谷氨酸钠损伤幼年豚鼠耳蜗螺旋神经节

我们曾报道过不同剂量谷氨酸钠腹腔注射对新生豚鼠死亡率、听力及耳蜗螺旋神经节 (spiralganglion ,SG)的影响。对于谷氨酸钠耳聋模型 ,2g/ (kg·d) ,ip 连续 7d是相对比较理想的方案[1] ,但仍伴有较高的死亡率。以往多采用新生啮齿类动物作为耳聋制作的模型动物[2 ] ,本实验利用血脑屏障已发育完善之幼年豚鼠对谷氨酸钠的耳蜗效应进行了研究。1　材料和方法1 1　材料 :豚鼠 ,山东省生物制品研究所 ,英国引种 ,普通级 ,证号 :2 0 0 2 0 6 10 ,日龄 4 5～ 5 0d ,雌雄不拘 ,共 4 0只 ,体重(2 74 3± 4 1 4 )g。随机分成四组 ,①生理盐水对照…     

谷氨酸钠对新生豚鼠耳蜗螺旋神经节的毒性损伤

探讨谷氨酸钠不同给药时程及不同存活时程对新生豚鼠耳蜗螺旋神经节细胞及听力损伤的程度。谷氨酸钠按 2g kg腹腔注射给予 ,每天一次。G2 1 0组 :连续用药 2 1d ;G7 14组 :连续用药 7d ,停药后饲养 14d ;G7 35组 :用药 7d ,后饲养 35d。随后检测其听力水平以及螺旋神经节细胞的形态变化。结果表明各组听阈均有升高 ,伴有神经节细胞的部分消失 ,G2 1 0组听阈为 :6 5 3± 2 1 5dB ,神经节细胞密度为 :1985 3± 10 2 1 7 mm2 ;G7 14组 :听阈 6 3 8±19 2dB ,细胞密度 314 5 4± 12 2 5 3 mm2 ;G7 35组 :听阈 5 4 6± 14 3dB ,细胞密度 2 990 4± 10 2 2 1 mm2 。提示谷氨酸钠对新生豚鼠螺旋神经节细胞毒性损伤程度与用药时程有关 ;受损细胞大部死亡 ,部分细胞随再存活时间逐渐自我修复 ;听力恢复与停药后存活时间及节细胞状态关联     

碱性成纤维细胞生长因子对豚鼠耳蜗螺旋神经节细胞谷氨酸钠毒性损伤的保护作用

目的　在体研究碱性成纤维细胞生长因子 (bFGF)对谷氨酸钠所致豚鼠耳蜗螺旋神经节细胞毒性损伤的拮抗作用。方法　谷氨酸钠组 :谷氨酸钠 2g·kg-1·d-1,ip× 18d ;bFGF组 :bFGF 80 0U·kg-1·d-1,ip ,谷氨酸钠 2g·kg-1·d-1,ip× 18d。停止用药后耳廓反射粗略测定动物听觉水平 ,后取耳蜗 ,固定包埋、切片染色 ,光镜观察。结果　谷氨酸钠组表现为基底转与顶转几乎均匀一致的耳蜗螺旋神经节细胞的消失 ,平均细胞密度为 (1976 3± 10 18 9)个 /mm2 ,占正常的 4 5 % ;bFGF组细胞密度为 (384 0 9± 373 1)个 /mm2 ,约为正常组的 89%。经方差分析 ,F 检验 ,谷氨酸钠组与bFGF组比较 ,耳蜗螺旋神经节存活细胞密度差异显著 ,P <0 0 5。结论　bFGF对豚鼠谷氨酸钠耳蜗旋神经节细胞在体毒性损伤具有保护作用     

