超短波影响神经源性疼痛时nNOS和NMDA受体表达的实验研究

目的：研究神经源性疼痛时兔背根神经节(dorsal root ganglia，DRG)和脊髓后角内神经元型一氧化氮合酶(neuronal nitric oxide synthase，nNOS)与N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor，NMDA受体)阳性表达的特点以及超短波对其的影响，从而探讨超短波治疗的分子作用机制。方法：新西兰纯种成年兔45只，随机分为空白对照组(n=15)，损伤模型组(n=15)和超短波治疗组(n=15)。采用免疫组化法结合图像分析，定量观察损伤后不同时间(10d、30d和90d)DRG和脊髓后角内nNOS和NMDA受体阳性表达的分布特点。结果：术后10d时模型组和治疗组手术侧DRG和脊髓后角内nNOS阳性表达增多，NMDA受体阳性表达在DRG内降低；90d时治疗组nNOS和NMDA受体表达已基本接近正常，而模型组nNOS和NMDA受体仍未恢复正常。结论：超短波治疗可改变神经源性疼痛时DRG和脊髓内神经元表达上调的nNOS和表达下调的NMDA受体水平，提示其可能通过影响NMDA受体-NO系统发挥缓解疼痛的满意疗效。     

Tat-LK15运载siRNA沉默RGC-5神经细胞nNOS基因的实验研究

目的探讨离体条件下细胞穿透肽Tat-LK15运载小干扰RNA(small interference RNA,siRNA)沉默RGC-5视神经节细胞(retinal ganglion cell line,RGC-5)神经元型一氧化氮合酶(neuronal nitric oxide synthase,n NOS)基因的可行性,为在体条件下研究Tat-LK15运载siRNA沉默n NOS表达治疗神经病理性疼痛提供理论依据。方法 1通过凝胶阻滞分析测定Tat-LK15与siRNA的最佳交联比。流式细胞术检测Tat-LK15/FAM-siRNA以最佳交联比转染RGC-5细胞的转染效率;不同剂量Tat-LK15(1、2.5、5、10和20μg)孵育RGC-5细胞24 h,流式细胞术检测细胞凋亡率。2制备n NOS高表达的RGC-5细胞模型。3将RGC-5细胞随机分为5组:对照组、模型组、Tat-S组(Tat-LK15运载n NOS/siRNA转染模型细胞)、Lipo-S组(LipofectamineTMRNAi MAX运载n NOS/siRNA转染模型细胞)及Tat-N组(Tat-LK15运载NCsiRNA转染模型细胞),通过Q-PCR及Western blot检测各组nN OS表达水平。结果 Tat-LK15与siRNA质量比为2∶1时可完全包裹siRNA,达到最佳交联,此时其转染效率为(84.4±3.9)%。当Tat-LK15剂量为20μg(6.1μmol·L-1)时才出现一定细胞毒性,细胞凋亡率高于对照组[(10.3±1.1)%vs(7.4±0.9)%,P<0.05]。造模后RGC-5细胞nN OS表达水平明显升高(P<0.05)。与模型组相比,Tat-S组nN OS mRNA及蛋白表达水平降低(P<0.05),Tat-S组与Lipo-S组相比无差异(P>0.05)。结论Tat-LK15能高效转染siRNA,细胞毒性低,离体条件下可有效运载siRNA沉默nN OS的基因表达。     

正常人肩关节囊内神经元型一氧化氮合酶阳性神经纤维的分布

目的 探讨肩周炎(冻结肩)的发病机理.方法 用免疫组织化学方法观察人肩关节囊内神经元型一氧化氮合酶(Nnos)阳性神经纤维存在的情况.结果 人肩关节囊的纤维层内有Nnos免疫反应阳性神经纤维存在,数量较多,多数神经纤维独立直行.结论 Nnos阳性神经纤维可能参与了人肩关节囊的伤害性感觉信息的传递.当人肩关节囊病变时,可能刺激Nnos阳性神经纤维,引起NO的释放和传递,这可能是引起肩周炎时肩痛的原因之一.     

nNOS expression in the brain of rats after burn and the effect of the ACE inhibitor captopril

Objective

To investigate the role of endogenous neuronal nitric oxide synthase (nNOS) on brain injury after burn and the effects of the captopril.

Methods

Wistar albino rats (200–250 g) were exposed on the dorsal surface to 90 °C (burn) or 25 °C (sham) water for 10 s. The ACE group was treated with intraperitoneal 10 mg/kg captopril immediately after burn and this treatment was repeated twice daily. At the end of the 24 h brain samples were taken. nNOS was studied in brain areas by immunohistochemistry.

