肾缺血引起大鼠儿茶酚胺神经元Fos表达

实验应用Fos蛋白和酪氨酸羟化酶（tyrosine hydroxylase,TH）的双重免疫组化方法,观察肾脏动脉阻断（renal artery occlusion,RAO）是否激活脑干中核团的儿荷酚胺能神经元。所得结果如下：（1）脑干中Fos样蛋白的基础性表达低;RAO可诱发孤束核（nucleus tractus solitarius,NTS）、最后区（area postrema,AP）、巨细胞旁外侧核（paragi-gantocellularis lateralis,PGL）和蓝斑（locus coeruleus,LC）核团中许多神经元显示Fos样免疫反应（Fos-like immunoreactivi-ty,FLI）。（2）NTS、AP、PGL和LC核团中含有较多的儿茶酚胺能神经元;RAO能激活其中的部分儿荷酚胺能神经元。（3）腺苷受体阻断剂8－苯茶碱可明显减弱RAO所致的上述效应。以上结果表明,肾脏短暂缺血能激活脑干内的一些神经核团以及其中的部分儿荷酚胺能神经元。此效应可能是肾缺血时腺苷释放作用于肾内腺苷受体后引起肾传入神经活动增加的结果。     

侧脑室注射肾上腺髓质素对大鼠脑内心血管相关核团儿茶酚胺神经元及c-fos表达的影响

目的和方法:利用Fos蛋白和酪氨酸羟化酶(TH)的双重免疫组化方法,观察侧脑室注射肾上腺髓质素对大鼠心血管相关核团中儿茶酚胺神经元及c fos表达的影响,以探讨肾上腺髓质素的中枢效应是否通过激活脑内儿茶酚胺能神经元而诱发。结果:①侧脑室注射肾上腺髓质素(3nmol/kg)诱发脑干、下丘脑及前脑等多个部位的心血管中枢出现大量Fos样免疫反应神经元。②侧脑室注射肾上腺髓质素引起最后区(AP)、孤束核(NTS)、巨细胞旁外侧核(PGL)和蓝斑核(LC)内Fos TH双标神经元明显增加。③降钙素基因相关肽受体拮抗剂CGRP8-37(30nmol/kg)可明显减弱肾上腺髓质素的效应。结论:肾上腺髓质素可兴奋脑干、下丘脑及前脑等多个部位心血管相关核团的神经元,其中枢效应通过激活儿茶酚胺能神经元而诱发,降钙素基因相关肽受体介导这一效应。     

胃伤害性刺激诱导大鼠脑干星形胶质细胞GFAP蛋白表达及其与神经元的关系

研究大鼠在福尔马林诱发胃伤害性刺激时脑干内星形胶质细胞及神经元的变化。应用免疫组织化学三重标记法在脑原位切片同时显示脑干内Fos蛋白,胶质原纤维酸性蛋白（GFAP）,酪氨酸羟化酶（TH）的表达,结果显示：1、在福尔马林诱发胃伤害性刺激后,脑干胶质细胞GFAP表达阳性,并表现出明显的核团或亚核定位特点,在延髓内脏带（MVZ0,中缝大核（RMg),蓝斑（LC）,臂旁外侧核（LPB）,中缝背核（DR）,中脑导水管周围灰质腹外侧区（vlPAG),上丘中灰层（IngSC)等脑区有较多的Fos阳性细胞,而且Fos阳性表达的分布与上述GFAP阳性分布基本一致;2、MVZ,LC,DR,vlPAG等部位有大量Fos及TH双标阳性神经元,周围有密集的GFAP阳性细胞;3、随着刺激后存活时间的变化,GFAP与Fos阳性细胞的反应均经历逐渐升高后又渐降低直至消失的变化。结果表明：上述核团的神经元和星形胶质细胞可能同时参与了内脏痛及其调节过程。     

辣椒素对大鼠延髓腹外侧头端区神经元电活动的影响

在35只切断两侧缓冲神经的麻醉大鼠,应用细胞外记录的电生理学方法,观察颈总动脉注射辣椒素(capsaicin)对延髓腹外侧头端区(RVLM)巨细胞旁外侧核(PGL)自发电活动的影响。所得结果如下:(1)颈动脉注射辣椒素(10μmol,01ml),MAP由1074±013升至1256±021kPa(P<0001);HR由374±4增至395±5bpm(P<0001);30个PGL神经元自发放电单位的放电频率由126±07增至209±11spikes/s(P<0001)。(2)在10个放电单位,应用辣椒素受体阻断剂钌红(rutheniumred;200mmol,01ml)后,明显抑制辣椒素的上述效应。以上结果提示,辣椒素可能通过激活RVLM神经元上的辣椒素受体,进而兴奋PGL神经元     

