慢性非细菌性前列腺炎大鼠腰6～骶1背神经节中蛋白激酶G及B型钠尿肽的表达水平

目的探讨慢性非细菌性前列腺炎(CNP)大鼠腰6~骶1背神经节中蛋白激酶G(PKG)、B型钠尿肽(BNP)的表达水平。方法(1)将120只大鼠随机分为实验组与对照组各60只,实验组大鼠给予前列腺腹叶注射完全弗氏佐剂0.1mL,对照组则予以注射等量生理盐水,均1次/d,持续14d,建模后按常规饲养。分别于模型建立后3d、7d、14d,每组各分离20只大鼠的腰6~骶1背神经节,检测其PKG、BNP蛋白及mRNA表达情况。(2)将另外40只大鼠分为研究组与参照组各20只,均给予前列腺腹叶注射完全弗氏佐剂0.1mL,1次/d,持续7d,建模同样按常规饲养。建模7d后,研究组大鼠神经鞘内注入50.0μL的BNP,参照组大鼠注入等量生理盐水。检测两组大鼠腰6~骶1背神经节中PKG蛋白表达情况。结果(1)实验组建模后3d、7d、14dPKG及BNPmRNA表达量均高于对照组建模后3d水平(均P<0.05);建模后3d、7d、14d实验组PKG及BNP蛋白表达量均高于对照组(均P<0.05)。(2)研究组腰6~骶1神经节中PKG蛋白表达水平高于参照组(P<0.05)。结论CNP大鼠腰6~骶1背神经节中PKG、BNP水平升高,两者可能参与CNP相关性疼痛的发生及发展。

胶原蛋白水解酶对背根神经节细胞亚细胞结构急性损伤的实验

目的:观察胶原蛋白水解酶(胶原酶)对大鼠脊神经背根神经节细胞亚细胞结构急性损伤的影响,以期探讨胶原酶应用的安全性,并进一步论证经皮椎间盘胶原蛋白水解酶化学髓核溶解术(胶原酶髓核溶解术)治疗方法的安全性。方法:实验于2002-07/09在中山大学基础医学院完成。SD健康雄性大鼠28只。随机分为3组:正常组9只;胶原酶急性实验组9只、急性假手术组10只。急性实验组和急性假手术组大鼠经腹腔注射戊巴比妥钠(45mg/kg)麻醉后,分离并辨认大鼠背根神经节,实验组局部滴注胶原酶1mL(4mL含1200U),急性假手术组局部滴注生理盐水1mL。于注药后1h行各组大鼠背根神经节细胞亚细胞结构的电镜检测。结果:各组大鼠背根神经节的神经元细胞、节内神经纤维检测结果:急性实验组背根神经节观测细胞种类、细胞数量、细胞大体形态、胞膜情况、节内神经纤维情况(有髓神经纤维有无肿胀,脱髓鞘,髓鞘松解等改变)、血管等情况与正常组、急性假手术组比较均无明显变化和差别。急性实验组背根神经节细胞亚细胞结构与正常组和急性假手术组比较均有明显变化和差别:①核仁部分偏向一侧。②线粒体大量肿胀,部分嵴断裂、空泡形成。各组均未见节细胞成群细胞坏死、细胞膜早期破裂、凋亡小体形成等节细胞凋亡相关表现和节细胞坏死相关表现。结论:临床应用于胶原酶髓核溶解术治疗浓度的胶原酶对背根神经节细胞是有损伤的。掌握胶原酶的用量和浓度,使胶原酶作用于精确合适的部位,才能提高胶原酶应用的安全性。

食指近节背侧带神经血管蒂岛状皮瓣临床应用

拇指作用非常重要,约占手功能50%,拇指外伤在手外伤中,特别重要,失去拇指意味着手功能丧失近半,没有感觉的手指亦等于无用.拇指指蹼短缩,活动度减小,关节僵硬,也都严重影响手的功能,食指近节背侧带神经血管蒂岛状皮瓣临床应用,较好的解决了上述问题,我们1996年2月～2002年11月应用本方法修复指背缺损16例,获较好疗效,报道如下:

神经病理性疼痛动物模型及其评价

对神经病理性疼痛发病机制的研究大多来源于动物模型;尽管模型还存在不少缺点,但是它为理解和探索人类神经病理性疼痛的发病机制提供了有用的工具。动物模型的缺点是动物无法语言交流,对动物的疼痛测量多基于主观行为反应,比如测量痛敏和异常痛敏。最常用的动物模型包括坐骨神经慢性压迫模型和坐骨神经部分损伤模型等,通过测量神经损伤侧肢体脚爪皮肤的感觉阈值即主要通过测评对热、机械刺激痛敏(hyperalgesia)和冷、触异常痛敏(allodynia)来确定模型是否成功。

