Induction of inducible nitric oxide synthase expression in activated microglia following domoic acid (DA)-induced neurotoxicity in the rat hippocampus

Neuronal degeneration followed by glial activation (microglia and astrocytes) and nitric oxide synthase (NOS) expression in the hippocampus was investigated at 3 months after domoic acid (DA) administration and compared with DA treated rats at 5 days time interval which was reported earlier. Massive degeneration with complete absence of neurons in the hippocampal CA1 and CA3 regions and hypertrophied microglial cells showing intense immunoreaction with the antibody OX-42 was observed at 3 months after DA administration. Sparsely distributed OX-42 positive microglial cells were observed in the hippocampus of control rats at 3 months after saline treatment No apparent changes could be observed in the immunoreactivity of GFAP at 3 months after saline and DA administration. Neuronal nitric oxide synthase immunoreactive neurons were completely absent in the hippocampus at 3 months after DA administration. In contrast, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemical analysis revealed absence of NADPH-d reactivity in the neurons, but positive reactivity in the microglial cells of CA1-CA3 regions in the hippocampus after DA treatment. Double immunofluorescense revealed co-expression of inducible nitric oxide synthase with immunoreactive OX-42 positive microglial cells in the hippocampal subfields at 3 months after DA administration. The microglia-produced NO appears to be a secondary phenomenon in the prolonged inflammatory process following DA-induced neuronal degeneration.     

The repeated immobilization stress increases IL-1beta immunoreactivities in only neuron, but not astrocyte or microglia in hippocampal CA1 region, striatum and paraventricular nucleus

The effect of repeated immobilization stress (RIS) on the expression of interleukin-1β (IL-1β) and types of cells that express IL-1β in hippocampal CA1 region, striatum and paraventricular nucleus (PVN) were investigated in ICR mice. The RIS was induced daily for 2 h for 4 consecutive days. In the immunohistochemical study, RIS increased IL-1β immunoreactivities (IR) in the hippocampal CA1 region and striatum and PVN. The RIS also increased glial fibrillary acidic protein (GFAP) IR and complement receptor type 3 (OX-42) IR in the hippocampal CA1 regions and striatum but not PVN. In confocal immunofluorescence study, the IL-1β IR increased by RIS were colocalized with only NeuN, but not GFAP or OX-42 in the hippocampal CA1 region, striatum and PVN. Our results indicate that RIS increases IL-1β IR on neuron, but not astrocyte or microglia in the hippocampal CA1 region, striatum and PVN, suggesting that the IL-1β IR on neuron may play an important role during RIS. In addition, GFAP and OX-42 increased by RIS may be involved indirectly in playing another role in the hippocampal CA1 region and striatum during RIS.     

海人酸、使君子酸致Huntington病鼠纹状体NOS阳性细胞的变化

为了研究海人酸和使君子酸对大鼠纹状体 NOS阳性细胞的影响 ,用神经毒海人酸、使君子酸破坏大鼠左侧尾壳核 ,将夜间活动超过 6 0 0次、主动回避试验阳性率在 35 %以下的大鼠作为成功的 Huntington舞蹈病模型。注射后 2个月 ,将动物脑切片进行 Nissl、NADPH-d组化和 GF AP免疫组化反应。结果表明 ,海人酸和使君子酸均可使纹状体 n NOS阳性神经元丢失、出现i NOS阳性胶质细胞和侧脑室扩大 ,两者无显著差异。在损伤中心区 n NOS阳性神经元减少甚至消失 ,但这种变化自中心向周围呈渐变趋势 ;i NOS阳性胶质细胞呈两种不同形态 :一类胞体略大而突起粗短 ,一类胞体小而突起相对较长。对侧纹状体及伤侧纹状体非损毁部均未见 NOS阳性胶质细胞 ;NADPH-d组化和 GFAP免疫组化双重反应表明一些 i NOS胶质细胞为 GFAP阳性胶质细胞。本研究提示 ,海人酸和使君子酸均可使纹状体 n NOS神经元减少消失 ,损毁区出现的 NOS阳性胶质细胞为诱导型神经胶质细胞 (i NOS)。海人酸和使君子酸所引起的 Huntington病模型鼠形态学及行为变化均无明显差异     

