神经降压素对大鼠脊髓背角胶状质内突触前神经递质释放的影响

目的:研究内源性神经肽神经降压素(neurotensin,NT)在脊髓背角胶状质(substantia gelatinosa,SG)内对突触前神经递质释放的影响。方法:采用全细胞电压膜片钳记录方法,在脊髓薄片上观察NT对SG内微小兴奋性突触后电流(mEPSCs)和微小抑制性突触后电流(mIPSCs)的频率和幅值的影响。结果:(1)灌流NT(2μmol/L)对SG内神经元mEPSCs的频率和幅值均无明显影响,说明NT不影响SG内兴奋性神经递质的释放;(2)灌流NT(2μmol/L)能增加SG内神经元mIPSCs的频率,但对幅值无明显影响,即NT可引起突触前抑制性神经递质的释放增加,但对突触后神经元无明显影响。结论:NT可通过增加SG内抑制性神经递质释放的途径抑制伤害性信息的传递,从而实现镇痛效应。     

氯胺酮在大鼠脊髓背角胶状质内对突触前神经递质释放的影响

为探讨临床有效浓度的氯胺酮(ketamine,KTM)在脊髓背角胶状质(substansia gelatinosa,SG)内对突触前神经递质释放的影响及其作用机制,本研究应用红外可视神经组织薄片全细胞膜片钳记录方法,在电压钳模式下,观察了KTM对自发性抑制性和兴奋性突触后电流(spontaneous inhibitory and excitatory postsynaptic currents,sIPSCs and sEPSCs)的频率和幅值的影响。结果显示:(1)钳制电压在0mV时,在人工脑脊液(artificial cerebrospinal fluid,ACSF)中加入10-5mol/LAP-V和10-6mol/LCNQX,可记录到sIPSCs。将此时记录到的频率和幅值都作为前对照组的基础值(100%)。给予10-4mol/LKTM后,与前对照组相比,sIPSCs频率为127.93%±25.17%(P<0.05),幅值为104.78%±11.35%(P>0.05,n=7);(2)钳制电压为-70mV时,在ACSF中加入3×10-7mol/L士的宁和10-6mol/L荷包牡丹碱后,可观察到sEPSCs。加入10-4mol/LKTM后,与前对照组相比,sEPSCs的频率和幅值分别为97.89%±4.06%和101.63%±7.66%(P>0.05,n=8)。以上结果提示:(1)KTM增加了sIPSCs的频率,而对幅值没有明显影响,即KTM引起突触前抑制性神经递质的释放增加,而对突触后神经元的作用不明显;(2)KTM对sEPSCs的频率和幅值均未见明显影响,说明KTM在SG内对兴奋性神经递质的释放无显著影响。由此我们推测KTM在脊髓SG内主要通过增强抑制性信息传递发挥作用,KTM增强SG内突触前抑制性神经递质释放可能与其在脊髓背角发挥麻醉和镇痛作用有关。     

内源性大麻素配体NADA在大鼠脊髓背角的镇痛作用

目的:探讨内源性大麻素配体NADA对脊髓背角II层胶状质(SG)神经元突触兴奋性的影响及其在神经病理性痛模型上的镇痛作用。方法:利用膜片钳技术,观察NADA(40μmol/L)对SG神经元自发性兴奋性突触后电流(sEPSC)及背根诱发兴奋性突触后电流(eEPSC)的影响。制备脊神经结扎(SNL)模型,观察鞘内注射20μgNADA对机械性缩足反射阈值(PWMT)的影响。结果:通过作用于CB1受体,NADA可以显著抑制由Aδ纤维及C纤维介导单突触eEPSC的幅值,并且显著减低sEPSC频率而对其幅度无改变。在神经病理性痛模型中,与对照侧相比,NADA可以显著增加手术侧机械缩足反射阈值(P0.0001),而溶剂组则无统计学意义(P0.05)。结论:内源性大麻素配体NADA能够抑制脊髓背角浅层Aδ纤维及C纤维介导的突触传递,并且可以减轻实验动物的神经病理性疼痛。     

