Changes in calbindin expression within the flocculus after unilateral labyrinthectomy in rats

The role of flocculus in vestibular compensation is still a controversial issue. Calbindin regulates intracellular signaling and has been reported to be a reliable marker of Purkinje cell. Expression of calbindin in flocculus was examined using immunohistochemistry following unilateral labyrinthectomy (UL) in rats. Both the staining intensity and number of calbindin-positive Purkinje cells in the ipsilateral flocculus to the lesion side decreased 6 h after UL compared to the control and contralateral side. Forty-eight hours after UL, the expression of calbindin returned to control levels and asymmetric expression in bilateral flocculus subsided. These transient reduction of calbindin expression in the ipsilateral flocculus may reflect a decrease in the GABAergic inhibition of the floccular Purkinje cell to the ipsilateral vestibular nuclei during vestibular compensation.     

一氧化氮合酶在坐骨神经夹伤后腓肠肌中的表达变化

目的:探讨外周神经损伤后同侧腓肠肌中3种一氧化氮合酶(nitric oxide synthase,NOS)的表达变化及定位。方法:采用H-E染色及Masson三色染色法分析大鼠坐骨神经夹伤后同侧腓肠肌的病理变化,NADPH-黄递酶组织化学研究其总NOS的改变,并利用Western印迹法、免疫荧光双标法,对3种NOS表达变化及定位进行分析。结果:神经夹伤后相应腓肠肌发生了明显的病理变化且总NOS发生改变,3种NOS变化不尽相同,其表达高峰均约在4周左右。nNOS与神经丝标记物NF-200有共定位,iNOS、eNOS则分别在巨噬细胞、血管内皮细胞中有表达。结论:3种NOS在坐骨神经夹伤后相应腓肠肌中表达变化不同,可能对肌肉损伤及再生修复发挥不同作用。     

Changes in pigment epithelium-derived factor expression following kainic acid induced cerebellar lesion in rat

Pigment epithelium-derived factor (PEDF) is a potent and broad-acting neurotrophic factor that protects various types of cultured neurons against glutamate excitotoxicity and induced apoptosis. The expression pattern and functions of PEDF in the central nervous system (CNS) remain largely undetermined. In this study, we analyzed the spatial and temporal expression of PEDF in normal and kainic acid (KA)-induced lesioned rat cerebellum using immunoblotting, immunohistochemistry and fluorescent in situ hybridization techniques. In normal rat cerebellum, PEDF protein and mRNA were mostly confined and co-localized with calbindin-positive cells in the Purkinje cell layer of the cerebellum, but not with glial fibrillary acidic protein (GFAP)-, 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase)-, and isolectin B4-positive cells. Injection of KA into the right cellebellum caused severe loss of calbindin-positive Purkinje neurons, and an increased number of GFAP-positive astrocytes and isolectin B4-positive microglia was observed on the ipsilateral side of the lesioned cerebellum. Although the PEDF level on the ipsilateral side of the cerebellum was dramatically decreased 2 days after KA treatment, significantly elevation of PEDF levels was observed at 7 days. In agreement with these results, PEDF protein and PEDF mRNA expression were co-localized with GFAP-positive reactive astrocytes in the ipsilateral side 7 days after KA treatment. Although the mechanism by which PEDF is induced in reactive astrocytes remains unclear, the increase in PEDF expression in injured brain may form part of a compensation mechanism against neuronal degeneration.     

