Neural-immune interactions have profound implications for the clinical management of neurological trauma or disease. Since maintenance of neuronal viability is an important first step in neural repair, a series of experiments was initiated to determine if peripheral immune cells normally associated with acquired immunity are able to regulate neuronal survival after injury. We combined the well-described facial nerve injury paradigm with the severe combined immunodeficient (scid) mouse model in which a gene mutation blocks lymphocyte development and causes a lack of functional T and B lymphocytes. We discovered that there is a dramatic reduction in facial motoneuron (FMN) survival 4 weeks after facial nerve transection in scid mice and that reconstitution of scid mice with splenocytes from wild-type mice restores FMN survival to that of wild-type controls. We replicated our findings in the recombinase activating gene-2 knockout (RAG-2 KO) mouse, in which there is a lack of functional T and B cells because the RAG-2 gene has been disrupted in T and B cells only. The RAG-2 KO mouse model, plus other targeted gene knockout mice, will be used in the proposed experiments. All mice will be on a C57B1/6 background. It is hypothesized that peripheral immune cells produce neurotrophic factors (NTF) that support FMN survival before target reconnection occurs. There are 4 specific aims designed to test this hypothesis.
Aim 1 is to elucidate the immune cells involved in FMN survival after peripheral nerve injury. Experiments with immune cell deficient mice and selective cell type reconstitution will be done to identify which immune cells are involved in FMN survival after injury.
Aim 2 is to determine if immune cells are present centrally in the facial motor nucleus or recruited there following peripheral nerve injury. Immunocytochemistry with immune cell-specific antibodies will be done to determine the phenotype of immune cells in the central brainstem.
Aim 3 is to determine if treatment with NTF will rescue axotomized FMN from cell death in RAG-2 KO mice. Experiments will be done to determine if injured FMN can be rescued by treatment of RAG-2 KO mice with NGF, BDNF, NT-3, CNTF, or LIF.
Aim 4 is to determine if NTF are produced by resting and/or activated peripheral immune cells. Experiments in vitro, utilizing immunofluorescence with anti-NTF antibodies and specific ELISA assays, will be done to determine if the aforementioned NTF proteins are present in, or secreted by, resting and/or activated peripheral immune cells. Experiments in vitro, utilizing semi-quantitative RT-PCR with NTF primers, will be done to determine if the NTF mRNAs are present in resting and/or activated peripheral immune cells.
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