【作者】 张国梁；

【导师】 石葛明；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2011， 博士

【摘要】 衰老过程中男性个体雄激素水平多呈现不同程度的降低,其运动及探索行为逐渐减弱、老年后常表现出平衡及运动协调的障碍。研究发现雄激素参与某些神经精神疾病的神经病理生理改变。伴有睾酮缺乏的帕金森病患者在睾酮替代治疗后,其静止性震颤及精细运动调控均得到显著改善,而且帕金森病运动症状的改善程度与血清睾酮水平有关;帕金森病患者皮下埋置睾酮能够缓解与睾酮缺乏相似的帕金森病非运动症状。目前针对睾酮缺乏所致相关疾病的睾酮替代治疗主要采用口服、肌肉注射及皮下植入(埋置)缓释囊等方法。但这些给药途径存在一定的缺点,口服睾酮制剂常因首过效应而降低睾酮的生物利用度;肌肉注射存在一定的伤害,会造成病人的一定痛苦,特别是需长期的睾酮替代治疗时;植入或皮下埋植法患者须经历小的手术创伤,且常发生植入缓释囊的膨出或因皮下植入非囊性斑块制剂导致患者的皮肤刺激症状。理想的睾酮替代治疗应提供安全、有效、经济、方便、剂量容易掌握且容易达到有效的血液生物利用度;如果期望睾酮作用于脑,改善脑的功能活动,则期望睾酮进入脑的同时,不引起或较少引起周围的副作用。作为呼吸系统一部分的鼻腔其被覆粘膜包括呼吸部和嗅部两部分。嗅部的嗅细胞属神经细胞则通过嗅丝与颅内的嗅脑相联系。由于鼻腔的粘膜特性,经鼻途径也成为近年某些药物制剂首选的给药方法。因此,本研究以健康雄性、去势(雄激素缺乏)及老年雄性大鼠为实验动物模型,通过分析大鼠行为的变化,探讨鼻腔给予雄激素对大鼠中枢神经系统的影响,并通过免疫细胞化学、免疫印迹以及高效液相色谱法探讨与行为密切相关的中脑多巴胺能神经元及5-HT能神经元所发挥的作用,期望所获结果为以鼻腔给予睾酮(Testosterone, T)或丙酸睾丸酮(Testosterone propionate, TP)治疗中枢神经系统相关疾病提供实验依据。第一部分:鼻腔给予T/TP对大鼠相关脑区c-Fos表达的影响目的:探讨鼻腔给予T/TP是否能够引起大鼠中枢神经系统c-Fos的表达、激活不同的脑区。方法:利用放射免疫分析法检测大鼠鼻腔给予TP后脑脊液和血清睾酮浓度的变化;以免疫细胞化学方法显示大鼠各脑区c-Fos的表达。结果:1.放射免疫分析法结果显示:去势组大鼠脑脊液和血清中睾酮含量比正常组分别降低了45%(P<0.01)和69%(P<0.01);去势大鼠皮下注射TP后只增加了血清中睾酮的含量;去势大鼠鼻腔给予TP后脑脊液和血清中睾酮的含量均明显增加,脑脊液中睾酮的含量明显高于去势皮下注射TP组(GDX-sc.TP)(P<0.01),而血清中的含量明显低于GDX-sc.TP组(P<0.05);与Intact组大鼠相比,正常大鼠鼻腔给予TP后也显著增加脑脊液和血清中睾酮的含量。2.免疫细胞化学结果显示:正常大鼠鼻腔给予TP或T增加嗅结节(Olfactory tubercle,Tu)、边缘前皮质(Prelimbic cortex, PrL)、运动皮质(Motor cortex, M1,M2)、扣带回(Cingulate cortex, Cg)、尾壳核背内侧(Dorsomedial caudate putamen, CPu-DM)、尾壳核背外侧(Dorsolateral caudate putamen, CPu-DL)、伏核(Accumbens nucleus, Acb)、下丘脑后区(Posterior hypothalamic area, PH)、黑质外侧部(Substantia nigra, lateral part, SNL)、中缝背核(Dorsal raphe nucleus, DR)的c-Fos免疫反应阳性神经元数目和嗅球(Olfactory bulb, OB)、Tu、PrL、M1、M2、Cg、CPu-DM、CPu-DL、Acb、海马(Hippocampus, HIP)、PH、SNL和DR的c-Fos免疫反应强度,而皮下注射TP只增加了Tu、PrL、CPu-DL和Acb脑区c-Fos免疫反应阳性神经元数目以及OB和Tu脑区的c-Fos免疫反应强度。结论:1.雄性大鼠去势后脑脊液和血清中睾酮的浓度明显降低;去势大鼠皮下注射TP仅增加血清中睾酮的浓度;去势大鼠和正常大鼠鼻腔给予TP后均能够增加脑脊液和血清中睾酮的浓度。2.皮下注射TP仅激活了少数脑区c-Fos蛋白的表达;鼻腔给予TP或T后能够激活多数脑区c-Fos蛋白的表达。第二部分:鼻腔给予睾酮改变大鼠的旷场行为目的:观察大鼠长期鼻腔给予睾酮后的旷场行为变化,推测鼻腔给予睾酮对中枢神经系统的影响。方法:通过旷场实验观察大鼠鼻腔给予睾酮后静止闻嗅行为、探索行为、趋触性行为、运动行为和理毛行为的变化。结果:1.正常大鼠鼻腔给予睾酮后静止闻嗅次数比intact增加了46%(P<0.01)。雄性大鼠去势后静止闻嗅次数比sham组显著降低44%(P<0.01),去势大鼠鼻腔给予睾酮后明显增加静止闻嗅次数(P<0.01),并且超过sham组水平88%(P<0.01)。2.正常大鼠大鼠鼻腔给予睾酮后walking、climbing、rearing和sniffing行为次数比intact组分别增加了82%(P<0.01)、54%(P<0.05)、85%(P<0.01)和65%(P<0.01)。雄性大鼠去势后四种行为次数比sham组分别降低了70%(P<0.01)、57%(P<0.01)、68%(P<0.01)和62%(P<0.01);去势大鼠鼻腔给予睾酮后能够恢复walking、climbing和sniffing三种行为到sham组水平,而Rearing的次数尽管比GDX组增加了68%(P>0.05),仍比sham组低47%(P<0.05)。3.正常大鼠鼻腔给予睾酮后垂直运动、水平运动和总径长比intact组分别增加了61%(P<0.01)、69%(P<0.05)和75%(P<0.01)。雄性大鼠去势后三种行为数量比sham组分别降低了60%(P<0.01)、60%(P<0.01)和59%(P<0.01);去势大鼠鼻腔给予睾酮后能够恢复三种行为到sham组水平。4.正常组大鼠鼻腔给予睾酮后理毛数量和理毛持续时间比intact组分别增加了72%(P<0.01)和61%(P<0.01)。雄性大鼠去势后理毛数量和理毛持续时间比sham组分别降低了54%(P<0.01)和55%(P<0.01),去势大鼠鼻腔给予睾酮后理毛数量和理毛持续时间恢复到sham组水平。5.去势大鼠皮下注射睾酮后只恢复了walking、水平运动和总径长到假手术皮下注射安慰剂(sham-sc)水平。结论:1.去势降低雄性大鼠的旷场行为。2.长期鼻腔给予睾酮增加正常大鼠和去势大鼠的旷场行为。3.鼻腔给予睾酮改善去势大鼠旷场行为的效果明显优于皮下注射。第三部分:鼻腔给予睾酮增加大鼠黑质-尾壳核、腹侧被盖区-伏核DA能神经元的功能活动目的:探讨长期鼻腔给予睾酮对黑质-尾壳核、腹侧被盖区-伏核DA能神经元的影响。方法:通过免疫细胞化学方法和免疫印记方法显示酪氨酸羟化酶(TH)和多巴胺转运体(DAT)的表达;利用HPLC法检测DA及其代谢产物二羟基苯乙酸(DOPAC)和高香草酸(HVA)的含量。结果:1.免疫细胞化学方法发现:1)正常大鼠鼻腔给予睾酮后SN的TH表达比intact组增加7%(P<0.05)、CPu的DM、VM、VL和DL四个亚群分别增加了27%(P<0.01)、30%(P<0.01)、17%(P<0.01)和18%(P<0.01)、VTA增加了5%(P<0.05)、Acb的Core和Shell两个亚群分别增加了18%(P<0.01)和15%(P<0.01);雄性大鼠去势后SN的TH表达比sham组降低13%(P<0.05)、CPu的DM、VM、VL和DL四个亚群分别降低18%(P<0.05)、16%(P<0.05)、11%(P<0.05)和14%(P<0.05),VTA降低了26%(P<0.05)、Acb的Core和Shell两个亚群分别降低17%(P<0.01)和16%(P<0.05);去势大鼠鼻腔给予睾酮后恢复上述脑区TH的表达到sham组水平,其中Acb的Core亚群TH的表达超过sham组水平(P<0.01)。