Ovary and ovulation: Cell-specific localization of nitric oxide synthases (NOS) in the rat ovary during follicular development, ovulation and luteal formation

Nitric oxide (NO) has emerged as one of several important intraovarianregulatory factors. In particular, NO has been implicated inthe processes of ovulation and atresia-related apoptosis. Theaim of the present study was to investigate the presence anddistribution of the NO-generating nitric oxide synthase (NOS)enzymes in the ovary during follicular development, ovulationand luteal formation of the equine chorionic gonadotrophin (ECG)/humanchorionic gonado-trophin (HCG)-primed rat NADPH diaphorase activitywas used as a histochemical marker for NOS within the ovary.Diaphorase reactivity was most abundant in the stroma (S) ofthe ovary and in the theca (T) layer of the follicle. In luteinizedovaries, weaker diaphorase reactivity was present within thecorpora lutea (CL). Two different isoforms of NOS, the constitutivelyexpressed endothelial NOS (eNOS) and the inducible isoform ofNOS (iNOS), were immunolocalized in ovaries of immature ratsand in ECG/HCG-primed rats during the periovulatory period fromHCG injection until 2 days after ovulation. In addition, ovarianconcentrations of eNOS and iNOS were quantified by immunoblotting.Immunoblotting with a monoclonal anti-eNOS antibody demonstratedthe presence of eNOS mainly in the residual ovary (ROV) duringthe periovulatory period. In luteinized ovaries, higher concentrationsof eNOS were seen in CL, while those in the ROV at this stagewere lower than in the periovulatory ovary. Immature ovariescontained diminutive amounts of eNOS, detectable mostly in theROV compartment. In contrast, iNOS was barely detectable duringfollicular development to the preovulatory stage. A slight elevationof iNOS was observed in the granulosa cells at 6 h after theHCG injection. The levels of iNOS during the luteal phase werealso low. Immunohistochemical analysis using polyclonal eNOSand iNOS antibodies revealed the localization of these two isoformsprimarily in the S and the T of the periovulatory ovary. Inluteinized ovaries, positive immunoreactivity was also seenwithin the CL. With a monoclonal antibody against eNOS, intenseimmunoreactivity was observed in the S, T and within CL. Therewas a particularly strong staining in blood vessels. These datademonstrate the presence of an intraovarian NO-generating system.The localization of this system to the S, T and CL suggestsa role for NO in the ovulatory process and in the regulationof CL function.     

Localization of nitric oxide synthase-like immunoreactivity in the developing nervous system of the snail Ilyanassa obsoleta

Production of nitric oxide (NO), an evolutionarily conserved, intercellular signaling molecule, appears to be required for the maintenance of the larval state in the gastropod mollusc Ilyanassa obsoleta. Pharmacological inactivation of endogenous nitric oxide synthase (NOS), the enzyme that generates NO, can trigger metamorphosis in physiologically competent larvae of this species. Neuropils in the brains of these competent larvae display histochemical reactivity for NADPH diaphorase (NADPHd), an indication of neuronal NOS activity. The intensity of NADPHd staining is greatest in the neuropil of the apical ganglion (AG), a region of the brain that contains the apical sensory organ and that innervates the bilobed ciliated velum, the larval swimming and feeding organ. Once metamorphosis is initiated, the intensity of NADPHd staining in the AG and presumably, concomitant NO production, decline. The AG is finally lost by the end of larval metamorphosis, some 4 days after induction. To determine if the neurons of the AG are a source of larval NO, we conducted immunocytochemical studies on larval Ilyanassa with commercially available antibodies to mammalian neuronal NOS. We localized NOS-like immunoreactivity (NOS-IR) to 3 populations of cells in competent larvae: somata of the AG and putative sensory neurons in the edge of the mantle and foot. Immunocytochemistry on pre-competent larvae demonstrated that numbers of NOS-IR cells in the AG increase throughout the planktonic larval stage.     

Localization of nitric oxide synthase and effects of nitric oxide donors on the human Fallopian tube.