Localization and characterization of nitric oxide synthase at the frog neuromuscular junction

Summary. The frog neuromuscular junction is sensitive to nitric oxide (NO), since exogenously applied NO reduces the release of transmitter by presynaptic terminals and the size of ATP-induced Ca²⁺ responses in perisynaptic Schwann cells. This study aimed at determining whether an NO synthase (NOS) is present at the neuromuscular junction, notably in perisynaptic Schwann cells, the glial cells at this synapse. The NADPH-diaphorase (NADPH-d) histochemical technique revealed the presence of NOS in cell bodies and presumed processes of perisynaptic Schwann cells. Incubation with NOS inhibitors, N^G-nitro-L-arginine methyl ester or N^G-monomethyl-L-arginine-acetate, abolished the NADPH-d staining. Moreover, L-arginine, the precursor of NO, impeded the blockade by NOS inhibitors, establishing the NOS specificity of NADPH-d staining in frog tissue. The pattern of labelling with a polyclonal antibody against the neuronal form of NOS was similar to the NADPH-d staining, also suggesting the presence of a neuronal NOS in perisynaptic Schwann cells. Using electron microscopy, the NOS immunostaining was found at the membrane and occasionally in the cytoplasm of perisynaptic Schwann cells and was not detected in the nerve terminal or muscle. There was no enzymatic or immunocytochemical labelling of NOS 6 days after denervation. It is concluded that NOS is present in frog perisynaptic Schwann cells. The presence of this endogenous NOS suggests that NO may act as a diffusible glial messenger to modulate synaptic activity and synapse formation at the neuromuscular junction.     

大鼠创伤性脑损伤后星形胶质细胞的变化

目的：探讨大鼠创伤性脑损伤后星形胶质细胞的形态学变化及GFAP和NOS的表达情况。方法：采用大鼠自由落体脑损伤模型，伤后1、3、7d取脑切片，行Nissl染色以及GFAP免疫组化和NADPH—d组化单标记及双标记染色。结果：损伤区周围皮质GFAP阳性细胞胞体增大、突起增粗增长，GFAP阳性细胞数量与正常侧及对照组相比，伤后1d即有明显增加，伤后3d、7d数量持续增加；损伤侧海马CAI～3区和DG各层GFAP阳性细胞排列紊乱，胞体增大、突起增粗增长，GFAP阳性细胞数量与正常侧及对照组相比则无明显变化。损伤区周围皮质、损伤侧海马NOS阳性细胞数量明显增加。伤后3d损伤区周围皮质和损伤侧海马中GFAP与NOS双标细胞分别占GFAP阳性细胞的14．2％和13．4％左右。结论：大鼠创伤性脑损伤后大量的星形胶质细胞活化、GFAP表达增加并且部分转化为NOS阳性细胞，提示其参与了脑组织的损伤与修复过程。     

大鼠额叶皮质损害后诱导型iNOS阳性细胞的变化

目的：一氧化氮在脑内的许多生功能中起重要作用,并参与脑损伤的病理生理过程。本研究旨在探讨实验性脑损伤后的NOS阳性细胞的来源及成份。方法：利用机械油吸法制成大鼠额叶皮质损伤动物模型,应用NADPH-d组织化学及GFAP免疫组化法观察损伤后1、3、7、14、21、30及60d皮质损害区NOS阳性细胞的类型和变化。结果：损害后1d即见皮质损害区底部和两侧nNOS阳性细胞增加,损害底部出现诱导型胶质细胞,尤以胼胝体中更为密集。这种反应3-7d时逐渐增强,2周时最明显,以后随时间推移及损害的修复而降低,研究发现部分iNOS阳性细胞与GFAP者共存,提示系反应性胶质细胞,未见eNOS阳性细胞上调现象。结论：皮质受损时,出现2种不同表型的NOS阳性细胞且上调,即出现诱导型神经元nNOS和诱导型iNOS胶质细胞。究其来源各不相同,但均能合成NO,NO主要集中在损伤部位的周围。     