大鼠中央杏仁核神经元对引发晕动病的旋转运动刺激的电生理学反应特性

目的:研究引起晕动病发生的双轴旋转运动刺激前庭感受器后,中央杏仁核神经元电学反应的变化,探讨中央杏仁核在晕动病(MS)发病中的作用。方法:10只幼年SD大鼠分成两组:对照组和旋转刺激组。旋转刺激组动物经2 h双轴旋转运动刺激(大、小转盘分别以168°/s和432°/s的角速度旋转)。取脑、切片(300μm),应用脑片膜片钳全细胞记录技术记录中央杏仁核神经元膜的主动反应特性以及自发的兴奋性突触后电流(sE PSCs)变化,统计分析、比较两组间的差异。结果:旋转运动刺激后,中央杏仁核神经元电学特性,如膜电位水平(对照组为(-62.80±2.69)mV,旋转组为(-61.37±1.50)mV)以及动作电位(AP)形态未发生显著变化。AP的幅值、半幅时程、最大上升/下降斜率、刺激阈电位和基强度等在两组间没有显著性差别;旋转刺激后,sE PSCs频率从正常组的(0.88±0.42)Hz增加到旋转组的(1.69±0.30)Hz(P<0.05)而幅值(正常组(20.07±3.01)pA;旋转组(21.03±1.44)pA)未发生变化。结论:引发晕动病的运动刺激可引起中央杏仁核神经元兴奋性突触活动增加,突触前谷氨酸释放增加;中央杏仁核参与晕动病反应。     

大鼠睁眼前初级视皮层2/3层锥体神经元突触自身稳态可塑性的变化特征

目的：通过对Wistar大鼠睁眼前初级视皮层2/3层锥体神经元突触AMPA（α-氨基-3-羧基-5-甲基异恶唑-4-丙酸）介导的微小兴奋性突触后电流 (mEPSCs)的测定分析,研究突触自身稳态可塑性在生后早期初级视皮层的作用特点。方法: 采用红外可视膜片钳技术全细胞模式记录生后4-11(d)（P4-11）Wistar大鼠初级视皮层脑片2/3层锥体神经元AMPA介导的mEPSCs,钳制电位-70 mV。人工脑脊液中加入河豚毒素（TTX）、荷包牡丹碱（BMI）及2-氨基-5-磷酸基戊酸（AP-5）分离出AMPA介导的mEPSCs,加入阻断剂6-氰基-7-硝基喹喔啉-2,3二酮 (CNQX）可消除mEPSCs。使用Clampfit 9.0进行数据分析。结果: P4至P11,大鼠初级视皮层2/3层锥体神经元AMPA介导的mEPSCs的波幅呈现上升趋势,频率自P7至P11逐渐增加,上升时间常数及下降时间常数均呈缩短趋势,以下降时间常数变化为著。P4至P7可见“单通道样”电流形态。结论: 在大鼠睁眼前初级视皮层2/3层锥体神经元亦存在突触自身稳态可塑性调节机制,其作用特点不同于睁眼后。     

体视学研究坐骨神经慢性限制性损伤对大鼠脊髓背角内突触数量的可塑性改变及COX-2抑制剂的作用

目的:探讨坐骨神经慢性限制性损伤(CCI)所致神经病理性疼痛是否伴有脊髓背角神经元和突触数量的可塑性变化以及帕瑞昔布干预的作用.方法:正常成年SD大鼠随机分为假手术组、CCI组及帕瑞昔布组.术后28d取第5腰段脊髓作石蜡包埋切片,分别用尼氏染色和突触素的免疫组织化学显色显示神经元和突触,采用体视学新技术--光学体视框估计脊髓背角内神经元和突触的数量.结果:与对侧未手术侧相比,CCI组手术侧单位长度脊髓背角内的突触数及突触数与神经元数之比分别增加了86%、98%;帕瑞昔布组手术侧单位长度脊髓背角内的突触数及突触数与神经元数之比分别增加了78%、68%.与假手术组手术侧相比,CCI组手术侧单位长度脊髓背角内的突触数及突触数与神经元数之比分别增加了78%、73%;帕瑞昔布组则分别增加了81%、71%.结论:CCI所致神经病理性疼痛伴有脊髓背角内突触数量增加的可塑性变化,COX-2抑制剂帕瑞昔布对CCI致突触数量的增加无作用.     

