一氧化氮对过氧化氢所致听力损失的保护作用

通过全耳蜗灌流法在体观察一氧化氮(N0)能否通过一氧化氮/环磷酸鸟苷(NO/cGMP)途径对抗过氧化氢这种氧自由基所致的听力损失。实验选用耳廓反射灵敏、无耳毒性药物使用史的健康杂色豚鼠(250-350 g)50只,雌雄不拘,随机分为5组,每组10只动物,分别行全耳蜗灌流人工外淋巴液;过氧化氢(H2O2);L-精氨酸(合成NO的底物);H2O2+L-精氨酸;H2O2+L-精氨酸+L-NNA(一氧化氮合成酶的抑制剂),均灌流2 h。通过圆窗龛电极,每隔30 min记录复合动作电位(compound action potential,CAP:由短声Click诱发)阈值,耳蜗微音器电位(cochlear microphonic,CM;由短纯音Tone Burst诱发)幅度,了解耳蜗功能的变化,并分离取出耳蜗基底膜并制备基底膜硬铺片,通过碘化毗啶(PI)和Hoecbst双染色方法,观察耳蜗组织各类细胞损伤情况。结果显示,灌流H2O2+L-精氨酸组的CAP阈移和CM下降幅度值明显低于单独灌流H2O2组,差异有显著性(氏P<0.05);前者形态学观察未见明显的细胞损伤,后者可见大量坏死红染的细胞。H2O1+L-精氨酸+L-NNA组CAP阈移和CM下降幅度与单独灌流H2O2组比较无统计学差异。实验结果提示NO可能通过NO/cGMP途径部分对抗过氧化氢所致的听力损失。     

用共聚焦显微镜观察ACh及ATP对豚鼠耳蜗外毛细胞Ca^2＋的调控

用激光扫描共聚焦显微镜研究了一般公认的耳蜗传出神经递质乙酰胆碱（ＡＣｈ）和三磷酸腺苷（ＡＴＰ）对豚鼠耳蜗外毛细胞（ＯＨＣｓ）胞内游离Ｃａ＾２＋浓度（Ｃａ＾２＋）的作用,ＯＨＣｓ用Ｃａ＾２＋敏感荧光染料Ｆｌｕｏ－３着色,胞内Ｃａ＾２＋的分布以细胞底部稍强。ＡＣｈ在ＯＨＣ底部引起Ｃａ＾２＋的缓慢上长并维持在一个较高水平。ＡＴＰ在整个ＯＨＣ引起一个急剧的Ｃａ＾２＋升高,升高幅度在ＯＨＣ顶部最大。随着ＡＴ     

卡那霉素致聋豚鼠耳蜗电极植入模型的建立及意义

目的:探讨人工耳蜗电极的插入对耳蜗功能的影响,为研究人工耳蜗植入建立相应的动物模型。方法:取听力正常的豚鼠8只,4只注射卡那霉素联合呋塞米致聋,为致聋组;4只仅注射生理盐水,为对照组。对两组动物行听性脑干反应(ABR)及耳声发射(DPOAE)检查后,将耳蜗电极植入左侧耳蜗。结果:致聋组术侧4个频率段ABR阈移随着时间的推移逐渐减小,术后24 h、48 h、72 h时间段比较无显著性差异(P0.05);对照组术侧ABR阈移随着时间的推移逐渐减小,32 kHz频率的三个时间段比较有显著性差异(P0.05),其余3个频率无显著性差异。此外,致聋组与对照组术侧耳ABR阈移比较均无显著性差异(P0.05)。致聋组术前5个频率的DPOAE无法引出,术后DPOAE仍无法引出;对照组术前DPOAE均可引出,术后术侧的DPOAE均无法引出。术后72 h可见电极周围有组织包绕,固定良好,局部未见明显炎症反应。结论:本实验成功建立了卡那霉素致聋的豚鼠耳蜗电极植入模型,可为人工耳蜗植入术后颞骨病理改变的研究提供实验基础。     

记录豚鼠耳蜗电位的外耳道慢性电极植入法

本文介绍用外耳道慢性电极植入法可以在较长时间内记录到清醒豚鼠的耳蜗电位(CAP和CM)。正常豚鼠的CAP(N_1)最大振幅平均达70—80μV。CAP(N_1)的潜伏期、阈值和最大振幅在4个月内均较稳定。因此用此法能远期观察清醒豚鼠耳蜗听功能的变化。     

