下丘脑室旁核CRH神经元激活在慢性充血性心力衰竭中的交感兴奋作用

目的:观察慢性心衰时下丘脑室旁核(PVN)内促肾上腺皮质激素释放激素(CRH)表达变化及其与交感神经活动之间的关系。方法:健康雄性SD大鼠,冠脉结扎制备心衰模型,侧脑室插管渗透压泵持续给药。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CRH抑制剂αh-CRH 15 mg/h。同时,选取健康雄性体内CRH合成不足的Lewis大鼠与同源纯种Fischer 344大鼠分别制备心衰模型和假手术对照进行对比研究。4周后,测定左室舒张末压(LVEDP)、左室内压最大上升和下降速率(±dp/dtmax)、右心室/体重比(RV/BW)、肺/体重比(lung/BW)、肾交感神经放电活动(RSNA)、血浆去甲肾上腺素(NE)浓度和PVN内CRH阳性神经元数目。血浆促肾上腺皮质激素(ACTH)含量。结果:与假手术组相比,SD心衰大鼠PVN内CRH阳性神经元数目明显增加,血浆ACTH浓度升高,RSNA增强,血浆NE浓度增加,LVEDP、lung/BW和RV/BW增加,±dp/dtmax降低;心衰模型后给予αh-CRH可明显逆转上述各种变化(P0.05)。Fisher 344大鼠心衰组和假手术对照相比,PVN内CRH阳性神经元数目明显增加,血浆ACTH浓度升高,RSNA增强,外周血NE浓度升高,LVEDP、RV/BW和lung/BW增加,±dp/dtmax下降(P0.05)。但Lewis大鼠心衰组和假手术对照相比,以上各指标改变均不明显。结论:慢性心衰时,下丘脑室旁核CRH神经元被激活,激活的CRH神经元可增强外周交感神经活动,加重心功能恶化。     

下丘脑室旁核小电导钙激活钾通道过表达降低慢性心衰大鼠肾交感神经兴奋性

 目的：研究下丘脑室旁核小电导钙激活钾通道亚型2（SK2）过表达对慢性心衰大鼠心率、血压和肾交感神经活性的影响,揭示心衰大鼠下丘脑室旁核对交感系统的调节机制。方法：构建SK2重组腺病毒穿梭载体pDC316-mCMV-EGFP-rKCNN2。采用SD大鼠,手术组用左冠状动脉前降支结扎术制作慢性心衰模型,假手术组大鼠左冠状动脉前降支下穿线但不结扎,术后6周用超声心动图测定心功能。心衰组和假手术组分别在室旁核微量注射 SK2 腺病毒pDC316-mCMV-EGFP-rKCNN2和对照腺病毒pDC316-mCMV-EGFP,7 d后通过超声心动图检测心功能的改变;运用免疫组织化学、RT-PCR和Western blotting方法检测SK2重组腺病毒转染情况及表达。室旁核微量注射SK通道阻滞剂apamin,通过PowerLab 8/30系统采集信号记录心率、血压和肾交感神经活性的变化。结果: 心衰大鼠SK2表达较假手术组减少,下丘脑室旁核微量注射SK2腺病毒导致心衰大鼠肾交感神经兴奋性明显降低,下丘脑室旁核微量注射对照腺病毒对假手术大鼠交感神经兴奋性的改变不明显。结论：SK通道蛋白在心衰状态下表达降低并可能导致其功能减弱,进而促成由SK2通道介导的中枢负性交感调节通路的降低,增加了交感神经兴奋性,从而加重心衰的发展。SK2过表达减弱了心衰大鼠的肾交感神经活性,为治疗心衰提供了新的方向。     

心衰大鼠室旁核微量注射AT1和AT2受体阻滞剂对肾交感神经活性的影响

目的: 研究慢性心衰大鼠室旁核微量注射血管紧张素II-1型和2型(AT₁和AT₂)受体阻滞剂对心率、血压和肾交感神经系统的影响,揭示心衰大鼠下丘脑室旁核对交感系统的调节机制。方法: 采用SD大鼠,手术组用左冠状动脉前降支结扎术制作心衰模型,假手术组大鼠左冠状动脉前降支下穿线但不结扎。术后4周,测定血流动力学评判心功能状态,测定心脏/体重比与肺/体重比,并进行心脏病理组织学观察。对符合标准的大鼠进行麻醉,经腹膜后途径暴露左肾,在手术显微镜下剥离肾交感神经,脑立体定位仪对大鼠室旁核定位,微量注射AT₁和AT₂受体阻滞剂(100 nL),POWERLAB 8/30系统采集信号,记录心率、血压和肾交感神经放电活动的改变,人工脑脊液组作为对照。结果: 肾交感神经放电:下丘脑室旁核微量注射AT₁受体阻滞剂导致肾交感神经兴奋性减弱,对心衰大鼠交感神经的兴奋性减弱较假手术组明显。下丘脑室旁核微量注射AT₂受体阻滞剂及人工脑脊液对心衰大鼠及假手术大鼠交感神经的兴奋性改变不明显。结论: 心衰时室旁核内注射AT₁、AT₂受体阻滞剂对交感神经的输出反应有差异。在中枢肾素-血管紧张素-醛固酮系统(RAAS),血管紧张素II主要通过AT₁受体起作用,而AT₂受体无相关介导作用。     