Kainic acid (KA)-induced Ca2+/calmodulin-dependent protein kinase II (CaMK II) expression in the neurons, astrocytes and microglia of the mouse hippocampal CA3 region, and the phosphorylated CaMK II only in the hippocampal neurons

In the present study, we investigated the role of Ca2+/calmodulin-dependent protein kinase II (CaMK II) and which types of neuronal cells contain CaMK II and phosphorylated CaMK II (p-CaMK II) in the CA3 hippocampal region of mice using confocal immunofluorescence study. KA increased the CaMK II, p-CaMK II, glial fibrillary acidic protein (GFAP) and complement receptor type 3 (OX-42) immunoreactivities (IR) at 30 min after KA treatment in mouse hippocampal area. In studies, nevertheless KA-induced CaMK II is expressed in neurons or astrocytes or microglia, p-CaMK II is expressed only in neurons. Thus, our results suggest that the activated CaMK II in early time may be performed important roles only in neurons but not in the astrocytes and microglia.     

异丙酚预处理对拟阿尔茨海默病模型大鼠神经行为学和神经原纤维缠结及胶质纤维酸性蛋白的影响

目的:观察异丙酚预处理对拟阿尔茨海默病(AD)模型大鼠的神经行为学及海马损伤神经原纤维缠结(NFT)和胶质纤维酸性蛋白(GFAP)表达的影响,分析异丙酚的脑保护作用。方法:大鼠海马CA1区微量注射冈田酸(OA),建立拟AD大鼠模型。异丙酚预处理组大鼠于拟AD模型制作前30min,腹腔注射异丙酚100mg/kg。并以Morris水迷宫实验观察大鼠行为学变化,Bielschowsky染色观察NFT及免疫组织化学方法观察GFAP表达的变化。结果:与对照组大鼠相比,模型组大鼠学习记忆能力显著降低;Bielschowsky染色观察海马CA1区域出现大量NFT的特征性病理变化,GFAP免疫组织化学阳性细胞明显增多。与模型组大鼠相比,异丙酚预处理组大鼠学习记忆能力明显提高;海马CA1区NFT明显减少或消失,GFAP免疫组织化学阳性细胞明显减少。结论:拟AD模型建立前30min异丙酚预处理能改善拟AD模型大鼠学习记忆能力,降低NFT和GFAP的表达,有明显的脑保护作用。     

PKC抑制剂灯盏花素乙对甲醛炎性痛大鼠脊髓NOS表达及NO含量的影响

目的：观察蛋白激酶C（PKC）选择性抑制剂灯盏花素乙（CH）对甲醛炎性痛时大鼠自发痛反应、脊髓一氧化氮合酶（NOS）表达和一氧化氮（NO）含量的影响，探讨炎性痛时脊髓内NO产生是否受PKC调控。 方法： 采用右后掌足底注射甲醛复制炎性痛模型；计数缩足反射次数反映自发痛程度；应用NADPH-d组织化学法测定脊髓NOS表达；硝酸/亚硝酸还原法测定脊髓腰膨大部位NO2-/NO3-含量。 结果： 甲醛炎性痛大鼠L5脊髓后角浅层和中央管周围灰质NADPH-d阳性细胞的数目、阳性细胞胞体及纤维的染色深度均明显大于正常对照组，脊髓腰膨大部位 NO2-/NO3-含量明显增高。预先鞘内给予CH，可明显抑制甲醛炎性痛诱导的大鼠第二相自发痛反应以及脊髓NOS表达和NO2-/NO3-的含量。 结论： 炎性痛时，脊髓伤害性感受神经元内PKC激活可以促进NOS表达和NO的产生。     

