视上核星形胶质细胞对急性腹腔高渗刺激引起的神经元反应的调节及其机制
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摘要
为探索腹腔渗透压感受器的信息传至视上核(SON)和室旁核(PVN)之通路,信息通路各站(脊髓背角〈DHSC〉、延髓孤束核〈NTS〉和SON内星形胶质细胞和神经元对腹腔急性高渗刺激的反应及其相互关系,星形胶质细胞在渗透压调节中的作用及调节机制,本研究进行了下列四组实验。
实验一外周和中枢神经元的轴突终末可直接终止于星形胶质细胞
目的:观察周围神经传入纤维终末和中枢的传入纤维终末是否直接终止于星形胶质细胞。
方法:10只健康成年雄性SD大鼠被分为2组:第1组为迷走神经切断组,切断左侧颈部迷走神经干,动物成活30天。动物按常规免疫电镜制片方法进行抗GFAP免疫电镜染色。第2组为BDA顺行示踪组,将顺行示踪剂定位导入右侧NTS内,5天后常规灌流固定,恒冷箱制片,取延髓切片观察BDA的注射区,2套SON切片分别进行抗Vasopressin(VP)和抗GFAP的免疫荧光染色。Confocal显微镜下观察计数。结果:(1)电镜下在NTS内观察到正常的Synapses(包括对称性和非对称性Synapses)和轴突终末与GFAP阳性的星形胶质细胞突起接触形成Synaptoid Contacts。在迷走神经切断侧NTS内发现许多变性的轴突终末,它可单独与神经元(胞体或树突)、GFAP阳性的星形胶质细胞或同时与两者接触,形成Synaptoid Contacts。
(2)BDA注射区局限于右侧NTS,可见许多BDA标记的胞体和密集的纤维。在SON发现40%的BDA标记的纤维可与VP阳性神经元接触,36%的BDA标记的纤维与GFAP阳性突起接触。电镜下也观察到轴突终末与GFAP星形胶质细胞形成Synaptoid Contacts。
结论:星形胶质细胞可直接与周围神经或中枢神经的轴突终末接触,形成Synaptoid Contacts。
实验二迷走神经至孤束核,孤束核至视上核的信息通路参与急性腹腔高渗刺激的调节
目的:探索腹腔渗透压信息传至SON和PVN的神经通路及相关部位星形胶质细胞和神经元对急性腹腔高渗刺激的反应。
方法:30只SD大鼠被分为6组(5只/组):第1组为正常对照组,大鼠不作任何处理;第2组为等渗刺激组(IS组),向腹腔注入4ml 0.15 mol/L盐水;第3组为高渗刺激组(HS组),注入4ml 1.54mol/L盐水;第4组为两侧膈下迷走神经切断加高渗刺激组(SDV plus HS组),在胃贲门处分离结扎并切断两侧迷走神经,7天后给予高渗刺激;第5组为两侧内脏大神经切断加高渗刺激组(SNL plus HS组),在腹后壁分离、结扎并切断两侧内脏大神经,7天后给予高渗刺激,所有动物常规固定,切断前脑(含SON)、延髓(含NTS)和脊髓胸段;第6组为假手术加高渗刺激组。切片分别进行各自的双重免疫荧光反应。
结果:(1)各组动物脊髓胸段背角内的星形胶质细胞和神经元未见到明显的反应。
(2)HS组延髓NTS和最后区(AP)内Fos阳性神经元和GFAP阳性星形胶质细胞明显增加,与IS组的差异显著,这种增加可被SDV所阻止,但不能被SNL所抑制。
(3)HS组SON内Fos阳性神经元显著增多,星形胶质细胞由静止型转化为激活型,胞体变大,突起变粗,GFAP深染,平均荧光强度增强,SON-VGL平均厚度增加,Fos/GFAP双标细胞增多。SDV可阻止HS引起的反应,SNL则不能。
结论:腹腔渗透压感受器的信息是经迷走神经传至NTS,由NTS发出的纤维至SON,不是经内脏大神经传导,脊髓背角不参与此信息的传递,而NTS可能起着重要作用。
实验三急性腹腔高渗刺激后SON内神经元的反应依赖于星形胶质细胞的活性
目的:观察急性腹腔高渗刺激后SON星形胶质细胞和神经元的反应及其相互关系。
方法:85只SD大鼠被分为5组:第1组正常对照组(n=5)处理;第2组为IS组(n=20);第3组HS组(n=20);第4组为氟代柠檬酸加高渗组(FCA plus HS组,n=20),预先向侧脑室缓慢注入1nmol/1μ的FCA,2h后再给高渗刺激;第5组为甘珀酸加高渗组(CBX plus HS组,n=20),预先向侧脑室缓慢注入50μg CBX (10μg/μl, W/V),2h后再给高渗刺激。以上动物除第1组外,其余各组均分别成活15,45,90和180 min,每一时间点5只。常规灌流固定、切片,SON切片进行抗VP,抗Fos,抗Connexin43(Cx43),抗GFAP的单一或双重免疫荧光染色。
