亚低温对大鼠局灶性脑缺血再灌注后不同脑区iNOS表达的影响

目的 探讨亚低温对大鼠局灶性脑缺血再灌注后不同脑区诱导型一氧化氮合酶(iNOS)表达的影响.方法 雄性SD大鼠,随机分为假手术组、常温缺血组和亚低温组.采用线栓法制作大脑中动脉闭塞再灌注模型,于缺血后48h,观察不同组间组织形态学变化,检测不同脑区iNOS蛋白表达、iNOS活性和产物NO含量.结果 常温缺血后48h,纹状体和皮质均检测到iNOS活性升高和免疫阳性反应,且皮质缺血半暗带区iNOS免疫反应明显强于纹状体和皮质缺血核心区.亚低温明显缩小梗死面积,抑制皮质和纹状体iNOS活性,明显下调半暗带区iNOS蛋白表达,减少NO产生.结论亚低温可能通过减少半暗带区iNOS蛋白表达,抑制iNOS活性,减少NO产生而起到脑保护作用.     

亚低温对大鼠局灶性脑缺血再灌注后半暗带区iNOS诱导细胞凋亡的影响

目的通过研究亚低温对大鼠局灶性脑缺血再灌注后诱导型一氧化氮合酶（iNOS）表达和细胞凋亡的影响，探讨亚低温脑保护的可能机制。方法雄性SD大鼠54只随机分为假手术组、常温缺血组和亚低温组。线栓法制备大脑中动脉闭塞再灌注模型。于缺血后48h取脑组织，邻片行HE染色，检测各组不同脑区iNOS蛋白表达和细胞凋亡情况，免疫双重染色研究iNOS蛋白表达与细胞凋亡间的关系，同时行NO含量测定。结果常温缺血组皮质缺血半暗带（IP）区iNOS免疫反应较强，TUNEL阳性细胞也主要位于皮质IP区，免疫双重染色发现TUNEL阳性细胞中存在着iNOS蛋白表达。亚低温组IP区iNOS蛋白表达明显下调，NO产生减少．IP区细胞凋亡的数目也减少。结论亚低温可能通过抑制IP区iNOS蛋白表达，减少NO产生．阻遏细胞凋亡，从而起到脑保护作用。     

脑室内注射乙酰胆碱受体抗体IgG对大鼠神经元凋亡及一氧化氮合酶表达的影响

目的研究乙酰胆碱受体抗体(AchRab)对大鼠脑内神经元的损害及一氧化氮合酶(NOS)在损害中所起的作用，探讨重症肌无力(MG)中枢神经系统损害的机制。方法将AchRab IgG或健康人的IgG注入大鼠侧脑室。HE染色、TUNEL法检测细胞凋亡；免疫组化方法观察大鼠皮质、海马及杏仁核神经元型一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)表达变化。结果2周后实验组皮质、海马及杏仁核凋亡细胞明显增多，对照组仅见少量凋亡。实验组皮质、海马及杏仁核nNOS神经元数目明显减少。实验组及对照组脑内细胞均来见iNOS表达。结论AchRab脑内注射可诱导神经元凋亡；损伤皮质。海马及杏仁核nNOS神经元；但未能诱导脑内细胞iNOS表达。神经元凋亡损害参与了AchRab对中枢神经损害的机制；nNOS神经元的减少，可能与MG认知功能障碍有密切关系；而神经元的损伤可能与NO的毒性作用无关。     

氨基胍对大鼠脑缺血-再灌流损伤保护作用的研究

目的:探讨诱生型一氧化氮合酶抑制剂氨基胍(Aminogunidine,AG)对脑缺血-再灌流损伤保护作用的机制。方法:将大鼠随机分为对照组、AG治疗组、假手术组,测定再灌流各时相点脑内一氧化氮含量,观察光镜下顶叶皮层尼氏染色、HE染色改变。结果:再灌流不同时相点AG治疗组NO含量,顶叶皮层神经元变性程序及数量、微血管数目明显低于对照组。结论:AG可以抑制iNOS活性,降低脑内NO含量,减轻半暗带区神经元变性、微循环障碍和血脑屏障破坏,具有脑保护作用。     