2)正常大鼠鼻腔给予睾酮SN的DAT表达比intact组增加7%(P<0.01)、CPu的DM、VM、VL和DL四个亚群分别增加了27%(P<0.01)、23%(P<0.05)、43%(P<0.01)和22%(P<0.05)、VTA增加了10%(P<0.01)、Acb的Core和Shell两个亚群分别增加了21%(P<0.01)和13%(P<0.05);雄性大鼠去势后SN的DAT表达比sham组降低13%(P<0.01)、CPu的DM、VM、VL和DL四个亚群分别降低15%(P<0.05)、16%(P<0.05)、15%(P<0.05)和15%(P<0.01)、VTA降低了15%(P<0.05)、Acb的Core和Shell两个亚群分别降低18%(P<0.01)和17%(P<0.05);去势大鼠鼻腔给予睾酮后恢复上述脑区DAT的表达到sham组水平。3)去势大鼠皮下注射睾酮后只恢复了SN和VTA的DAT表达。2.免疫印记方法发现:1)正常大鼠鼻腔给予睾酮后SN、CPu、VTA和Acb脑区TH的表达比intact组分别增加123%(P<0.01)、8%(P<0.01),27%(P<0.01)和51%(P<0.01);雄性大鼠去势后上述四个脑区TH的表达比sham组分别降低39%(P<0.01)、11%(P<0.01)、35%(P<0.01)和43%(P<0.01);去势大鼠鼻腔给予睾酮后增加了上述脑区TH的表达。2)DAT的表达显示两个条带,一条位于80KDa处为成熟糖基化DAT(Glycosylated-DAT),另一条位于50KDa处为未成熟的非糖基化DAT(Non-Glycosylated-DAT)。正常大鼠鼻腔给予睾酮增加了SN两种DAT的表达比intact组分别增加了14%(P<0.01)和10%(P<0.01)、CPu分别增加了40%(P<0.01)和11%(P<0.01)、Acb分别增加了22%(P<0.01)和10%(P<0.05)、VTA糖基化DAT增加了53%(P<0.01);雄性大鼠去势后SN两种DAT的表达比sham组分别降低了20%(P<0.01)和23%(P<0.01)、VTA分别降低了58%(P<0.01)和23%(P<0.01)、Acb分别降低了51%(P<0.01)和17%(P<0.01)、CPu非糖基化DAT降低了12%(P<0.05);去势大鼠鼻腔给予睾酮后增加了上述脑区降低的两种DAT的表达。3)去势大鼠皮下注射睾酮后只增加了SN的TH(P<0.01)、CPu糖基化DAT(P<0.01)和非糖基化DAT(P<0.01)的表达。3. HPLC检测发现,正常大鼠鼻腔给予睾酮后SN和CPu的DA含量无明显变化(P>0.05),CPu的DOPAC和HVA的含量以及DOPAC+HVA/DA的比值比Intact组分别增加了38%(P<0.01)、60%(P<0.01)和35%(P<0.05);雄性大鼠去势后SN和CPu的DA含量,CPu的DA代谢产物和代谢率无明显变化(P>0.05),去势大鼠鼻腔给予睾酮后CPu的HVA的含量比GDX组增加了23%(P<0.05),DOPAC和HVA的含量分别超过Sham组水平的18%(P<0.01)和51%(P<0.01)。结论:1.大鼠去势显著降低了大鼠SN、VTA、CPu和Acb四个脑区TH和DAT的表达。2.长期鼻腔给予睾酮能够增加正常大鼠和去势大鼠SN、VTA、CPu和Acb四个脑区TH和DAT的表达,增加了DA能神经元的代谢。3.睾酮改善去势所致中脑DA能神经元功能活动降低的效果,鼻腔途径优于皮下注射。第四部分:鼻腔给予睾酮增加大鼠中脑5-羟色胺能神经元的功能活动目的:探讨长期鼻腔给予睾酮对中缝背核(DR)5-HT能神经元的影响。方法:以免疫细胞化学方法显示大鼠中缝背核TPH、5-HT和SERT的表达;利用HPLC方法检测DR、CPu和Acb脑区5-HT及其代谢产物五羟吲哚乙酸(5-HIAA)的含量。结果:1.免疫细胞化学方法发现:1)正常大鼠鼻腔给予睾酮后中缝背核TPH、5-HT和SERT的表达比intact组分别增加3%(P<0.05)、9%(P<0.05)和15%(P<0.01);雄性大鼠去势后DR上述三种物质的表达比sham组分别降低21%(P<0.01)、22%(P<0.01)和29%(P<0.05);去势大鼠鼻腔给予睾酮后恢复DR的TPH、5-HT和SERT表达至sham组水平,其中SERT的表达超过sham组水平(P<0.05)。2)去势大鼠皮下注射睾酮后增加了TPH、5-HT和SERT的表达,但仍低于sham-sc组水平。2. HPLC法发现:正常大鼠鼻腔给予睾酮后DR的5-HT含量比intact组增加32%(P<0.01),CPu的5-HIAA和5-HIAA/5-HT比值分别增加了27%(P<0.05)和26%(P<0.05),Acb的5-HIAA和5-HIAA/5-HT比值分别增加了29%(P<0.05)和28%(P<0.05);雄性大鼠去势后DR、CPu和Acb的5-HT含量比Sham组分别降低了26%(P<0.05)、26%(P<0.05)和12%(P<0.05),CPu和Acb的5-HIAA含量分别降低了13%(P<0.05)和23%(P<0.05);去势大鼠鼻腔给予睾酮后能够恢复上述脑区各指标到sham组水平。结论:1.去势降低雄性大鼠中缝背核TPH、5-HT和SERT的表达。2.长期鼻腔给予睾酮增加大鼠中缝背核TPH、5-HT和SERT的表达,其效果优于皮下注射。3.去势降低雄性大鼠DR、CPu和Acb的5-HT及其代谢产物的含量,长期鼻腔给予睾酮逆转上述改变。第五部分:鼻腔给予丙酸睾丸酮对老年大鼠行为及其脑内DA和5-HT能神经元的影响目的:观察长期鼻腔给予TP后老年大鼠旷场行为、平衡反应能力、肌张力和运动协调能力的变化,以及对脑内DA和5-HT能神经元的影响,期望所获结果为鼻腔给予睾酮(或TP)治疗中枢神经系统相关疾病提供实验依据。方法:利用旷场实验、倾斜面实验、水平绳实验和粘附物去除实验观察大鼠的行为;以免疫细胞化学和免疫印迹显示TH、DAT、TPH、5-HT、SERT的表达。结果:1.旷场实验发现,老年24月龄大鼠的静止闻嗅次数、Walking、Climbing、Rearing、Sniffing、垂直运动、水平运动、总径长、理毛数量和理毛持续时间比6Mon组分别降低了39%(P<0.01)、55%(P<0.01)、39%(P<0.01)、74%(P<0.01)、53%(P<0.01)、53%(P<0.05)、58%(P<0.01)、58%(P<0.01)、56%(P<0.01)和57%(P<0.01),理毛潜伏期比6Mon组延长了506%(P<0.01);老年大鼠长期鼻腔给予TP后能够增加或恢复上述降低的行为,缩短理毛潜伏期时间。2.倾斜面实验发现,老年24月龄大鼠的倾斜面下滑角度比6月龄组降低了27%(P<0.05),50°夹角滑下次数增加了660%(P<0.01);老年大鼠鼻腔给予TP后,增加了倾斜面下滑的角度,减少了50°角滑下次数。3.水平绳实验发现,老年24月龄大鼠的悬挂时间比6月龄大鼠缩短69%(P<0.01);老年大鼠鼻腔给予TP后延长了悬挂时间。4.粘附物去除实验发现,老年24月龄大鼠的左鼻和右鼻粘附物去除时间比6月龄大鼠分别延长了435%(P<0.05)和656%(P<0.05);老年大鼠鼻腔给予TP后左鼻和右鼻粘附物去除时间恢复到6月龄大鼠水平。5.免疫细胞化学方法发现:1)老年24月龄大鼠SN的TH表达比6月龄组降低了22%(P<0.05)、CPu的DM、VM、VL和DL四个亚群分别降低21%(P<0.05)、25%(P<0.05)、20%(P<0.05)和24%(P<0.01)、VTA降低了12%(P<0.05)、Acb的Core和Shell两个亚群分别降低20%(P<0.05)和15%(P<0.01),老年大鼠鼻腔给予TP后增加了上述脑区TH的表达。