Results

There was no difference between the cerebellar and hypothalamic areas the nNOS expression of all groups. nNOS expression increased in the frontal cortex, striatum and midbrain in the burn group compared to the control group. In the frontal cortex, nNOS expression significantly decreased after ACE inhibitor treatment (p < 0.05). The striatal nNOS of the ACE group significantly increased when compared to the control group (p = 0.001). In the midbrain of the animals, nNOS decreased in the ACE group. Hippocampal nNOS expression did not change after burn and significantly increased after ACE inhibitor therapy (p < 0.05).

Conclusions

Our data showed that the pathophysiological events following burn appear to be related to an acute inflammatory reaction which is associated with nNOS in the frontal cortex, striatum and midbrain, and captopril treatment abrogates the nNOS response in the frontal cortex and midbrain.     

Lens cholesterol biosynthesis inhibition: A common mechanism of cataract formation in laboratory animals by pharmaceutical products

CJ‐12,918, a 5‐lipoxygenase (5‐LO) inhibitor, caused cataracts during a 1‐month safety assessment studies in rats whereas the structurally similar ZD‐2138 was without effect. For CJ‐12,918 analogs, blocking different sites of metabolic liability reduced (CJ‐13,454) and eliminated (CJ‐13,610) cataract formation in both rats and dogs. Using this chemical series as a test set, models and mechanisms of toxicity were first explored by testing the utility of ex vivo rat lens explant cultures as a safety screen. This model overpredicted the cataractogenic potential of ZD‐2138 due to appreciably high lens drug levels and was abandoned in favor of a mechanism‐based screen. Perturbations in lens sterol content, from a decline in lathosterol content, preceded cataract formation suggesting CJ‐12,918 inhibited lens cholesterol biosynthesis (LCB). A 2‐day bioassay in rats using ex vivo LCB assessments showed that the level of LCB inhibition was correlated with incidence of cataract formation in animal studies by these 5‐LO inhibitors. Thereafter, this 2‐day bioassay was applied to other pharmaceutical programs (neuronal nitric oxide synthase, sorbitol dehydrogenase inhibitor, squalene synthetase inhibitor and stearoyl‐CoA desaturase‐1 inhibitors/D₄ antagonists) that demonstrated cataract formation in either rats or dogs. LCB inhibition >40% was associated with a high incidence of cataract formation in both rats and dogs that was species specific. Bioassay sensitivity/specificity were further explored with positive (RGH‐6201/ciglitazone/U18666A) and negative (tamoxifen/naphthalene/galactose) mechanistic controls. This body of work over two decades shows that LCB inhibition was a common mechanism of cataract formation by pharmaceutical agents and defined a level of inhibition >40% that was typically associated with causing cataracts in safety assessment studies typically ≥1 month.     

老年小鼠突触可塑性和记忆功能变化与nNOS表达的关系

目的 对老年小鼠突触可塑性和记忆能力变化与海马nNOS表达变化之间的关系进行探讨.方法 2月龄(青年)和16月龄(老年)昆明小鼠分别用Y型迷宫测试自发交替和活动能力,离体脑片细胞外观测海马长时程增强(LTP)的变化,免疫组化检测海马nNOS阳性细胞表达的变化.结果 老年小鼠的自发交替的百分率和活动能力较青年小鼠明显下降(P＜0.01),离体海马脑片LTP诱发成功率和群峰电位振幅增大率明显下降(P＜0.01),海马CA1区、齿状回的nNOS阳性细胞的染色强度减弱(P＜0.01).结论 小鼠在衰老过程中会伴有突触可塑性和记忆功能降低,其病理机制和nNOS神经元丧失有关.     

铝中毒AD模型鼠海马神经元nNOS的表达

目的 探讨铝中毒对小鼠学习记忆功能及海马nNOS表达的影响。方法 采用口服氯化铝方法建立小鼠AD模型，用跳台试验和避暗试验检测小鼠的学习记忆行为，并用免疫组化ABC法检测小鼠海马CA2～3区nNOS表达的变化。结果 口服氯化铝高剂量组及低剂量组小鼠均表现为跳台和避暗试验错误次数增多，nNOS表达下降．结论 铝中毒可导致小鼠学习记忆能力下降，其原因可能与nNOS表达下降有关．     

The Claustrum in the Squirrel Monkey

The claustrum (CLA) is a subcortical structure that is reciprocally and topographically connected with the cerebral cortex. The complexity of the cerebral cortex varies dramatically across mammals, raising the question of whether there might also be differences in CLA organization, circuitry, and function. Species variations in the shape of the CLA are well documented. Studies in multiple species have identified subsets of neurochemically distinct interneurons; some data suggest species variations in the nature, distribution, and numbers of different neurochemically identified neuronal types. We have studied the CLA in a smooth-brained primate, the squirrel monkey, using Nissl-stained sections and immunohistochemistry. We found that the shape of the CLA is different from that in other primates. We found several different neurochemically defined populations of neurons equally distributed throughout the CLA. Immunoreactivity to GAD_65/67 and GABA_A receptors suggest that GABAergic interneurons provide widespread inhibitory input to CLA neurons. Immunoreactivity to glutamate transporters suggests widespread and overlapping excitatory input from cortical and possibly subcortical sources. Comparison of CLA organization in different species suggests that there may be major species differences both in the organization and in the functions of the CLA. Anat Rec, 303:1439–1454, 2020. © 2019 American Association for Anatomy     