豚鼠脑缺血再灌注损伤后螺旋神经节细胞形态改变及BDNF的表达

目的 观察豚鼠脑缺血再灌注损伤(IRI)后螺旋神经节细胞形态、结构的改变和脑源性神经营养因子(BDNF)表达的变化,探讨BDNF对脑IRI后螺旋神经节细胞的营养保护作用.方法 将48只健康豚鼠随机分成正常组、假手术组和IRI 6 h、12 h、24 h、48 h组,用灼断两侧椎动脉、夹闭一侧颈总动脉的方法建立豚鼠IRI模型.光镜下观察螺旋神经节的病理改变,透射电镜观察螺旋神经节细胞超微结构的改变;免疫组织化学法和Western blot检测BDNF表达部位和表达量的变化.结果 光镜和透射电镜观察表明脑IRI导致螺旋神经节细胞数量减少和细胞损伤,IRI 24 h细胞的损伤最为严重;Western blot检测显示各组豚鼠螺旋神经节细胞都有BDNF表达,正常组、假手术组分别为0.390±0.021和0.381±0.019,两组表达差异无统计学意义(P＞0.05),IRI6h组(0.423±0.041)表达增加,差异有统计学意义(P＜0.05),随后各组表达量减少,IRI 24 h组最少(0.218±0.081),差异有统计学意义(P＜0.05);免疫组织化学法与Western blot检测结果一致,并显示BDNF表达部位在细胞浆.结论 脑IRI可导致耳蜗螺旋神经节细胞损伤和BDNF表达量的改变,BDNF可能参与了 IRI引发的螺旋神经节细胞损伤的病理过程.     

组胺在豚鼠颈上神经节中的分布

目的 检测组胺在外周交感神经元中的分布。方法 应用原位杂交组织化学技术检测组氨酸脱羧酶(HDC)mRNA的表达,用荧光双标免疫组织化学技术观察组胺和酪氨酸羟化酶(TH)在神经细胞中的共存。结果豚鼠颈上神经节中有较多的神经元呈．HDC阳性,HDC mRNA阳性信号在大、小细胞的细胞浆内均有分布,在细胞核内未检测到阳性信号;组胺的阳性细胞和TH的阳性细胞在豚鼠颈上神经节中均有大量分布,在大型和小型神经元内均发现有双标阳性信号;6．羟多巴胺(6-OHDA)化学损毁颈上神经节的交感神经细胞后阳性细胞大量减少。结论 提示在颈上神经节细胞的胞浆内有组胺的合成,组胺与单胺类神经递质在外周交感神经元中共存。     

大鼠背根神经节内kv2.1 mRNA和kv4.2 mRNA的表达

目的　了解kv2 1和kv4 2型钾离子通道在背根神经节的表达。方法　应用原位杂交方法观察背根神经节内细胞膜钾离子kv2 1和kv4 2型通道的mRNA阳性神经结构的表达。结果　腰 3～ 5背根神经节内均有kv2 1和kv4 2mRNA阳性神经元的表达 ,kv4 2mRNA阳性神经元的数量和总面积明显多于kv2 1阳性神经元。结论　kv2 1和kv4 2型钾离子通道可能参与了背根神经节对外周传入信息的传递和调制。     

组胺在豚鼠颈上神经节投射到心脏交感神经纤维中的表达

目的 探讨组胺在豚鼠颈上神经节投射到心脏交感神经纤维中的表达,及其与去甲肾上腺素的共存,为组胺可能是心脏交感神经系统新发现的递质提供形态学证据. 方法生物索化葡聚糖胺(BDA)顺行追踪结合组胺和去甲肾上腺素免疫荧光三标技术. 结果将BDA注入颈上神经节后,左、右心房和心室均有其投射的交感神经纤维,同时在这些示踪纤维中亦有组胺和组胺/去甲肾上腺素共存的免疫阳性表达. 结论豚鼠颈上神经节投射到心脏交感神经纤维中有组胺的表达并与去甲肾上腺紊共存.     

