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处理的启示。 因为维持神经元的活性是神经学的重要的第一步 修复，启动了一系列实验来确定外周免疫 通常与获得性免疫有关的细胞能够调节神经元 受伤后存活。我们结合了描述良好的面神经损伤 严重联合免疫缺陷(SCID)小鼠模型的范例 基因突变阻碍淋巴细胞发育并导致功能性T细胞缺乏 和B淋巴细胞。我们发现面部的数量急剧减少。 SCID小鼠面神经切断后运动神经元存活4周 用野生型小鼠的脾细胞重建SCID小鼠 将FMN存活率恢复到野生型对照组的水平。我们重复了我们的发现 在重组酶激活基因-2基因敲除(RAG-2KO)小鼠中，存在 是由于RAG-2基因被破坏而导致功能T和B细胞的缺乏 仅限于T和B细胞。RAG-2KO小鼠模型，外加其他靶向基因 基因敲除小鼠，将用于拟议的实验。所有的鼠标都将在一个 C57B1/6背景。据推测，外周免疫细胞产生 靶基因重联前支持FMN存活的神经营养因子(NTF) 发生。有四个特定的目标被设计来检验这一假设。目标1是 免疫细胞在周围神经损伤后FMN存活中的作用 受伤。免疫细胞缺陷小鼠和选择性细胞类型的实验 将进行重建以确定哪些免疫细胞参与了FMN 受伤后存活。目标2是确定是否存在免疫细胞 集中于面神经运动核或在外周后在那里募集 神经损伤。免疫细胞化学与免疫细胞特异性抗体将 以确定中央脑干中免疫细胞的表型。目标3 是确定NTF治疗是否会从细胞中挽救轴突切断的FMN RAG-2 KO小鼠死亡。将进行实验以确定损伤的FMN 可以通过用NGF、BDNF、NT-3、CNTF或 生活。目的4是确定NTF是否是通过静息和/或激活产生的 外周免疫细胞。体外实验，利用免疫荧光 用抗NTF抗体和特异性的酶联免疫吸附试验来确定 如果上述NTF蛋白存在于休眠状态或由休眠状态分泌 和/或激活外周免疫细胞。体外实验，利用 用NTF引物进行半定量RT-PCR，以确定NTF是否 MRNAs存在于静息和/或激活的外周免疫细胞中。

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.