鞘内注射丹参酮ⅡA对神经病理性疼痛大鼠的镇痛作用

目的:研究丹参酮ⅡA对坐骨神经慢性压迫大鼠的的镇痛效果及大鼠脊髓背角内半胱氨酸天冬氨酸蛋白酶3(caspase-3)和胶质纤维酸性蛋白(GFAP)表达的影响.方法:检测大鼠在手术前及术后14d的机械痛阈和热痛阈;PCR及免疫组织化学分别检测大鼠脊髓背角内caspase-3和GFAP基因及蛋白的表达,TUNEL法检测脊髓背角内的细胞凋亡.结果:与假手术组比较,模型组大鼠的机械痛阈和热痛阈明显降低,caspase3和GFAP的表达均增多,凋亡染色阳性细胞数目也增多;与模型组比较,丹参酮ⅡA处理组大鼠的机械痛阈和热痛阈明显升高,脊髓背角内caspase-3和GFAP的表达均下降,凋亡染色阳性细胞数目也减少,差异均有统计学意义.结论:鞘内注射丹参酮ⅡA对坐骨神经慢性压迫模型大鼠有镇痛作用,其机制可能与降低脊髓背角内caspase-3和GFAP的表达有关.     

神经病理性痛大鼠脊髓背角缝隙连接蛋白表达变化及鞘内注射甘珀酸的镇痛作用

目的 研究慢性神经病理性痛大鼠脊髓背角内缝隙连接蛋白家族(Cx)中Cx43和Cx32的表达变化,以及鞘内注射缝隙连接阻断剂甘珀酸（CBX）的镇痛作用。 方法 成年SD大鼠50只,分为正常对照组、假手术组和坐骨神经分支选择性损伤组(SNI)。手术前1d、术后3d、5d、10d、20d和30d,观察大鼠行为并检测机械刺激缩足反射阈值(PWMT)。15只大鼠于术后10d、20d、30d取脊髓腰段进行免疫印迹检测,另15只大鼠于术后10d、20d、30d取脊髓腰段进行免疫荧光染色,检测腰段脊髓背角内Cx43和Cx32表达的变化。有10只大鼠先进行鞘内插管,后行SNI手术,术后20d向鞘内注射生理盐水或CBX,观察大鼠PWMT的变化。 结果 SNI大鼠手术侧PWMT阈值较非手术侧或假手术组明显降低,术后20d达最低值。SNI大鼠手术侧脊髓背角内Cx43、32表达增多,明显高于非手术侧和假手术组背角。鞘内注射CBX 3h后,PWMT平均阈值由(2.5±1.0)g上升到(20.0±3.2)g,有抑制效应, 而生理盐水组则无抑制效应。 结论 脊髓背角内的缝隙连接在因外周神经损伤引起的神经病理性痛中起重要作用。     

吗啡对坐骨神经钳夹伤大鼠脊髓Ⅱ板层初级传入纤维终末突触重建的影响

目的 探讨吗啡能否促进大鼠受损伤的初级传人纤维终末在脊髓Ⅱ板层再生及其突触重建。方法 采用电镜定量方法计数吗啡组、生理盐水组和纳络酮吗啡组坐骨神经损伤后15d和30d,L4脊髓Ⅱ板层背根无髓传人纤维来源的Ⅰ型复合终末、背根有髓传人纤维来源的Ⅱ型复合终末和由中间神经元轴突和下行传导束轴突形成的简单终末。结果 吗啡组大鼠脊髓Ⅱ板层的Ⅰ型复合终末数比生理盐水组和纳络酮吗啡组明显增多,然而,Ⅱ型复合终末却比生理盐水组和纳络酮吗啡组少。3组大鼠脊髓Ⅱ板层的突触性终末总数,复合终末数和简单终末数没有差异。结论 吗啡能够促进大鼠脊髓Ⅱ板层受损伤的初级无髓传人纤维终末再生及其突触重建,这可能通过阿片受体介导。     

