The primary goal of this proposal is to understand the role of the cytokine Interleukin-1beta in the pathophysiology of acute brain injury in the developing mammalian nervous system. This revised application includes two complementary experimental approaches in which, on the one hand, IL-1beta production in response to injury is to be evaluated, while, on the other hand, the influence of blockade of the IL-1 receptors by IL-1ra during these events is also to be evaluated. These strategies of assessing IL-1beta influences during acute brain trauma are to be applied to two models of brain injury in P7 perinatal rats: One model involves overactivation of the N-methyl-D-aspartate (NMDA)-type excitatory amino acid (EAA) agonist receptors by intra-cerebral injection of NMDA that results in irreversible neuronal injury. The second hypoxia-ischemia model involves carotid vessel ligation, followed by exposure to 8 percent oxygen, which leads to neuronal injury and perinatal rodent stroke. The overall hypothesis of this project is that acute excitotoxic and hypoxic-ischemic brain injury stimulate synthesis of IL-1beta in the developing nervous system. Further, IL-1beta is a potent mediator of progressive brain injury, and pharmacological antagonism of IL-1 with IL-1ra will limit the extent of irreversible injury by interference with the post-injury inflammatory cascade. Thus, the Specific Aims of the proposal are:
Specific Aim 1 : Determine the temporal and neuroanatomic distribution of IL-1beta in the developing nervous system. IL-1beta is to be monitored following excitotoxic or hypoxic-ischemic lesioning of rat brain. The time-course and histological location of IL-1beta production is to be assessed using whole-tissue ELISA analysis for the quantification of IL-1beta and by immunocytochemistry to identify the cellular sources of IL-1beta production (identified by morphology, enzyme-associated activities, and monoclonal antibody staining such as for ED1+ microglial cells or GFAP+ astrocytes). In the case of NMDA-induced injury and IL-1 responses, issues of specificity are to be approached by the use non-NMDA agonists such as AMPA, as well as NMDA receptor antagonists such as MK-801. In the case of hypoxic-ischemic injury, factors peculiar to the model to be considered are to include the duration of 8 percent O2 exposure and the temperature during hypoxia.
Specific Aim 2 : Use replication-deficient adenovirus vectors to induce over-expression of IL-1ra in immature rat brain to determine if these adenoviruses exert deleterious effects in the developing brain. Preliminary Data indicated that over the time period of interest for these experiments, the encoded proteins are expressed and there is no apparent virus- or protein-induce toxicity. However, additional evaluations of such possible confounding factors are to be performed. As stated, anatomic distribution, time course, and consistency and longevity of expression of beta-galactosidase and IL-1ra are to be determined. The possibility of induction of local or systemic inflammation and/or injury, or the production of endogenous IL-1 caused by adenovirus infection is also to be considered. With regard to influences of IL-1ra production on the development and function of brain-derived cells of interest to this study, microglial, astroglial, and neuronal cell types are to be examined so as to reveal baseline characteristics for purposes of comparison in the aims that follow.
Specific Aim 3 : Induce over-expression of IL-1ra in immature rat brain by intra-cerebral administration of replication-deficient adenovirus constructs encoding IL-1ra to determine the influence of IL-1ra on outcome of excitotoxic and hypoxic-ischemic brain injury. This is a merging of Aims 1 and 2, designed to determine if the course of injury aggravated by IL-1beta that follows initial injury caused by the two lesioning methods describe above, can be altered by the over-expression of IL-1ra. In order to optimize any neuroprotection produced by IL-1ra, the timing and longevity of observed effects are to be assessed, as well as effects produced when IL-1ra is combined with other neuroprotective agents such as MK-801.
Specific Aim 4 : Delineate the cellular and molecular mechanisms whereby over-expression of IL-1ra is neuroprotective in vivo. Experiments designed to satisfy this aim are intended to identify specific cellular and molecular targets of IL-1beta in the acutely injured brain, and to determine the mechanism(s) of IL-1ra-mediated neuroprotection. Attention is to be focused primarily on production of IL-1beta and influences of IL-1ra in the NMDA-lesioning model. A spectrum of factors are to be monitored in these studies including increases in the numbers of microglial cells or monocytes that are activated, neutrophil infiltration monitored as myeloperoxidase positive cells, production of IL-1beta and the IL-1-inducible chemokine monocyte chemoattractant protein-1 (MCP-1), and the induction of genes that encode enzymes responsible for the production of toxic metabolite, that is, nitric oxide synthase (generates NO) and cyclooxygenase-2 (involved in the production of prostaglandins and related mediators).
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