神经生长因子对哮喘豚鼠内脏感觉传入部位MMP-2表达的影响

目的探讨基质金属蛋白酶-2(MMP-2)在哮喘豚鼠内脏感觉传入部位的表达及神经生长因子(NGF)对MMP-2的调节作用。方法应用免疫组织化学方法检测豚鼠内脏感觉传入部位MMP-2免疫反应的变化。用W estern b lot蛋白印迹方法检测豚鼠C7~T5段脊神经节和相应节段脊髓背角NGF和MMP-2的表达。用Luzex-F实时图像分析系统和凝胶成像分析系统分别对结果进行图像分析。结果①免疫组织化学结果显示:豚鼠C7~T5段脊神经节和相应节段脊髓背角内MMP-2阳性免疫反应产物的平均灰度值(0-深色,255-浅色),生理盐水组(175.50±4.67、166.47±6.20)与单纯致敏组(172.63±9.51、165.20±5.14)比较没有显著差异(P>0.05);哮喘组(139.87±4.88、125.93±4.49)明显低于生理盐水组和单纯致敏组(P<0.01);哮喘+NGF抗体组(鼻腔吸入NGF抗体,169.73±6.24、152.37±8.67)则明显高于哮喘组(P<0.01)。②W estern b lot结果显示:与生理盐水组和单纯致敏组比较,哮喘组豚鼠C7~T5段脊神经节及相应节段脊髓样品中MMP-2和NGF阳性产物的IDV(Integrated Density Value)与β-actin IDV的比值均明显升高(P<0.01),而哮喘+NGF抗体组则明显低于哮喘组(P<0.01),表明NGF可上调豚鼠内脏感觉传入部位MMP-2的表达。结论C7~T5段脊神经节以及相应节段脊髓背角内的MMP-2可能也参与哮喘的发病过程,NGF诱发MMP-2的表达可能是NGF参与哮喘发病的机制之一,NGF抗体抑制哮喘时MMP-2的表达有可能延缓气道重塑的发生,可能是治疗哮喘的新途径。     

NGF对哮喘豚鼠下呼吸道及内脏感觉传入部位TNF-αmRNA表达的上调作用

目的　探讨神经生长因子 (NGF)在哮喘发病中的作用及其神经免疫调节机制。　方法　应用原位杂交方法结合显微图像分析 ,研究实验性哮喘豚鼠下呼吸道和内脏感觉传入部位肿瘤坏死因子 αmRNA(TNF αmRNA)表达的变化以及NGF抗体对其的影响。　结果　生理盐水组与单纯致敏组TNF αmRNA的表达没有显著差异 (P >0 0 5 ) ;与这两个对照组相比 ,哮喘豚鼠TNF αmRNA的表达在气道上皮、肺内炎性细胞、C7～T5段脊神经节及脊髓后角内均明显上调 (P <0 0 1) ;在哮喘 +NGF抗体组 ,上述部位内TNF αmRNA的表达明显低于哮喘组 (P <0 0 1)。　结论　NGF诱发TNF α的表达可能是NGF参与哮喘发病的机制之一 ,NGF抗体抑制哮喘时下呼吸道和内脏感觉传入部位TNF αmRNA的表达可能是治疗哮喘的新途径。     

氨茶碱对哮喘豚鼠嗜酸细胞凋亡及bcl—2 mRNA表达的影响

目的 观察氨茶碱对体外不同密度嗜酸细胞（Eos）凋亡及bcl-2 mRNA表达的影响,探讨其促进哮喘Eos凋亡的机制。方法 卵蛋白激发哮喘豚鼠动物模型48h后行支气管肺泡灌洗,分离不同密度Eos。氨茶碱（AM）（10^-6-10^-3mol/L)干预24h,原位杂交检测Eos的bcl-2 mRNA表达,TUNL法检测细胞凋亡。结果 AM干预24h,可见Eos凋亡增加,以低密度Eos(HEos)为明显,bcl-2 mRNA表达,呈剂量依赖性。bcl-2 mRNA的表达与Eos凋亡呈负相关。结论 AM可抑制bcl-2 mRNA表达,促进Eos凋亡。bcl-2参与了AM对Eos凋亡的调节。     

水杨酸钠静脉滴注对家兔EP性发热的解热效应观察

本研究用新西兰白色家兔50只,观察了不同剂量水杨酸钠(SS)静脉滴注对家兔正常体温及EP性发热的影响。结果表明:SS静脉滴注对家兔正常体温没有影响;不同剂量SS不同程度地抑制了家兔EP性发热效应,解热程度与SS剂量大小有关。其中,起始量为1.8mM,维持量为15μM/min的SS静脉滴注可充分抑制静注EP所致的体温升高。以上实验结果对于临床治疗或实验研究时选择解热剂剂量具有重要参考价值。     