结果:光镜结果:(1)高渗刺激后SON星形胶质细胞反应明显,表现为细胞被激活,胞体变大,突起变粗,GFAP深染,平均荧光强度增强,SON-VGL厚度增厚,Fos/GFAP双标细胞增加,Cx43表达明显增加,以上反应在刺激后45min达到高峰。(2)高渗刺激后SON MNCs也被激活,Fos阳性胞核明显增加,其高峰为90min。(3)FCA可明显干扰星形胶质细胞和神经元两者的反应,而CBX只抑制神经元的反应,不干扰星形胶质细胞的反应。
结论:高渗刺激后SON内星形胶质细胞反应敏感,在45min达到高峰,神经元的Fos表达90min为高峰。神经元的活性依赖于星形胶质细胞的活性。
实验四腹腔急性高渗刺激后SON星形胶质细胞经Cx43 hemi channel释放谷氨酸调节神经元的活性
目的:观察高渗刺激后SON星形胶质细胞对神经元的调节及其机制。方法:(1)光镜制片,25只SD雄性大鼠被分为5组(5只/组):第1组为正常对照组;第2组为IS组;第3组为HS组;第4组为FCA plus HS组;第5组为CBX plus HS组。所有动物成活45min,常规固定、切片,切片进行抗NMDAR-2,抗glutamate,抗GFAP,抗Cx43,抗Cx32,抗Fos,抗VP等单一,双重或三重免疫荧光染色。
(2)星形胶质细胞培养,取18天胎鼠的下丘脑,常规分离、纯化培养星形胶质细胞2周,将培养细胞分为4组:第1组为等渗DMEM培养组;第2组为高渗DMEM处理组;第3组先用CBX处理30min后再移至等渗DMEM;第4组先用CBX处理30min后再移至高渗DMEM。各组均处理1,3,5,10和15min。
(3)分别收上述各组培养液用HPLC检测Glutamate和Taurine的含量。
(4)电镜制片,另有10只大鼠分为IS组(5只)和HS组(5只),进行抗Cx43和抗NMDAR-2免疫电镜染色。
(5)用3H放免测定检测正常对照组,IS,HS,FCA plus HS,CBX plusHS,SDV plus HS,SNL plus HS各组血浆中VP的含量。
结果:(1)腹腔高渗刺激后SON星形胶质细胞内glutamate表达明显上调,FCA可干扰其上调,而CBX则不干扰。
(2)腹腔高渗刺激后SON神经元上的NMDAR-2的表达明显上调,FCA和CBX均可阻止其上调。
(3)腹腔高渗刺激后SON神经元内VP的表达上升,FCA,CBX,SDV均抑制VP的上升,但SNL不抑制。
(4)培养的星形胶质细胞高渗刺激后,胞内glutamate的表达明显上升,3min达到高峰,以后下降。CBX加高渗刺激后glutamate的表达上升明显,并一直维持在高位。
(5)HPLC检测高渗刺激后培养液中glutamate含量明显上升,而牛磺酸不上升。
(6)腹腔高渗刺激SON内Cx43 hemi channels出现率明显增加。
结论:腹腔高渗刺激激活SON星形胶质细胞,被激活的星形胶质细胞增加合成glutamate,并通过Cx43 hemi channels释放,作用于神经元上的NMDAR-2,兴奋神经元,引起VP的合成释放增加。
To study a route of signal from the peripheral osmotic receptors reaches the supraoptic nucleus (SON) and paraventricular nucleus (PVN), and the response and relationship each other of the astrocytes and neurons in the dorsal horn of spinal cord (DHSC), nucleus of tract solitarius (NTS) and SON to acute hypertonic stimulation via peritoneal , and the role and mechanism of astrocytes regulating the activation of neurons induced by hypertonic stimulation. The flowing four experiments were performed.