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。     

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。     

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。     

不同游泳训练方式对大鼠下丘脑室旁核c-fos表达的影响

为了观察游泳运动后大鼠下丘脑室旁核(PVN)内c-fos mRNA及Fos免疫阳性神经元的表达变化,探讨下丘脑PVN对不同形式运动的调节机制,本实验将60只雄性SD大鼠随机分为对照组(n=10)和运动组(n=50),建立大鼠游泳运动模型(持续训练和间歇训练)。在运动结束后0、0.5、1、2、4h等不同时间点,冰水浴分离新鲜脑组织或用多聚甲醛灌注固定取脑,前者经RT-PCR反应,半定量分析运动后下丘脑PVN内c-fos mRNA的含量变化,后者经冰冻切片、免疫组织化学染色,观察Fos免疫阳性神经元的定位和分布情况。结果显示:(1)对照组大鼠下丘脑PVN内c-fos mRNA表达极低,而运动结束后c-fos mRNA表达明显升高。持续运动组逐渐升高,1h时达到最大值;间歇训练结束后2h,c-fos mRNA表达最强,然后回落,运动结束4h组c-fos mRNA仍较对照组显著增多(P<0.05);(2)运动大鼠Fos免疫阳性神经元的数目明显增多,特别是在PVN处。在室旁核小细胞部(pPVN),持续组游泳结束后1hFos阳性神经元的数目显著升高达峰值,而间歇组在运动结束后2h达峰值,同一时刻间歇组的表达高于持续运动组;室旁核大细胞部(mPVN)内,持续组Fos阳性神经元数在运动结束后持续升高,2h后显著下降,而间歇组各时刻阳性神经元的表达变化无统计学差异(P>0.05)。以上结果提示,下丘脑PVN在运动后机体调节中起重要作用,不同的运动方式可以产生不同的影响,且pPVN对不同形式运动性应激反应具有较高灵敏度。     

白细胞介素—1对大鼠下丘脑室旁核Fos癌蛋白和CRF的诱导作用

将白细胞介素-1(IL)注入大鼠侧脑室,用Fos癌蛋白抗体免疫组化法检测了下丘脑室旁核的激活神经元:大量位于含促肾上腺皮质激素释放因子(CRF)相应区域的室旁核小细胞部神经元呈Fos免疫强阳性。Fos和CRF的免疫双染色显示了许多Fos免疫阳性神经元也呈CRF免疫阳性。此外,在IL-1注射动物中,CRF的免疫阳性显著加强,提示CRF合成增加。     

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。     

妊高征大鼠脑内室旁核、视上核及孤束核内血管紧张素Ⅱ1型受体的表达

目的观察妊娠高血压大鼠(PIH)与正常妊娠大鼠下丘脑室旁核(PVN)、视上核(SON)及孤束核(NTS)内血管紧张素Ⅱ1型受体(AT1)免疫阳性神经元的分布。方法用免疫荧光染色技术对PIH大鼠AT1分布的神经元进行了研究,并与正常大鼠进行比较。结果两组大鼠PVN、SON、NTS内AT1免疫阳性神经元的分布与形态基本类似。数据经统计学处理,结果表明,PIH组大鼠PVN和NTS内AT1阳性细胞数目较对照组多,而两组大鼠SON内AT1的表达无明显差异。结论实验结果提示AT1在下丘脑PVN和NTS内的血压神经内分泌调节活动中起着重要的介导作用,中枢AT1受体的异常增加可能与母体妊高征的发病有关。     

Endogenous hydrogen peroxide in paraventricular nucleus mediates sympathetic activation and enhanced cardiac sympathetic afferent reflex in renovascular hypertensive rats

