中枢前列腺素E_2在慢性心力衰竭交感兴奋中的作用

目的:观察中枢前列腺素E_2(PGE_2)在慢性心力衰竭(心衰)交感神经兴奋中的作用,并探讨其机制。方法:雄性SD大鼠冠脉结扎制备心衰模型,侧脑室渗透压泵持续给药,假手术组和心衰组给予人工脑脊液(0.25μL/h),心衰给药组给予塞来考昔(CLB;20 mg/h)。4周后,检测各组大鼠脑脊液内PGE_2浓度、交感神经兴奋性和心功能指标,同时测定下丘脑室旁核(PVN)内促肾上腺皮质激素释放激素(CRH)神经元激活指标和神经递质的变化。结果:与假手术组相比,心衰组大鼠脑脊液内PGE_2含量增加,肾交感神经放电活动增强,外周血去甲肾上腺素升高,左心室舒张末期压力、肺/体质量比和右心室/体质量比均增加,左室内压最大上升和下降速率均下降,PVN内CRH阳性神经元数目增多,外周血促肾上腺皮质激素浓度升高(P0.05);心衰大鼠侧脑室给予CLB后,脑脊液内PGE_2明显下降,PVN内CRH神经元激活减少,交感神经兴奋性减弱,心功能得到改善(P0.05)。与假手术组相比,心衰大鼠PVN内谷氨酸含量较高,γ-氨基丁酸含量和谷氨酸脱羧酶67阳性神经元数目较低(P0.05);侧脑室给予CLB后,以上各指标均被逆转(P0.05)。结论:慢性心衰时,中枢内升高的PGE_2可以激活下丘脑室旁核CRH神经元从而增强交感神经活动,而该作用可能是通过改变下丘脑室旁核神经递质系统来实现的。     

脑室注射组胺对下丘脑室旁核促皮质素释放激素神经元的作用

目的:观察第三脑室注射组胺对下丘脑室旁核促皮质素释放激素(CRH)神经元活动的影响。方法:Fos癌蛋白免疫组化LSAB法结合双抗原标记法;半定量逆转录聚合酶链反应(RT-PCR)方法。结果:第三脑室注射组胺后,(1)下丘脑室旁核Fos阳性神经元数目明显增加(P＜0.05);(2)室旁核内的Fos阳性神经元中约有31.78%同时呈CRH阳性反应;(3)室旁核CRHmRNA含量明显升高,且有量效关系。结论:中枢组胺可以激活下丘脑室旁核的CRH神经元,并使CRH基因表达增加。     

下丘脑室旁核神经元多重神经支配的电镜研究

为了探讨下丘脑神经内分泌的突触调控机制，本文用电镜细胞化学与免疫电镜双标技术相结合的方法，研究了大鼠下丘脑室旁核神经元的多重神经支配。即先用６－ＯＨＤＡ损毁ＣＡ能神经末梢，再于振动切片上用包埋前免疫电镜法，分别以ＤＡＢ和ＴＡＢ为呈色剂先后对肽能（ＯＴ或ＳＰ）神经元和ＧＡＢＡ神经元进行双重标记。电镜观察结果表明：在下丘脑室旁核内存在肽能（ＯＴ，ＳＰ）和氨基酸（ＧＡＢＡ）能神经元及ＣＡ神经末梢；ＯＴ神     

大鼠下丘脑室旁核神经元的超微结构特征

在透射电镜下,我们观察到PVN大细胞具有四种不同的形态,认为属于同一类细胞分泌活性的不同时相:(1)合成相,粗面内质网非常丰富,(2)加工相:Golgi复合体发达,周围分布着一些神经分泌颗粒;(3)储存相:神经分泌颗粒明显增多:(4)转运相:细胞质内神经分泌颗粒很少,而在细胞周围可见一些充满神经分泌颗粒的突起。免疫电镜的结果也证实了VP阳性神经细胞具有类似的四种不同形态,其中在合成相,粗面内质网囊膜呈免疫阳性反应,还有一些PAP免疫复合物沉积在细胞质中。而在加工,储存和转运相,神经分泌颗粒呈免疫阳性反应。同样,小细胞亦具有三种不同形态特证,可能代表着不同的功能状态。     