卡那霉素耳慢性中毒对豚鼠耳蜗毛细胞中Bcl-2表达的影响

目的:探讨卡那霉素耳慢性中毒对豚鼠耳蜗毛细胞中Bcl-2表达的影响。方法:取20只豚鼠随机分为2组,实验组连续14d肌肉注射硫酸卡那霉素,200mg/(kg.d),对照组连续14d等量肌肉注射生理盐水,停药14d处死动物后制作耳蜗标本,处死前检测其ABR的变化,免疫组化及原位杂交法测定Bcl-2的表达。结果:豚鼠卡那霉素耳慢性中毒后,ABR阈值较对照组明显上升,Bcl-2阳性表达减低,与对照组比较差异有显著性(P<0.05)。结论:卡那霉素耳慢性损害可能与抑制Bcl-2的表达有关。     

庆大霉素对豚鼠耳蜗一氧化氮合酶活性的影响

目的：研究庆大霉素（ＧＭ） 致聋豚鼠耳蜗一氧化氮合酶（ＮＯＳ） 的分布及其变化,探讨一氧化氮（ＮＯ） 与ＧＭ 耳毒机制的关系。方法：ＮＡＤＰＨｄ 酶组化方法及显微图像分析技术。结果：ＧＭ 致聋后,ＮＯＳ 阳性反应物密度在血管纹、内毛细胞、外毛细胞、螺旋神经节、听神经纤维的分布均比对照组明显增高（ Ｐ＜ ０ ．００１）;上述部位的ＮＯＳ平均灰度值降低与对照组相比有显著差异（ Ｐ＜ ０ ．００１）;ＡＢＲ 听阈变化与ＮＯＳ平均密度及灰度值变化高度相关（ 上述各部位ｒ 值范围,ｒ密度听阈：０ ．９１４０～０．８６９８;ｒ灰度听阈：－０ ．７８９２ ～－ ０．９３４７;Ｐ＜ ０．０１）。结论：①ＮＯ 可能与ＧＭ的耳毒作用机制有关,是耳毒作用的重要因素之一;②ＧＭ 耳毒作用部位不仅发生在血管纹和外毛细胞,而且还涉及到内毛细胞、螺旋神经节及听神经纤维     

传出神经递质ACh及耳蜗活性物质ATP对耳蜗外毛细胞的作用

本文综述了耳蜗传出神经对耳蜗外毛细胞的调控作用。从解决和组织化学等形态学角度分析了传出神经纤维在耳蜗的分布特点,并从形态和生理两方面进一步证实了ＡＣＨ是耳蜗传出神经递质之一。近年来,应用离体耳蜗毛细胞的膜片钳和荧光测钙技术,人们对于ＡＣＨ,ＡＴＰ对外毛细胞的调控作用,Ｃａ＾２＋在其中的介导作用及其ＡＣＨＲ和Ｐ２Ｒ的药理分型有了更多的认识,但尚未有统一明确的结论。     

正常豚鼠耳蜗核一氧化氮合酶阳性神经元的投射

本文采用辣根过氧化物酶（ＨＲＰ）逆行追踪技术结合硫辛酰胺脱氨酸（ＮＡＤＰＨ－ｄ）组织化学方法,研究正常豚鼠耳蜗核一氧化氮合酶（ＮＯＳ）阳性神经元的上行投射特点。探讨耳蜗核ＮＯＳ阳性神经元在听觉信号传递中的可能作用。结果表明,一侧上橄榄复合体加压注射ＨＲＰ后,两侧耳蜗核均出现ＨＲＰ标记细胞,同侧耳蜗核ＮＯＳ－ＨＲＰ双标细胞较多占８２．６３％,并可见ＨＲＰ阳性纤维和终末包绕ＮＯＳ阳性胞体,对侧耳蜗核ＮＯＳ－ＨＲＰ双标细胞相对较少,仅占１４．８７％。一侧下丘加压注入ＨＲＰ后两侧耳蜗核均无ＨＲＰ－ＮＯＳ双标细胞。结果提示,耳蜗核ＮＯＳ阳性神经元向上橄榄复合体投射,可能具有调节听觉声信号传递的作用     

P物质、脑啡肽在豚鼠耳蜗的分布——免疫组织化学观察

本研究应用免疫组织化学方法系统地观察了P物质(SP)、亮氨酸脑啡肽(L-ENK)在豚鼠耳蜗的分布以及SP、L-ENK免疫反应阳性神经纤维与Corti's器毛细胞之间的关系,结果表明:SP的免疫反应活性(SP-IR)存在于耳蜗螺旋神经节的部分神经细胞及传入神经纤维中,在Corti's器的毛细胞下方亦可见SP免疫反应阳性纤维;L-ENK的免疫反应活性(ENK-IR)存在于耳蜗的传出神经纤维中。节内螺旋束、内螺旋束、隧道螺旋束、横贯纤维均含有大量的L-ENK免疫反应阳性纤维,Cort's器中的L-ENK免疫反应阳性终末与毛细胞之间具有密切接触,由此提示,SP可能为听觉初级传入神经递质之一;L-ENK作为传出神经递质或调质对听觉传入起调控作用。     