The objective of the study was to assess the plausible existence of a nitric oxide (NO) system within the human Fallopian tube and to examine the effects of NO on tubal contractility. Tissue was obtained from fertile women at operations due to non-tubal diseases. Production of NO and sites of nitric oxide synthase (NOS) activity were assessed by the use of NADPH diaphorase staining and by immunoblots as well as immunohistochemistry for the isoforms of NOS. Effects of NO on tubal contractility in vitro were examined by adding either of two NO donors (nitroglycerin, spermine NONOate) or an analogue of its second messenger (8-bromo cyclic GMP). The production of NO was indicated by positive NADPH diaphorase staining. In immunoblots, endothelial and inducible NOS were demonstrated in all samples analysed. By immunohistochemistry, moderate staining for endothelial NOS was demonstrated in the luminal epithelial cells and in the endothelial cells of blood vessels. Moderate staining for inducible NO synthase was seen in smooth muscle cells and weak staining in epithelial cells. Nitroglycerin, spermine NONOate and 8-bromo cGMP all resulted in a concentration-dependent inhibition of contractility with significant contractility inhibition at 10(-7) mol/l, 10(-6) mol/l and 10(-5) mol/l respectively. The study demonstrates the existence of an endogenous NO system, which may be of physiological importance in Fallopian tube function.     

Induction of heat shock proteins and motor function deficits after focal cerebellar injury

A weight drop model of focal cerebellar injury was used to identify heat shock protein induction and motor function deficits in the anesthetized, adult male, Sprague-Dawley rat. All animals were trained on a beam walking test prior to surgery. Groups of animals received severe, mild or sham weight drop injury to the lateral/paravermal region of the cerebellum. The mild and sham-injured animals showed no motor deficits in the beam walking test, whereas animals with severe cerebellar injury showed significant motor deficits in the beam walking test that approached recovery of motor function 20 days after injury. Following severe injury, induction of heat shock protein of 27kDa was observed in Purkinje cells and in neurons of the deep cerebellar nuclei, as well as Bergmann glial cells, glial cells located in the granule cell layer and the underlying white matter. Following mild injury, heat shock protein of 27kDa induction was observed in Purkinje cells and glial cells, but not in neurons of the deep cerebellar nuclei. The labeled Purkinje cells were widely distributed in the ipsilateral cerebellar cortex. Many of the glial cells that were immunostained with heat shock protein of 27kDa co-localized with cells immunoreactive for glial fibrillary acidic protein. After severe injury, heat shock protein of 72kDa was localized mainly in granule cells at the site of the trauma and in the ipsilateral deep cerebellar nuclei whereas, after mild injury, light labeling was observed only in the granule cell layer. The results demonstrate that focal cerebellar injury has profound effects on motor behavior and induces different families of heat shock proteins in specific groups of neurons and glial cells in the cerebellum.     

2型糖尿病模型db/db小鼠海马NOS阳性神经元变化

目的　观察人类 2型糖尿病模型———C5 7BL/KsJdb/db(db/db)小鼠海马NOS阳性神经元变化。方法　糖尿病组 :6周龄C5 7BL/KsJ(db +db +)小鼠 5只 ,尾静脉空腹血糖高于 11.1mmol/L且肥胖。对照组 :非糖尿病小鼠C5 7BL/KsJ(?+) 5只 ,尾静脉空腹血糖低于 6 .0mmol/L体重正常 ,于 30周龄 (成模第 6月末 )时 ,灌注固定取脑 ,以NADPH d组化法显示海马NOS阳性神经元。结果　与正常对照组相比 ,糖尿病组小鼠海马齿状回NOS阳性神经元密度显著减少 (P <0 0 1)。结论　糖尿病时NOS阳性神经元数量减少 ,NO的合成降低表明NO可能参与糖尿病中枢神经系统功能障碍     

Nitric oxide synthase immunoactivity and NADPH diaphorase enzyme activity in neurons of the gastrointestinal tract of the toad, Bufo marinus.

