Faye S. Silverstein, M.D. - US grants

Perinatal hypoxic-ischemic encephalopathy is a major cause of chronic neurologic disorders in children and adults. Developing motor systems in the brain are particularly vulnerable to perinatal injury. Unilateral carotid artery ligation and subsequent exposure to 8% 02 in 7 day old rats leads to reproducible ischemic neuronal damage limited to forebrain ipsilateral to the ligation. This is a useful small animal model for study of perinatal brain injury. Using this well-characterized model, the proposal will focus on examination of synaptic mechanisms in the pathogenesis of ischemic neuronal injury, in particular the role of the excitatory neurotransmitter glutamate, and on identification of features of ischemic injury which are relevant in the immature brain. Hypoxia-ischemia induced alterations in the distribution and pharmacology of post-synaptic glutamate receptors will be analyzed using in vitro 3H-glutamate autoradiography. The impact of hypoxia-ischemia on pre-synaptic high affinity glutamate uptake will be examined in synaptosomes derived from striatum, a major target for injury. The pattern of hypoxia-ischemia induced glutamate release will be studied using regional in vivo microdialysis in lesioned rat pups. This work will provide a better understanding of the neurobiology of perinatal ischemic brain injury and may provide a basis for development of more specific and effective therapeutic interventions.

Perinatal hypoxic-ischemic encephalopathy is a major cause of chronic neurologic disorders in children and adults. Developing motor systems in the brain are particularly vulnerable to perinatal injury. Unilateral carotid artery ligation and subsequent exposure to 8% oxygen in 7 day old rats leads to reproducible ischemic neuronal damage limited to forebrain ipsilateral to ligation. This is a useful small animal model for study of perinatal brain injury. Using this well characterized model, the proposal will focus on examination of the role of the excitatory neurotransmitter glutamate in the pathogenesis of ischemic neuronal injury and on identification of features of ischemic injury which are relevant in the immature brain. Hypoxia-ischemia induced alterations in the distribution and pharmacology of post-synaptic glutamate receptors will be analyzed using in vitro 3H-glutamate autoradiography. Animals treated with the neuroprotective glutamate antagonist MK -801 will be used to dissect -the relationship between receptor changes and evolution of neuronal injury. Biochemical factors that regulate high affinity glutamate uptake(HAGU) in the developing nervous system will be examined and the impact of hypoxia-ischemia on HAGU will be assessed in synaptosomes derived from lesioned brain. Chemical modulation of striatal glutamate release in immature brain and the effects of hypoxia- ischemia on glutamate release will be studied with in vivo microdialysis. This work will provide a better understanding of the neurobiology of perinatal hypoxic-ischemic brain injury and may provide a basis for development of more specific and effective therapeutic interventions.

The overall goal of this project is to delineate the mechanisms that contribute to the neurotoxicity of HIV-1 derived peptides in the developing brain. Our primary focus will be to assess the influence of the HIV-1 envelope protein gp120 on excitatory amino acid (EAA)-mediated brain injury. This proposal will test the hypothesis that gp120 is neurotoxic in the developing brain, and that neurotoxicity is mediated, at least in part, by enhanced activation of EAA receptors. Our preliminary data demonstrate that in 7 day old (P7) rats:(i) intra-hippocampal injection of gp120 together with the EAA agonist N-methyl-D-aspartate (NMDA) selectively increases the extent of neuronal loss and tissue atrophy, in comparison with injection of the same dose of NMDA alone; and (ii) in a perinatal rodent stroke model in which NMDA receptor over-activation by endogenous EAA contributes to irreversible neuronal injury, preceding intra-hippocampal injection of gp120 markedly increases the extent of ischemic brain injury. The first Specific Aim includes assessment of intrinsic gp 120 neurotoxicity, evaluation of the impact of concurrent gpl20 administration on the severity of EAA agonist-mediated brain injury, and identification of the neurotoxic moiety of gp120 in P7 rats. The second Specific Aim is to dissect the mechanisms by which gp120 may influence NMDA receptor activation: we will determine if intracerebral injection of gp120 alters the distribution or density of NMDA-type EAA receptors or high affinity EAA re-uptake activity, and if EAA antagonists attenuate gpl20 neurotoxicity. The third Specific Aim is to determine if gp120 increases susceptibility to brain injury resulting either from pathophysiologic insults that stimulate synaptic accumulation of endogenous EAA (e.g. hypoxia-ischemia, hypoglycemia) or from pharmacologic interventions that stimulate synaptic EAA accumulation (e.g. treatment with EAA uptake inhibitors). Understanding these potential mechanisms of HIV neurotoxicity in the perinatal brain may provide the scientific foundation for development of more effective neuroprotective interventions for HIV- infected infants.

