enhance our understanding of the cellular and molecular events following trauma and may lead to the development of novel targeted therapies for the treatment of traumatic brain injury.