内质网应激蛋白BiP在姜黄素抗2型糖尿病神经病理性疼痛大鼠的作用

目的: 探讨姜黄素对2型糖尿病诱导的神经病理性疼痛(DNP)大鼠机械痛敏(MWT)、热痛敏(TWL)、脊髓背角和背根神经节(DRG)的保护作用及机制。方法: 高脂高糖饲料喂养8周诱导胰岛素抵抗后,继以单次小剂量链脲佐菌素(STZ)35 mg/kg腹腔注射,在注射STZ前和14 d后采用Electronic Von Fery触觉测痛仪和甩尾/足底测试仪测大鼠MWT和TWL,注射STZ 3 d后血糖≥16.7 mmol/L且在注射STZ 14 d后痛阈下降至基础值85%以下者入选为D组(81只)。将D组随机分为3组(n=27): DNP组、DNP+姜黄素100 mg·kg^-1·d^-1组(DCur组)和DNP+溶剂组(DSC组),另取27只为正常对照组(C组),给予普通饲料喂养。给姜黄素3、7、14 d后测血糖、MWT与TWL,并在同时点取大鼠L4~L6脊髓和DRG,用免疫组化和免疫印迹法测定脊髓背角和DRG中免疫球蛋白重链结合蛋白(BiP)的表达。结果: 与C组相比,DNP组各时点MWT降低,TWL缩短,血糖值升高,脊髓背角和DRG中BiP均出现表达上调(P<0.05)。与DNP组相比,DCur组在给姜黄素7 d后MWT升高,TWL延长;给姜黄素14 d后脊髓背角和DRG中BiP表达下调(P<0.05)。DSC组和DNP组相比差异无统计学意义。结论: BiP参与了2型糖尿病神经病理性疼痛的发病机制。姜黄素减轻2型糖尿病大鼠神经病理性疼痛的机制可能与抑制BiP表达有关。     

Comparison of responses of warm and nociceptive C-fiber afferents in monkey with human judgments of thermal pain.

1. Radiant-heat stimuli of different intensities were delivered every 28 s to the thenar eminence of the hand of human subjects and to the receptive fields (RFs) of 58 "mechanothermal nociceptive" and 16 "warm" C-fibers, most of which innervated the glabrous skin of the monkey hand. A CO2 infrared laser under control via a radiometer provided a step increase in skin temperature to a level maintained within +/- 0.1 degrees C over a 7.5-mm-diameter spot. 2. Human subjects categorized the magnitude of warmth and pain sensations evoked by stimuli that ranged in temperature from 40 to 50 degrees C. The scale of subjective thermal intensity constructed from these category estimates showed a monotonically increasing relation between stimulus temperature and the magnitude of warmth and pain sensations. 3. The mechanothermal fibers had a mean RF size of 18.9 +/- 3.2 mm2 (SE), a mean conduction velocity of 0.8 +/- 0.1 m/s, mean thresholds of 43.6 +/- 0.6 degrees C for radiant heat and 5.95 +/- 0.59 bars for mechanical stimulation, and no spontaneous activity. In contrast, warm fibers had punctate RFs, a mean conduction velocity of 1.1 +/- 0.1 m/s, heat thresholds of less than 1 degrees C above skin temperature, no response to mechanical stimulation, and a resting level of activity in warm skin that was suppressed by cooling. 4. The cumulative number of impulses evoked during each stimulation in the nociceptive afferents increased monotonically as a function of stimulus temperature over the range described by humans as increasingly painful (45-50 degrees C). Nociceptive fibers showed little or no response to stimulus temperatures less than 45 degrees C that elicited in humans sensations primarily of warmth but not pain. In contrast, the cumulative impulse count during stimulation of each warm fiber increased monotonically with stimulus temperature over the range of 39-43 degrees C. However, for stimuli of 41-49 degrees C the cumulative impulse count in warm fibers was nonmonotonic with stimulus temperature. Warm-fiber response to stimuli of 45 degrees C or greater usually consisted of a short burst of impulses followed by cessation of activity. 5. The subjective magnitude of warmth and pain sensations in humans and the cumulative impulse count evoked by each stimulus in warm and nociceptive afferents varied inversely with the number, delivery rate, and intensity of preceding stimulations. 6. The results of these experiments suggest the following: a) that activity in the mechanothermal nociceptive C-fibers signals the occurrence of pain evoked by radiant heat, and that the frequency of discharge in these fibers may encode the intensity of painful stimulation; b) that activity in warm fibers may encode the intensity of warmth at lower stimulus temperatures, but is unlikely to provide a peripheral mechanism for encoding the intensity of painful stimulation at higher stimulus temperatures.     