2)老年24月龄大鼠SN的DAT表达比6月龄组降低了10%(P<0.05)、CPu的DM、VM、VL和DL四个亚群分别降低17%(P<0.05)、16%(P<0.05)、15%(P<0.05)和17%(P<0.05)、VTA降低了9%(P<0.05)、Acb的Core和Shell两个亚群分别降低19%(P<0.05)和17%(P<0.01);老年大鼠鼻腔给予TP后增加了SN、CPu的DM、VL和DL三个亚群,VTA和Acb的Shell亚群DAT的表达。3)老年24月龄大鼠DR的TPH、5-HT和SERT表达比6月龄组分别降低10%(P<0.05)、14%(P<0.01)和12%(P<0.01);老年大鼠鼻腔给予TP后增加TPH、5-HT和SERT的表达。6.免疫印记方法发现:1)老年24月龄大鼠SN、CPu、VTA和Acb四个脑区TH的表达比6月龄大鼠分别降低25%(P<0.01)、18%(P<0.01)、41%(P<0.01)和24%(P<0.01);老年大鼠鼻腔给予TP后增加了上述脑区TH的表达。2)老年24月龄大鼠SN的糖基化DAT和非糖基化DAT的表达比6月龄大鼠分别降低10%(P<0.05)和40%(P<0.01)、CPu分别降低了36%(P<0.01)和16%(P<0.05)、VTA分别降低了70%(P<0.01)和10%(P<0.05)、Acb分别降低了12%(P<0.05)和14%(P<0.01);老年大鼠鼻腔给予TP后SN、CPu和VTA非糖基化的DAT恢复到6Mon组水平。结论:1. 24月龄大鼠的旷场行为、平衡反应能力、肌张力和运动协调能力比6月龄明显降低;老年大鼠长期鼻腔给予TP后能够改善上述行为障碍。2. 24月龄大鼠黑质-尾壳核和腹侧被盖区-伏核DA能神经元TH和DAT的表达、以及中缝背核TPH、5-HT和SERT的表达明显降低;老年大鼠长期鼻腔给予TP后能够改善上述脑区各物质的表达。更多还原

【Abstract】 In the senile process, testosterone (T) level decreased gradually and the motor behavior and exploratory behavior were impaired in aged male organisms. Aged men or aged male rats often displayed the disturbances of the body balance and motor coordination. Testosterone is involved in the neuropathophysiology of some neuropsychiatric disorders. The resting tremor and fine motor control were significantly improved after testosterone administration in a parkinsonian with testosterone deficiency. The improvement in motor symptoms correlated with serum testosterone levels. Nonmotor symptoms of Parkinson disease, which are virtually identical to the testosterone deficiency symptoms, were ameliorated by transdermal testosterone gel of a daily dose.Currently, testosterone replacement therapies include oral therapy, intramuscular injections and subcutaneous testosterone implants. The administration routes above have been shown some disadvantages. Orally administered testosterone has lower bioavailability because of first-pass metabolism. Intramuscular injections are painful and inappropriate to be used, especially in long-term testosterone replacement. The insertion of T implants requires minor surgery and can be painful, and extrusion of the pellets is common. Transdermal testosterone non-scrotal patches often result in skin irritation in patients.The ideal testosterone replacement therapy should offer safety, efficacy, cost-effectiveness, convenience, a good release profile, dosing flexibility, and effective normalization of T levels. Targeting testosterone to the central nervous system should allow the administration of lower doses of testosterone and fewer peripheral side effects.Nasal cavity was a part of respiratory system. The lining mucosa of nasal cavity includes respiratory region and olfactory region. The olfactory cells of olfactory region belong to nerve cells, which contact with rhinencephalon through fila olfactoria. Based on the mucous membrane characteristics, intranasal administration of drugs has become preferred method in recent year.Therefore, the intact male rats, gonadectomized (GDX) rats and aged male rats were used as experimental animal models in present study. The influence of intranasal administration of T upon the central nervous system was studied by analyzing the open field behavior of rats after long-term intranasal administration of T and the effects of intranasal delivery of T on mensencephalic dopaminergic and serotoninergic neurons were analyzed by immunocytochemistry, immunoblotting and HPLC. It is hoped that intranasal administration of T/TP could be used in adjutant