坐骨神经结扎后大鼠背根神经节和脊髓CGRP表达的变化

目的研究大鼠坐骨神经结扎后降钙素基因相关肽(calcitoningene-relatedpeptide,CGRP)表达变化。方法SD大鼠随机分为假手术对照组和坐骨神经结扎组,实验组结扎后分别存活1、3、5、7、14、21和28d(n=8),免疫荧光(双标法)和免疫组织化学(SABC法)观察术后不同时间点CGRP和NGF在坐骨神经、背根神经节(dorsalrootganglion,DRG)和脊髓的表达变化,Westernblot结合图像分析技术对不同时间的变化进行定量测定。结果术后1d结扎远端坐骨神经内NGF大量堆积,持续到28d仍高于正常。结扎后7dDRG内CGRP阳性细胞百分率减少,持续到28d仍低于正常;结扎后14d脊髓后角CGRP下降,28d仍低于正常,各时间点脊髓前角CGRP表达未见明显变化。结论神经结扎可导致DRG和脊髓后角的CGRP表达下调,可能与靶源性的NGF来源减少有关。     

大鼠坐骨神经结扎后脊髓钙结合蛋白Calbindin D-28k的表达变化

目的:研究大鼠坐骨神经结扎模型的钙结合蛋白(Calbindin D-28k,CB)在脊髓的时空变化规律,为探讨其在神经再生中的作用与机制提供实验依据。方法:SD大鼠随机分为假手术对照组和坐骨神经结扎组,实验组结扎后分别存活1,3,7,14或21d,免疫组化结合图像分析技术观察CB在脊髓的表达变化。结果:在对照组,CB阳性神经元主要分布于腰髓背角Ⅰ、Ⅱ层,Ⅲ～Ⅵ层只观察到少量散在分布的CB样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见少量多极的大型阳性神经元。术后各时间点CB样阳性神经元表达下降,14d下降最显著,21d表达有所上升,但还是低于7d组。脊髓后角CB免疫阳性产物灰度值测定结果显示:术后14d后角CB表达最低,与对侧和对照组以及1、3d组相比有统计学意义(P<0.05)。结论:坐骨神经结扎后CB表达变化呈现一定的时空模式,为进一步揭示CB在神经系统疾病中的作用提供实验依据。     

大鼠坐骨神经结扎后脊髓钙结合蛋白PV的表达变化

目的:研究大鼠坐骨神经结扎模型钙结合蛋白Parvalbumin(PV)在脊髓的时空变化规律,为探讨其在神经再生中的作用与机制提供实验依据。方法:SD大鼠随机分为假手术对照组和坐骨神经结扎组,实验组结扎后分别存活1,3,7,14或21d,采用免疫组化结合图像分析技术观察PV在脊髓的表达变化。结果:在对照组,PV免疫阳性神经元主要分布于腰髓背角Ⅱ层,Ⅲ～Ⅵ层只观察到少量散在分布的PV样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见少量多极的大型阳性神经元。术后各时间点PV样阳性神经元表达下降,14d下降最显著,21d表达有所上升,但还是低于7d组。脊髓后角PV免疫阳性产物灰度值测定结果显示:术后14d后角PV表达最低,与对侧和对照组以及1、3d组相比有统计学意义(P<0.05)。结论:坐骨神经结扎后PV表达变化呈现一定的时空模式,为进一步揭示PV在神经系统疾病中的作用提供实验依据。     

大鼠坐骨神经结扎后疼痛受体P2X3在背根神经节内的表达变化

目的:研究坐骨神经结扎损伤后疼痛受体P2X3在相应背根神经节(dorsal root ganglia,DRG)内的表达变化情况。方法:选取健康成年SD大鼠35只,建立右侧坐骨神经结扎损伤模型,采用免疫组织化学和图像分析技术检测相应L4-6DRG内P2X3的表达情况。结果:正常大鼠L4-6DRG内有大量P2X3免疫阳性神经元,坐骨神经结扎后3d P2X3表达即下调,3,7,14,21和28d其表达呈进行性下降趋势,各时间点与正常和假手术对照比较差异均有统计学意义(P＜0．05)。结论:坐骨神经结扎后P2X3在L4-6DRG内表达明显下调,提示其可能在神经源性疼痛中发挥一定的作用。     