蛋白酶活化受体4在外周痛觉信号调节作用的研究进展

蛋白酶活化受体4(PAR4)是G蛋白偶联受体家族成员之一,可被凝血酶、胰蛋白酶活化。PAR4是血小板活化和炎症的强力调节受体,活化的PAR4可作为潜在的内源性镇痛因子,在正常和炎症条件下参与调节外周疼痛反应。PAR4通过致敏TRPV1、细胞内Ca2+的释放和刺激周围感觉神经末梢释放P物质和CGRP参与了炎症和疼痛的周围机制。PAR4直接与电压门控Ca2+通道的相互作用可能是PAR4对细胞除极诱发的Ca2+信号产生抑制作用和PAR4参与镇痛的作用机制。     

Activation of 5-hydroxytryptamine type 3 receptor-expressing C-fiber vagal afferents inhibits retrotrapezoid nucleus chemoreceptors in rats

Retrotrapezoid nucleus (RTN) chemoreceptors are regulated by inputs from the carotid bodies (CB) and from pulmonary mechanoreceptors. Here we tested whether RTN neurons are influenced by 5-hydroxytryptamine type 3 receptor-expressing C-fiber vagal afferents. In urethan-anesthetized rats, selective activation of vagal C-fiber afferents by phenylbiguanide (PBG) eliminated the phrenic nerve discharge (PND) and inhibited RTN neurons (n = 24). PBG had no inhibitory effect in vagotomized rats. Muscimol injection into the solitary tract nucleus, commissural part, reduced inhibition of PND and RTN by PBG (73%), blocked activation of PND and RTN by CB stimulation (cyanide) but had no effect on inhibition of PND and RTN by lung inflation. Bilateral injections of muscimol into interstitial solitary tract nucleus (NTS) reduced the inhibition of PND and RTN by PBG (53%), blocked the inhibitory effects of lung inflation but did not change the activation of PND and RTN neurons by CB stimulation. PBG and lung inflation activated postinspiratory neurons located within the rostral ventral respiratory group (rVRG) and inhibited inspiratory and expiratory neurons. Bilateral injections of muscimol into rVRG eliminated PND and partially decreased RTN neuron inhibition by PBG (32%). In conclusion, activation of cardiopulmonary C-fiber afferents inhibits the activity of RTN chemoreceptors. The pathway relays within a broad medial region of the NTS and involves the rVRG to a limited degree. The apnea triggered by activation of cardiopulmonary C-fiber afferents may be due in part to a reduction of the activity of RTN chemoreceptors.     

Responses of bronchial C-fiber afferents of the rabbit to changes in lung compliance

Rapidly adapting receptors (RAR) in the lung are stimulated when the lung compliance is reduced. The present investigation was undertaken to determine whether bronchial C-fibers are also activated when lung compliance is decreased since both RAR and bronchial C-fibers are influenced by extra-vascular fluid in the airways. Action potentials (AP) were recorded from bronchial C, pulmonary C, RAR and slowly adapting receptor (SAR) afferents in the cervical vagus in open chest New Zealand White rabbits ventilated at a constant rate and tidal volume. AP were recorded during (a) positive end-expiratory pressure (PEEP) of 2-3 cmH2O (control), (b) zero end-expiratory pressure (ZEEP), (c) negative end-expiratory pressure (NEEP) of -4 cmH2O, (d) restoration of PEEP and (e) final control after hyper-inflation. Both RAR and bronchial C-fiber activity increased significantly compared with control when lung compliance was decreased (bronchial C-fibers: 35 +/- 5 vs. 66 +/- 13 impulses per 30 sec and RAR: 3 +/- 1 vs. 94 +/- 14 impulses per 30 sec).     