硒的抗癌机制研究——亚硒酸钠对环磷鸟苷的作用

 AFB_1和NDEA与正常小鼠肝切片培养5min即可使cGMP分别升高一倍及一倍半(p<0.001);当有Na_2SeO_3(1μg/mL培养液)存在时,此作用可被消除,单独加入Na_2SeO_3对cGMP无影响。培养介质中存在磷酸二酯酶抑制剂茶碱时,除cGMP基础水平提高外,上述作用均相同。荷腹水型肝癌(HepA)小鼠腹水细胞中cGMP比正常肝中cGMP水平高三倍,连续4天腹腔给硒(Na_2SeO_3、1mg/kg体重)可使癌细胞中cGMP下降一倍。此剂量对正常小鼠肝中cGMP无影响;Na_2SeO_3(1μg/mL)与腹水细胞在体外短时间培养时,对细胞存活无影响,却可使cGMP含量下降。     

In Vivo NO/cGMP Signalling in the Hippocampus

In the hippocampus of freely-moving rats, basal extracellular levels of cGMP are inhibited by L-NARG or ODQ whereas they are increased by NO donors or phosphodiesterase inhibitors. Activation of NMDA receptors also augments cGMP dialysate levels in a MK-801 and L-NARG sensitive manner, an effect dramatically diminished during ageing. Experiments with AMPA, AMPA receptor antagonists and cyclothiazide revealed complex relationships with GABAergic circuits that potently control the NO/cGMP pathway. Furthermore, the activity of this neurochemical cascade is also modulated by hippocampal nicotinic receptors via enhancement of endogenous glutamate release and stimulation of NMDA receptors. From a behavioural point of view, increased hippocampal excitation leads to the appearance of epileptic-like manifestations that, however, seem unrelated to the increase of NO/cGMP formation.     

Nitric oxide/cGMP pathway components in the Leydig cells of the human testis

In this study we sought to determine whether the main components of the nitric oxide (NO) pathway are localized within the Leydig cells of the human testis and whether the soluble guanylyl cyclase (sGC), the enzyme that accounts for NO effects, is functionally active in these cells. Using an amplified immunocytochemical technique, immunoreactivity for nitric oxide synthase (NOS-I), sGC and cyclic guanosine monophosphate (cGMP) was detected within the cytoplasm of human Leydig cells. Distinct differences in staining intensity were found between individual Leydig cells, between cell groups and between Leydig cells of different patients. By means of a specific cGMP-RIA, a concentration-dependent increase in the quantity of cGMP was measured in primary cultures of human Leydig cells following exposure to the NO donor sodium nitroprusside. In addition, NOS-I immunoreactivity was seen in Sertoli cells, whereas cGMP and sGC immunoreactivity was found in Sertoli cells, some apically situated spermatids and residual bodies of seminiferous tubules. Dual-labelling studies and the staining of consecutive sections showed that there are several populations of Leydig cells in the human testis. Most cells were immunoreactive for NOS-I, sGC and cGMP, but smaller numbers of cells were unlabelled by any of the antibodies used, or labelled for NOS-I or cGMP alone, for sGC and cGMP, or for NOS-I and sGC. These results show that the Leydig cells possess both the enzyme by which NO is produced and the active enzyme which mediates the NO effects. There are different Leydig cell populations that probably reflect variations in their functional (steroidogenic) activity. Received: 27 March 1996 / Accepted: 14 July 1996     

Endogenous nitric oxide/cGMP signalling in the guinea pig bladder: evidence for distinct populations of sub-urothelial interstitial cells