鞘内注射Akt特异性抑制剂GSK690693抑制骨癌诱导的大鼠痛相关行为的研究

目的:探讨在大鼠乳腺癌骨转移诱导癌症疼痛的调控过程中Akt信号通路的作用。方法:将Wistar成模wistar大鼠随机分为Model组、Model+GSK690693组及Model+Saline组;Model组大鼠不进行鞘内注射,Model+GSK690693组及Model+Saline组大鼠,于手术后数天(post-cancer cell implantation day,PID)PID 13 d、14 d及21 d鞘内分别注入Akt特异抑制剂GSK690693、等量生理盐水组,分别于PID 0 d、7 d、14 d及21 d,观察大鼠痛相关行为学检查,PID 21 d取DRG行免疫印迹检查p-Akt表达。结果:在鞘内注射Akt特异性抑制剂GSK690693后,Model+GSK690693组大鼠的机械缩足反射阈值上升,自发性疼痛行为值及患侧DRG中p-Akt表达下降。在PID 14 d、21 d等不同时间节点,Model+GSK690693组大鼠的疼痛行为学与Model+Saline组大鼠、Model组大鼠经统计学比较有显著的统计学差异(P0.01);PID 21d Model+GSK690693组大鼠患侧DRG中p-Akt表达与Model组的比较有显著统计学意义(P0.01),与Model+Saline组比较有统计学意义(P0.05);结论:鞘内注射Akt特异性抑制剂GSK690693可抑制大鼠乳腺癌骨转移诱导的大鼠痛相关行为。     

生长激素对实验大鼠牙齿移动过程中VEGF表达的影响

目的:探讨生长激素(growth hormone,GH)对实验大鼠牙齿移动过程中血管内皮生长因子(vascular endothelial growth factor,VEGF)在牙周组织中表达的影响。方法:将40只7周龄大的雄性Wistar大鼠根据是否注射生长激素分为实验组(E)和对照组(C)。将50 g力值加于近中左侧上颌第一磨牙。E组和C组分别腹部皮下注射GH(0.15IU/公斤/天)及等剂量的生理盐水。大鼠分别在第1、3、7、14和21天处死。上颌第一磨牙及其牙周组织切片行VEGF免疫组织化学染色,并进行图像分析和统计。结果:无论张力侧及压力侧VEGF在实验组表达的量均高于对照组,第3天组中张力侧及压力侧实验组平均光密度值分别为174.47±7.53和345.80±25.46与其各自对照组相比较数据具有统计学意义(P0.05),两侧实验组的VEGF的表达强度峰值均出现在第7天,分别为669.97±3.22和923.77±18.41差异具有统计学意义(P0.05),VEGF表达峰值及峰值之前压力侧无论实验组和对照组VEGF的表达强度要明显高于张力侧相应分组。结论:短期注射生长激素促进了牙周组织中VEGF的表达,对于正畸牙齿移动具有一定的促进作用。     

CCI大鼠坐骨神经损伤区内中终球与重链神经微丝蛋白的检测

为探寻CCI大鼠坐骨神经损伤区内终球的存在,了解其与重链神经微丝蛋白(heavy neurofilaments,NF-H)分布、表达的联系.通过对雄性SD大鼠的坐骨神经慢性压迫损伤(chronic constriction injury,CCI)组与对照组(对侧正常坐骨神经)进行不同时间点电镜、免疫组化及Western-blotting等方法的观察与检测.研究发现,与对照组相比,CCI模型组大鼠术后4 d损伤坐骨神经中NF的分布较非手术侧有明显变化,术后7、14 d电镜观察到损伤区中枢侧终球样结构,术后4、7、14、28 d CCI模型组大鼠坐骨神经中NF-H表达水平有动态变化(P<0.01).结果表明CCI大鼠模型损伤坐骨神经中NF-H其分布有明显变化,且分布于终球样结构中,其表达随损伤时间有动态变化,这些变化可能参与终球的形成以及与病理性疼痛机制相关联.     

钙结合蛋白CR在坐骨神经压榨模型脊髓内的表达

目的:研究大鼠坐骨神经压榨模型的钙结合蛋白Calretinin(CR)在脊髓的时空变化规律,为探讨其在神经再生中的作用提供实验依据。方法:36只SD大鼠随机分为假手术对照组和坐骨神经压榨组,实验组压榨后分别存活1d到21d,免疫组化结合图像分析技术观察CR在脊髓分布和含量的变化。结果:在对照组,CR样阳性神经元主要分布于腰髓背角Ⅰ,Ⅱ层,Ⅲ～Ⅵ层只观察到一些散在分布的CR样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见一些多极的中间型阳性神经元。坐骨神经压榨1d后,分布于腰髓背角Ⅱ层内的CR样阳性神经元比对照组有轻微增加。3d后,CR样阳性神经元与对照组相比没有明显改变。7d后,CR样阳性神经元有轻微的减少;14d后,CR的表达显著下降;至21d,CR的表达有所恢复,但仍低于7d组。脊髓后角CR免疫阳性产物灰度值测定结果显示:术后14d后角CR表达最低,与对侧和对照组相比有统计学意义(P<0.05)。结论:坐骨神经压榨后CR表达变化呈现一定的时空模式,为进一步揭示CR在神经系统疾病中的作用提供实验依据。     