The first experiment The peripheral and central neural axonal terminals directly termite on the astrocytes
Objective To investigate whether the axonal terminals form peripheral or central nerve directly end on the astrocytes.
Methods Ten SD rats were divided into two groups: The rats of first group were performed cut left vagal trunk in the neck under an operating microscope and survived for 30 days. The tissues from the brain and spinal cord of experimental animals were performed according to immunoelectro microscopic methods. The rats in second group, 10 % Biotinylated dextran amine (BDA, Vector, diluted with 0.9 % NaCl, 0.2μl/per rat) was injected into right NTS (n=5) through microsyringe (0.47 mm in diameter) over a period of 10 min, as BDA anterograde tracing model, after 5 days were fixed, cut sections and performed immunofluorescent staining and observed under confocal microscope.
Results (1) The typical neuronal synapses (including symmetrical and asymmetrical synapses) and synaptoid contacts formed by axonal terminals and GFAP positive astrocytic processes in NTS were found. (2) Some (64%) of the degenerate axonal terminals induced vagotomy directly terminate on the neurons, or astrocytes labeled with GFAP, or both neurons and astrocytes and formed a synaptoid contacts. (2) BDA injection area located in right NTS, which revealed many BDA labeled neurons and dense fibers. In SON 40 % BDA labeled fibers directly contacted with VP positive neurons, and 36 % BDA positive fibers contacted with astrocytes labeled by GFAP. Immunoeletromicroscope observed that the axonal terminals directly contacted with GFAP positive processes and formed synaptoid contacts.
Conclusion the astrocytes directly contacted with afferent fibers from peripheral or central neural system and formed a synaptoid contacts.
The second experiment The route from the peripheral osmoreceptors reaching supraoptic nucleus involved in regulating osmolarity
Objective To research the neural route of transmitting osmotic signal from the peripheral osmotic receptors to SON and PVN, and the response of astrocytes and neurons in interrelated areas to acute hypertonic stimulation. Methods Thirty SD rats were divided into six groups:①The rats in the first group did not receive any treatment, as a normal control model.②The second group was isotonic saline (IS) model, 4 ml isotonic saline (0.15 mol/L) was injected into peritoneaus.③The third group was hypertonic saline (HS) model, 4 ml hypertonic saline (1.54 mol/L) was injected into the peritoneaus, these animals survived for 45 min.④The fourth group was bilateral subdiaphragmatic vagotomy (SDV) plus HS model.⑤The fifth group was bilateral splanchnic nerve lesion (SNL) plus HS.⑥The sixth group was sham-operated plus HS. 7 days after SDV, SNL, or sham-operared, these rats were gave HS and survived 45 min. The sections from dorsal horn of spinal cord (DHSC), or NTS of medullary oblongata, or SON were preformed immunofluorescent staining.
Results (1) The clear response of astrocytes and neurons in DHSC of above mentioned experimental animals did not observe. (2) HS stimulation induced Fos positive neurons and GFAP positive astrocytes significantly increased in NTS and the area postrema (AP), and revealed marked difference as compared with IS group. SDV blocked the response, but SNL can not block. (3) HS induced that the astrocytes were activated and GFAP expression increased and the mean fluorescent intensity of GFAP intensified, the mean thickness of SON-VGL thickened, the mean number of Fos/GFAP double labeled astrocytes significantly increased. These response was blocked by SDV, but SNL can not block. Conclusion The signal from peripheral osmotic receptors transmitted to NTS, AP via vagus, the fibers from NTS projected to SON. The splanchnic nerve did not involve in osmotic signal transmitting. The NTS and AP play important role in osmotic signal transmitting, but DHSC did not play role.
The third experiment Acute hyperosmotic stimulus induces activation in the neurons depends on the regulation of astrocytes in supraoptic nucleus
Objective To investigate the response and relationship each other of the astrocytes and neurons in SON induced by acute hypertonic stimulation.