大鼠下丘脑室旁核与orexin神经元的纤维联系及其在癫痫中的活性研究

目的:观察大鼠下丘脑室旁核(PVN)神经元和下丘脑orexin神经元在癫痫发作后的活性变化,以及相互之间的纤维联系。方法:制作匹罗卡品癫痫大鼠模型,免疫组织化学方法检测PVN内神经元和orexin神经元表达c-Fos情况; BDA顺行神经示踪实验观察PVN内神经元的纤维向orexin神经元分布区域的投射情况;CTb逆行神经示踪实验观察orexin神经元向PVN的投射情况。结果:PVN内神经元和orexin神经元在癫痫大鼠中的活性明显提高; PVN神经元向orexin集中分布的区域有大量的神经纤维投射; orexin神经元也可以投射到PVN。结论:PVN神经元和orexin神经元参与癫痫发作,PVN与orexin神经元之间存在相互神经纤维联系,可能在癫痫发作及伴发功能性障碍中发挥着重要的作用。     

慢性心衰大鼠下丘脑室旁核瞬时外向钾通道蛋白Kv4.2和Kv4.3低表达促进肾交感神经兴奋性

 目的: 观察慢性心衰大鼠下丘脑室旁核内瞬时外向钾通道蛋白Kv4.2和Kv4.3的变化及其对交感神经活性的影响。方法: 采用冠状动脉左前降支结扎术建立大鼠心衰模型或假手术模型,造模4周后超声心动图测定心功能;酶联免疫吸附法测定血浆去甲肾上腺素(NE)及血清N端前脑钠肽(NT-proBNP)含量;Western blot和real-time PCR法测定室旁核内Kv4.2和Kv4.3的表达情况;室旁核部位注射钾通道阻滞剂4-氨基吡啶(4-AP),电生理记录仪记录血压、心率和肾交感神经放电的变化。结果: 与假手术组比,心衰组大鼠心功能明显降低,血浆NE及血清NT-proBNP明显升高,室旁核内Kv4.2和Kv4.3表达明显下调;注射4-AP后导致血压、心率和交感神经放电升高,但心衰组的升高幅度小于假手术组。结论: 心力衰竭时室旁核内Kv4.2和Kv4.3表达下调并伴有交感神经放电增加,促进心衰进展。     

大鼠下丘脑室旁核中一氧化氮合酶阳性神经元的生后发育

本文用NADPH－d组织化学方法观察NOS阳性神经元在大鼠下丘脑室旁核生后发育各阶段（1、7、14、21、28和90d）的形态及分布特征。结果显示，1d时下丘脑室旁核内已有密集的NOS阳性神经元分布，但随生长发育，室旁核的截面积逐渐变大，其中的NOS神经元主要集中在该核的外侧大细胞部以及腹侧部，单位面积中的NOS神经元的密度逐渐降低。到21d，下丘脑室旁核NOS阳性神经元的分布密度较1d时下降50％。28d以后至成年鼠（90d），此密度维持在一定水平．提示下丘脑室旁核中NOS神经元的生后发育主要在生后1d至21d之间，并提示胚胎时期该核中已有NOS神经元存在。     

大鼠延髓至下丘脑室旁核的儿茶酚胺能投射神经元在胃伤害性刺激后的FOS表达

用ＨＲＰ注入下丘脑室旁核逆行追踪与抗Ｆｏｓ和抗酪氨酸羟化酶（ＴＨ）双重免疫细胞化学相结合的三重标记方法，对大鼠孤束核和延髓腹外侧区至下丘脑室旁核的儿茶酚胺能投射神经元对胃伤害性刺激后的ｃ－ｆｏｓ表达进行了观察，发现孤束核和延髓腹外侧区有７种不同的标记细胞：ＨＲＰ、Ｆｏｓ、ＴＨ单标细胞，Ｆｏｓ／ＨＲＰ、Ｆｏｓ／ＴＨ、ＨＲＰ／ＴＨ双标细胞，Ｆｏｓ／ＨＲＰ／ＴＨ三标细胞。上述７种标记细胞主要分布在延髓中、尾段孤束核的内侧亚核、连合亚核和延髓腹外侧区以及两者之间的网状结构。ＨＲＰ标记细胞以注射侧为主，对侧有少量分布。本文结果证明，大鼠孤束核和延髓腹外侧区至下丘脑室旁核投射的部分儿茶酚胺能神经元可能参与胃伤害性刺激的传导和调控。     