表没食子儿茶素没食子酸酯保护阿米卡星诱导耳蜗螺旋神经节神经元的凋亡

透射电镜观察SD大鼠耳蜗螺旋神经节神经元形态变化,发现阿米卡星可以诱导耳蜗螺旋神经节神经元凋亡。免疫组化染色和RT-PCR检测发现,阿米卡星诱导耳蜗螺旋神经元Bcl-2 蛋白表达下调,Bax、Caspase-3蛋白及Caspase-6 mRNA表达增强。表没食子儿茶素没食子酸酯可以抑制耳蜗螺旋神经元Bax、Caspase-3蛋白及Caspase-6 mRNA的表达,同时增强Bcl-2蛋白表达,从而降低耳蜗螺旋神经元凋亡率。证实表没食子儿茶素没食子酸酯对阿米卡星损伤的耳蜗螺旋神经节具有保护作用。     

Aβ1—40海马注射对大鼠脑内一氧化氮合酶表达的影响

目的探讨一氧化氮合酶(NOS)在β淀粉样蛋白(Aβ)神经毒性及阿尔茨海默病(AD)病理机制中的作用.方法应用免疫组化方法,观察大鼠海马齿状回Aβ1-40注射后神经元型一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)表达变化.结果正常大鼠海马齿状回区含nNOS神经元计数为8.96±0.35个/视野;生理盐水注射后局部含nNOS神经元无明显变化(8.97±0.29个/视野);Aβ1-40注射后,注射区周围含nNOS神经元数目显著减少(2.98±0.24个/视野).正常及生理盐水注射组脑内未见iNOS表达;Aβ1-40注射后2d、10d和30d,注射区持续出现大量含iNOS的胶质细胞(主要为星形胶质细胞),反应面积分别为0.905±0.082、0.962±0.161、0.935±0.125mm2.结论Aβ1-40海马注射可损伤局部含nNOS神经元及诱导胶质细胞iNOS表达,NOS在Aβ神经毒性和AD发病中有重要作用.     

人参皂甙对β淀粉样蛋白诱导THP-1单核细胞一氧化氮合酶和一氧化氮的影响

目的研究人参皂甙对β淀粉样蛋白(βamyloid,Aβ)1-40诱导THP-1细胞表达诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和一氧化氮(nitric oxide,NO)的影响。方法用Western blotting测定THP-1细胞iNOS表达,以Griess法测定细胞上清中NO的浓度。结果Aβ模型组iNOS的表达明显高于对照组(P<0.01)。与模型组比较,人参皂甙可明显减少THP-1细胞iNOS的表达和NO的产生(P<0.05)。随着人参皂甙剂量的增大,人参皂甙可引致THP-1细胞iNOS的表达和NO的产生明显减少(P<0.05),且有剂量依赖性。结论人参皂甙可抑制THP-1细胞iNOS的表达,进一步影响NO的产生。     

Aβ_(1-40)海马注射对大鼠脑内一氧化氮合酶表达的影响

目的　探讨一氧化氮合酶 (NOS)在 β淀粉样蛋白 (Aβ)神经毒性及阿尔茨海默病 (AD)病理机制中的作用。方法　应用免疫组化方法 ,观察大鼠海马齿状回Aβ1 4 0 注射后神经元型一氧化氮合酶 (nNOS)和诱导型一氧化氮合酶 (iNOS)表达变化。结果　正常大鼠海马齿状回区含nNOS神经元计数为 8 96± 0 35个 /视野 ;生理盐水注射后局部含nNOS神经元无明显变化 (8 97± 0 2 9个 /视野 ) ;Aβ1 4 0 注射后 ,注射区周围含nNOS神经元数目显著减少 (2 98± 0 2 4个 /视野 )。正常及生理盐水注射组脑内未见iNOS表达 ;Aβ1 4 0 注射后 2d、10d和 30d ,注射区持续出现大量含iNOS的胶质细胞 (主要为星形胶质细胞 ) ,反应面积分别为 0 90 5± 0 0 82、0 96 2± 0 16 1、0 935± 0 12 5mm2 。结论　Aβ1 4 0 海马注射可损伤局部含nNOS神经元及诱导胶质细胞iNOS表达 ,NOS在Aβ神经毒性和AD发病中有重要作用。     