L-NAME对沙土鼠脑缺血再灌流后海马区星形胶质细胞活化的作用机制研究

目的 探讨一氧化氮合酶(NOS)抑制剂左旋硝基精氨酸甲酯(L-NAME)对沙土鼠脑缺血再灌注后海马区胶质纤维酸性蛋白(GFAP)合成的影响。方法 钳夹沙土鼠的双侧颈总动脉制造脑缺血模型，应用免疫荧光法染色，观察GFAP密度的变化及分布。结果 脑缺血再灌流后海马区GFAP合成增加，GFAP阳性细胞主要分布在放射层及分子层，L-NAME能减少海马区GFAP的合成。结论 L-NAME是NOS强有力的抑制剂，L-NAME可能通过抑制NO的产生抑制了GFAP的合成。     

Ultrastructural localisation of NADPH-d/nNOS expression in the superior cervical ganglion of the hamster

This study examined NADPH-d and nNOS expression in the SCG of hamsters. By light microscopy, numerous NADPH-d/NOS positive processes were widely distributed in the ganglion. Ultrastructurally, the NADPH-d reaction product was associated with the membranous organelles of neuronal soma, dendrites, myelinated fibres, small granular cells, and axon profiles bearing agranular vesicles. The NOS immunoreaction product, on the other hand, was localised in the cytoplasm of principal neurons and dendrites. Some of the NADPH-d/NOS labelled processes formed junctional contacts including synapses or zonulae adherentia. Compared with the neurons, the nonneuronal cells in the ganglion, namely, macrophages, satellite cells and endothelial cells were labelled by NADPH-d but devoid of nNOS immunoreaction product. The results suggest that the NADPH-d/NOS positive fibres in the SCG originate not only from the projecting fibres of the lateral horns of thoracic spinal cord, but also from the principal neurons and small granular cells; some may represent visceral afferent fibres. Electron microscopic morphometry has shown that about 67% of the principal neurons contain NADPH-d reaction product, and that the majority were small to medium sized neurons based on cross-sectional areas in image analysis. On the basis of the present morphological study, it is concluded NO is produced by some local neurons and possibly some nonneuronal cells in the SCG as well as some fibres of extrinsic origin. In this connection, NO may serve either as a neurotransmitter or neuromodulator.     

Cytokines participate in neuronal death induced by trimethyltin in the rat hippocampus via type II glucocorticoid receptors

We investigated the role of IL-1alpha and IL-1beta expressed in the reactive gliosis following hippocampal damage induced by trimethyltin (TMT). IL-1alpha immunoreactivity was expressed earlier in small glial cells on day 4 post-TMT, while IL-1beta expression was obvious in large swollen glial cells on day 14 post-TMT. Both IL-1alpha and IL-1beta immunoreactivities were double-labeled with astrocyte marker, vimentin, but not with a microglia marker, OX-42. The expression of both IL-1alpha/beta was enhanced by adrenalectomy (ADX) prior to TMT administration. Corticosterone (CORT) or dexamethasone (DEX) supplementation not only cancelled effects of ADX, but also partially reversed TMT-induced enhancement of IL-1alpha/beta expressions. These changes coincided with TMT-induced neuronal death in CA3 pyramidal cells of the hippocampus. It is suggested that IL-1alpha/beta expressed in reactive astrocytes participate in TMT neurotoxicity via type II glucocorticoid receptors.     

Cytokines participate in neuronal death induced by trimethyltin in the rat hippocampus via type II glucocorticoid receptors

We investigated the role of IL-1alpha and IL-1beta expressed in the reactive gliosis following hippocampal damage induced by trimethyltin (TMT). IL-1alpha immunoreactivity was expressed earlier in small glial cells on day 4 post-TMT, while IL-1beta expression was obvious in large swollen glial cells on day 14 post-TMT. Both IL-1alpha and IL-1beta immunoreactivities were double-labeled with astrocyte marker, vimentin, but not with a microglia marker, OX-42. The expression of both IL-1alpha/beta was enhanced by adrenalectomy (ADX) prior to TMT administration. Corticosterone (CORT) or dexamethasone (DEX) supplementation not only cancelled effects of ADX, but also partially reversed TMT-induced enhancement of IL-1alpha/beta expressions. These changes coincided with TMT-induced neuronal death in CA3 pyramidal cells of the hippocampus. It is suggested that IL-1alpha/beta expressed in reactive astrocytes participate in TMT neurotoxicity via type II glucocorticoid receptors.     