D_2受体激动剂R(一)—NPA对DRG神经元GABA-激活电流的抑制作用

应用全细胞膜片钳记录技术在大鼠新鲜分离DRG神经元上,观察预加多巴胺D_2受体选择性激动剂R(—)—NPA对GABA—激活电流的调制作用.绝大部分受检细胞86.2%(50/58)对外加GABA敏感.10^(-6)～10^(-3)mol/LGABA 引起一剂量依赖性有明显去敏感作用的内向电流.对GABA敏感的50个受检细胞中13个细胞引起一较大的无明显去敏感的内向电流,其他的无反应.预加R(一)—NPA30秒后再加GABA,则对GABA—激活电流幅值的影响如下:抑制的为76%(38/5O),增强的为2%(1/50),无明显作用的为22%(11/5O).在其浓度为10^(-4)mol/L、10^(-5)mol/L、10^(-6)mol/L、10^(-7)moV/L时抑制(X±SE)分别为19.1%12.8(n=6)、42.49%±3.2(n=6)、83.6%±1.2(n=6)、8.69%±1.8(n=6).细胞内加蛋白激酶抑制剂H7后,R(—)—NPA对GABA的抑制作用完全消除(n=16).

美西律对大鼠背根神经节细胞钠电流的抑制作用

应用膜片钳技术,全细胞钳位方式可见Mex对大鼠背根神经节细胞Na^+电流有明显的抑制作用。其临界作用浓度为5 μmol·L^(-1),50％抑制浓度为31 μmol·L^(-1),完全抑制浓度为150 μmol·L^(-1)。其抑制作用具有电压和频率依赖性。抑制剂量与小鼠抗脑缺血缺氧作用量相近。提示Mex抗脑缺血缺氧作用的机制之一可能是通过对Na^+电流的抑制。

脊髓背侧神经节切除术——一种定量性评价周围神经再生与修复效果的实验模型

选用成年犬12条,其中6条切除一侧脊髓背侧神经节(DRGs),双侧胫神经分支对比染色,证实犬的胫神经皮支含有100%的感觉纤维,运动支内含有65%的运动纤维。另外6条胫神经横断后外膜修复,14周时二次手术切除DRGs,待感觉纤维变性后取材,观察再生的运动纤维在胫神经运动和感觉神经支内的分布,结果显示:再生神经纤维均匀分布在胫神经各分支内,运动神经纤维错接率为30.5%,有近1/3的轴索是无功能性再生,这也是临床神经修复效果差的主要原因,此模型可对神经再生做出更客观的功能评价。

短时程脊髓电刺激术与背根神经节脉冲射频配合电针治疗带状疱疹神经痛疗效对比

观察短时程脊髓电刺激术与背根神经节脉冲射频配合电针治疗带状疱疹后顽固性神经疼痛患者的治疗效果。方法:选取40例胸腰段带状疱疹顽固性神经痛患者,常规治疗效果不佳,病程小于三个月患者,分A、B两组,A组行短时程脊髓电刺激术,B组行CT引导下行背根神经脉冲射频配合每日电针治疗,观察两组术前,术后1天,1周,2周,1月,3月患者视觉模拟评分(VAS)和匹兹堡睡眠质量指数(PSQI)。结果:术前两组病人VAS和PSQI无显著差异(P>0.05),A组和B组在术后1天、1周、2周、1月、3月疼痛较术前明显改善(P<0.05),术后2周两组病人VAS和PSQI无明显差异(P>0.05);两组病人术后3月PSQI均与术前存在统计学差异(P<0.05),且A组PSQI明显优于B组(P<0.05)。结论:短时程脊髓电刺激术与CT引导下背根神经节脉冲射频配合电针治疗带状疱疹后顽固性神经痛都能够有效缓解疼痛,且短时程脊髓电刺激术临床效果优于CT引导下背跟神经节脉冲射频配合电针治疗。

Roles of TRESK,a novel two-pore domain K+channel,in pain pathway and general anesthesia

TRESK is the most recently reported two-pore domain K^＋ channel, and different from other two-pore domain channels in gene, molecular structure, electrophysiological and pharmacological properties. Although the current knowledge of this potassium channel is inadequate, researches have demonstrated that TRESK is remarkablely linked to acute and chronic pain by activation of calcineurin. The fact that TRESK is sensitive to volatile anesthetics and localization in central nerve system implies that TRESK may play a very important role in the mechanism mediating general anesthesia. The further research of TRESK may contribute to explore the underlying mechanism of some pathological conditions and yield novel treatments for some diseases.