Methods Eighty-five rats were divided into five groups: The first group was normal control group (n=5). The second was IS group (n-20). The third group was HS group (n-20). The fourth group, a glial metabolic inhibitor, fluorocitrate (FCA, 1μl /rat, 1nmol/μl, W/V, n=20) and the fifth group, a gap junction blocker, carbenoxolone (CBX, 30μg/rat, 10μg/μl, W/V, n=20) was pre-injected into lateral ventricle respectively followed by HS 2 h later as FCA or CBX plus HS models. All experimental animals, ecepted normal contol group, were survived for 15, 45 90 and 180 min respectively, n=5/per time point, fixation, cut section and anti-Fos, anti-GFAP immunofluorescent staining were preformed by usual methods.
Results (1) Hypertonic stimulation induced SON astrocytes clearly responding and revealed marked cellular hypertrophy with thickened processes, the mean fluorescent intensity of GFAP staining intensified, the mean thickness of SON-VGL thickened, the mean number of Fos/GFAP double labeled astrocytes significantly increased. At 45 min after stimulation the response peaked. (2) Fos positive neurons significantly increased and peaked at 90 min after stimulation. (3) FCA markedly blocked the response both astrocytes and neurons, while CBX only inhibited neuronal response, was not astrocytes.
Conclusion Hypertonic stimulation induced the response of SON astrocytes was early than neurons. HS induces Fos expression in SON neurons depends on the activation of astrocytes.
The fourth experiment The glutamate released from astrocytes via connexin 43 hemichannels is required for regulating hypertonicity induced activation of the supraoptic neurons
Objective To research mechanisms of SON astrocytes regulating the activation of neurons induced by hypertonic stimulation.
Methods (1) For immunofluorescent staining, twenty-five SD rats were divided into five groups (5 rats/per group): the first group was normal control group, the second group was IS group, the third group was HS group, the fourth group was FCA plus HS group and the fifth group wa CBX plus HS group. All animals were survived for 45 min, fixation, cut section and anti-glutamate, anti-NMDAR-2, anti-VP immunofluorescent staining were preformed by usual methods. (2) Cultured astrocytes from the hypothalamus of rat embryos (E18) were prepared and maintained in culture as described previously (Ye et al., 2003). Astrocytes were plated on 60-mm Primaria plastic culture dishes (Nunc Clone) with or without glass coverslips (1×105 cells/cm2 24 h before the experiment) and kept at 37oC in a 5% CO2/95% air atmosphere at nearly 100% relative humidity. Cultures were maintained in growth medium (Dulbecco’s modified Eagle’s medium, DMEM) and reached confluence within 2 weeks. All cultured cells were divided into four groups: the first group was IS DMEM group, the second group was group treated by HS DMEM for 1, 3, 5, 10 and 15 min, the third group was pre-treated by CBX for 30 min next moved to IS DMEM, the fourth group was pre-treated by CBX for 30 min next moved to HS DMEM for 1, 3, 5, 10 and 15 min. These cells were fixed and anti-glutamate and anti-GFAP double immunofluorescent staining. (3) Glutamate and taurine measurement, using HPLC measured concentration of glutamate and taurine in astrocytes medium. (4) For immunoelectro-microscope ten rats were divide into IS group and HS group, anti-connexin 43 (Cx43) and anti-NMDAR 2 double immunoelectromicroscopic staining was performed. (5) Plasma VP content radioimmunoassay. The blood collected from the femoral veins of rats as described above, was centrifuged at 1500×g for 15 min at 4oC. The plasma was separated and stored as aliquots in plastic tubes at–70oC until used. All samples were measured using the vasopressin 125I RIA kit (DiaSorin Company). Assays were performed blindly.
Results (1) Hypertonic stimulation significantly increased glutamate expression in SON strocytes, FCA markedly inhibited both glutamate and GFAP signals in astrocytes, but CBX did not inhibit. (2) Hypertonic stimulation induced the expression of NMDAR 2 in neurons markedly up-regulated, FCA and CBX inhibited its up-regulation. (3) Hypertonic stimulation increased the expression of VP in SON neurons, FCA, CBX and SDV inhibited its increasing, but SNL can not inhibit. (4) Hypertonic stimulation induced the expression of glutamate and GFAP in cultured astrocytes significantly up-regulated, at 3min peaked. The cells pre-treated by CBX, hypertonic stimulation increased glutamate expression and maintained high level. (5) Hypertonic stimulation significantly increased the concentration of glutamate in medium, but the concentration of taurine did not increase. (6) After hypertonic stimulation, the number of Cx43 hemichannels in SON significantly increased.