DESCRIPTION: (Adapted from Applicant's Abstract): 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).

DESCRIPTION (adapted from applicant's abstract): The long-term goal of the research is to understand the roles of inflammatory mediators in the pathogenesis of neonatal brain injury. The investigators' research focuses primarily on understanding how inflammation, initiated by acute ischemic brain injury, influences the ultimate expression of tissue damage, and how critical components of this inflammatory response can be modulated to improve neurological outcome. The primary hypothesis underlying this research proposal is that pro-inflammatory mediators play pivotal roles in determining the impact of hypoxic-ischemic insults on the developing brain. Experiments will be performed in well characterized neonatal rodent (rat and mouse) models of hypoxic-ischemic brain injury (unilateral carotid artery ligation + timed exposure to moderate hypoxia). The original research focus was on the role of the proinflammatory cytokine Interleukin-1beta in neonatal brain injury. Their findings in the initial funding period prompted them to broaden the scope of the work to include studies of several related pathogenetically relevant inflammatory mediators. They propose to delineate specific mechanisms of inflammation-mediated neuronal and oligodendroglial injury in the neonatal brain, and to determine if anti-inflammatory treatment interventions can reduce neonatal hypoxic-ischemic brain injury. Aim 1 will focus on the pathogenetic roles of 2 beta-chemokines (monocyte chemoattractant protein-1 and monocyte inflammatory protein 1-alpha) and their cellular targets. Aim 2 will evaluate the contribution of complement system activation in the brain in the pathogenesis of neonatal hypoxic-ischemic brain injury. Aim 3 will evaluate the influence of pro-inflammatory mediators on the vulnerability of oligodendroglia to hypoxia-ischemia. Aim 4 will evaluate the long-term outcome of anti-inflammatory interventions with respect to tissue integrity and behavioral measures. The proposed experiments could identify some of the critical cellular/molecular mechanisms in the acute post injury inflammatory cascade that determine the extent of neuronal and glial injury, and ultimately provide novel treatment approaches to improve long-term neuro-developmental outcome.

DESCRIPTION: (PI's abstract) This application is submitted in response to PAS 99-080, "Exploratory Grants in Pediatric Brain Disorders: Integrating the Science." The proposal will lay the foundation for a new inter-institutional collaboration (Univ. Of Michigan and Wayne State Univ.) between clinician scientists with expertise in the pathogenesis of brain injury (Drs. Silverstein and Barks), and a neuroanatomist with expertise in oligodendroglial development (Dr. Skoff). The long-term goal of the proposed research is to develop novel approaches for understanding the pathogenesis of hypoxic-ischemic white matter injury in the immature brain. Studies of neonatal ischemic brain injury in experimental models have focused on mechanisms of neuronal injury and neuroprotection, and have largely neglected white matter injury. Yet, in human neonates and young infants the white matter, particularly in periventricular regions, is highly susceptible tp ischemic injury. In preliminary experiments, in situ hybridization and RT-PCR assays were developed to examine injury-induced changes in expression of 2 oligodendroglia (OL)-specific genes, proteolipid protein (PLP), and myelin basic protein (MBP); preliminary findings provide the impetus for this application. Experiments performed in immature rodent stroke models (in neonatal rats and mice) revealed that hypoxic ischemic injury results in acute disruption of OL gene expression within periventricular white matter; and also alters expression of OL-specific genes in cells in the sub-ventricular zone; in neonatal rats, intraventricular injection of the excitatory amino acid agonist AMPA results in a similar pattern of acute disruption of OL gene expression. This application proposes to build upon these finding to develop novel approaches for understanding mechanisms of OL injury in the immature brain. Aim 1 will: evaluate the influence of hypoxia-ischemia (HI) on PLP and MBP gene expression; determine whether acute HI induced suppression of OL-gene expression predicts the severity of chronic OL damage; and evaluate the impact of specific interventions that could exacerbate or attenuate HI-induced OL injury. Aim 2 will examine the influence of AMPA lesioning on PLP and MBP gene expression and OL injury. Aim 3 focuses upon the impact of HI on OL precursors in the ventricular and subventricular zones; and specifically will analyze whether these OL precursors undergo apoptosis, whether the loss of OL precursors is permanent, and whether mild HI insults can elicit a proliferative response in these cells. Taken together, results of these experiments will improve our understanding of the impact of HI on OL in the immature brain and enhance development of more effective therapeutic approaches to limit or prevent neonatal ischemic white matter injury.