组成型一氧化氮合成酶及其酪氨酸磷酸化过程对LPS介入的鼠肺动脉张力的调节作用

目的 研究组成型NO合成酶及其酪氨酸磷酸化过程对LPS介入的鼠肺动脉张力的影响。方法 将取自Sprague-Dawley鼠的肺动脉环悬挂在含有Krebs溶液的游离器官槽中以备作血管反应实验之用。结果 经LPS处理的肺动脉环对去氧肾上腺素的收缩反应明显降低，但此反应的减弱可以被L-NAME，非选择性NO合成酶抑制剂和7-NINA，选择性神经元NO合成酶抑制剂弥补。LPS同时可以降低肺功能对乙酰胆碱的内皮依赖舒张反应，但此减弱由于钒酸钠，选择性酪氨酸磷酸酶抑制剂的存在而缓解，结论 神经元NO合成酶参与了LPS介入的NO过度产生而形成的肺动脉张力降低，而在这个过程中，内皮NO合成酶的催化活性是受损的，但这种损害因触发了另一种通过酪氨酸磷酸化途径的内皮依赖舒张反应而得到补偿。     

胃电刺激对大鼠海马胃扩张反应神经元及胃动素和nNOS表达的影响

目的:观察胃电刺激(GES)对大鼠海马CA1区胃扩张(GD)相关神经元放电活动的影响及脑内一氧化氮合酶(nNOS)和胃动素(MTL)表达的改变,初步探讨GES的中枢作用机制.方法:选用成年Wistar大鼠58只,采用细胞外记录神经元单位放电方法,记录海马CA1区神经元自发放电活动,根据神经元对胃扩张刺激反应的不同,分为胃扩张兴奋性神经元(GD-E)和胃扩张抑制性神经元(GD-I).观察3组不同参数的胃电刺激(GES-A,GES-B和GES-C)对CA1区内GD-E和GD-I放电频率的影响:GES-A(6 mA,0-3 ms,40 Hz,2 s-on,3 S-off)为标准参数;GES-B的串刺激波宽增至3 ms;GES-C的串刺激频率减少至20 Hz,其他参数均同GES-A.采用放射免疫法检测胃电刺激对大鼠不同脑区MTL含量的影响;并采用免疫荧光组织化学染色方法观察胃电刺激2 h对大鼠海马内nNOS免疫阳性神经元的表达.结果:CA1区记录的87个神经元中有79个神经元(90.8%)对胃扩张刺激(GD,3-5 mL,10-30 s)有反应,其中40个(50.6%)为GD-E神经元,39个(49.4%)为GD-I神经元.GES-A,-B,-C分别兴奋了62.5%,100%和62.3%的GD-E神经元,GES-B对GD-E神经元的作用明显强于GES-C (神经元兴奋率GES-B 100%vs GES-C 62.3%,P<0.05).在GD-I神经元中有63.6%,85.7%和50%的GD-I神经元分别被GES-A,-B和-C所兴奋,其中GES-C的兴奋作用较弱(P=0.041,vs GES-A:P=0.021,vs GES-B).GES-A刺激胃窦部2h,下丘脑(48.93±6.98 fmol/mg vs 96.23±12.93 fmol/mg,P<0.01)、中脑(30.96±4.86 fmol/mg vs 53.17±8.96 fmol/mg,P<0.05)、延脑(46.27±7.83 fmol/mg vs 73.86±9.37 fmol/mg,P<0.05)和海马(32.23±6.51 fmol/mg vs 62.72±10.07 fmol/mg,P<0.05)中MTL免疫反应物(MTL-IR)的含量与假手术对照组相比明显减少,但脑桥内MTL-IR的含量与假手术对照组相比无显著差异;且海马CA1区(16.75±0.91 cells/mm~2 vs 20.46±1.30 cells/mm~2,P<0.05)和CA2-3区(14.91±1.17 cells/mm★★★2 vs 18.73±1.10 cells/mm~2,P<0.05)nNOS免疫阳性神经元表达明显减少.结论:GES可兴奋海马CA1区内胃扩张反应性神经元,电刺激作用的强弱与GES刺激的频率和单个电刺激的持续时间有关;脑内MTL和海马内nNOS可能参与了GES的中枢作用机制.