We have examined structures that may operate by using nitric oxide (NO)/soluble guanylyl cyclase (sGC) signalling in the lamina propria of the guinea pig bladder. Cells on the luminal surface of the urothelium and sub-urothelial interstitial cells (SU-ICs) responded to NO with a rise in cGMP. The distribution of these different cells varied between the base, lateral wall and dome. In the base, two regions were identified: areas with sparse surface urothelial cells and areas with a complete covering. A layer of cGMP-positive (cGMP⁺) cells (up to 10 cells deep) was found in the base. cGMP⁺/SU-ICs were also observed in the lateral wall. However, here, the cGMP⁺ cells were confined to a layer of only 1–2 cells immediately below the basal urothelial layer (basal cGMP⁺/SU-ICs). Below these cGMP⁺/SU-ICs lay cells that had a similar structure but that showed little cGMP accumulation (deep cGMP^–/SU-ICs). Both basal and deep SU-ICs expressed the β1 subunit of sGC and the cGMP-dependent protein kinase I (cGKI), suggesting that the deep SU-ICs can sense NO and signal via cGMP. By using BAY 41-2272, a sensor of endogenous NO production, NO-dependent cGMP synthesis was observed primarily in the basal SU-ICs. A third population of cGKI⁺/cGMP⁻ cells was seen to lie immediately below the basal urothelial layer. These cells (“necklace” cells) were less numerous than SU-ICs and extended linking processes suggesting a network. The specific functions of these structures are not known but they may contribute to the emerging multiple roles of the urothelium associated with the generation of bladder sensation.     

丹参注射液对庆大霉素耳中毒豚鼠耳蜗氧自由基生成的影响

目的:研究丹参注射液(SM)对庆大霉素(GM)耳中毒豚鼠耳蜗氧自由基生成的影响,探讨SM对GM耳毒性损伤的保护作用及其机制.方法:检测豚鼠耳蜗组织中超氧化物歧化酶(SOD)活力和丙二醛(MDA)含量,结合听性脑干反应(ABR)测试及透射电镜技术.结果:经GM处理的耳蜗组织中SOD活力明显下降,MDA含量则明显增加(P＜0.01),且与ABR阈值升高高度相关(|r|＞0.8,P＜0.05).同时接受SM的动物,其耳蜗组织中SOD活力明显升高(P＜0.01),MDA含量则明显减少(P＜0.05),且听功能显著改善.电镜观察显示耳蜗形态学改变与听力变化相一致.结论:氧自由基及其引发的脂质过氧化参与了GM耳中毒过程,SM可通过提高耳蜗组织中SOD活力,防止脂质过氧化,减轻GM的耳蜗毒性,改善听功能.     

Modulation of cAMP level by tedisamil in guinea pig heart

Tedisamil is antiarrhythmic class III drug with antifibrillating/defibrillating potency linked to enhancement of intermyocyte gap junctional electrical coupling most likely via its sympathomimetic cAMP-related mechanisms. This study was designed to examin the effect of tedisamil on cAMP level in guinea pig hearts in vivo and in vitro in Langendorff preparation. The drug was administered either as a bolus into vena jugularis in dosage 1.0 and 1.5 mg/kg or into the perfusion solution at a concentration of 1.5 × 10^-6 mol/l. In additional experiments, this period was followed by brief 10 min global ischemia, induced by clamping of the aorta or perfusion. After 10 min from the onset of tedisamil administration as well as after 10 min of ischemia the ventricular tissue was immediately frozen for cAMP immunoassay Tedisamil caused in normal heart small but significant dose-dependent increase of myocardial cAMP (pmol/mg) level in vivo 1.8 and 2.5 vs. 1.4 as well as in vitro 1.1 vs. 0.8 (p < 0.05) conditions. Ischemia itself induced accumulation of cAMP in both, in vitro and in vitro experiments, 2.6 vs. 1.4 and 1.3 vs. 0.8, respectively. The preischemic elevation of cAMP by tedisamil was not potentiated by following ischemia, on the contrary, decline of the cyclic nucleotide was detected comparing to ischemia itself. In conclusion, tedisamil increased cAMP level in normal heart and prevented additional ischemia-related elevation of this nucleotide. The results indicate modulation of myocardial cAMP level by tedisamil, which may account for its protective effect on gap junctional electrical coupling.     

Effects of hydrogen peroxide on relaxation through the NO/sGC/cGMP pathway in isolated rat iliac arteries