Regulatory effect of nerve growth factor on release of substance P in cultured dorsal root ganglion neurons of rat

Objective To investigate the regulatory effects of nerve growth factor （NGF） on basal and capsaicin-induced release of neuropeptide substance P （SP） in primary cultured embryonic rat dorsal root ganglion （DRG） neurons. Methods DRGs were dissected from 15-day-old embryonic Wistar rats. DRG neurons were dissociated and cultured, and then exposed to different concentrations of NGF （10 ng/mL, 30 ng/mL, or 100 ng/mL） for 72 h. The neurons cultured in media without NGF served as control. RT-PCR were used for detecting the mRNAs of SP and vanilloid receptor 1 （VR1） in the DRG neurons. The SP basal and capsaicin （100 nmol/L）-induced release in the culture were measured by radioimmunoassay （RIA）. Results SP mRNA and VR1 mRNA expression increased in primary cultured DRG neurons in a dose-dependent manner of NGF. Both basal release and capsaicin-evoked release of SP increased in NGF-treated DRG neurons compared with in control group. The capsaicin-evoked release of SP also increased in a dose-dependent manner of NGF. Conclusion NGF may promote both basal release and capsaicin-evoked release of SP. NGF might increase the sensitivity of nociceptors by increasing the SP mRNA or VR1 mRNA.

Identification of differentially expressed genes in dorsal root ganglion in early diabetic rats

Objective To screen and identify differentially expressed genes in the dorsal root ganglion （DRG） in early experimental diabetic rats. Methods Diabetic model rats were induced by single intraperitoneal injection of streptozotocin （STZ）. At the second week after STZ injection, the sensory nerve conduction velocities （SNCV） of sciatic nerve were measured as an indicator of neuropathy. The technique of silver-staining mRNA differential display polymerase chain reaction （DD-PCR） was used to detect the levels of differentially expressed genes in rat DRG. The cDNA fragments that displayed differentially were identified by reverse-hybridization, cloned and sequenced subsequently, and then confirmed by Northern blot. Results The SNCV in the diabetic model group [n = 9, （45.25±10.38） m/s] reduced obviously compared with the control group [n = 8, （60.10± 11.92） m/s] （P 〈 0.05）. Seven distinct cDNA clones, one was up-regulated gene and the others were downregulated ones, were isolated by silver-staining mRNA differential display method and confirmed by Northern blot. According to the results of sequence alignment with GenBank data, majority of the clones had no significant sequence similarity to previously reported genes except only one that showed high homology to 6-pyruvoyl-tetrahydropterin synthase mRNA （accession No., BC059140）, which had not been reported to relate to diabetic neuropathy. Conclusion These differentially expressed genes in the diabetic DRG may contribute to the pathogenesis of diabetic peripheral neuropathy.

Isolation and Evaluation of Nerve Growth Factor

Objective: to obtain the high purified and active nerve growth factor (NGF) from mouse submaxillary glands. Methods: NGF was prepared from mouse submaxillary glands by the way of elution with CM 52 column. The molecular weight and purification of NGF were detected by SDS-PAGE polyacrylamide gel electrophoresis. The biological activity of NGF was verified thorough culturing DRG. Results: About 14 kDa stained band was observed on SDS-PAGE and it promoted proliferation of dorsal root gang lia (DRG). Conclusion: Good quality of NGF could be obtained with these methods.

挑治法加艾灸治疗带状疱疹

关于针刺治疗带状疱疹已有不少报道,笔者用挑治法加艾灸治疗该病取得较满意的疗效。介绍如下: 一、治疗方法: 1.病人俯卧或反坐在靠背椅上,暴露背部。取与病变相应部位的夹脊穴。

Purification and Bioassay of Mouse β NGF Antibody

Anti β NGF antiserum was prepared by immunizing rabbits with purified mouse β NGF. It presented immunopositivity to β NGF on Western blot, resulting in a single band approximately at the molecular weight of 14 kDa. The antiserum was further purified into anti β NGF IgG by affinity chromatography. Chicken embryonic dorsal root ganglia (DRG) were cultured to test the biological activities of β NGF and its antibody.