Conclusion Hypertonic stimulation activated SON astrocytes, activated astrocytes synthesized glutamate and released it via Cx43 hemichannels, glutamate signal activated neurons via NMDAR and increased synthesis and release of VP.
实验一外周和中枢神经元的轴突终末可直接终止于星形胶质细胞
目的:观察周围神经传入纤维终末和中枢的传入纤维终末是否直接终止于星形胶质细胞。
方法:10只健康成年雄性SD大鼠被分为2组:第1组为迷走神经切断组,切断左侧颈部迷走神经干,动物成活30天。动物按常规免疫电镜制片方法进行抗GFAP免疫电镜染色。第2组为BDA顺行示踪组,将顺行示踪剂定位导入右侧NTS内,5天后常规灌流固定,恒冷箱制片,取延髓切片观察BDA的注射区,2套SON切片分别进行抗Vasopressin(VP)和抗GFAP的免疫荧光染色。Confocal显微镜下观察计数。结果:(1)电镜下在NTS内观察到正常的Synapses(包括对称性和非对称性Synapses)和轴突终末与GFAP阳性的星形胶质细胞突起接触形成Synaptoid Contacts。在迷走神经切断侧NTS内发现许多变性的轴突终末,它可单独与神经元(胞体或树突)、GFAP阳性的星形胶质细胞或同时与两者接触,形成Synaptoid Contacts。
(2)BDA注射区局限于右侧NTS,可见许多BDA标记的胞体和密集的纤维。在SON发现40%的BDA标记的纤维可与VP阳性神经元接触,36%的BDA标记的纤维与GFAP阳性突起接触。电镜下也观察到轴突终末与GFAP星形胶质细胞形成Synaptoid Contacts。
结论:星形胶质细胞可直接与周围神经或中枢神经的轴突终末接触,形成Synaptoid Contacts。
实验二迷走神经至孤束核,孤束核至视上核的信息通路参与急性腹腔高渗刺激的调节
目的:探索腹腔渗透压信息传至SON和PVN的神经通路及相关部位星形胶质细胞和神经元对急性腹腔高渗刺激的反应。
方法:30只SD大鼠被分为6组(5只/组):第1组为正常对照组,大鼠不作任何处理;第2组为等渗刺激组(IS组),向腹腔注入4ml 0.15 mol/L盐水;第3组为高渗刺激组(HS组),注入4ml 1.54mol/L盐水;第4组为两侧膈下迷走神经切断加高渗刺激组(SDV plus HS组),在胃贲门处分离结扎并切断两侧迷走神经,7天后给予高渗刺激;第5组为两侧内脏大神经切断加高渗刺激组(SNL plus HS组),在腹后壁分离、结扎并切断两侧内脏大神经,7天后给予高渗刺激,所有动物常规固定,切断前脑(含SON)、延髓(含NTS)和脊髓胸段;第6组为假手术加高渗刺激组。切片分别进行各自的双重免疫荧光反应。
结果:(1)各组动物脊髓胸段背角内的星形胶质细胞和神经元未见到明显的反应。
(2)HS组延髓NTS和最后区(AP)内Fos阳性神经元和GFAP阳性星形胶质细胞明显增加,与IS组的差异显著,这种增加可被SDV所阻止,但不能被SNL所抑制。
(3)HS组SON内Fos阳性神经元显著增多,星形胶质细胞由静止型转化为激活型,胞体变大,突起变粗,GFAP深染,平均荧光强度增强,SON-VGL平均厚度增加,Fos/GFAP双标细胞增多。SDV可阻止HS引起的反应,SNL则不能。
结论:腹腔渗透压感受器的信息是经迷走神经传至NTS,由NTS发出的纤维至SON,不是经内脏大神经传导,脊髓背角不参与此信息的传递,而NTS可能起着重要作用。
实验三急性腹腔高渗刺激后SON内神经元的反应依赖于星形胶质细胞的活性
目的:观察急性腹腔高渗刺激后SON星形胶质细胞和神经元的反应及其相互关系。
方法:85只SD大鼠被分为5组:第1组正常对照组(n=5)处理;第2组为IS组(n=20);第3组HS组(n=20);第4组为氟代柠檬酸加高渗组(FCA plus HS组,n=20),预先向侧脑室缓慢注入1nmol/1μ的FCA,2h后再给高渗刺激;第5组为甘珀酸加高渗组(CBX plus HS组,n=20),预先向侧脑室缓慢注入50μg CBX (10μg/μl, W/V),2h后再给高渗刺激。以上动物除第1组外,其余各组均分别成活15,45,90和180 min,每一时间点5只。常规灌流固定、切片,SON切片进行抗VP,抗Fos,抗Connexin43(Cx43),抗GFAP的单一或双重免疫荧光染色。