The presence of nitric oxide synthase (NOS) was demonstrated immunohistochemically, and NADPH diaphorase was demonstrated by enzyme histochemistry in neurons throughout the gastrointestinal tract of the anuran amphibian, Bufo marinus. Successive staining showed that NOS immunoreactivity and NADPH diaphorase activity occurred in precisely the same subgroup of enteric neurons. Subsequent detailed studies of the distribution of these neurons were made using NADPH diaphorase histochemistry. Numerous reactive nerve cell bodies and fibres were found in the myenteric plexus from the esophagus to the cloaca. A dense innervation of the longitudinal and circular muscle layers occurred throughout the gastrointestinal tract. The lamina muscularis mucosae was only prominent in the stomach, where it was sparsely innervated. Reactive nerve cell bodies were common in the submucosa of the large intestine, less common in the small intestine and extremely rare in the stomach and esophagus. Reactive fibres contributed to subepithelial plexuses in the esophagus, colon, rectum and cloaca. It is concluded that NOS/NADPH diaphorase is conserved amongst vertebrate classes and that NO is a likely neurotransmitter in the toad gastrointestinal tract.     

Transient expression of NADPH diaphorase activity in the mouse whisker to barrel field pathway

Summary Development of the topographic map of the somatosensory cortex of rodents appears to depend on fine-tuned patterns of neuronal activity. Nitric oxide (NO) has been described as a potent messenger in the modulation of neural activity associated with synaptic plasticity. To evaluate the role of NO in the murine somatosensory pathway, we investigated NO synthase activity by NADPH diaphorase histochemistry at crucial developmental stages. At birth, NADPH diaphorase activity was detected in the cortical plate of the developing somatosensory cortex. At day 3, diffuse NADPH diaphorase activity increased within the emerging layer 4 in the future barrel field hollows. This staining was most intense at day 6 in the barrel field hollows and became undetectable by the end of the second postnatal week. The appearance of the diffuse NADPH diaphorase staining pattern was also observed in a similar time course and topography in the ascending relays of the somatosensory cortex, specifically in the barreloids within the ventrobasal nucleus of the thalamus and the barrelettes of the trigeminal nucleus of the brainstem. Lesioning the C row of whiskers at day 1 (i.e. during the critical period of barrel formation) led to fused C barrels of diffuse NADPH diaphorase activity in the barrel fields. In addition, highly NADPH diaphorase activity-positive individual cells present in the deeper layers of the somatosensory cortex at days 0 and 3 became visible in the upper layers at day 6 and remained until day 15. In layer 4, these cells were predominantly localized in the septa at day 6 and 9. No positive individual cells were detected in barrelettes or barreloids at any age. We conclude that NADPH diaphorase activity is present during experience-dependent consolidation of synaptic contacts in the somatosensory pathway.     

Input to the vestibulo cerebellum and triceps brachii motoneurons during natural vestibular stimulation in frog

The response of cerebellar Purkinje cells and nerve triceps brachii was recorded in paralyzed frogs during natural vestibular stimulation. The response from about 63% of the Purkinje cells (mossy fiber input) recorded in the vestibulo cerebellum and of the triceps nerve during triangular wave roll oscillation consisted of activity increase during the ipsilateral side-down half of the cycle and decrease during the contralateral side-down half. It was shown that this activity, which originates partially from ipsilateral vertical canals, can be added to, or suppressed by, otolithic activity, depending on head position and direction rotation.The fact that the response of Purkinje cells was similar to that of triceps nerve implies that the vestibulo-cerebellum receives information of vestibular signals passing to the motor system. The characteristics of otolithic-canal interaction recorded in triceps nerve may explain the motor disturbances that result from lesions of otolithic receptors.     