Cyclophosphamide-induced cystitis in rats: Involvement of capsaicin-sensitive primary afferents

The involvement of capsaicin-sensitive primary afferent neurons has been investigated in a rat model of cyclophosphamide (CYP)-induced cystitis. CYP (150 mg/kg i.p., 48 h before) was administered in both vehicle- and capsaicin-(50 mg/kg s.c., 4 days before)-treated rats. Some experiments were performed 96 h after bilateral removal of pelvic ganglia (bladder denervation). CYP produced a marked detrusor hyperreflexia which was abolished by capsaicin pretreatment. CYP produced a marked increase in bladder weight and plasma protein extravasation (PPE, measured by Evans blue leakage technique): CYP-induced PPE was reduced by bladder denervation and was aggravated by capsaicin pretreatment. PPE aggravation by capsaicin was abolished by ganglionectomy. The bladder content of calcitonin gene-related peptide, was unaffected CYP. We conclude that CYP-induced decrease in bladder capacity is entirely mediated through stimulation of capsaicin-sensitive bladder afferents. At the time chosen as end point in these experiments, capsaicin-sensitive afferents exert an antiinflammatory influence on CYP-induced cystitis.     

5-HT acts on nociceptive primary afferents through an indirect mechanism to induce hyperalgesia in the subcutaneous tissue

We have recently demonstrated that s.c.-injected 5-hydroxytryptamine (5-HT) induces nociception by an indirect action on the primary afferent nociceptor in addition to its previously described direct action. Although the mechanisms mediating hyperalgesia can be quite separate and distinct from those mediating nociception, the aim of this study was to test the hypothesis that 5-HT induces mechanical hyperalgesia by mechanisms similar to those mediating nociception. s.c. injection of 5-HT induced a dose-dependent mechanical hyperalgesia measured by the mechanical paw withdrawal nociceptive threshold test in the rat. 5-HT-induced hyperalgesia was significantly reduced by local blockade of the 5-HT(3) receptor by tropisetron, by the nonspecific selectin inhibitor fucoidan, by the cyclooxygenase inhibitor indomethacin, by guanethidine depletion of norepinephrine in the sympathetic terminals, and by local blockade of the beta(1)- or beta(2)-adrenergic receptor by atenolol or ICI 118,551, respectively. Taken together, these findings indicate that like nociception, hyperalgesia induced by the injection of 5-HT in the s.c. tissue is also mediated by an indirect action of 5-HT on the primary afferent nociceptor. This indirect hyperalgesic action of 5-HT is mediated by a combination of mechanisms involved in inflammation such as neutrophil migration and the local release of prostaglandin and norepinephrine. However, in contrast to nociception, hyperalgesia induced by 5-HT in the s.c. tissue is mediated by a norepinephrine-dependent mechanism that involves the activation of peripheral beta(2) adrenoceptors.     

Eseroline depresses the responses of dorsal horn neurons to C-fiber afferents in the spinal rat

Eseroline not only has some structural features in common with morphine but also has a specific antinociceptive action like opioid drugs. The effects of eseroline on the responses of rat dorsal horn lamina V neurons to C-fiber-related noxious stimuli were investigated. The data obtained showed that 15 min after eseroline administration, the neuronal responses to C-fiber-related afferents were almost totally suppressed. Morphine was used as reference drug. The postulated action of eseroline on opioid receptors was confirmed by reversal of eseroline-driven cell activity after naloxone injection.     

Bidirectional synaptic plasticity at nociceptive afferents in the rat central amygdala

Glutamatergic inputs arising from the parabrachial nucleus to neurons in the lateral sector of the central amygdala were studied in vitro . Tetanic stimulation of these inputs led to LTP that did not require activation of NMDA receptors or a rise of postsynaptic calcium. LTP was accompanied by a reduction in the paired-pulse ratio, indicating that LTP results from an increase in transmitter release probability. Activation of adenylyl cyclase with forskolin potentiated these inputs with a similar reduction in paired-pulse facilitation and occluded LTP induction. LTP was inhibited by the protein kinase A blocker H89. Low-frequency stimulation led to LTD that required activation of postsynaptic NMDA receptors and a rise in postsynaptic calcium. There was no change in paired-pulse facilitation with LTD. LTD was blocked by protein phosphatase blockers calyculin and okadaic acid. We conclude that parabrachial inputs to the lateral sector of the central amygdala show presynaptic LTP that requires activation of a presynaptic protein kinase A via a calcium-dependent adenylyl cyclase while LTD at the same synapses is postsynaptic and requires a rise in postsynaptic calcium and activation of protein phosphatase.