The failure of recent clinical trials in traumatic brain injury (TBI) demonstrates the critical need for more comprehensive and mechanistic preclinical studies. After TBI, cognitive deficits are associated with damage to the hippocampus, which is selectively vulnerable to TBI. To determine the molecular mechanisms of selective vulnerability of the rat hippocampus, our recent studies have focused on the molecular determinants of neuronal injury and survival. The goal of the present proposal is to test the feasibility of using RNA interference (RNAi) in vivo to effectively knockdown TBI-induced gene expression in rat hippocampal neurons. We will use short interfering RNA(siRNA) in vivo to address the hypothesis that silencing of deleterious injury-induced genes will reduce the numbers of injured hippocampal neurons and improve functional outcome after TBI and, conversely, that silencing of protective injury-induced genes will increase the numbers of injured hippocampal neurons and worsen functional outcome after TBI. Our preliminary in vivo siRNA studies suggest that knocking down nNOS decreased numbers of injured neurons in the rat hippocampus 24 hours after TBI and improved functional outcome (spatial memory deficits assessed by Morris Water Maze test) two weeks after injury.
Specific Aim 1. To examine the role of nNOS and IL-1(3 in the pathophysiology of TBI, we will use siRNA adenoviral constructs targeting these genes to knockdown their expression in rat hippocampus before TBI and quantify the influence on downstream gene expression, on numbers of injured neurons 24 hours and 7 days after TBI (at 7 days only for nNOS) using established stereological methods and on functional (assessment of cognitive dysfunction, spatial memory deficits) outcome after TBI.
Specific Aim 2. To evaluate the consequences of silencing genes that are implicated in endogenous neuroprotective responses, we will use siRNA adenoviral constructs targeting these genes to knockdown expression of GPx-1 and BDNF in the rat hippocampus and quantify the influence on downstream gene expression, on numbers of injured neurons 24 hours and 7 days after TBI (at 7 days only for GPx-1) using established stereological methods and assess neurobehavioral outcome as in Aim 1.
Specific Aim 3. To determine the feasibility and assess the effectiveness of post-trauma treatment with siRNA, we will inject the siRNA adenoviral constructs for nNOS and IL-1Pinto the injured rat hippocampus at 4 and 24 hours after TBI and evaluate the effect on neuronal injury 7 days after TBI using established stereological methods and on functional outcome after TBI as described in Aim 1. Although RNAi represents a powerful therapeutic possibility for human diseases, the therapeutic potential of RNAi has not been studied in animal models of TBI. The ultimate goal of these proof-of-principle studies is to demonstrate that an in vivo gene silencing approach can effectively modify gene expression in living animals and result in a desired functional outcome. In turn, this may open up avenues for therapeutic manipulation of deleterious gene expression in human patients.