Immunohistochemical localization of peripheral nitric oxide synthase-containing nerves using antibodies raised against synthesized C- and N-terminal fragments of a cloned enzyme from rat brain

Antibodies were raised in rabbits against C- or N-terminal fragments of a cloned nitric oxide synthase (NOS) enzyme from rat cerebellum, and used for demonstration of NOS-immunoreactive (NOS-IR) nerves in different tissues from the rat (colon, duodenum, adrenal gland, aorta, caval vein, penis and urethra). Both antisera demonstrated the same neuronal elements, although with differences in intensity in the immunoreaction in some tissues. Sections incubated with antisera preabsorbed with excess of the antigens showed no NOS immunoreactivity. In duodenum and colon, NOS-immunoreactivity was found in the cytoplasm of numerous cell bodies in myenteric ganglia and in some nerve cell bodies in the submucosa. NOS-IR nerve fibres were numerous in the circular muscle layer, while few were found in the longitudinal layer or the mucosa and submucosa. In the penis, strong NOS immunoreactivity was found in nerves surrounding the deep penile and dorsal arteries, and in nerves in the stroma of the cavernous tissue. In the urethra, NOS immunoreactivity was found in nerves in the mucosa. No NOS immunoreactivity was found in the urothelium. The adrenal medulla, and occasionally the cortex, contained nerve cell bodies with strong cytoplasmic NOS immunoreactivity as well as scattered nerve fibres. No NOS immunoreactivity was found in the abdominal aorta or inferior caval vein. Combined NOS immunostaining and NADPH diaphorase staining showed that virtually all NOS-IR nerve structures were also NADPH diaphorase-positive. However, thin nerve fibres and cell linings were sometimes better visualized by NOS-immunohistochemistry. Furthermore, the adrenal cortex, which only occasionally showed NOS immunoreactivity, was strongly NADPH diaphorase-positive. A positive NADPH diaphorase reaction, but a negative NOS immunoreactivity, was also found in other structures, such as urothelium, epithelial cells in duodenum and colon, and endothelium of some vessels. It is concluded that the antibodies raised against the synthesized sequences of neuronal NOS are highly specific and may be used in immunohistochemistry in order to detect neuronal NOS.     

一氧化氮合酶阳性神经元在小鼠脑内的分布

目的 :研究一氧化氮合酶 (NOS)在小鼠脑内的分布。方法 :用NADPH 黄递酶组织化学技术 ,观察了NOS阳性神经元在小鼠脑内的分布和形态。结果 :在大脑皮质、纹状体、基底前脑、杏仁核、下丘脑和脑干等处有较多一氧化氮合酶阳性神经元的分布。结论 :表明NO与中枢神经系统的诸多功能有关     

应激性防御反应过程中脑内一氧化氮合酶阳性神经元分布的变化

本实验采用 NADPH-d方法研究发现 :在应激的早期 ( 1～ 6d) ,一氧化氮合酶阳性神经元在大脑皮质、基底前脑、纹状体、间脑和脑干内出现的部位增多 ,一氧化氮合酶阳性神经元的数量也增多 ;而在应激的晚期 ( 9d以后 ) ,一氧化氮合酶阳性神经元出现的部位及数量明显减少。提示在慢性应激的早期一氧化氮合酶活性增高 ,合成一氧化氮的能力增强 ;而在应激的晚期一氧化氮合酶活性降低 ,合成一氧化氮的能力降低     

兔急性上颌窦炎早期一氧化氮合酶的表达及意义

目的 :探讨一氧化氮合酶 (NOS)在兔急性上颌窦炎 (AMS)窦粘膜中的表达及其意义。方法 :健康新西兰白兔 36只 ,分为空白、阴性对照组和AMS组。通过阻塞窦口并注入 1.0ml的肺炎链球菌悬液 (10 9CFU)建立AMS模型 ,观察时间点为自手术第 5、10天。应用黄递酶———NADPH组织化学技术 ,以NADPH脱氢酶特异性测定NOS在空白、阴性对照组和AMS组兔上颌窦粘膜中的分布及不同时间点AMS组NOS活性表达。结果 :正常和急性上颌窦炎兔窦粘膜酶化学染色均有反应 ,主要分布于粘膜上皮、血管内皮和腺体细胞 (染色程度与正常组相比 ,P<0 .0 5或P <0 .0 1)。结论 :正常兔上颌窦粘膜存在NOS ,一氧化氮 (NO)与上颌窦急性炎症有关 ,炎症时NOS活性明显增高 ,过多的NO会对组织或细胞产生损伤 ,提示NO在AMS发病机制中有重要意义     