Sodium current kinetics in freshly isolated neostriatal neurones of the adult guinea pig

Neurones of the neostriatum were freshly dispersed from the adult guinea pig brain. A fast, transient inward Na⁺ current (I _Na) was analysed using the whole-cell patch-clamp technique. Upon depolarizations, I _Na developed with a sigmoidal time course, which was described by m ³ kinetics. I _Na showed an activation threshold of about –60 mV, a peak current at –30 to –20 mV, and a reversal of polarity at +60 mV. The steady-state activation (m) curve for I _Na had a slope factor of about 9 mV with a mid-point potential of about –26 mV. The voltage dependence of the activation time constant, _m , had a bell-shaped configuration with a maximum value at –60 mV. The forward rate constant for I _Na activation ( _m ) increased as the membrane was depolarized (about 9 mV for a change in the rate constant by a factor of e) in the range between –50 mV and –20 mV. Conversely, the backward rate constant ( _m) decreased as the membrane was depolarized (about 31 mV for an e-fold change). The steady-state inactivation (h ) curve was well expressed by the Boltzmann's equation with a half-inactivation potential of –62 mV and a slope parameter of 6 mV. The time course of I _Na decay followed a second-order process, whereas the recovery from inactivation was described as a first-order process. The _h curve showed a bell-shaped configuration with a maximum value at –60 mV. The forward rate constants ( _h ) decreased as the membrane was depolarized (about 17 mV for an e-fold change) in the range between –50 mV and –20 mV. The backward rate constants ( _h) increased as the membrane was depolarized (about 10 mV for an e-fold change). There was a significant overlap between m and h curves, suggesting a steady influx of Na⁺ (window current).     

Effects of heating on electrical activities of guinea pig olfactory cortical slices

We examined the effect of heating on electrical activity of neurons in the guinea pig olfactory cortex slice. At the control temperature (37° C) the potential evoked by stimulation of the lateral olfactory tract consisted of an initial spike (IS) potential and a negative (N) potential. The IS potential is considered to be presynaptic and the other transsynaptic.The IS potential decreased in amplitude on heating and completely disappeared at 49° C. However, it recovered when the temperature was lowered to 37° C after five minutes of incubation at 49° C. In contrast, the N potential increased in amplitude at 39° C, was completely suppressed at 47° C and did not recover when the temperature was dropped to the control temperature. The maximum temperature from which the N potential recovered was 43° C.Unit activity was extracellularly recorded from neurons in the slice. On heating the brain slice some neurons showed an increase in activity, others a decrease, and the rest were unaffected. We conclude that neurons in the olfactory cortex have different thermal sensitivities.     

Uptake of inorganic phosphate by the maternal border of the guinea pig placenta

Mechanism of uptake of inorganic phosphate (P_i) by the maternal border of the dually perfused guinea pig placenta was studied using the paired-tracer dilution technique with³²P-phosphate and¹⁴C-sucrose being the tracers. Placental uptake of radioactive phosphate increased when the concentration of P_i in the perfusion fluid was reduced, and it decreased during anoxia, in presence of CN or during perfusion with low-Na or Na-free fluids. Iodoacetate was without effect. These observations are consistent with placental uptake of P_i being effected by a carrier mediated process dependent on external Na and, partly, on placental metabolism. Unidirectional flux of P_i from the maternal vascular space into the cell compartment of the placenta, estimated from the values of instantaneous extraction of³²P, correlated significantly with foetal weight. The flux per unit weight of the foetus was 17.0±1.0 nmol · min^–1 g^–1.     

阿米卡星致毒豚鼠耳蜗中p-JNK表达增强

目的 研究阿米卡星(AMK)对豚鼠耳蜗磷酸化C-JUN氨基末端激酶(JNK)表达的影响及其耳毒性机制.方法 将豚鼠分为对照组、AMK 3、7和11d组.AMK组分别每日肌肉注射AMK(400 mg/kg),对照组肌肉注射等量0.9%氯化钠注射液.用听脑干反应(ABR)测试和耳蜗铺片异硫氰酸四甲基罗丹明(TRITC)标记...     