therapy for some CNS diseases.Prat 1: Effects of intranasal administration of T/TP on c-Fos in rat brainObjective: To study whether the intranasal administration of T/TP could induce the expression of c-Fos in rat brain and activate different brain regions.Methods: Radioimmunoassay (RIA) was used to detect testosterone concentration in cerebrospinal fluid and in serum after intranasal administration of TP. Immunocytochemistry was used to detect the expression of c-Fos protein in different brain regions.Results: 1. RIA: Testosterone in cerebrospinal fluid and in serum was decreased by 45% (P<0.01) and 69% (P<0.01) respectively in GDX rats compared to intact rats. Subcutaneous injection of TP in GDX rats (GDX-sc.TP) only increased testosterone in serum. Testosterone in cerebrospinal fluid and in serum was increased after intranasal administration of TP in GDX rats (GDX-TP). Testosterone in cerebrospinal fluid of GDX-TP rats was higher than that in GDX-sc.TP rats (P<0.01), however testosterone in serum of GDX-TP rats was lower than that in GDX-sc.TP rats (P<0.05). Intranasal administration of TP in intact rats also increased testosterone in cerebrospinal fluid and in serum compared to intact rats. 2. Immunocytochemistry: After intranasal administration of TP or testosterone, the number of c-Fos-positive neurons was increased in Tu, PrL, M1,M2, Cg, CPu-DM, CPu-DL, Acb, PH, SNL and DR brain regions and the c-Fos immunoreactive intensity was enhanced in OB, Tu, PrL, M1,M2, Cg, CPu-DM, CPu-DL, Acb, HIP, PH, SNL and DR brain regions. However, after subcutaneous injection of TP, the number of c-Fos-positive neurons was increased only in Tu, PrL, CPu-DL and Acb brain regions and the c-Fos immunoreactive intensity was enhanced only in OB and Tu.Conclusions: 1. Testosterone in cerebrospinal fluid and in serum was significantly decreased after GDX. Subcutaneous injection of TP in GDX rats only increased the testosterone in serum. Testosterone in cerebrospinal fluid and in serum was increased after intranasal administration of TP in intact rats and GDX rats. 2. The expression of c-Fos was incresead only in a few brain regions after subcutaneous injection of TP, however, more brain regions were activated after intranasal administration of TP or testosterone.Prat 2: Behavior in open field test was affected after intranasal administration of testosterone to ratsObjective: To study the effects of long-term intranasal administration of testosterone on the central nervous system by analyzing the open field behavior of rats.Methods: The open field test was used to analyze five types of behavioral patterns in present study: immobile-sniffing, exploratory behavior, thigmotaxic behavior, motor behavior and grooming behavior.Results: 1. Immobile-sniffing: The intranasal administration of testosterone in intact rats significantly increased the number of immobile-sniffing events by 46% (P<0.01). Gonadectomy decreased the immobile-sniffing events, with a 44% (P<0.01) reduction and the intranasal administration of testosterone in GDX rats significantly increased the number of immobile-sniffing events (P<0.01), which was even more than in sham rats by 88% (P<0.01). 