加压素在自发性高血压大鼠与正常大鼠下丘脑视上核、室旁核神经元内表达

目的观察加压素(AVP)在自发性高血压大鼠(SHR)与正常大鼠下丘脑视上核(SON)、室旁核(PVN)内的分布。方法应用光镜和免疫细胞化学技术。结果SHR的AVP阳性细胞内分泌颗粒密集呈棕黄色,正常大鼠组则染色较浅。SHR大鼠SON内AVP阳性神经元百分数(69.30±18.10%)明显多于正常大鼠(59.53±16.97%,P<0.05),而两组大鼠PVN内AVP的表达无明显差异。结论AVP在下丘脑的血压调节活动中起着重要的介导作用,中枢AVP含量的异常增加可能与高血压的发病有关。     

大鼠下丘脑室旁核的神经支配

本文应用ＨＲＰ追踪、免疫细胞化学与电镜方法研究了大鼠下丘脑室旁核的神经支配。结果显示，中缝背核向室旁核投射的神经元中，部分为５－ＨＴ免疫反应阳性；被盖背外侧核的部分５－ＨＴ神经元也发出纤维投射至室旁核。将ＣＢ－ＨＲＰ注入第三脑室后，电镜下发现室旁核内ＥＶＫ免疫反应阳性树突接受ＨＲＰ反应阳性轴突形成突触，ＨＲＰ免疫反应阳性的树突与阴性轴突的传入形成突触，提示室旁核内ＥＮＫ神经元受触液神经元的突触调控，同时触液神经元又受到其他神经元的突触调控。     

室旁核内花生四烯酰乙醇胺对心衰大鼠的心脏功能和交感神经活动的影响

目的:探究慢性心衰大鼠下丘脑室旁核(PVN)内花生四烯酰乙醇胺(AEA)对心脏功能和交感神经活动的影响。方法:采用冠状动脉结扎法构建大鼠心衰模型,超声心动图检测心功能;PVN内连续4周分别灌注AEA、钙/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)选择性抑制剂KN-93、瞬时受体电位香草酸亚型1(TRPV1)通道特异性阻断剂辣椒平(CPZ)、Ca~(2+)螯合剂BAPTA-AM和小电导钙激活钾通道(SK通道)阻断剂apamin后,检测交感驱动及心功能指标;同时采用不同浓度AEA孵育NG108细胞,荧光测定法检测细胞内钙离子浓度([Ca~(2+)]_i);Western blot检测CaMKII、SK_2及磷酸化TRPV1蛋白水平。结果:与假手术组相比,心衰组左心室舒张末期压(LVEDP)明显升高,左室压力最大上升、下降速率(±dp/dt_(max))和射血分数(EF)明显下降;PVN内AEA含量、[Ca~(2+)]_i及CaMKII、SK_2和磷酸化TRPV1蛋白水平均显著降低;与溶剂组相比,心衰组PVN内灌注AEA可显著降低心衰大鼠死亡率和交感驱动指标,并改善心功能;然而,PVN内分别灌注KN-93、CPZ、BAPTA-AM和apamin均显著增强交感驱动指标并恶化心功能;AEA可剂量依赖性增加NG108细胞内[Ca~(2+)]_i及CaMKII、SK_2和磷酸化TRPV1蛋白水平。结论:室旁核内CaMKII/TRPV1/Ca~(2+)/SK_2信号通路可能参与了AEA对心衰大鼠心脏功能和交感神经活动的影响。     