PAd-shRNA-PTN reduces pleiotrophin of pancreatic cancer cells and inhibits neurite outgrowth of DRG

AIM:To investigate the silencing effects of pAdshRNA-pleiotrophin(PTN) on PTN in pancreatic cancer cells,and to observe the inhibition of pAd-shRNA-PTN on neurite outgrowth from dorsal root ganglion(DRG) neurons in vitro.METHODS:PAd-shRNA-PTN was used to infect pancreatic cancer BxPC-3 cells;assays were conducted for knockdown of the PTN gene on the 0th,1st,3rd,5th,7th and 9th d after infection using immunocytochemistry,real-time quantitative polymerase chain reaction(PCR),and Western blotting analysis.The morphologic changes of cultured DRG neurons were observed by mono-culture of DRG neurons and co-culture with BXPC-3 cells in vitro.RESULTS:The real-time quantitative PCR showed that the inhibition rates of PTN mRNA expression in the BxPC-3 cells were 20%,80%,50% and 25% on the 1st,3rd,5th and 7th d after infection.Immunocytochemistry and Western blotting analysis also revealed the same tendency.In contrast to the control,the DRG neurons co-cultured with the infected BxPC-3 cells shrunk;the number and length of neurites were significantly decreased.CONCLUSION:Efficient and specific knockdown of PTN in pancreatic cancer cells and the reduction in PTN expression resulted in the inhibition of neurite outgrowth from DRG neurons.

Expression of three distinct families of calcium-activated chloride channel genes in the mouse dorsal root ganglion

Objective A calcium-activated chloride current （ICl（Ca）） has been observed in medium-sized sensory neurons of the dorsal root ganglion （DRG）. Axotomy of the sciatic nerve induces a similar current in the majority of medium and large diameter neurons. Our aim is to identify the molecule（s） underlying this current. Methods Using conventional and quantitative RT-PCR, we examined the expression in DRG of members of three families of genes, which have been shown to have latch） current inducing properties. Results We showed the detection of transcripts representing several members of these families, i.e. chloride channel calciumactivated （CLCA）, Bestrophin and Tweety gene families in adult DRG, in the normal state and 3 d after sciatic nerve section, a model for peripheral nerve injury. Conclusion Our analysis revealed that that mBestl and Tweety2 appear as the best candidates to play a role in the injury-induced Icl（Ca） in DRG neurons.

Expression of the tyrosine kinase receptor EphA5 and its ligand ephrin-A5 during mouse spinal cord development

Objectives To study the expression patterns of two Eph family molecules, the receptor EphA5, and the ligand ephrin-A5, during spinal cord development. Methods The receptor expression was analyzed using beta-galactosidase knockin mice, and affinity ligand probe binding. The ligand expression was assessed using two different affinity probes, and knockout mouse tissues as controls. Results EphA5 was expressed in the ventral spinal cord, while ephrin-A5 was located in the dorsolateral regions of the spinal cord throughout development. Conclusions These results show that EphA5 and ephrin-A5 are expressed over broad developmental stages and may play important roles in establishing the dorsoventral organization of the spinal cord.

Gene expression profiling and endothelin in acute experimental pancreatitis

AIM:To analyze gene expression profiles in an experimental pancreatitis and provide functional reversal of hypersensitivity with candidate gene endothelin-1 antagonists.METHODS:Dibutyltin dichloride(DBTC) is a chemical used as a polyvinyl carbonate stabilizer/catalyzer,biocide in agriculture,antifouling agent in paint and fabric.DBTC induces an acute pancreatitis flare through generation of reactive oxygen species.Lewis-inbred rats received a single i.v.injection with either DBTC or vehicle.Spinal cord and dorsal root ganglia(DRG) were taken at the peak of inflammation and processed for transcriptional profiling with a cDNA microarray biased for rat brain-specific genes.In a second study,groups of animals with DBTC-induced pancreatitis were treated with endothelin(ET) receptor antagonists [ET-A(BQ123) and ET-B BQ788)].Spontaneous pain related mechanical and thermal hypersensitivity were measured.Immunohistochemical analysis was performed using anti-ET-A and ET-B antibodies on sections from pancreatic tissues and DRG of the T10-12 spinal segments.RESULTS:Animals developed acute pancreatic inflammation persisting 7-10 d as confirmed by pathological studies(edema in parenchyma,loss of pancreatic architecture and islets,infiltration of inflammatory cells,neutrophil and mononuclear cells,degeneration,vacuolization and necrosis of acinar cells) and the painrelated behaviors(cutaneous secondary mechanical and thermal hypersensitivity).Gene expression profile was different in the spinal cord from animals with pancreatitis compared to the vehicle control group.Over 260 up-regulated and 60 down-regulated unique genes could be classified into 8 functional gene families:circulatory/acute phase/immunomodulatory;extracellular matrix;structural;channel/receptor/transporter;signaling transduction;transcription/translation-related;antioxidants/chaperones/heat shock;pancreatic and other enzymes.ET-1 was among the 52 candidate genes upregulated greater than 2-fold in animals with pancreatic inflammation and visceral pain-related behavior.Treatments with the ET-A(BQ123) and ET-B(BQ-788) antagonists revealed significant protection against inflammatory pain related mechanical and thermal hypersensitivity behaviors in animals with pancreatitis(P < 0.05).Open field spontaneous behavioral activity(at baseline,day 6 and 30 min after drug treatments(BQ123,BQ788) showed overall stable activity levels indicating that the drugs produced no undesirable effects on normal exploratory behaviors,except for a trend toward reduction of the active time and increase in resting time at the highest dose(300 μmol/L).Immunocytochemical localization revealed that expression of ET-A and ET-B receptors increased in DRG from animals with pancreatitis.Endothelin receptor localization was combined in dual staining with neuronal marker NeuN,and glia marker,glial fibrillary acidic protein.ET-A was expressed in the cell bodies and occasional nuclei of DRG neurons in na ve animals.However,phenotypic expression of ET-A receptor was greatly increased in neurons of all sizes in animals with pancreatitis.Similarly,ET-B receptor was localized in neurons and in the satellite glia,as well as in the Schwann cell glial myelin sheaths surrounding the axons passing through the DRG.CONCLUSION:Endothelin-receptor antagonists protect against inflammatory pain responses without interfering with normal exploratory behaviors.Candidate genes can serve as future biomarkers for diagnosis and/or targeted gene therapy.