S-甲基异硫脲对脂多糖诱导多巴胺能神经元变性的保护作用

目的　 探讨诱导型一氧化氮合酶 (iNOS)抑制剂S 甲基异硫脲 (S methylisourea ,SMT)对脂多糖(lipopolysaccharide ,LPS)诱导多巴胺 (DA)能神经元变性的保护作用及其机制。 方法　 4 8只大鼠随机分成 4组(n =12 ) :磷酸缓冲液 (PBS)对照组、LPS组、生理盐水治疗对照组和SMT治疗组 ;此 4组又各分成 1,7d两个亚组。立体定向注射 5 μgLPS致大鼠脑黑质建立PD炎症模型 ,采用化学比色法检测 1d亚组黑质内NO释放量以及iNOS活性改变 ;RT PCR检测iNOSmRNA的表达 ;酪氨酸羟化酶 (tyrosinehydroxylase ,TH)免疫组织化学染色观察 7d亚组黑质DA能神经元的损伤情况。结果　 与PBS对照组相比 ,LPS注入黑质导致TH阳性细胞数下降至 35 % (P <0 .0 5 ) ;同时黑质内NO含量 ,iNOS活性及iNOSmRNA表达明显增高 ;SMT治疗组NO释放量 ,iN OS活性及iNOSmRNA表达显著降低 (P <0 .0 1) ;TH阳性细胞数亦明显增多 ,达 70 % (P <0 .0 5 ) ,生理盐水治疗组对此无明显改善。结论　iNOS上调是LPS诱导DA能神经元变性机制中重要的因素之一 ,iNOS特异性抑制剂SMT可以显著抑制LPS诱导DA能神经元变性及iNOSmRNA的高表达     

苯丁酸钠诱导分化治疗可移植性人脑胶质瘤实验研究

目的 探讨诱导分化剂苯丁酸钠与化疗药顺铂联合治疗可移植性人脑胶质瘤的剂量及疗效。方法 将低分化人脑胶质瘤体外细胞系SHG-44-9移植于裸小鼠,制作可移植性实体瘤模型后,用不同剂量的苯丁酸钠单独或与顺铂联合治疗,观察实体瘤的体积、组织细胞形态和胶质纤维酸性蛋白表达量的变化。结果 当苯丁酸钠增加至300mg·kg-1,每天2次腹腔注射时,肿瘤生长明显受抑（P<0.001）,组织细胞形态和胶质纤维酸性蛋白表达与对照组相比显示其分化程度增高,但与顺铂联用未显示出疗效的增加。结论 苯丁酸钠具有明显的促进人脑胶质瘤细胞分化的作用。     

Substance P inhibits potassium and calcium currents in inner ear spiral ganglion neurons