背根神经节P2X3受体表达上调在糖尿病神经痛中的作用

目的本研究拟观察注射链脲佐菌素(streptozotocin,STZ)后大鼠不同时期背根神经节(dorsal root ganglion,DRG)上嘌呤受体(purinergic receptor subtype,P2X3)的表达情况及P2X3受体拮抗剂TNP-ATP对糖尿病神经痛(diabetic neuropathic pain,DNP)大鼠的干预作用。方法 (1) 20只健康雄性SD大鼠随机选取6只选取作为正常组,其余14只大鼠予以腹腔注射STZ,剔除未成模的2只大鼠,分别观察造模前(Base),造模后7 d、14 d、21 d的机械缩爪阈值(paw withdrawal threshold,PWT)变化情况;并在上述各时间点取大鼠L4-L6 DRG,采用免疫荧光法检测L4-L6 DRG上P2X3阳性细胞表达情况。(2)将25只健康雄性SD大鼠随机选取6只作为正常+生理盐水(Control+NS)组,其余19只予以STZ注射,剔除未成模大鼠1只,成功建立DNP的大鼠随机分为模型+生理盐水(DNP+NS)组,模型+50 nmol P2X3抑制剂TNP-ATP组(DNP+50 nmol TNP-ATP),模型+100 nmol P2X3抑制剂TNP-ATP组(DNP+100 nmol TNP-ATP),每组6只; STZ注射14 d后,DNP+TNP-ATP组分别按照上述剂量予以足背注射TNP-ATP溶液,其余两组分别予以注射等量NS,观察注射后0.5、1、1.5 h大鼠PWT变化。同时观察连续注射7 d药物对大鼠PWT的影响。结果 (1)与正常组比较,模型组大鼠第7天、第14天及第21天空腹血糖显著升高;与正常组比较,模型组大鼠Day 7 PWT无明显改变,第14天、第21天PWT显著降低。免疫荧光结果显示,与正常组相比,STZ注射7、14、21 d后,DNP大鼠L4、L5 DRG上P2X3的阳性细胞的表达显著升高,STZ注射14、21 d后,DNP大鼠L6 DRG上P2X3的阳性细胞的表达显著升高。(2) TNP-ATP干预前,DNP+NS组与DNP+50 nmol TNP-ATP组、DNP+100 nmol TNP-ATP组PWT无显著差异;干预0.5 h后,与DNP+NS组、DNP+50 nmol TNP-ATP组相比,DNP+100 nmol TNP-ATP组PWT明显升高,效果持续至1 h。(3)连续注射7 d TNP-ATP后,DNP+100 nmol TNP-ATP组PWT较DNP+NS组与DNP+50 nmol TNPATP组明显升高。结论腹腔注射STZ可成功建立DNP大鼠模型,背根神经节P2X3表达上调参与糖尿病神经痛的调节。     

SNAP-25在大鼠蛛网膜下腔出血后脑组织中的表达研究

目的:观察蛛网膜下腔出血(SAH)后突触小体相关蛋白(SNAP-25)在大鼠脑组织中的表达变化.方法:42只成年健康雄性SD大鼠,随机分为正常组、对照组、SAH后1、3、5、7、14天共7组,经大鼠自体股动脉(尾动脉)非肝素化动脉血注入视交叉池致蛛网膜下腔出血模型.蛛网膜下腔出血后1、3、5、7、14天后处死大鼠,取颞叶皮层、海马、小脑做标本,Western blot及免疫组化测定SNAP-25,采用SPSS16.0软件分析.结果:与正常组相比,对照组无明显差异(P＞0.05);SAH组中SAH后1天的SNAP-25蛋白水平降低明显(P＜0.05);SAH后3天蛋白水平达到最低值(P＜0.01);SAH后5、7天的SNAP-2蛋白水平虽降低但呈逐渐上升趋势(P＜0.05);SAH后14天的SNAP-2蛋白水平与正常组相比无明显差异(P＞0.05).结论:SNAP-2在SAH后72小时明显下降,后逐渐上升,至14天时基本恢复正常.     