Prokaryotic expression of recombinant human p75NTR-Fc fusion protein and its effect on the neurite outgrowth of dorsal root ganglia neuron

Objective： To clone, express, and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fe （hp75NTR-Fc） in prokaryotic expression system, and investigate the effect of the recombinant protein on dorsal root ganglia （DRG） neuron neurites. Methods： The hp75NTR-Fc coding sequence was amplified from pcDNA-hp75NTR-Fc by polymerase chain reaction （PCR） and subcloned into vector pET30a （＋）, in which hp75NTR-Fc expression was controlled under the T7 promoter. The recombinant vectors were amplified in E. coli DH5α and identified by PCR, enzyme digestion and sequencing, and then transformed into E. coli BL21 （DE3）. The expression product was analyzed with SDS-PAGE and Western blot. Then after the recombinant protein purified with Protein A affinity chromatograph, and renaturated with dialysis, respectively, the effect of the recombinant protein on DRG neuron neuritis was further investigated. Results： The results of PCR, enzyme digestion, and sequencing demonstrated the success of inserting the hp75NTR-Fc fragment into vector pET30a （＋）. SDS-PAGE and Western blot showed a positive protein band with molecular weight about 50 kD in the expression product, which is accordant with the interest protein, and this band could be specifically recognized by rabbit anti-NGFRp75 antibody. The purified infusion protein following dialysis could promote neurite outgrowth of DRG neurons cultured with myelin-associated glycoprotein （MAG）. Conclusion： The hp75NTR-Fc coding sequence was subcloned into the expression vector pET30a （＋） correctly and expressed successfully in the prokaryotie expression system. The infusion protein could promote neurite outgrowth of DRG neurons cultured with MAG.

Proteome profiling of spinal cord and dorsal root ganglia in rats with trinitrobenzene sulfonic acid-induced colitis

AIM: To investigate proteomic changes in spinal cord and dorsal root ganglia (DRG) of rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. METHODS: The colonic myeloperoxidase (MPO) activity and tumor necrosis factor-(TNF- ) level were determined. A two-dimensional electrophoresis (2-DE)-based proteomic technique was used to profile the global protein expression changes in the DRG and spinal cord of the rats with acute colitis induced by intracolonic injection of TNBS. RESULTS: TNBS group showed significantly elevated colonic MPO activity and increased TNF-level. The proteins derived from lumbosacral enlargement of the spinal cord and DRG were resolved by 2-DE; and 26 and 19 proteins that displayed significantly different expression levels in the DRG and spinal cord were identified respectively. Altered proteins were found to be involved in a number of biological functions, such as inflammation/immunity, cell signaling, redox regulation, sulfate transport and cellular metabolism. The over-expression of the protein similar to potassium channel tetramerisation domain containing protein 12 (Kctd 12) and low expression of proteasome subunit type-1 (psma) were validated by Western blotting analysis. CONCLUSION: TNBS-induced colitis has a profound impact on protein profiling in the nervous system. This result helps understand the neurological pathogenesis of inflammatory bowel disease.