Association study of the INPP1, 5HTT, BDNF, AP-2beta and GSK-3beta GENE variants and restrospectively scored response to lithium prophylaxis in bipolar disorder

The mesencephalic dorsolateral periaqueductal gray (dlPAG) mediates different modalities of aversive behaviors including pain and nociception and is anatomically delineated from other columns of the PAG by its content of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d). In many brain regions, neuronal NADPH-d is a nitric oxide (NO) synthase (NOS) and NO production mediates many nociceptive and aversive behavioral responses. The aim of this study was to determine how the noxious stimulant capsaicin affects intracellular dynamics in the dlPAG evidenced by Fos protein immunoreactivity (index of intracellular activation) and the NADPH-d reactivity. The basic hypothesis tested was that the effect of systemic capsaicin administration involved activation of the NO-producing machinery in the dlPAG. Compared to vehicle, capsaicin (50mg/kg, subcutaneous) significantly increased NADPH-d reactivity and Fos expression along the dlPAG neuraxis. However, less than one percent of the capsaicin-induced Fos activation occurred in NADPH-d-positive cells. This suggests that different intracellular mechanisms involving NO and activation of at least one other transmitter substance underlie the effects of capsaicin in the dlPAG. Although NADPH-d is a marker for constitutive NOS, only about two-thirds of the NADPH-d-positive neurons in the dlPAG were colocalized with neuronal NOS immunoreactive cells. This observation suggests that in contrast to other brain regions, neuronal NOS is unlikely to account for all NADPH-d activity in the dlPAG. Taken together, the present results show that the effect of capsaicin requires activation of at least one other transmitter and NADPH-d-dependent NO synthesis involving, but not limited to, the neuronal NOS isoform.     

Expression of NADPH-diaphorase and nitric oxide synthase in lumbosacral motoneurons after knee joint immobilisation in the guinea pig

The expression of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and nitric oxide synthase (NOS) in spinal ventral horn neurons was studied in the guinea pig after right knee joint immobilisation (RKJI). At 1 wk after RKJI, neurons in the ipsilateral ventral horn from L4 to S1 segments showed a moderate reactivity for NADPH-d staining. At 2 wk, NADPH-d labelled neurons were also observed in the contralateral ventral horn. Ipsilateral NOS immunoreactive cells were not detectable until wk 2. The intensity of NADPH-d and NOS labelled neurons in the bilateral ventral horns was sustained, peaking at the 4th wk after RKJI. In guinea pigs subjected to 4 wk of RKJI and subsequently released from the immobilisation for 2 and 4 wk, NADPH-d and NOS reactivity in ventral horn neurons diminished. The expression of NADPH-d positive neurons differed from that of NOS labelled neurons in terms of time interval, cell number and staining intensity, the latter being later, fewer and weaker. It is suggested that the induction and upregulation of NADPH-d and NOS are attributable to reduced activity of muscles acting on the knee joint after RKJI; the changes are reversible. It is speculated that increased levels of NO production are involved in protective mechanisms against possible neuronal degeneration as a consequence of target dysfunction.     

Expression of nitric oxide synthase in the developing rat hippocampus

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry was used to study the development of neurons synthesizing nitric oxide (NO) in the postnatal rat hippocampus. We show that NADPH-d reactive somata and processes are present from the day of birth until adulthood in the Ammon's horn. The dentate gyrus, however, has a more delayed period of nitric oxide synthase (NOS) expression with the staining appearing only by the end of the first postnatal week. Our results suggest that the time course of NOS expression correlates with the developmental sequence of events described in the hippocampus and that NO could be involved in the development of connections in this structure.     