结果:光镜结果:(1)高渗刺激后SON星形胶质细胞反应明显,表现为细胞被激活,胞体变大,突起变粗,GFAP深染,平均荧光强度增强,SON-VGL厚度增厚,Fos/GFAP双标细胞增加,Cx43表达明显增加,以上反应在刺激后45min达到高峰。(2)高渗刺激后SON MNCs也被激活,Fos阳性胞核明显增加,其高峰为90min。(3)FCA可明显干扰星形胶质细胞和神经元两者的反应,而CBX只抑制神经元的反应,不干扰星形胶质细胞的反应。
结论:高渗刺激后SON内星形胶质细胞反应敏感,在45min达到高峰,神经元的Fos表达90min为高峰。神经元的活性依赖于星形胶质细胞的活性。
实验四腹腔急性高渗刺激后SON星形胶质细胞经Cx43 hemi channel释放谷氨酸调节神经元的活性
目的:观察高渗刺激后SON星形胶质细胞对神经元的调节及其机制。方法:(1)光镜制片,25只SD雄性大鼠被分为5组(5只/组):第1组为正常对照组;第2组为IS组;第3组为HS组;第4组为FCA plus HS组;第5组为CBX plus HS组。所有动物成活45min,常规固定、切片,切片进行抗NMDAR-2,抗glutamate,抗GFAP,抗Cx43,抗Cx32,抗Fos,抗VP等单一,双重或三重免疫荧光染色。
(2)星形胶质细胞培养,取18天胎鼠的下丘脑,常规分离、纯化培养星形胶质细胞2周,将培养细胞分为4组:第1组为等渗DMEM培养组;第2组为高渗DMEM处理组;第3组先用CBX处理30min后再移至等渗DMEM;第4组先用CBX处理30min后再移至高渗DMEM。各组均处理1,3,5,10和15min。
(3)分别收上述各组培养液用HPLC检测Glutamate和Taurine的含量。
(4)电镜制片,另有10只大鼠分为IS组(5只)和HS组(5只),进行抗Cx43和抗NMDAR-2免疫电镜染色。
(5)用3H放免测定检测正常对照组,IS,HS,FCA plus HS,CBX plusHS,SDV plus HS,SNL plus HS各组血浆中VP的含量。
结果:(1)腹腔高渗刺激后SON星形胶质细胞内glutamate表达明显上调,FCA可干扰其上调,而CBX则不干扰。
(2)腹腔高渗刺激后SON神经元上的NMDAR-2的表达明显上调,FCA和CBX均可阻止其上调。
(3)腹腔高渗刺激后SON神经元内VP的表达上升,FCA,CBX,SDV均抑制VP的上升,但SNL不抑制。
(4)培养的星形胶质细胞高渗刺激后,胞内glutamate的表达明显上升,3min达到高峰,以后下降。CBX加高渗刺激后glutamate的表达上升明显,并一直维持在高位。
(5)HPLC检测高渗刺激后培养液中glutamate含量明显上升,而牛磺酸不上升。
(6)腹腔高渗刺激SON内Cx43 hemi channels出现率明显增加。
结论:腹腔高渗刺激激活SON星形胶质细胞,被激活的星形胶质细胞增加合成glutamate,并通过Cx43 hemi channels释放,作用于神经元上的NMDAR-2,兴奋神经元,引起VP的合成释放增加。
To study a route of signal from the peripheral osmotic receptors reaches the supraoptic nucleus (SON) and paraventricular nucleus (PVN), and the response and relationship each other of the astrocytes and neurons in the dorsal horn of spinal cord (DHSC), nucleus of tract solitarius (NTS) and SON to acute hypertonic stimulation via peritoneal , and the role and mechanism of astrocytes regulating the activation of neurons induced by hypertonic stimulation. The flowing four experiments were performed.