Substance P (SP), a member of the tachykinin family of neurotransmitters and neuromodulators, has been identified on spiral ganglion neurons (SGNs) in the inner ear; however, its high affinity receptor, neurokinin-1 (NK1), has not been identified and the physiological effects of SP on SGNs are not well understood. To address these issues, immunolabeling, RT-PCR, Western blots and whole-cell patch-clamp recordings were made from SGNs in P0-P5 mouse cochlear organotypic cultures. The NK1 receptor was detected on SGNs by immunocytochemistry, the protein was detected in cochlear tissues by Western blots, and the mRNA for the NK1 receptor was also found in cochlear tissues of postnatal mice (P2) by RT-PCR. Application of SP (1 to 25 microM) significantly increased the latency of SGN action potentials (APs) (mean increase 7.8 +/- 4 ms; 25 microM of SP), prolonged the duration of the action potential and made the resting potential (RP) more positive (mean 9.0 +/- 7 mV) relative to normal values (-54 +/- 6 mV). SP (1 to 25 microM) also suppressed voltage-activated potassium currents (IK+) and calcium currents (ICa2+). Puffing 25 microM of SP onto SGNs suppressed IK+ by 43 +/- 9% (n = 7) and ICa2+ by 40.6 +/- 5.6% (n = 7); both currents recovered when SP was washed out. A SP antagonist blocked the SP-induced suppression of IK+ and ICa2+. These results indicate that SP acting through NK1 receptors can have direct neuromodulatory effects on SGNs.     

Ouabain-Induced Cochlear Degeneration in Rat

Ouabain, a potent inhibitor of the Na+/K+-ATPase pump, selectively destroys spiral ganglion neurons (SGNs) in gerbils and mice, whereas in guinea pigs it preferentially damages cochlear hair cells. To elucidate the effects of ouabain on the rat inner ear, a species widely used in research, 5 μl of 1 or 10 mM ouabain was applied to the round window membrane. Distortion product otoacoustic emissions (DPOAE) and auditory brainstem responses (ABR) were used to identify functional deficits in hair cells and neurons, respectively, and histological techniques were used to characterize cochlear pathologies. High-frequency ABR thresholds were elevated after treatment with 1 mM ouabain, whereas DPOAEs remained normal. In contrast, 10 mM ouabain increased ABR thresholds and reduced DPOAE amplitudes. Consistent with the physiological changes, 1 mM ouabain only damaged the SGNs and auditory nerve fibers in the basal turn of the cochlea whereas 10 mM ouabain destroyed both SGNs and cochlear hair cells; damage was greatest near the base and decreased toward the apex. The nuclei of degenerating SGNs and hair cells were condensed and fragmented and many cells were TUNEL-positive, morphological features of apoptotic cell death. Thus, ouabain-induced cochlear degeneration in rats is apoptotic and concentration dependent; low concentrations preferentially damage SGNs in the base of the cochlea, producing an animal model of partial auditory neuropathy, whereas high concentrations damage both hair cells and SGNs with damage decreasing from the base toward the apex.     

Astrocytes protect cultured neurons from degeneration induced by anoxia

Neurons grown in cultures of dissociated brain cells degenerate when exposed to anoxia and deprived of glucose. We have developed culture systems in which neurons can be grown in the presence or absence of astrocytes and have used them to study the influence of astrocytes on the neuronal degeneration induced by anoxia and glucopenia. Cultures were prepared from fetal rat forebrains. Mixed cultures contained neurons (identified by immunocytochemical staining of neuron-specific enolase, NSE) and about an equal number of non-neuronal cells (identified by glial fibrillary acid protein). Pure neuronal cultures were prepared by adding a cytostatic compound (cytosine arabinoside) to the medium. Treated cultures were exposed for 4 h to glucose-free medium and an atmosphere of 95% N₂ and 5% CO₂, whereas control cultures were left in the usual medium containing glucose and in an atmosphere composed of 95% air and 5% CO₂. After an interval of 24 h, cultures were fixed, taken for NSE staining, and the number of surviving neurons was counted. Exposure to anoxia and glucopenia reduced the number of surviving neurons in pure neuronal cultures to 5–10% of control levels. In contrast, in mixed cultures 40–60% of the neurons survived these conditions. Anoxia without glucose deprivation reduced the number of surviving neurons in both types of cultures to the same extent as anoxia combined with glucopenia. Glucose deprivation alone was ineffective. The findings suggest a protective influence of astrocytes on neurons under anoxic conditions. γ-d-Glutamylglycine protected neurons in both types of cultures from anoxia-induced degeneration. However, no evidence was obtained for an involvement of excitatory amino acids in the protective influence of astrocytes.     