An enhancement of the cardiac sympathetic afferent reflex (CSAR) contributes to sympathetic activation in renovascular hypertension. Angiotensin II in the paraventricular nucleus (PVN) augments the CSAR and increases sympathetic outflow and blood pressure. The present study aimed to determine whether endogenous hydrogen peroxide in the PVN mediated the enhanced CSAR, sympathetic activity and the effects of angiotensin II in the PVN in renovascular hypertension induced by the two-kidney, one-clip method (2K1C) in rats. At the end of the fourth week, the rats underwent sino-aortic and vagal denervation under general anaesthesia with urethane and α-chloralose. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded. The CSAR was evaluated by the RSNA response to epicardial application of bradykinin. Microinjection of polyethylene glycol-catalase (PEG-CAT), an analogue of endogenous catalase, into the PVN decreased the RSNA and MAP and abolished the CSAR in both sham-operated and 2K1C rats. Microinjection into the PVN of the catalase inhibitor, aminotriazole, increased the RSNA and MAP and enhanced the CSAR. The effects of PEG-CAT or aminotriazole were greater in 2K1C rats than in sham-operated animals. The effects of angiotensin II in the PVN were abolished by pretreatment with PEG-CAT in both sham-operated and 2K1C rats; however, aminotriazole failed to potentiate the effects of angiotensin II. The catalase activity was decreased but the H(2)O(2) levels were increased in the PVN of 2K1C rats. These results indicate that endogenous H(2)O(2) in the PVN not only mediates the enhanced sympathetic activity and CSAR, but also the effects of angiotensin II in the PVN in renovascular hypertensive rats.     

Prostaglandins and food intake of rats: A component of energy balance regulation?

Modulation of food intake by signals arising in adipose tissue has been an important component of theories concerning energy balance regulation. Our experiments tested the effects of some prostaglandins (PG) which are produced in adipose tissue, on food and water intakes in rats. Rats were injected intrahypothalamically with 1 μg of either PGE₁ or PGB₁ in 1 μl bilaterally prior to a 2-hr daily meal. PGE₁, but not PGB₁, reduced food intake of the rats in groups with lateral hypothalamic (LH) and anterior commissure (AC) cannulas, but did not reduce food intake in groups with perifornical hypothalamic (PFH) or mammillary body (MB) cannulas. Water intake was reduced in all groups injected with PGE₁ except the LH and MB groups. Anterior and medial hypothalamic groups showed a sustained 2°C rise in rectal temperature following injections of PGE₁. Subcutaneous injections of PGE₁ reduced food intake of three different groups of rats at different levels of adiposity. We hypothesize that some PG's may be components of a signal relating fat depots and energy balance regulation.     

Regulation of tonic GABA inhibitory function, presympathetic neuronal activity and sympathetic outflow from the paraventricular nucleus by astroglial GABA transporters

Neuronal activity in the hypothalamic paraventricular nucleus (PVN), as well as sympathetic outflow from the PVN, is basally restrained by a GABAergic inhibitory tone. We recently showed that two complementary GABA_A receptor-mediated modalities underlie inhibition of PVN neuronal activity: a synaptic, quantal inhibitory modality (IPSCs, I _phasic) and a sustained, non-inactivating modality ( I _tonic). Here, we investigated the role of neuronal and/or glial GABA transporters (GATs) in modulating these inhibitory modalities, and assessed their impact on the activity of RVLM-projecting PVN neurons (PVN-RVLM neurons), and on PVN influence of renal sympathetic nerve activity (RSNA). Patch-clamp recordings were obtained from retrogradely labelled PVN-RVLM neurons in a slice preparation. The non-selective GAT blocker nipecotic acid (100–300 μ m ) caused a large increase in GABA_A I _tonic, and reduced IPSC frequency. These effects were replicated by β-alanine (100 μ m ), but not by SKF 89976A (30 μ m ), relatively selective blockers of GAT3 and GAT1 isoforms, respectively. Similar effects were evoked by the gliotoxin l -α-aminodipic acid (2 m m ). GAT blockade attenuated the firing activity of PVN-RVLM neurons. Moreover, PVN microinjections of nipecotic acid in the whole animal diminished ongoing RSNA. A robust GAT3 immunoreactivity was observed in the PVN, which partially colocalized with the glial marker GFAP. Altogether, our results indicate that by modulating ambient GABA levels and the efficacy of GABA_A I _tonic, PVN GATs, of a likely glial location, contribute to setting a basal tone of PVN-RVLM firing activity, and PVN-driven RSNA.     