DESCRIPTION (provided by applicant): This is a substantially revised R21 application, designed to address a clearly defined clinical question in a well-established preclinical model. The goal of this proposal, submitted in response to PAR-06-189, is to identify clinically feasible interventions that increase the neuroprotective efficacy of induced hypothermia after neonatal hypoxic-ischemic brain injury. In both infants and adults, moderate hypothermia improves neurological outcome after acute asphyxial brain injury. However, in two recent neonatal hypothermia trials, even with cooling, over 40% of treated infants had abnormal neurological outcomes at 18 months. Thus, it is a very high clinical priority to develop therapeutic interventions to improve the neuroprotective efficacy of cooling. We hypothesize that early treatment with anticonvulsants will augment the efficacy of therapeutic hypothermia. Experiments will be carried out in a well-characterized neonatal rodent model of hypoxic- ischemic brain injury, unilateral carotid artery ligation, followed by timed exposure to moderate hypoxia (8% oxygen, 1.5 hours) in 7 day old (P7) rats of both genders, and will incorporate functional (sensorimotor and cognitive) and neuropathological outcome measures. One critical factor that limits the neuroprotective efficacy of post-asphyxial cooling is the inevitable time interval between the hypoxic-ischemic event and the opportunity to begin treatment. Our goal is to identify drugs that extend the therapeutic window for initiation of hypothermic neuroprotection. The rationale stems from a study in which we replicated this clinically important lag period in the animal model, by delaying onset of cooling until 3 hours after the insult. In this model, 3 hours of cooling, initiated immediately after hypoxia-ischemia, reduces acute brain injury;in contrast, if cooling is initiated 3 hours later ("delayed- cooling") it is ineffective. However, the combination of treatment with topiramate, an anticonvulsant with multiple modes of action, within 15 min after hypoxia-ischemia and delayed cooling resulted in long-term neuroprotection. Yet, since no parenteral formulation of topiramate is available, these findings provide a compelling impetus for identification of other drugs with similar efficacy. Since seizures are common in asphyxiated neonates, and there is evidence that they exacerbate brain injury, we propose to evaluate other antiepileptic drugs. In Aim 1, we will evaluate 4 clinically available anticonvulsants, and determine which agents extend the therapeutic window for initiation of hypothermia after neonatal hypoxic-ischemic brain injury. We will also determine if selected drugs are more effective if there is a shorter delay period (one hour) before the onset of cooling. In Aim 1 sensorimotor and pathological outcomes will be evaluated after a one week recovery period. In Aim 2 we will incorporate functional (sensorimotor and cognitive) and pathological outcomes and determine if neuroprotection is sustained one month later. Results of these experiments can be readily translated to other pre-clinical and ultimately to clinical applications. The aim of this study is to determine if early treatment with anticonvulsants can extend the time window for initiation of effective and safe hypothermia-mediated neuroprotection in neonates. The proposed research will use a well-characterized neonatal rodent model of hypoxic-ischemic brain injury. Our long term goal is to identify effective, safe, and feasible treatment strategies that can be rapidly translated into clinical practice and that will limit the adverse impact of hypoxic-ischemic brain injury in neonates and infants.