室旁核内脂肪酸酰胺水解酶上调介导慢性心衰大鼠交感神经兴奋亢进

目的:探讨慢性心衰大鼠室旁核(PVN)内脂肪酸酰胺水解酶(FAAH)表达失衡导致中枢交感传出增加的机制。方法:雄性Wistar大鼠左冠状动脉结扎8周后,用超声心动图检测心功能,并通过组织病理学测定心肌梗死面积,鉴定大鼠心衰模型构建成功;酶联免疫吸附法测血浆去甲肾上腺素(NE)水平;Western blot测定PVN内FAAH蛋白表达量;高效液相色谱法测定PVN内N-花生四烯酰乙醇胺(AEA)的生成量;PVN微量注射AEA、FAAH抑制剂PF3845或r AAV2-FAAH sh RNA病毒,记录交感驱动和心功能指标的变化。结果:与假手术组相比,心衰组心功能显著降低,血浆NE显著增加,PVN内FAAH表达量显著上调,AEA生成量显著减少(P0.05);PVN微量灌注PF3845、AEA或r AAV2-FAAH sh RNA病毒后,心衰组交感驱动指标显著降低,心功能明显改善。结论:心衰状态下PVN内FAAH蛋白表达上调可能引起AEA生成量的减少,从而增强交感神经兴奋性,恶化心衰。     

生长激素释放肽对心力衰竭大鼠血清和心肌组织中脂联素的影响

目的研究生长素释放肽(GHRP)对脂联素(APN)的影响,探讨GHRP在慢性充血性心力衰竭(CHF)发生、发展中的作用,以期为临床更好地防治CHF提供理论依据。方法采用腹腔注射阿霉素(Adriamycin,ADR)方法建立大鼠CHF模型,采用酶联免疫吸附剂测定(ELISA)检测大鼠血清和心肌组织匀浆中APN含量的变化。结果各组间血清和心肌组织匀浆中APN的含量比较结果显示:模型组APN明显低于对照组和治疗组(P<0.05);治疗组APN比对照组轻度降低,但无明显差异(P>0.05)。结论APN在CHF发生过程中有着重要作用,GHRP具有保护和改善CHF大鼠心功能的作用。     

The paraventricular nucleus of hypothalamus mediates the pressor response to noradrenergic stimulation of the medial prefrontal cortex in unanesthetized rats

The medial prefrontal cortex (MPFC) is a structure that is also involved in cardiovascular modulation. The injection of norepinephrine (NE) into the prelimbic (PL) area of the MPFC of unanesthetized rats evokes a pressor response which is mediated by acute vasopressin release. Vasopressin is synthesized by magnocellular cells of the paraventricular (PVN) and supraoptic nucleus (SON) of the hypothalamus. In the present study, we endeavored to determine which vasopressin-synthesizing hypothalamic nucleus is involved in the pressor pathway activated after NE injection into the PL area of the MPFC. We report here that lidocaine microinjection into the SON did not change the pressor response evoked by NE injection into the PL. However, the response to NE was blocked by prior injection of lidocaine or CoCl₂ into the PVN, indicating that this area is responsible for the mediation of this pressor response. A neuroanatomic experiment in which the neuronal tracer biotinylated dextran amine (BDA) was microinjected into the MPFC showed a lack of axons or neuronal cell bodies in the PVN, indicating that there are no direct connections between the PL area of the MPFC and the PVN. The results suggest that the PVN is involved in the mediation of the pressor response to NE in the PL area and that this pathway must relay in other brain structures before reaching the PVN.     