精氨酸加压素翻转脂多糖引起大鼠发热及其对痛觉敏感性的影响

目的:研究外周给精氨酸加压素(AVP)对脂多糖(LPS)引起的大鼠发热和痛觉过敏的影响,以及与血清中IL-1β和PGE_2水平变化的关系。方法:实验用成年雄性SD大鼠,在23℃环境温度下,明暗时间各12 h。用无线遥测系统连续测量大鼠体核温度(Tc)、棕色脂肪温度(T_(BAT))和活动。10:00或11:30分别给大鼠腹腔注射LPS(50μg/kg)、AVP(10μg/kg)或V1a受体阻断剂(30μg/kg)。用ELISA法测定血清IL-1β和PGE_2的含量。用足底痛觉测试仪(Hargreaves test)测试大鼠热痛缩爪潜伏期的变化。结果:(1)腹腔注射LPS引起大鼠双相发热过程伴有痛觉过敏现象。(2)AVP能够翻转LPS引起的Tc和T_(BAT)升高反应,降低发热引起的痛觉敏感性。(3)外周给V1a受体阻断剂能提高LPS引起的发热反应,但不影响发热引起的痛觉敏感性变化。(4)AVP能抑制LPS引起的发热大鼠血液中IL-1β和PGE_2水平升高。结论:(1)外周给予AVP可通过抑制棕色脂肪产热以及降低血液中IL-1β和PGE_2的浓度而翻转LPS发热反应并降低发热伴随的痛觉敏感性升高现象。(2)内源性AVP也有限制LPS发热的作用,但可能不影响发热引起的痛觉阈值降低现象。     

The Effect of Prostaglandin E1 or E2 OH the in Vitro Blastogenic Response of Lymphocytes from Normal and Tumor-Bearing Mice

The in vitro effect of exogenously added prostaglandin (PG) El or E₂ over concentrations ranges of from 1 × 10^--⁴ to 1 × 10-9 M were studied in order to determine their effect on the in vitro lymphocyte proliferation of thymic and splenic T and B cells from normal and tumor-bearing CD₂ F ₁ mice. It was found that PGE₁ generally caused greater inhibition of blastogenesis than did PGE₂ when reacted with splenic lymphocytes from normal mice. Indeed, PGE₂ was found to be stimulatory for both Con A^- and LPS-sensitive normal splenic lymphocytes. Both PGE_l and PGE₂ caused potent inhibition of Con A^- and PHA-sensitive splenic lymphocytes from the tumor-bearing mice. Additionally, PGE₂ was found to stimulate the LPS-reactive lymphocytes from the tumored mice. PGE₁ and PGE₂ both inhibited the Con A- and PHA-reactive thymic lymphocytes from normal mice at the lower concentrations studied, i.e., 10^--⁴ to 10^--⁶ M. Thereafter, at concentration ranoes of from 10^--⁷ to 10^--⁹ M both PGE₁ and PGE₂ were both found to be stimulatory. Finally, both PGE₁ and PGE₂ at all concentrations studied, strongly inhibited the thymic lymphocytes from tumored mice.     

Vitamin E Exposure Modulates Prostaglandin and Thromboxane Production by Avian Cells of the Mononuclear Phagocytic System

The production of Prostaglandin E₂ (PGE₂) and Thromboxane B₂ (TXB₂) by turkey blood monocytes and a chicken mononuclear phagocytic cell line MQ-NCSU after exposure to vitamin E (VE) was examined. Turkey embryos were exposed in ovo to 0 and 10 international units (IU) of VE; blood monocytes were collected at 2 weeks of age and cultured. MQ-NCSU macrophage monolayers were exposed to 0, 0.1, 0.25, and 0.5 IU VE. The monocyte/macrophage cultures were exposed to 1 μg/mL bacterial lipopolysaccharide (LPS). Non-stimulated parallel cultures were maintained as controls. The PGE₂ and TXB₂ levels were quantitated in culture supernatants by a competitive ELISA. Blood monocytes from the 10 IU VE poults produced lower PGE₂ levels as compared with the 0 IU VE controls. Upon stimulation with LPS, monocytes from the 10 IU VE group exhibited levels of PGE₂ that were higher than the 0 IU VE group. Levels of TXB₂ were not quantitated in the poult blood monocyte culture supernatants. The PGE₂ and TXB₂ levels in the supernatant of the VE treated MQ-NCSU macrophage cultures were lower than the 0 IU VE controls. Stimulation with LPS resulted in increased PGE₂ and TXB₂ production by the VE-exposed macrophages. The results from this study suggest that in ovo or in vitro exposure with VE may either upregulate or downregulate PGE₂ and TXB₂ production by monocytes/macrophages, and that this production may be dependent upon the exposure to a variety of external stimuli and/or the state of macrophage activation.     

虾青素提高血管性痴呆小鼠学习记忆能力

目的 探讨腹腔给药虾青素对于血管性痴呆(VaD)小鼠学习记忆能力的影响及其作用机制.方法 采用双侧颈总动脉夹闭法建立小鼠VaD模型后随机分成假手术组、模型组、虾青素低剂量组及虾青素高剂量组,分别给予相应形式的药物治疗.Morris水迷宫观察各组小鼠的学习记忆及空间探索能力,尼氏染色、免疫组织化学染色、Western b...