The first experiment The peripheral and central neural axonal terminals directly termite on the astrocytes
Objective To investigate whether the axonal terminals form peripheral or central nerve directly end on the astrocytes.
Methods Ten SD rats were divided into two groups: The rats of first group were performed cut left vagal trunk in the neck under an operating microscope and survived for 30 days. The tissues from the brain and spinal cord of experimental animals were performed according to immunoelectro microscopic methods. The rats in second group, 10 % Biotinylated dextran amine (BDA, Vector, diluted with 0.9 % NaCl, 0.2μl/per rat) was injected into right NTS (n=5) through microsyringe (0.47 mm in diameter) over a period of 10 min, as BDA anterograde tracing model, after 5 days were fixed, cut sections and performed immunofluorescent staining and observed under confocal microscope.
Results (1) The typical neuronal synapses (including symmetrical and asymmetrical synapses) and synaptoid contacts formed by axonal terminals and GFAP positive astrocytic processes in NTS were found. (2) Some (64%) of the degenerate axonal terminals induced vagotomy directly terminate on the neurons, or astrocytes labeled with GFAP, or both neurons and astrocytes and formed a synaptoid contacts. (2) BDA injection area located in right NTS, which revealed many BDA labeled neurons and dense fibers. In SON 40 % BDA labeled fibers directly contacted with VP positive neurons, and 36 % BDA positive fibers contacted with astrocytes labeled by GFAP. Immunoeletromicroscope observed that the axonal terminals directly contacted with GFAP positive processes and formed synaptoid contacts.
Conclusion the astrocytes directly contacted with afferent fibers from peripheral or central neural system and formed a synaptoid contacts.
The second experiment The route from the peripheral osmoreceptors reaching supraoptic nucleus involved in regulating osmolarity
Objective To research the neural route of transmitting osmotic signal from the peripheral osmotic receptors to SON and PVN, and the response of astrocytes and neurons in interrelated areas to acute hypertonic stimulation. Methods Thirty SD rats were divided into six groups:①The rats in the first group did not receive any treatment, as a normal control model.②The second group was isotonic saline (IS) model, 4 ml isotonic saline (0.15 mol/L) was injected into peritoneaus.③The third group was hypertonic saline (HS) model, 4 ml hypertonic saline (1.54 mol/L) was injected into the peritoneaus, these animals survived for 45 min.④The fourth group was bilateral subdiaphragmatic vagotomy (SDV) plus HS model.⑤The fifth group was bilateral splanchnic nerve lesion (SNL) plus HS.⑥The sixth group was sham-operated plus HS. 7 days after SDV, SNL, or sham-operared, these rats were gave HS and survived 45 min. The sections from dorsal horn of spinal cord (DHSC), or NTS of medullary oblongata, or SON were preformed immunofluorescent staining.
Results (1) The clear response of astrocytes and neurons in DHSC of above mentioned experimental animals did not observe. (2) HS stimulation induced Fos positive neurons and GFAP positive astrocytes significantly increased in NTS and the area postrema (AP), and revealed marked difference as compared with IS group. SDV blocked the response, but SNL can not block. (3) HS induced that the astrocytes were activated and GFAP expression increased and the mean fluorescent intensity of GFAP intensified, the mean thickness of SON-VGL thickened, the mean number of Fos/GFAP double labeled astrocytes significantly increased. These response was blocked by SDV, but SNL can not block. Conclusion The signal from peripheral osmotic receptors transmitted to NTS, AP via vagus, the fibers from NTS projected to SON. The splanchnic nerve did not involve in osmotic signal transmitting. The NTS and AP play important role in osmotic signal transmitting, but DHSC did not play role.
The third experiment Acute hyperosmotic stimulus induces activation in the neurons depends on the regulation of astrocytes in supraoptic nucleus
Objective To investigate the response and relationship each other of the astrocytes and neurons in SON induced by acute hypertonic stimulation.
Methods Eighty-five rats were divided into five groups: The first group was normal control group (n=5). The second was IS group (n-20). The third group was HS group (n-20). The fourth group, a glial metabolic inhibitor, fluorocitrate (FCA, 1μl /rat, 1nmol/μl, W/V, n=20) and the fifth group, a gap junction blocker, carbenoxolone (CBX, 30μg/rat, 10μg/μl, W/V, n=20) was pre-injected into lateral ventricle respectively followed by HS 2 h later as FCA or CBX plus HS models. All experimental animals, ecepted normal contol group, were survived for 15, 45 90 and 180 min respectively, n=5/per time point, fixation, cut section and anti-Fos, anti-GFAP immunofluorescent staining were preformed by usual methods.
