项目申请人曾报导大鼠腰骶脊髓节段的内脏神经中枢出现衰老相关的形态学结构：老年相关NADPH-diaphorase(N-d)小体(ANB)。ANB随衰老而累积，并且分布的骶副交感核和背联合核等区域是调控盆腔泌尿生殖器官的中枢。腰骶脊髓节段也是性激素作用的靶区，我们认为老年尿控障碍和便秘等泌尿生殖器官功能障碍及其疾病与ANB相关。预实验结果表明电镜观察相应区域，ANB为神经退行性变，补充雄激素可减缓老年大鼠ANB生成。我们将应用比较组织学验证ANB发生的普遍性，再进行N-d染色结合神经追踪，免疫组化多重标记的普通或双光子共聚焦显微镜观察和免疫电镜研究其形态学特征，以及离体脊髓厚片电生理以及记录后标记，以去势和补充性激素揭示功能相关作用,研究ANB和盆腔脏器功能形态学及其电生理变化。ANB发生机制可成为研究老年泌尿生殖器官功能减退和疾病的新视角。本研究对老年人补充性激素减缓衰老作用提供新的依据。

The project applicant has reported a new finding of age-related NADPH-diaphorase positive body (ANB) located in the lumbosacral spinal cord segments which is a part of the autonomic nervous system and regulates pelvic urogenital organs. Acumulated with aging， ANBs were occured in the region of the sacral parasympathetic nuclei and dorsal commisural nucleus, etc. These nuclei are a part of autonomic nervous system to regulate the urogenital organs. The neurons in the sacral spinal cord innervation of the urogenital organs are also sex hormone dependent. Sex hormones contribute to modulating the functions and morphology of the lumbosacral spinal cord innervated to pelvic viscera throughout the life span. Meanwhile, lumbosacral spinal segment is also a link of neuroendocrine regulation and a region targeted by stress. ANB may be implicated in the neurodegenerative mechanism of the aging dysfunctions of pelvic organs such as dysfunction of micturition control and constipation，and the pathogenesis of its related disorders. Recently, our preliminary experiments of supplemental androgens can attenuate the development of ANB in aged rats and an obvious neurodegeneration was subjected to ANB under electron microscopy. The comparative histological study will be conducted to identify ANB consistently developing during aging in different experimental animals. Staining of N-d and Immunocytochemistry combined with neural tracing and multiple labeling will also be examined under confocal microscopy and/ or two photon microscopy and its morphological characteristics will be studied with immuno-electron microscopy study. To test the effect of sex hormones, the rats will be divided into castration and replacement sex hormones group. The effects of exogenous acetylcholine or carbachol on the smooth muscles of urinary bladder, distal colon and bulbospongiosus muscle will be tested by means of morphological and functional approaches in aging, castration and age-matched rats with or without androgen replacement. Intracellular recording and whole cell patch clamp will be used in a slice of spinal cord and the cells will be labelled by neuronal marker after recording. The mechanism of ANB development can be a new perspective to study urogenital organ dysfunction and disease with aging. We hypothesize that ANB could be an adequate pathogenesis marker in an experimental aging model. These investigations will suggest a novel theory for aging neurodegeneration of the lumbosacral spinal cord and new strategy of treatment.