Immune Regulation of Neuronal Injury and Repair

描述（申请人摘要）：神经免疫相互作用具有深刻的 对神经创伤或疾病的临床管理的影响。 由于维持神经元的活力是神经功能恢复的重要的第一步， 修复后，启动一系列实验以确定外周免疫 通常与获得性免疫相关的细胞能够调节神经元免疫， 受伤后的生存我们将面部神经损伤 严重联合免疫缺陷（SCID）小鼠模型的范例，其中 基因突变阻断淋巴细胞发育，导致功能性T细胞缺乏， 和B淋巴细胞。我们发现，在人类的面部， scid小鼠面神经切断后4周运动神经元（FMN）存活 用野生型小鼠脾细胞重建SCID小鼠 使FMN存活恢复到野生型对照的水平。我们复制了我们的发现 在重组酶激活基因-2敲除（RAG-2 KO）小鼠中，其中 缺乏功能性T和B细胞，因为RAG-2基因被破坏 仅在T和B细胞中。RAG-2 KO小鼠模型，加上其他靶向基因 基因敲除小鼠将被用于拟议的实验。所有小鼠将在 C57B1/6背景。假设外周免疫细胞产生 神经营养因子（NTF）支持FMN在靶点重新连接前存活 发生。有4个具体目标旨在验证这一假设。目标1： 阐明外周神经损伤后FMN存活所涉及的免疫细胞 损伤免疫细胞缺陷小鼠和选择性细胞类型的实验 将进行重建以鉴定哪些免疫细胞参与FMN 受伤后的生存目的2是确定是否存在免疫细胞 集中在面部运动核或招募后，周边 神经损伤免疫细胞特异性抗体的免疫细胞化学将 以确定中枢脑干中免疫细胞的表型。目标3 确定用NTF治疗是否将从细胞中拯救轴突切断FMN RAG-2 KO小鼠死亡。将进行实验，以确定是否受伤的FMN 可以通过用NGF、BDNF、NT-3、CNTF或 LIF。目的4是确定NTF是否通过静息和/或激活产生 外周免疫细胞体外实验，利用免疫荧光 用抗NTF抗体和特异性ELISA测定，将进行测定 如果上述NTF蛋白存在于静息细胞中或由静息细胞分泌， 和/或活化的外周免疫细胞。体外实验，利用 用NTF引物进行半定量RT-PCR，以确定NTF是否 mRNA存在于静息和/或活化的外周免疫细胞中。

DESCRIPTION (applicant's abstract): 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.