Separate populations of neurons within the paraventricular hypothalamic nucleus of the rat project to vagal and thoracic autonomic preganglionic levels and express c-Fos protein induced by lithium chloride

The role of different hypothalamic nuclei, particularly the paraventricular nucleus (PVN), in the control of food intake and feeding behaviour is well known. It is also well established that lithium chloride (LiCl) causes various disorders in feeding behaviour. In this study, we analyzed the precise distribution of hypothalamic neurons activated by i.p. LiCl administration (LCA neurons) and compared it to that of hypothalamic neurons which project to autonomic preganglionic levels (HAP neurons). We also analysed the possibility that some neurons belong to both populations of nerve cells. To this end, a multiple-labelling technique, using two retrograde fluorescent tracers together with c-Fos-like immunohistochemistry, was performed. Fast Blue was injected in the dorsal motor nucleus of the vagus and Fluorogold (FG) in the thoracic intermedial-lateral cell column, to trace parasympathetic and sympathetic pathways, respectively. LiCl was used as stimulus for c-Fos-like immunohistochemistry. HAP neurons were located mainly in the dorsal, ventral and lateral regions of the parvocellular PVN, while LCA neurons were observed predominantly in the magnocellular region of the PVN rostrally to HAP neurons. A significant number of FG/Fos double-labelled neurons were located in the dorsal parvocellular subnucleus of the PVN (dp) in the LiCl-stimulated rats. We concluded that there is a clear segregation of LCA neurons from HAP neurons within the PVN. The presence of FG/Fos double-labelled neurons in the dp suggests that this nucleus could mediate a sympathetic response after LiCl administration.     

Defective excitation‐contraction coupling in hearts of rats with congestive heart failure

Aim: We examined the cellular basis for depressed cardiac contractility in rats with congestive heart failure (CHF) secondary to myocardial infarction. Methods: Six weeks after ligation of the left coronary artery, CHF was confirmed by haemodynamic measures and echocardiographic demonstration of reduced myocardial contractility in vivo. Papillary muscles from CHF animals developed less force than those from sham operated (SHAM) animals. Cell shortening was measured in isolated ventricular myocytes voltage-clamped with high resistance electrodes. Ca²⁺ transients were measured in fluo-4 loaded myocytes. Results: Contractions triggered by depolarizing test steps from a post conditioning potential of −70 mV were significantly smaller and had significantly reduced velocity of shortening in CHF compared with SHAM myocytes. However, contractions initiated from −40 mV, were similar in amplitude and velocity of shortening in CHF and SHAM cells. L-type Ca²⁺ current was not significantly different between CHF and SHAM cells, whether activated from −70 or −40 mV. Therefore, in SHAM cells, excitation-contraction coupling exhibited higher gain when contractions were initiated from negative (−70 mV), as compared with depolarized potentials (−40 mV). However, in CHF myocytes, excitation-contraction coupling gain was selectively depressed with steps from −70 mV. This depression of gain in CHF was not accompanied by a significant reduction in sarcoplasmic reticulum Ca²⁺ content. Isoproterenol increased Ca²⁺ transients less in CHF than SHAM myocytes. Conclusion: In this post-infarction model of CHF, the contractile deficit was voltage dependent and the gain of excitation-contraction coupling was selectively depressed for contractions initiated negative to −40 mV.     

Increased NADPH-diaphorase reactivity in the hypothalamic paraventricular nucleus and tanycytes following systemic administration of leptin in rats

Leptin, a hormone mainly produced by adipocytes in proportion to fat mass, is a key component in the regulation of energy homeostasis and reproductive, neuroendocrine, immune, and metabolic functions. Leptin binds to the leptin receptor, which is expressed throughout the central nervous system but particularly in neurons of several nuclei of the hypothalamus, such as the arcuate nucleus (ARC) and paraventricular nucleus (PVN). It has been found that nitric oxide (NO) plays an important role in mediating effects of leptin. Since PVN and ARC neurons are known to express leptin receptors, we investigated the effects of leptin on nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in the PVN and ARC of male Wistar rats. Our results have shown that systemic administration of leptin resulted in increased NADPH-d positive cell number in the PVN and ARC, suggesting that both the PVN and ARC may be important centers in the hypothalamus for the leptin action, mediated by increased NO production. In addition, we have also observed that hypothalamic tanycytes in the ventral portion of the third ventricle were NADPH-d positive. We speculate that leptin may affect the release of neurohormones and hypothalamic neurogenesis by activating nitric oxide synthase in hypothalamic tanycytes.