STEROID HORMONES AND NEURONAL REGENERATION

这一建议是我们正在进行的 神经营养剂治疗作用的研究， 丙酸睾酮（TP），促进神经再生。模型 我们一直在使用的系统是仓鼠面部运动神经元（HFMN）， 含有雄激素受体的外周神经元群体。 上一笔赠款目标的实现表明， 显著加快面神经再生的速度， 缩短萌芽发生前的延迟，TP触发神经细胞 身体对伤害的反应，通过加速发病和增加 核仁反应的大小，内在的性别差异存在， 面神经再生及雄激素对面神经的影响 再生是性别特异性的。这种神经营养剂 调节再生运动神经元的分子程序将是 这是此次申请的重点。具体目标是测试 五个假设。首先，TP加快了面神经的速率， 通过细胞骨架基因的差异调节再生 轴突切开术后的表达。TP对大鼠血清中 编码轴突再生所必需的蛋白质的细胞骨架mRNA将被 用原位杂交和北方 用适当的细胞骨架cDNA探针进行印迹分析。第二，TP 加速了面部神经的再生速度 轴突运输的慢组分B的速率。立体定位 将标记的氨基酸注射到面部核群中， 以及TP对标记蛋白转运和数量的影响 评估再生轴突节段内的细胞骨架蛋白， 确定面神经损伤后的时间点。第三，TP诱导 加速面神经细胞体对损伤的反应 通过核糖体RNA的快速转录激活发生 基因两种核糖体DNA探针的串联原位杂交 初始转录物（前体）或稳定（产物rRNA）将在 选择损伤后的时间点，以确定是否rRNA基因表达， 轴突切断的HFMN由TP转录调节。第四，性别-- TP对仓鼠面神经再生特异性影响涉及性别 HFMN中雄激素受体mRNA水平和/或调节的差异。在 用雄激素受体mRNA探针进行原位杂交， 确定雄激素受体mRNA水平的内在差异 男性和女性之间的HFMN存在，以及建立是否性别- 受体mRNA水平的特异性调节发生在轴突切断后， 有/没有TP。第五，TP可以以积极的方式改变 含雄激素受体中枢神经系统的再生反应模式 运动神经元rDNA和β Ⅱ-微管蛋白cDNA原位杂交 将使用探针来确定睾丸激素是否会引发 损伤的仓鼠红核脊髓神经元的再生反应模式 与HFMN中观察到的相似。这些研究应该提供信息 与神经元损伤中类固醇作用的机制有关， 关于内在分子程序差异的信息， 损伤的PNS与CNS神经元。

This proposal represents a direct continuation of our ongoing investigation of the therapeutic role of the neurotrophic agent, testosterone propionate (TP), in enhancing nerve regeneration. The model system we have been utilizing is the hamster facial motoneuron (HFMN), a population of peripheral neurons which contain androgen receptors. Accomplishment of the aims of the previous grant has established that TP significantly accelerates the rate of facial nerve regeneration without shortening the delay before sprouting occurs, TP triggers the nerve cell body response to injury by accelerating the onset and increasing the magnitude of the nucleolar reaction, intrinsic sex differences exist in facial nerve regeneration, and the effects of androgens on facial nerve regeneration are sex-specific. How this neurotrophic agent differentially regulates the molecular program of regenerating motoneurons will be the focus of this continuation application. The specific aims are to test the following 5 hypotheses. First, TP accelerates the rate of facial nerve regeneration through differential regulation of cytoskeletal gene expression following axotomy. The effects of TP on the levels of cytoskeletal mRNAs encoding proteins essential for axonal regrowth will be determined in axotomized HFMN using in situ hybridization and northern blot analyses with the appropriate cytoskeletal cDNA probes. Second, TP accelerates the rate of facial nerve regeneration through an acceleration of the rate of slow component b of axonal transport. Stereotaxic injections of labeled amino acids into the facial nuclear groups will be done and the effects of TP on the transport and quantity of labeled cytoskeletal proteins within segments of regenerating axons assessed at defined timepoints after facial nerve injury. Third, TP-induced acceleration of the onset of the facial nerve cell body response to injury occurs through the rapid transcriptional activation of the ribosomal RNA gene. Tandem in situ hybridization with 2 ribosomal DNA probes specific to the initial transcript (precursor) or stable (product rRNA will be done at selected timepoints after injury to determine if rRNA gene expression in axotomized HFMN is transcriptionally regulated by TP. Fourth, the gender- specific effects of TP on hamster facial nerve regeneration involve sex differences in androgen receptor mRNA levels and/or regulation in HFMN. In situ hybridization with androgen receptor mRNA probes will be done to determine if intrinsic differences in androgen receptor mRNA levels between male and female HFMN exist, as well as to establish whether sex- specific regulation of receptor mRNA levels occurs following axotomy, with/without TP. Fifth, TP can alter, in a positive manner, the regenerative response pattern in androgen receptor-containing central motoneurons. In situ hybridization with rDNA and betaII-tubulin cDNA probes will be used to determine if testosterone can trigger a regenerative response pattern in injured hamster rubrospinal neurons similar to that observed in HFMN. These studies should provide information relevant to the mechanism of steroid action in neuronal injury, and new information concerning differences in the intrinsic molecular programs of injured PNS vs. CNS neurons.