辣椒素引起脑干内心血管活动相关核团中c-fos的表达

在１６只切断两侧缓冲神经的大鼠,观察颈总动脉注射辣椒素对脑干内心血管活动相关核团ｃ－ｆｏｓ原癌基因表达的影响。在剂对照组大鼠脑干,仅见少数Ｆｏｓ蛋白样免疫反应（ＦＬＩ）神经元。与对照组相比,颈总动脉注射辣椒素（１０μｍｏｌ,０．１ｍｌ）时,脑干内巨细胞旁外侧核（ＰＧＬ）、蓝斑（ＬＣ）、最后区（ＡＰ）和孤束核（ＮＴＳ）等部位的ＦＬＩ神经元显著增加,而中脑中央灰质（ＰＡＧ）和中缝核群（ＲＮ）的ＦＬＩ神     

侧脑室注射肾上腺髓质素增加大鼠心血管相关核团中c-fos的表达并激活脑内一氧化氮神经元

本研究利用Fos蛋白和一氧化氮合酶(nNOS)双重免疫组化方法,观察侧腑脑室注射肾上腺髓质素(adrenomedullin,ADM)对大鼠心血管相关核中c-fos表达及一氧化氮神经元的影响,以探讨ADM在中枢的作用部位并研究其在中枢的作用是否有NO神经元参与。侧脑室注射ADM(1nmol／kg,3nmol／kg)诱发脑干的孤束核、最后区、蓝斑核、臂旁核和外侧巨细胞旁核,下丘脑的室旁核、视上核才腹内侧核以及前脑的中央杏仁核和外侧缰核等多个部位的心血管中枢出现大量Fos样免疫反应神经元。侧脑室注射ADM(3nmol／kg),引起脑干的孤束核、外侧巨细胞旁核,下丘脑的室旁核、视上核内的Fos-nNOS双标神经元增加;ADM(1nmol／kg)亦可引起室旁核、视上核内的Fos-nNOS双标神经元增加,而对孤束核、外侧巨细胞旁核内的Fos-nNOS双标神经元无影响。降钙素基因相关肽(calcitonin gene—related peptide,CGRP)受体拈抗剂CGRP8-37(30nmol／kg)可明显减弱此效应。以上结果表明,ADM可兴奋脑内多个心血管相关核闭的神经元并激活室旁核、视上核、孤束核及外侧巨细胞核内一氧化氮神经元,此效应可能部分山CGRP受体介导。     

鞘内注射M受体和GDNF反义寡脱氧核苷酸抑制吗啡戒断大鼠蓝斑c-Fos表达

应用免疫组化方法观察鞘内注射毒蕈碱型乙酰胆碱(muscarinic acetylcholine receptor,M) 受体和胶质细胞源性神经营养因子(glial cell derived neurotrophic factor,GDNF)反义寡脱氧核苷酸对吗啡戒断大鼠蓝斑(locus coeruleus,LC)区内Fos表达的影响。结果显示,鞘内注射M_2受体和GDNF反义寡脱氧核苷酸明显减少大鼠吗啡戒断症状评分值(n=6,P<0.05)。正常大鼠LC区神经元Fos基础表达较低,吗啡依赖大鼠LC区神经元Fos表达增加,吗啡依赖大鼠纳酪酮(4mg/Kg,ip)催促戒断后,Fos表达进一步增加;鞘内注射M_2受体和GDNF反义寡脱氧核苷酸处理后均减少吗啡戒断大鼠LC区神经元Fos表达(n=5,P<0.05)。而鞘内注射M_1受体反义寡脱氧核苷酸处理组LC 区神经元Fos表达较吗啡戒断组没有显著差异(n=5,P>O.05)。结果提示:脊髓M_2受体调节吗啡戒断时LC区的神经元激活,而这种神经上行性激活涉及神经元与胶质细胞之间的适应性调节。     

冷应激诱导的脾脏NK细胞活性下降和脑内c-fos表达

目的 :观察单一冷应激对大鼠脾脏NK细胞活性的影响以及脑内Fos表达。方法 :大鼠置于 4℃的冷室 4h。采用YAC 1细胞51Cr释放法测定脾脏NK细胞的活性。用免疫细胞化学观察脑内有关核团的Fos表达 ,以及PVN和LC中Fos表达阳性的神经元的性质。结果 :单一冷应激能使脾脏NK细胞活性明显下降 ,下丘脑室旁核 (PVN)和脑干蓝斑 (LC)出现明显的Fos表达。双染实验表明 ,PVN中有部分神经元同时表达精氨酸加压素 (AVP) ,LC中大多数神经元同时表达酪氨酸羟化酶 (TH)。结论 :PVN中的AVP能神经元和LC中的儿茶酚胺能神经元很可能参与冷应激诱发的脾脏NK细胞活性的下降     