Effect of minocycline on cerebral ischemia- reperfusion injury

Minocylcine, a tetracycline derivate, has been shown to cross the blood-brain barrier and enter the central nervous system. In this study, cerebral ischemia-reperfusion injury models were established using the suture method, and minocycline was immediately injected intraperitoneally after cerebral ischemia-reperfusion (22.5 mg/kg, initially 45 mg/kg) at a 12-hour interval. Results showed that after minocycline treatment, the volume of cerebral infarction was significantly reduced, the number of surviving cell in the hippocampal CA1 region increased, the number of apoptotic cells decreased, the expression of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 protein was down-regulated, and the escape latency in the water maze test was significantly shortened compared with the ischemia-reperfusion group. Our experimental findings indicate that minocycline can protect against neuronal injury induced by focal ischemia-reperfusion, which may be mediated by the inhibition of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 protein expression.     

Cisplatin ototoxicity to the rat inner ear: a role for HMG1 and iNOS

Cisplatin is a chemotherapeutic agent that causes toxic damage to the inner ear (ototoxicity). Although much attention has been directed at identifying ways to protect the inner ear against cisplatin ototoxicity, little is known about the mechanisms by which cisplatin causes damage to the inner ear. Binding of high-mobility group (HMG1) protein to cisplatin-modified DNA participates in mediating the antitumor effects of cisplatin. This study seeks to determine if HMG1 may also participate in the ototoxicity of cisplatin. To address this, patterns and levels of expression of HMG1 have been evaluated in the rat cochlea in response to cisplatin chemotherapy. Our findings demonstrate a marked upregulation of HMG1 protein in the spiral (auditory) ganglion cells of cisplatin-treated rats in comparison to levels of expression of HMG1 in the spiral ganglion cells of untreated control animals. Increased levels of HMG1 were observed in the cisplatin-treated kidney, a peripheral target tissue of cisplatin, but not in the heart, a tissue not typically affected by cisplatin chemotherapy, suggesting HMG1 specificity in cisplatin toxicity. Furthermore, levels of inducible nitric oxide synthase (iNOS), an HMG-regulated enzyme associated with cochlear pathology, are increased in the spiral ganglion cells of cisplatin-treated rats 1 day post the cisplatin-mediated upregulation in HMG1. This increase in HMG1 and iNOS can be prevented in the cochleae of cisplatin-treated rats by administration of l-methionine, an established method of protection against cisplatin ototoxicity. Our results support a role for HMG1 and iNOS in mechanisms of cisplatin ototoxicity in the rat inner ear.     

Opposing expression gradients of calcitonin‐related polypeptide alpha (Calca/Cgrpα) and tyrosine hydroxylase (Th) in type II afferent neurons of the mouse cochlea

Type II spiral ganglion neurons (SGNs) are small caliber, unmyelinated afferents that extend dendritic arbors hundreds of microns along the cochlear spiral, contacting many outer hair cells (OHCs). Despite these many contacts, type II afferents are insensitive to sound and only weakly depolarized by glutamate release from OHCs. Recent studies suggest that type II afferents may be cochlear nociceptors, and can be excited by ATP released during tissue damage, by analogy to somatic pain‐sensing C‐fibers. The present work compares the expression patterns among cochlear type II afferents of two genes found in C‐fibers: calcitonin‐related polypeptide alpha (Calca/Cgrpα), specific to pain‐sensing C‐fibers, and tyrosine hydroxylase (Th), specific to low‐threshold mechanoreceptive C‐fibers, which was shown previously to be a selective biomarker of type II versus type I cochlear afferents (Vyas et al., 2016 ). Whole‐mount cochlear preparations from 3‐week‐ to 2‐month‐old CGRPα‐EGFP (GENSAT) mice showed expression of Cgrpα in a subset of SGNs with type II‐like peripheral dendrites extending beneath OHCs. Double labeling with other molecular markers confirmed that the labeled SGNs were neither type I SGNs nor olivocochlear efferents. Cgrpα starts to express in type II SGNs before hearing onset, but the expression level declines in the adult. The expression patterns of Cgrpα and Th formed opposing gradients, with Th being preferentially expressed in apical and Cgrpα in basal type II afferent neurons, indicating heterogeneity among type II afferent neurons. The expression of Th and Cgrpα was not mutually exclusive and co‐expression could be observed, most abundantly in the middle cochlear turn.     