睡眠剥夺后GFAP在大鼠视交叉上核星形胶质细胞的表达

为探索大鼠视交叉上核星形胶质细胞对睡眠剥夺及睡眠恢复的反应,本研究采用水环境小平台法建立大鼠睡眠剥夺模型,12只大鼠随机分为睡眠剥夺24h组,睡眠剥夺24h后恢复睡眠3h组,大平台对照组和正常单独饲养组,每组3只。用免疫组化的方法检测神经胶质酸性蛋白(GFAP)在视交叉上核的表达变化。结果如下:睡眠剥夺后GFAP在视交叉上核表达明显增强,睡眠恢复后表达降低。以上结果提示:星形胶质细胞可能参与睡眠的调节。     

长时间应用L-精氨酸增强大鼠学习记忆功能和大脑皮质、海马nNOS或c-fos的表达及神经元数目的增加(英文)

为探讨长时间应用一氧化氮(NO)对学习记忆功能和神经系统可塑性的影响,以及NO与c-fos基因表达的关系,分别给初断乳的大鼠灌胃NO前体左旋-精氨酸(L-Arg)或一氧化氮合酶抑制剂L-NAME,用Morris水迷宫检测大鼠的学习记忆功能,用免疫组化技术和HE染色检测大脑皮质和海马CA1、CA3、DG区的神经元型一氧化氮合酶(nNOS)和c-fos基因的表达以及神经细胞数目的变化。将大鼠随机分成L-Arg组、L-NAME组和对照组。大鼠断乳后分别每天用L-Arg[200mg/(kg.d)]、L-NAME[50mg/(kg.d)]和等剂量的蒸馏水灌胃,持续3个月。结果显示:长期应用NO前体L-Arg可明显缩短大鼠的寻台潜伏期,促进nNOS和c-fos基因的表达,同时大脑皮质和海马CA1、CA3、DG区的神经元数目增加;而长期应用一氧化氮合酶抑制剂L-NAME可抑制大鼠寻台潜伏期的缩短和nNOS、c-fos基因的表达,同时大脑皮质和海马CA1、CA3、DG区的神经元数目减少。因此我们认为长时间应用NO可促进神经系统c-fos基因的表达和幼鼠神经系统的发育,即可塑性的变化,继而影响大鼠的学习记忆功能。     

Cellular proliferation in the cerebral cortex following neural excitation in rats

Cortical spreading depression (SD) is characterized by propagation of neuronal/glial membrane depolarization throughout the unilateral cerebral cortex and has been linked to several neurological disorders, including migraine aura and epilepsy. SD induction resulted in a dramatic increase in BrdU-incorporated cells in the ipsilateral cortical hemisphere that was dependent on the number of elicited SD. Immunohistochemical studies revealed that 53% of the BrdU-labeled cells in the SD-generated cortex were NG2 immunopositive and 25% were OX-42 immunopositive. The remaining 22% of BrdU-incorporated cells showed no immunoreactivity to GST-rr, GFAP, NeuN, NG2 or OX-42.These data indicate that functional excitation of the cerebral cortex induces proliferative response in cortical cells, which may subsequently differentiate into glial progenitor or microglia within 3 days after stimulation.     

肝性脑病大鼠海马CA3区神经元形态和一氧化氮合酶表达的变化

目的观察肝性脑病模型组和正常对照组大鼠脑海马CA3区神经元的变化及一氧化氮合酶(NOS)的表达;探讨海马CA3区神经元的形态学改变及一氧化氮(NO)在肝性脑病发病机制中的作用。方法雄性大鼠50只,实验开始前所有动物均进行莫里斯水迷宫测试,之后将动物分为对照组和实验组。9周后建立CCL4肝性脑病模型,分别取两组大鼠海马组织进行尼氏染色及烟酰胺腺嘌呤二核苷酸-黄递酶(NADPH-d),染色。结果尼氏染色发现,实验组大鼠海马神经元数目减少、染色较浅,胞质内尼氏体减少或消失;NADPH-d染色发现,实验组可见粗大轴突着色,树突联系广泛;对照组则少有粗大轴突着色,树突间联系不如实验组广泛。实验组NOS阳性神经元染色较对照组深,为紫蓝或深蓝色(强阳性及阳性),且阳性神经元数目较多;而对照组染色浅淡,呈浅蓝或与背景同色,为弱阳性。结论肝性脑病时海马受到损伤,NO可能介导了神经元的损伤并参与了肝硬化和肝性脑病的发病,血氨升高是肝性脑病(HE)致病因素之一。