Localization of dopamine receptor subtypes in the rat spiral ganglion

Although dopaminergic neurons are thought to exist in the lateral olivocochlear efferent system and modulate the afferent nerve activity, the distribution of dopamine (DA) receptor subtypes is still obscure. In the present study, we investigated the localization of five subtypes of DA receptor (D1-5) by immunocytochemical analysis and the gene expression of D1-5 using RT-PCR procedure in the rat cochlea. Most, but not all, spiral ganglion neurons were immunolabeled with all the anti-DA receptor subunit antibodies and faint punctuate immunoreactivities were observed in inner hair cell regions. Gene expression for all receptors was detected. These results suggest that all DA receptor subtypes are present in spiral ganglion cells, and potentially regulate afferent neurotransmission.     

Autoradiographic quantitation and anatomical mapping of GTP sensitive-galanin receptors in the guinea pig central nervous system

Galanin is a 29-amino acid peptide widely distributed in the mammalian central nervous system. Galanin receptors in the guinea pig brain were visualized using [¹²⁵I]galanin by in vitro receptor quantitative autoradiography. Scatchard analysis of [¹²⁵I]galanin binding to slide-mounted sections revealed saturable binding to a single class of high affinity receptors with a K_D of approximately 1 nM. Specific [¹²⁵I]galanin binding sites were detected in a large number of brain areas (concentration range: from non detectable to 99.32 fmol/mg of tissular proteins). The anatomical mapping revealed high densities essentially in the telencephalon (e.g. lateral septal nuclei, amygdala, hippocampal dentate gyrus) and the diencephalon (e.g. the anterodorsal and medial habenular thalamic nuclei, the paraventricular, dorsomedian and median mammillary hypothalamic nuclei, the posterior lobe of the pituitary). Addition of Mg²⁺ and GTP increased binding in some areas such as the zona incerta, the median eminence and the arcuate nucleus, and decreased it in other areas such as the amygdala, the hippocampus and the mammillary nuclei. This regional heterogeneity in the effect of Mg²⁺ and GTP can be interpreted as: (1) different rates of galanin receptor occupancy by endogenous peptide; (2) a differential coupling of GTP binding proteins to galanin receptors in the brain structures; and (3) a different nature of receptors. At any rate, this study provides evidence for a specific GTP-sensitive galanin receptor in guinea pig brain with an extensive distribution suggesting various physiological implications. Comparison with studies performed in several mammals shows that the overall distribution of galanin receptors is well preserved among species. These data suggest that galanin may posses similar functional properties in the different species tested so far. Nevertheless, very distinct differences were found in some areas like the cortex, the hippocampus and the pituitary.     

COX-2 expression in the guinea pig cochlea is partly altered by moderate sound exposure

The cyclooxygenase-2 isoform (COX-2) was found recently to be constitutively expressed in the guinea pig inner ear. To gain knowledge about its role in sound perception, alterations in the COX-2 level of moderate noise-stimulated cochleae were determined. Staining intensities were quantified in different regions using an immunohistochemical staining procedure and computer-assisted system. After 70 dB and 90 dB noise exposure for 1 h at 8000 Hz, COX-2 downregulation was observed in the organ of Corti, which was most prominent in Deiters' cells near Hensen cells and outer hair cells. In pillar cells, COX-2 levels were only slightly reduced after 70 dB but strongly diminished after 90 dB exposure. In Hensen cells, COX-2 was downregulated after 70 dB stimulation, revealing a decreasing COX-2 content from the third to the first turn of the cochlea and a homogeneously reduced enzyme expression in all three turns after 90 dB. The COX-2 content in inner hair cells was nearly identical to unexposed cochleae after 70 dB exposure but significantly reduced after 90 dB stimulation. In spiral ganglion cells, stria vascularis, spiral ligament and limbus, COX-2 expression was unchanged after 70 dB and 90 dB. We suggest that alterations in COX-2 expression might contribute to diminished sensitivity at the cochlea after noise exposure to reduce subsequent noise distress, termed sound conditioning.