Zonal organization of the vestibulo-cerebellum in the control of horizontal extraocular muscles using pseudorabies virus: I. Flocculus/ventral paraflocculus

Much literature has studied the relationship between the organization of neurons in the flocculus/ventral paraflocculus and vestibulo-ocular reflex pathways. Although activation of a flocculus central zone produces ipsilateral horizontal eye movement, anatomical tracing evidence in rats suggests that there may not be a simple one-to-one correspondence between flocculus/ventral paraflocculus zones and control of single extraocular muscles or coplanar pairs of antagonistic extraocular muscles. This study used the retrograde transynaptic transport of pseudorabies virus to identify the topographical organization of Purkinje cells in the flocculus/ventral paraflocculus that control the lateral rectus (LR) and medial rectus (MR) muscles in rats. A survival time of 80 h and 84 h was necessary to observe consistent transynaptically labeled cells in the flocculus/ventral paraflocculus following injections of pseudorabies virus into the MR and LR, respectively. The organization of Purkinje cells in the dorsal flocculus and ventral paraflocculus abided by the traditional boundaries, whereas the labeling pattern in the ventral flocculus showed a more complex, interdigitated arrangement. In agreement with prior studies, transynaptically labeled neurons were also observed in specific vestibular nuclear regions within the medial and superior vestibular nuclei and dorsal Y group. The distribution of labeled neurons in ipsilateral and contralateral vestibular nuclei was associated with features of ipsilateral and contralateral retrograde labeling of Purkinje cells in flocculus/ventral paraflocculus. Importantly, this study provides the first evidence of vestibulo-cerebellar zones controlling individual extraocular muscles and also overlapping distribution of neurons in flocculo-vestibular zones that influence the LR and MR motoneuron pools. This suggests that some of these neurons may be responsible for controlling both muscles.     

Timing of bilateral cerebellar output evoked by unilateral vestibular stimulation in the frog

Electrical stimulation of one VIIIth nerve evoked simple spike activity in Purkinje cells located on either side of the cerebellum. This cerebellar output was delayed by ca. 10 ms with respect to its mossy fiberparallel fiber input. The onset of the cerebellar output occurs on the average simultaneously on either side of the corpus cerebelli. The delay is explained by slowly rising EPSPs in PC induced by primary afferent and by second and higher order vestibular fibers. The latter inputs are stronger and terminate ipsi- and contralaterally in the granular layer.Supported by Deutsche Forschungsgemeinschaft (Pr. 158/1)     

Recombinant L7/L12 ribosomal protein and gamma-irradiated Brucella abortus induce a T-helper 1 subset response from murine CD4+ T cells.

Nitric oxide (NO) has become recognized as a multifunctional mediator, with roles in vascular physiology, neurotransmission and non-specific immune defense. The histochemical marker associated with the neural and endothelial form of NO synthase (NOS), reduced nicotinamide adenine dinucleotide diaphorase (NADPHd), has enabled the indirect localization of potential sites of NO production. Innervation of the thymus and its immunological functions made this tissue a candidate for utilization of various NO systems. In the present study on adult rat thymus, multiple cellular sites expressing NADPHd activity, thereby implicated as sites of NOS activity, have been identified using morphological criteria alone: blood vessel endothelium, dendritic cells, deep cortical or medullary stromal cells, intrinsic neuron-like profiles, granulocytes (possibly neutrophils) and fat cells. In addition, the availability to the thymic microenvironment of another form of NOS in macrophages, which is not stained by the diaphorase technique, was supported by the observation of these cells at corticomedullary and cortical locations. These results indicate that a wide variety of possible immunomodulatory roles can be expected for NO in the thymus including the induction of tolerance, major histocompatibility complex (MHC) restriction, lymphocyte trafficking and regulation of thymic endocrine output.