刺激室旁核及加压素对大鼠胃缺血-再灌注损伤的保护作用

采用夹闭大鼠腹腔动脉30min,松开动脉夹血流复灌1h的胃缺血-再灌注损伤(gastric ischemia-reper-fusion injury,GI-RI）模型,观察了电或化学刺激室旁核（paraventricular nucleus,PVN）及外源性加压素（arginine-va-sopression,AVP）对GI-RI的影响,并对PVN的调控通路进行了初步分析。结果表明：电或化学刺激PVN后,GI-RI显著减轻;损毁双侧孤束核（nucleus tractus solitarius,NTS）或一侧NTS内注射AVP-V1受体阻断剂,均能取消电刺激PVN对GI-RI的效应;去除脑垂体后不影响PVN的作用;切断膈下迷走神经或切除腹腔交感神经节,则能加强电刺激PVN对GI-RI的影响;PVN内注射不同剂量的AVP同样能减轻大鼠GI-RI损伤。结果提示：PVN及AVP对大鼠GI-RI具有保护作用;PVN的这种作用可能是因电或化学刺激后,激活了其中的加压素能神经元,经其下行投射纤维释放AVP作用于NTS神经元的VAP-V1受体,并通过迷走和交感神经介导,从而影响GI-RI;而似与PVN-垂体通路关系不大。     

窦内压升高和灌流腺苷激活颈动脉窦压力感受器时延髓内c-fos蛋白的表达

在14 只隔离灌流颈动脉窦区的大鼠, 观察了窦内压(ISP) 升高和灌流腺苷 (adenosine, Ado) 激活压力感受器时延髓内c-fos蛋白的表达.结果显示: 在孤束核、最后区、延髓腹外侧头端区和中缝苍白核可见Fos蛋白样免疫阳性反应(FLI)神经元分布, 且其数量随ISP升高而增多.在给定ISP下, 颈动脉窦内灌流Ado, 可使上述区域中FLI表达明显增多.根据以上结果, 得出如下结论: c-fos在压力感受器反射延髓通路中的表达, 可由ISP增高和灌流Ado而增强, 表明Ado对压力感受器反射有易化作用.     

Altered setting of the pituitary-thyroid ensemble in hypocretin-deficient narcoleptic men

Hypo- or hyperthyroidism is associated with autonomic disorders. We studied Fos expression in the medullary dorsal motor nucleus of the vagus (DMV), nucleus tractus solitarii (NTS), and area postrema (AP) in four groups of rats with different thyroid states induced by a combination of drinking water and daily intraperitoneal injection for 1-4 wk: 1) tap water and vehicle; 2) 0.1% propylthiouracil (PTU) and vehicle; 3) PTU and thyroxine (T4; 2 microg/100 g); and 4) tap water and T4 (10 microg/100 g). The numbers of Fos immunoreactive (IR) positive neurons in the DMV, NTS, and AP were low in euthyroid rats but significantly higher in the 4-wk duration in hypothyroid rats, which were prevented by simultaneous T4 replacement. Hyperthyroidism had no effect on Fos expression in these areas. There were significant negative correlations between T4 levels and the numbers of Fos-IR-positive neurons in the DMV (r = -0.6388, P < 0.008), NTS (r = -0.6741, P < 0.003), and AP (r = -0.5622, P < 0.004). Double staining showed that Fos immunoreactivity in the DMV of hypothyroid rats was mostly localized in choline acetyltransferase-containing neurons. Thyroid hormone receptors alpha1 and beta2 were localized in the observed nuclei. These results indicate that thyroid hormone influences the DMV/NTS/AP neuronal activity, which may contribute to the vagal-related visceral disorders observed in hypothyroidism.     

Nitric oxide synthase (NOS) coexists with activated neurons by skeletal muscle contraction in the brainstem of cats

Contraction of skeletal muscle evokes increases in arterial blood pressure and heart rate. Some regions of the brainstem have been implicated for expression of the cardiovascular responses to muscle contraction. Previous studies have reported that static muscle contraction induced c-Fos protein in the nucleus of tractus solitarii (NTS), lateral reticular nucleus (LRN), lateral tegmental field (FTL), subretrofacial nucleus (SRF), A1 region and periaqueductal gray (PAG) of the brainstem. Furthermore, neuronal NADPH-diaphorase (NADPH-d), which is considered as a marker of neuronal nitric oxide synthase (nNOS), has been localized in those same regions. In this study, static muscle contraction was induced by electrical stimulation of the L7 and S1 ventral roots in anaesthetized cats. Distribution of c-Fos protein within neurons containing nNOS was evaluated by double labeling methods in order to determine if nNOS containing neurons in the brainstem were activated during muscle contraction. The results indicate that c-Fos protein colocalized with NADPH-d positive staining within the neurons of the SRF and PAG, but not within the NTS neurons. Distinct number of neurons with c-Fos protein was in close proximity to NADPH-d positive staining in the NTS, SRF, and PAG. Coexisting of c-Fos protein and NADPH-d positive staining was not observed in the LRN, FTL and A1 region. These findings demonstrate that nNOS containing neurons were activated by muscle contraction in the selective regions of the brainstem, and nNOS positive staining had close anatomic contacts with the neurons activated by contraction. This result provides neuroanatomic evidence suggesting that nitric oxide modulates the cardiovascular responses to muscle contraction within the NTS, SRF and PAG of the brainstem.     