2. Exploratory behavior: The intranasal administration of testosterone significantly increased the amount of walking, climbing, rearing and sniffing in intact rats by 82% (P<0.01), 54% (P<0.05), 85% (P<0.01) and 65% (P<0.01) respectively. Gonadectomy decreased the amount of above four behaviors by 70% (P<0.01), 57% (P<0.01), 68% (P<0.01) and 62% (P<0.01) respectively and that the amount of above behaviors in GDX rats was restored to the level of sham rats after intranasal administration of testosterone, except for the rearing. The amount of rearing in GDX-T rats was still lower than in the sham rats by 47% (P<0.05), even though the amount was increased 68% (P>0.05) compared to GDX rats. 3. Motor behavior: The intranasal administration of T significantly increased the amount of vertical activity and horizontal activity as well as the total path length in intact rats by 61% (P<0.01), 69% (P<0.05) and 75% (P<0.01) respectively. The amount of vertical activity and horizontal activity in GDX rats was significantly lower than in the sham rats by 60% (P<0.01) and 60% (P<0.01) respectively, the total path length in GDX rats was significantly less than in the sham rats by 59% (P<0.01) and the amount of vertical activity and horizontal activity as well as the total path length in GDX rats were restored to the level of sham rats after intranasal administration of testosterone. 4. Grooming behavior: The intranasal administration of T significantly increased the number of grooming events and the duration of grooming in intact rats by 72% (P<0.01) and 61% (P<0.01) respectively. The number of grooming events in GDX rats was significantly less than in the sham rats by 54% (P<0.01), the duration of grooming in GDX rats was significantly shorter than in the sham rats by 55% (P<0.01) and the number of grooming events as well as the duration of grooming in GDX rats was restored to the level of sham rats after intranasal administration of testosterone. 5. Subcutaneous injection results: The amount of walking and horizontal activity, the total path length was only restored to the level of sham-sc rats after subcutaneous injection of testosterone. Conclusions: 1. Significant decreases in open field activity were observed in GDX rats. 2. The open field activity scores was significantly increased in GDX rats and in intact rats compared with the corresponding GDX rats and intact rats after intranasal administration of testosterone. 3. Intranasal route improved the impaired behaviors better than subcutaneous injection.Prat 3: Intranasal administration of testosterone increased the dopaminergic activity in SN-CPu and VTA-Acb neurons of ratsObjective: To sduty the effects of long-term intranasal administration of testosterone on dopaminergic neurons in SN-CPu and VTA-Acb of rats.Methods: Immunohistochemistry and immunoblotting were used to detect the expression of TH and DAT. HPLC was used to detect DA and its metabolites DOPAC and HVA.Results: 1. Immunocytochemistry: 1) The intranasal administration of testosterone significantly increased the expression of TH in intact rats by 7% (P<0.05) in SN, increased by 27% (P<0.01), 30% (P<0.01), 17% (P<0.01) and 18% (P<0.01) in dorsomedial CPu (CPu-DM), ventromedial CPu (CPu-VM), ventrolateral CPu (CPu-VL) and dorsolateral CPu (CPu-DL) respectively, increased by 5% (P<0.05) in VTA, increased by 18% (P<0.01) and 15% (P<0.01) in core and shell of Acb respectively. Gonadectomy decreased the expression of TH above eight brain regions by 13% (P<0.05), 18% (P<0.05), 16% (P<0.05), 11% (P<0.05), 14% (P<0.05), 26% (P<0.05), 17% (P<0.01) and 16% (P<0.05) and that the expression of TH above eight brain regions in GDX rats was restored to the level of sham rats after intranasal administration of testosterone, even though the expression of TH was higher than sham rats (P<0.01). 