Stimulation of the rat's sciatic nerve regeneration by local treatment with Xymedon

1. The possibility of a neuro-protective effect of Xymedon as a pharmacological stimulator of nerve regeneration has been studied through Schwann cells (SCs) located in the potential area of regenerating nerve fibers' growth. 2. Xymedon was injected into the silicone chamber connecting the central and peripheral stumps of the rat's sciatic nerve. Carboxymethyl cellulose was used as a depositioned medium. 3. A 0.95% concentration of Xymedon increased the sciatic nerve functional index (SFI) values on the 14th, 21st and 28th day after the operation. By day 30, the total number of survival neurons in the L5 dorsal root ganglion (DRG) on the ipsilateral side increased with the following changes in Xymedon concentration: [see text] The number of surviving sensory neurons in the group with 0.95% Xymedon increased by 36% (p < 0.05) compared with animals with depositioned medium but Xymedon free. 4. It is suggested that the positive effects of Xymedon on neural regeneration and recovery of motor function support the potential use of Xymedon for the treatment of peripheral nerve injuries.     

Experimental Study of Low Dose Ultrashortwave Promoting Nerve Regeneration after Acellular Nerve Allografts Repairing the Sciatic Nerve Gap of Rats

Objectives To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. Methods Sixteen Wistar rats weighing 180–220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. Results Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. Conclusions USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.     

Role of Thy-1 in in vivo and in vitro neural development and regeneration of dorsal root ganglionic neurons

We have examined the expression of Thy-1, an abundant glycosylphosphatidylinositol (GPI)-anchored glycoprotein, in dorsal root ganglia (DRG) and associated nerve fascicles, during postnatal development and following a nerve crush. The expression levels of Thy-1 in DRG neurons, dorsal roots, and central processes in spinal cord were rather low at postnatal day 2, and gradually increased as DRG neurons matured. During early development, the expression of Thy-1 within DRG neurons was low and equally distributed between plasma membrane and cytosol. With maturation, the staining intensities of Thy-1 in both the plasma membrane and the cytosol of DRG neurons became increased. We also studied Thy-1 expression in the regeneration of mature DRG neurons following the crush injury of sciatic nerve. Two days after the crush injury, Thy-1 expression dramatically decreased in the DRG neurons on the lesion side. Between 4 and 7 days after the injury, the expression of Thy-1 gradually increased and returned to a normal level 1 week after the sciatic nerve crush. The time course of the up-regulation of Thy-1 expression during regeneration matched that of the recovery of sensory functions, such as pain withdraw reflex, placing reflex, and the score of Basso-Beattie-Bresnahan Locomotor Rating Scale. Taken together, our results suggest that Thy-1 expression is developmentally regulated and is closely associated with the functional maturation of DRG neurons during both postnatal development and nerve regeneration. Furthermore, perturbation of Thy-1 function with anti-Thy-1 antibodies promoted neurite outgrowth from primary cultured DRG neurons, again confirming the inhibitory role of Thy-1 on neurite outgrowth.     

Altered Expression of Inducible Nitric Oxide Synthase After Sciatic Nerve Injury in Rat

Nitric oxide is known to contribute to neuronal damage as well as to peripheral neuronal regeneration following injury. Sciatic nerve injury is a common and serious complication of intramuscular injections. In order to ascertain the role of inducible nitric oxide synthase (iNOS) in the injured sciatic nerve, we studied the expression of this enzyme by RT-PCR and immunohistochemistry, in a rat model of sciatic nerve injury. In sham-operated control rats iNOS expression was undetectable by immunohistochemistry and its mRNA level was also very low. In contrast, in the experimental group that was subjected to sciatic nerve injury, both mRNA and protein of iNOS were found to be significantly elevated. The protein level of iNOS, as revealed by positive immunostaining, peaked at 7 days post-surgery followed by a decrease. Similarly, the iNOS mRNA levels remained elevated at 1, 3, 7 days but declined to very low level by day 21, after surgery. This study indicates that the increased expression of iNOS after sciatic nerve injury in rats may contribute to nerve regeneration. Thus our results suggest that excessive expression of iNOS after nerve injury is not conducive to nerve regeneration.     