Gastric distension-induced release of 5-HT stimulates c-fos expression in specific brain nuclei via 5-HT3 receptors in conscious rats

We examined c-fos expression in specific brain nuclei in response to gastric distension and investigated whether 5-HT released from enterochromaffin (EC) cells was involved in this response. The role of 5-HT3 receptors in this mechanism was also addressed. Release of 5-HT was examined in an ex vivo-perfused stomach model, whereas c-fos expression in brain nuclei induced by gastric distension was examined in a freely moving conscious rat model. Physiological levels of gastric distension stimulated the vascular release of 5-HT more than luminal release of 5-HT, and induced c-fos expression in the nucleus of the solitary tract (NTS), area postrema (AP), paraventricular nucleus (PVN), and supraoptic nucleus (SON). The c-fos expression in all these brain nuclei was blocked by truncal vagotomy as well as by perivagal capsaicin treatment, suggesting that vagal afferent pathways may mediate this response. Intravenous injection of 5-HT3 receptor antagonist granisetron blocked c-fos expression in all brain nuclei examined, although intracerebroventricular injection of granisetron had no effect, suggesting that 5-HT released from the stomach may activate 5-HT3 receptors located in the peripheral vagal afferent nerve terminals and then induce brain c-fos expression. c-fos Positive cells in the NTS were labeled with retrograde tracer fluorogold injected in the PVN, suggesting that neurons in the NTS activated by gastric distension project axons to the PVN. The present results suggest that gastric distension stimulates 5-HT release from the EC cells and the released 5-HT may activate 5-HT3 receptors located on the vagal afferent nerve terminals in the gastric wall leading to neuron activation in the NTS and AP and subsequent activation of neurons in the PVN and SON.     

疼痛刺激对大鼠脊髓上水平神经元NADPH—d和Fos表达的影响

目的：研究一氧化氮合酶（NOS）阳性神经元在中脑导水管周围灰质（APG）和中缝背核（DR）内的分布,探讨脊髓上水平一氧化氮（NO）与外周痛的关系。方法：SD大鼠20只,随机分为2组,每组10只。实验组大鼠单侧后肢足底皮下注射5％福尔马林0.1ml,对照组大鼠于相同部位注射生理盐水0.1ml。2h后常规灌注、固定、取材大鼠中脑所在段、切片。切片按序分为Ⅰ、Ⅱ、Ⅲ三套。全部切片先作NADPH－d组织化学反应,镜检阳性切片,Ⅰ套继续作中性红染色,Ⅱ、Ⅲ套作c-fos免疫细胞化学反应,其中Ⅲ套用0.01mol/LPBS代替c-fos抗体。封片后镜下观察。结果：NADPH－d阳性神经元主要分布于PAG腹外侧核（CGLV）、背外侧核（CGLD）和DR;大鼠足底注射福尔马林后可在上述部位发现NADPH－d、Fos和NADPH－d/Fos三种标记的阳性神经元。结论：脊髓上水平NO可能在PAG和DR的痛觉调制中起作用。     

NO参与介导吗啡戒断大鼠脊髓神经元敏感化

运用Fos免疫组织化学、NADPH－d组织化学、F/NADPH－d双标、鞘内注射和反义寡核苷酸技术,观察吗啡戒断大鼠脊髓神经元活动变化及NO在其中的作用,结果发现：非吗啡依赖大鼠急性应用纳洛酮和吗啡依赖大鼠脊髓水平Fos-LI和NADPH－d阳性神经元表达与对照组相比无明显变化,二者也无Fos／NADPH－d双标神经元表达;吗啡依赖纳洛酮催促戒断大鼠脊髓Fos-LI、NADPH－d阳性神经元、纤维和终末表达明显增加,且出现Fos／NADPH－d双标神经元表达。Fos-LI和Fos／NADPH－d双标神经元呈现双侧脊髓全层分布,NADPH－d阳性神经元、纤维和终末主要位于双侧脊髓背角浅层。鞘内注射NOS抑制剂L－NA和nNOS反义寡核苷酸均明显降低吗啡依赖大鼠纳洛酮催促戒断症状评分,减少吗啡戒断大鼠脊髓Fos-LI表达。上述结果提示：NO参与介导吗啡戒断大鼠脊髓神经元敏感化。