Results (1) Hypertonic stimulation induced SON astrocytes clearly responding and revealed marked cellular hypertrophy with thickened processes, the mean fluorescent intensity of GFAP staining intensified, the mean thickness of SON-VGL thickened, the mean number of Fos/GFAP double labeled astrocytes significantly increased. At 45 min after stimulation the response peaked. (2) Fos positive neurons significantly increased and peaked at 90 min after stimulation. (3) FCA markedly blocked the response both astrocytes and neurons, while CBX only inhibited neuronal response, was not astrocytes.
Conclusion Hypertonic stimulation induced the response of SON astrocytes was early than neurons. HS induces Fos expression in SON neurons depends on the activation of astrocytes.
The fourth experiment The glutamate released from astrocytes via connexin 43 hemichannels is required for regulating hypertonicity induced activation of the supraoptic neurons
Objective To research mechanisms of SON astrocytes regulating the activation of neurons induced by hypertonic stimulation.
Methods (1) For immunofluorescent staining, twenty-five SD rats were divided into five groups (5 rats/per group): the first group was normal control group, the second group was IS group, the third group was HS group, the fourth group was FCA plus HS group and the fifth group wa CBX plus HS group. All animals were survived for 45 min, fixation, cut section and anti-glutamate, anti-NMDAR-2, anti-VP immunofluorescent staining were preformed by usual methods. (2) Cultured astrocytes from the hypothalamus of rat embryos (E18) were prepared and maintained in culture as described previously (Ye et al., 2003). Astrocytes were plated on 60-mm Primaria plastic culture dishes (Nunc Clone) with or without glass coverslips (1×105 cells/cm2 24 h before the experiment) and kept at 37oC in a 5% CO2/95% air atmosphere at nearly 100% relative humidity. Cultures were maintained in growth medium (Dulbecco’s modified Eagle’s medium, DMEM) and reached confluence within 2 weeks. All cultured cells were divided into four groups: the first group was IS DMEM group, the second group was group treated by HS DMEM for 1, 3, 5, 10 and 15 min, the third group was pre-treated by CBX for 30 min next moved to IS DMEM, the fourth group was pre-treated by CBX for 30 min next moved to HS DMEM for 1, 3, 5, 10 and 15 min. These cells were fixed and anti-glutamate and anti-GFAP double immunofluorescent staining. (3) Glutamate and taurine measurement, using HPLC measured concentration of glutamate and taurine in astrocytes medium. (4) For immunoelectro-microscope ten rats were divide into IS group and HS group, anti-connexin 43 (Cx43) and anti-NMDAR 2 double immunoelectromicroscopic staining was performed. (5) Plasma VP content radioimmunoassay. The blood collected from the femoral veins of rats as described above, was centrifuged at 1500×g for 15 min at 4oC. The plasma was separated and stored as aliquots in plastic tubes at–70oC until used. All samples were measured using the vasopressin 125I RIA kit (DiaSorin Company). Assays were performed blindly.
Results (1) Hypertonic stimulation significantly increased glutamate expression in SON strocytes, FCA markedly inhibited both glutamate and GFAP signals in astrocytes, but CBX did not inhibit. (2) Hypertonic stimulation induced the expression of NMDAR 2 in neurons markedly up-regulated, FCA and CBX inhibited its up-regulation. (3) Hypertonic stimulation increased the expression of VP in SON neurons, FCA, CBX and SDV inhibited its increasing, but SNL can not inhibit. (4) Hypertonic stimulation induced the expression of glutamate and GFAP in cultured astrocytes significantly up-regulated, at 3min peaked. The cells pre-treated by CBX, hypertonic stimulation increased glutamate expression and maintained high level. (5) Hypertonic stimulation significantly increased the concentration of glutamate in medium, but the concentration of taurine did not increase. (6) After hypertonic stimulation, the number of Cx43 hemichannels in SON significantly increased.
Conclusion Hypertonic stimulation activated SON astrocytes, activated astrocytes synthesized glutamate and released it via Cx43 hemichannels, glutamate signal activated neurons via NMDAR and increased synthesis and release of VP.
引文
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