Bilirubin-induced neurotoxic and ototoxic effects in rat cochlear and vestibular organotypic cultures

Exposure to high levels of bilirubin in hyperbilirubinemia patients and animal models can result in sensorineural deafness. However, the mechanisms underlying bilirubin-induced damage to the inner ear, including the cochlear and vestibular organs, remain unknown. The present analyses of cochlear and vestibular organotypic cultures obtained from postnatal day 3 rats exposed to bilirubin at varying concentrations (0, 10, 50, 100, or 250 μM) for 24 h revealed that auditory nerve fibers (ANFs) and vestibular nerve endings were destroyed even at low doses (10 and 50 μM). Additionally, as the bilirubin dose increased, spiral ganglion neurons (SGNs) and vestibular ganglion neurons (VGNs) exhibited gradual shrinkage in conjunction with nuclei condensation or fragmentation in a dose-dependent manner. The loss of cochlear and vestibular hair cells (HCs) was only evident in explants treated with the highest concentration of bilirubin (250 μM), and bilirubin-induced major apoptosis most likely occurred via the extrinsic apoptotic pathway. Thus, the present results indicate that inner ear neurons and fibers were more sensitive to, and exhibited more severe damage following, bilirubin-induced neurotoxicity than sensory HCs, which illustrates the underlying causes of auditory neuropathy and vestibulopathy in hyperbilirubinemia patients.     

In vitro and in vivo induction of heme oxygenase-1 in rat glial cells: Possible involvement of nitric oxide production from inducible nitric oxide synthase

To determine whether heme oxygenase-1 (HO-1) protein is induced by endogenous nitric oxide (NO) in rat glial cultures, we examined the effects of lipopolysaccharide (LPS), interferon-γ (IFN-γ), and NO donors such as S-nitroso-N-acetylpenicillamine (SNAP), in mixed glial cells and in vivo rat hippocampus. In cultured glial cells, treatment with LPS induced the expression of 130-kd inducible NO synthase (iNOS) after 6 h, and NO₂⁻accumulation and enhancement of the protein level of 33-kd HO-1 after 12 h. In addition, treatment with SNAP induced HO-1 expression after 6 h. Although NOS inhibitors such as N^G-nitro-L-arginine (NNA) and N^G-methyl-L-arginine did not change LPS-induced iNOS expression, these inhibitors suppressed both NO₂⁻ accumulation and the enhancement of HO-1. Immunocytochemistry showed that treatment with LPS for 24 h induced iNOS immunoreactivity predominantly in ameboid microglia, while this treatment induced HO-1-immunoreactivity in both microglia and astrocytes. In in vivo rat hippocampus, microinjection of LPS plus IFN-γ, or SNAP after 24 h also induced HO-1 immunoreactivity in reactive microglia and astrocytes. In addition, intraperitoneal administration of NNA inhibited HO-1 immunoreactivity induced by the microinjection of LPS plus IFN-γ. These results suggest that endogenous NO production by iNOS in microglia causes autocrine and paracrine induction of HO-1 protein in microglia and astrocytes in vitro and in rat brain. GLIA 22:138–148, 1998.© 1998 Wiley-Liss, Inc.