髓核突出模型大鼠背根神经节中Wnt5a的表达

目的:观察Wnt5a在大鼠髓核突出模型的表达变化,探索Wnt5a在神经根病中的作用。方法:将80只成年雄性大鼠随机分为假手术组(Sham组,n=20)、髓核突出组(NP组,n=20)、髓核突出+生理盐水组(NP+Saline组,n=20)、髓核突出+益赛普组(NP+Etanercept组,n=20)。从大鼠尾椎椎间盘中取髓核,将髓核种植于L5背根神经节旁,制作髓核突出大鼠模型。采用免疫组织化学法分别检测各组大鼠背根神经节中的Wnt5a蛋白的表达,利用RT-PCR的方法分别检测各组大鼠背根神经节中Wnt5a m RNA的表达。结果:免疫组化结果显示,各组大鼠背根神经节的大中小神经元均有Wnt5a的表达,NP组较Sham组在3天、7天时Wnt5a表达量均增加(P0.01),3天和7天时NP+Etanercept组大鼠背根神经节中Wnt5a的表达量较NP+Saline组均减少(P0.01)。RT-PCR的结果与免疫组化的结果相符,表现为NP组较Sham组在3天、7天时Wnt5a mRNA的相对表达量增加(P0.01),NP+Etanercept组大鼠背根神经节中Wnt5a的相对表达量较NP+Saline组均减少(P0.01)。结论:髓核突出并压迫背根神节时,可引起背根神经节Wnt5a的表达增加,且阻断肿瘤坏死因子α(TNF-α)时,背根神经节的Wnt5a表达减少。     

HCN2在大鼠外周神经病理性疼痛发生中的作用

目的：初步探讨超极化激活的环核苷酸门控通道2型（HCN2）在外周神经病理性疼痛发生中的作用。方法：将24只健康成年大鼠进行随机分组（n=12）：假手术组（Sham）大鼠仅分离左侧L4、L5脊神经,模型组（SNL）分离脊神经后进行相应的结扎处理,手术7 d后用行为学方法进行模型评价;将造模成功的大鼠进行随机分组（n=6）：①阴性对照组（Saline）,左侧足底注射生理盐水;②阳性对照组（GBPT）,腹腔注射加巴喷丁;③实验组（ZD7288）,左侧足底注射HCN非特异性阻断剂ZD7288。在给药前以及给药后1 h、4 h、24 h、48 h用疼痛行为学实验检测其对神经病理性疼痛的作用;分别取手术前对照组（Control）、假手术组（Sham）和模型组（SNL）大鼠的背根神经节（DRG）（n=6）,利用qPCR和Western blot的方法研究造模前后大鼠DRG内HCN2的表达的变化情况。结果：①成功建立大鼠神经痛模型;②与Saline组比较,GBPT组和ZD7288组在注射1 h后,均能明显的减轻大鼠神经病理性疼痛的症状（P<0.01）,而GBPT组和ZD7288组之间比较则无差异;③与Control组和Sham组相比较,SNL组大鼠DRG内的HCN2 mRNA表达量明显增加（P<0.01）;与Control组和Sham组相比较,SNL组大鼠DRG内的HCN2通道蛋白表达量显著增加（P<0.05）。结论：HCN2参与外周神经病理性疼痛的发生,并有可能成为治疗神经病理性疼痛一个潜在的新靶点。     

Enhancement of nerve regeneration and orientation across a gap with a nerve graft within a vein conduit graft: a functional, stereological, and electrophysiological study

In this study, the right sciatic nerves of 40 rats were used to determine whether a nerve graft within a vein graft might accelerate and facilitate axonal regeneration, compared with a nerve graft alone. The animals were separated into four groups, as follows: group 1, sham control; group 2 (control), segmental nerve resection and no repair; group 3, segmental nerve resection and nerve grafting; group 4, segmental nerve resection and reconstruction with a nerve graft within a vein conduit graft. For all groups, sciatic functional indices were calculated before the operation and on postoperative days 7 and 90. On postoperative day 90, the sciatic nerves were reexposed and nerve conduction velocities were recorded. The sciatic nerves were harvested from all groups for counting of the myelinated axons with a stereological method. No statistically significant differences with respect to return of gait function, axon count, or nerve conduction were noted between groups 3 and 4 (p > 0.05). However, functional recovery in group 4 on postoperative day 90 was significant, compared with group 2 (p < 0.05); the recovery difference between groups 2 and 3 was not significant (p > 0.05). This study was not able to demonstrate any functional benefits with the use of a nerve graft within a vein graft, compared with standard nerve grafting.