2) The intranasal administration of testosterone significantly increased the expression of DAT in intact rats by 7% (P<0.01) in SN, increased by 27% (P<0.01), 23% P<0.05), 43% (P<0.01) and 22% (P<0.01) in CPu-DM, CPu-VM, CPu-VL and CPu-DL respectively, increased by 10% (P<0.01) in VTA, increased by 21% (P<0.01) and 13% (P<0.05) in core and shell of Acb respectively. Gonadectomy decreased the expression of DAT above eight brain regions by 13% (P<0.01), 15% (P<0.05), 16% (P<0.05), 15% (P<0.05), 15% (P<0.01), 15% (P<0.05), 18% (P<0.01) and 17% (P<0.05) and that the expression of DAT above eight brain regions in GDX rats was restored to the level of sham rats after intranasal administration of testosterone. 3) The expression of DAT in SN and VTA was only restored to the level of sham-sc rats after subcutaneous injection of testosterone. The expression of TH in CPu-DL and VTA and the expression of DAT in CPu-DM, CPu-DL and shell of Acb were improved after subcutaneous injection of testosterone. 2. Immunoblotting: 1) The intranasal administration of testosterone significantly increased the expression of TH in intact rats by 123% (P<0.01), 8% (P<0.01), 27% (P<0.01) and 51% (P<0.01) in SN, CPu, VTA and Acb respectively. Gonadectomy decreased the expression of TH above four brain regions by 39% (P<0.01), 11% (P<0.01), 35% (P<0.01) and 43% (P<0.01) and that the expression of TH above four brain regions in GDX rats was improved after intranasal administration of testosterone. 2) DAT bands included Glycosylated-DAT (80KDa) and Non-Glycosylated-DAT (50KDa). The intranasal administration of testosterone significantly increased the expression of two kinds of DAT in intact rats by 14% (P<0.01) and 10% (P<0.01) in SN respectively, increased by 40% (P<0.01) and 11% (P<0.01) in CPu respectively, increased by 22% (P<0.01) and 10% (P<0.05) in Acb respectively, the expression of Glycosylated-DAT increased by 53% in VTA (P<0.01). Gonadectomy decreased the expression of two kinds of DAT by 20% (P<0.01) and 23% (P<0.01) in SN respectively, decreased by 58% (P<0.01) and 23% (P<0.01) in VTA respectively, decreased by 51% (P<0.01) and 17% (P<0.01) in Acb respectively, the expression of Non-Glycosylated-DAT decreased by 12% (P<0.05) in CPu. The expression of DAT above four brain regions in GDX rats was improved after intranasal administration of testosterone. 3) The expression of TH in SN and the expression of two kinds of DAT in CPu were only improved after subcutaneous injection of testosterone. 3. HPLC: There are no significant differences on DA concentration in SN and CPu after intranasal administration of testosterone (P>0.05). The intranasal administration of testosterone significantly increased the concentration of DOPAC and HVA by 38% (P<0.01) and 60% (P<0.01) in CPu respectively, increased the ratio of DOPAC+HVA/DA by 35% (P<0.05) in CPu. There are no significant differences on DA concentration in SN and CPu, and the metabolites and metabolic rate in CPu after gonadectomy (P>0.05). The intranasal administration of testosterone in GDX rat increased the concentration of HVA by 23% (P<0.05) in CPu, the concentration of DOPAC and HVA increased by 18% (P<0.01) and 51% (P<0.01) compared to sham group respectively.Conclusions: 1. The expression