Abstract

The production of reactive oxygen species, including hydrogen peroxide (H₂O₂), is increased in diseased blood vessels. Although H₂O₂ leads to impairment of the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP signaling pathway, it is not clear whether this reactive molecule affects the redox state of sGC, a key determinant of NO bioavailability. To clarify this issue, mechanical responses of endothelium-denuded rat external iliac arteries to BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator), nitroglycerin (NO donor), acidified NaNO₂ (exogenous NO) and 8-Br-cGMP (cGMP analog) were studied under exposure to H₂O₂. The relaxant response to BAY 41-2272 (pD₂: 6.79?±?0.10 and 6.62?±?0.17), BAY 60-2770 (pD₂: 9.57?±?0.06 and 9.34?±?0.15) or 8-Br-cGMP (pD₂: 5.19?±?0.06 and 5.24?±?0.08) was not apparently affected by exposure to H₂O₂. In addition, vascular cGMP production stimulated with BAY 41-2272 or BAY 60-2770 in the presence of H₂O₂ was identical to that in its absence. On the other hand, nitroglycerin-induced relaxation was markedly attenuated by exposing the arteries to H₂O₂ (pD₂: 8.73?±?0.05 and 8.30?±?0.05), which was normalized in the presence of catalase (pD₂: 8.59?±?0.05). Likewise, H₂O₂ exposure impaired the relaxant response to acidified NaNO₂ (pD₂: 6.52?±?0.17 and 6.09?±?0.16). These findings suggest that H₂O₂ interferes with the NO-mediated action, but the sGC redox equilibrium and the downstream target(s) of cGMP are unlikely to be affected in the vasculature.     

Exploring the regulatory role of nitric oxide (NO) and the NO-p38MAPK/cGMP pathway in larval settlement of the bryozoan Bugula neritina

The bryozoan Bugula neritina is a cosmopolitan marine fouling species that causes major fouling problems in sub-tropical waters. Settlement of B. neritina larvae can be triggered without an obvious external cue. Here, the negative regulatory role of nitric oxide (NO) during larval settlement of B. neritina was demonstrated to be mediated by cyclic guanosine monophosphate (cGMP). Although the regulatory role of the NO-p38 MAPK signaling axis in larval settlement was not evident, inhibition of nitric oxide synthase (NOS) led to the deactivation of p38 MAPK. Exclusive localization of NO and NO signaling components in sensory-related organs of the larvae is consistent with its signal transduction function in metamorphosis. Overall, this study provides new insights into the regulatory roles of the NO-p38MAPK/cGMP pathway in B. neritina settlement.     

Protective effect of prior immunization on ocular paracoccidioidomycosis in guinea pigs

The present study reproduced the experimental model of ocular paracoccidioidomycosis in guinea pigs, by the intracardiac inoculation of yeast-forms of P. brasiliensis. Ocular involvement was observed in 80% of the infected animals. The uvea, ciliary body, choroid, iris, lids and the conjunctiva were the structures most commonly affected. To protect the animals against the infection, an immunization protocol was standardized utilizing a P. brasiliensis soluble antigen in Freund's complete adjuvant, administered weekly, during 3 weeks, by the subcutaneous route. Two weeks later, previously immunized guinea pigs were challenged by the intracardiac route with yeast-forms of P. brasiliensis (vaccinated group). When compared with a control group (infection in the absence of prior immunization), the vaccinated animals developed higher levels of anti-P. brasiliensis cellular and humoral immune response and a three times lower frequency of ocular involvement (85.7% vs 28.5%). In addition, the ocular lesions were significantly more localized and contained less fungal cells. The data demonstrated that the subcutaneous immunization was effective in decreasing the frequency and extent of ocular lesions, as well as in blocking fungal multiplication.     

Dual Effect of Diazepam on cGMP Levels in Rat Brain Slices

The effect of diazepam on NO-mediated cGMP synthesis was studied in rat brain slices. It was found that diazepam dose-dependently decreased cGMP synthesis in cerebellar slices, with an inhibition of 90% at 1 mM diazepam. cGMP levels in the presence of diazepam were not restored to control levels by the addition of 0.1 mM sodium nitroprusside, whereas the decrease in cerebellar cGMP levels induced by 0.1 mM L-NAME was restored by the simultaneous application of NO-donors. In addition to the decrease of cGMP levels in neuronal structures induced by 1 mM diazepam, we observed increased cGMP immunoreactivity in glial cells in the cerebellum, the hippocampus, and the cerebral cortex. The significance of this observation is discussed.     

Postnatal development of the shape of cerebellar Purkinje cells in guinea pig ontogenesis

In sagittal cerebellum sections, morphometrical study of cerebellum of mature-born animals—guinea pigs—was performed using Nissl’s procedure. A change of shape and volume of Purkinje cells and their nuclei in the course of the guinea pig postnatal ontogenesis was studied. It has been shown that both the growth process itself and the rate of formation of the definite form of Purkinje cells and of their nuclei in the course of ontogenesis proceeds non-uniformly. The most intensive growth of vertical and horizontal diameters of Purkinje cells and of their nuclei is observed during the 1st and 4th weeks of postnatal life. Especially rapid is an increase of horizontal diameters of Purkinje cells and of their nuclei, which impairs the ovoid-bear-like shape to the cerebellar Purkinje cells of adult guinea pigs.