of TH and DAT was significantly decreased in SN, CPu, VTA and Acb in GDX rats. 2. Long-term intranasal administration of testosterone significantly increased both the expression of TH and DAT in SN, CPu, VTA and Acb and the metabolites of dopaminergic neurons in GDX rats and Intact rats. 3. Intranasal route improved the reduced dopaminergic neurons activity in SN-CPu and VTA-Acb better than subcutaneous injection.Prat 4: Intranasal administration of testosterone increased the serotoninergic activity in dorsal raphe nucleus of rat.Objective: To study the effects of long-term intranasal administration of testosterone on the serotoninergic neurons in DR of rats.Methods: Immunohistochemistry was used to detect the expression of TPH, 5-HT and SERT in DR. HPLC was used to detect 5-HT and its metabolites 5-HIAA in DR, CPu and Acb.Results: 1. Immunocytochemistry: 1) The intranasal administration of testosterone significantly increased the expression of TPH, 5-HT and SERT in intact rats by 3% (P<0.05), 9% (P<0.05) and 15% (P<0.01) in DR respectively. Gonadectomy decreased the expression of TPH, 5-HT and SERT by 21% (P<0.05), 22% (P<0.01) and 29% (P<0.05) compared to sham rats and that the expression of TPH, 5-HT and SERT in GDX rats was restored to the level of sham rats after intranasal administration of testosterone, and the expression of SERT was even more than in sham rats (P<0.05). 2) The expression of TPH, 5-HT and SERT were improved, however, which were lower compared to sham-sc rats after subcutaneous injection of testosterone. 2. HPLC: The intranasal administration of testosterone significantly increased the concentration of 5-HT by 32% (P<0.01) in DR, increased the concentration of 5-HIAA and the ratio of 5-HIAA/5-HT by 27% (P<0.05) and 26% (P<0.05) in CPu respectively, increased the concentration of 5-HIAA and the ratio of 5-HIAA/5-HT by 29% (P<0.05) and 28% (P<0.05) in Acb respectively. Gonadectomy decreased the concentration of 5-HT by 26% (P<0.05), 26% (P<0.05) and 12% (P<0.05) in DR, CPu and Acb respectively, decreased the concentration of 5-HIAA by 13% (P<0.05) and 23% (P<0.05) in CPu and Acb respectively compared to sham rats and that were restored to the level of sham rats after intranasal administration of testosterone.Conclusions: 1. The expression of TPH, 5-HT and SERT was significantly decreased in DR of GDX rats. 2. Long-term intranasal administration of testosterone significantly increased the expression of TPH, 5-HT and SERT in DR of GDX rats and intact rats. Intranasal route improved the decreased serotoninergic neurons activity better than subcutaneous injection. 3. The concentration of 5-HT and 5-HIAA was significantly reduced in DR, CPu and Acb in GDX rats, and long-term intranasal administration of testosterone could restore them to sham level. Prat 5: Influence of intranasal administration of TP upon the behaviors, dopaminergic and serotoninergic neurons in aged ratsObjective: To study the influence of long-term intranasal administration of TP on behaviors and dopaminergic and serotoninergic neurons in aged rats.Methods: Open field test, tilting-plane test, horizontal-wire test and adhesive removal test were used to detect behaviors. Immunohistochemistry and immunoblotting were used to detect the expression of TH, DAT, TPH, 5-HT and SERT.Results: 1. Open field test: The amount of immobile-sniffing events, walking,climbing,rearing,sniffing, vertical activity, horizontal activity, total path length, number of grooming events and the duration of grooming were decreased in 24Mon rats by 39% (P<0.01), 55% (P<0.01), 39% (P<0.01), 74% (P<0.01), 53% (P<0.01), 53% (P<0.05), 58% (P<0.01), 58% (P<0.01), 56%(P<0.01) and 57% (P<0.01) respectively, and the latency of grooming were increased by 506% (P<0.01) compared to 6Mon rats. All behaviors above were significantly improved after intranasal administration of TP compared to 24Mon rats. 2. Tilting-plane test: The angle of sliding off decreased by 27% (P<0.05) and the number of sliding off at 50°angle increased by 660% (P<0.01) in 24Mon rats compared to 6Mon rats. Intranasal administration of TP increased the angle of sliding off and decreased the number of sliding off at 50°angle. 3. Horizontal-wire test: The duration time of hanging was shorter by 69% (P<0.01) in 24Mon rats compared to 6Mon rats and intranasal administration of TP improved the duration time of hanging compared to 24Mon rats. 4. Adhesive removal test: The latency to remove adhesive paper on left nose and right nose increased by 435% (P<0.05) and 656% (P<0.05) in 24Mon rats compared to 6Mon rats respectively and the latency to remove adhesive paper were restored to the level of 6Mon rats after intranasal administration of TP. 5. Immunocytochemistry: 1) The expression of TH was decreased by 22% (P<0.05) in SN, 21% (P<0.05), 25% (P<0.05), 20% (P<0.05) and 24% (P<0.01) in CPu-DM, CPu-VM, CPu-VL and CPu-DL respectively, 12% (P<0.05) in VTA, 20% (P<0.05) and 15% (P<0.01) in core and shell of Acb respectively in 24Mon rats compared to 6Mon rats. Intranasal administration of TP improved the expression of TH in brain regions above. 2) The expression of DAT was decreased by 10% (P<0.05) in SN, 17% (P<0.05), 16% (P<0.05), 15% (P<0.05) and 17% (P<0.05) in CPu-DM, CPu-VM, CPu-VL and CPu-DL respectively, 9% (P<0.05) in VTA, 19% (P<0.05) and 17% (P<0.01) in core and shell of Acb respectively in 24Mon rats compared to 6Mon rats. Intranasal administration of TP improved the expression of DAT in brain regions above except for CPu-VM. 3) The expression of TPH, 5-HT and SERT was decreased by 10% (P<0.05), 14% (P<0.01) and 12% (P<0.01) in 24Mon rats compared to 6Mon rats respectively. Intranasal administration of TP improved the expression of TPH, 5-HT and SERT. 6. Immunoblotting: 1) The expression of TH was decreased by 25% (P<0.01), 18% (P<0.01) 41% (P<0.01) and 24% (P<0.01) in SN, CPu, VTA and Acb in 24Mon rats compared to 6Mon rats respectively and intranasal administration of TP improved the expression of TH four brain regions above. 2) The expression of two kinds of DAT were decreased by 10% (P<0.05) and 40% (P<0.01) in SN respectively, 36% (P<0.01) and 16% (P<0.05) in CPu respectively, 70% (P<0.01) and 10% (P<0.05) in VTA respectively, 12% (P<0.05) and 14% (P<0.01) in Acb respectively. The expression of Non-Glycosylated-DAT in SN, CPu and VTA were restored after long-term intranasal administration of TP.Conclusions: 1. Open field behavior, balancing reactions, muscular strength performance and motor coordination ability were significantly reduced in 24Mon rats compared to 6Mon rats, which were improved after long-term intranasal administration of TP. 2. The expression of TH, DAT, TPH, 5-HT and SERT were significantly reduced in 24Mon rats compared to 6Mon rats, which were improved after long-term intranasal administration of TP.更多还原