The Double Projecting Neuron of the Cat Spinal Dorsal Horn——A Study with Double Retrograde Fluorescent Tracing Technique

The new double projecting neurons were found in the cat spinal dorsal horn by the double retrograde fluorescent tracing technique.Fast Blue(FB)was injected into unilateral dorsal column nucleus(DCN)of adult cats anesthetized with pentobarbital.Nuclear Yellow(NY)was injected ipsilaterally into the lateral cervical nucleus(LCN)8-9 days later.After an additional 18-30 hrs.

The mechanisms underlying long-term potentiation of C-fiber evoked field potentials in spinal dorsal horn

Long-term potentiation(LTP) of C-fiber evoked field potentials in spinal dorsal horn is first reported in 1995.Since then,the mechanisms underlying the long-lasting enhancement in synaptic transmission between primary afferent C-fibers and neurons in spinal dorsal horn have been investigated by different laboratories.In this article,the related data were summarized and discussed.

High-frequency spinal cord stimulation produces longlasting analgesic effects by restoring lysosomal function and autophagic flux in the spinal dorsal horn

High-frequency spinal cord stimulation(HF-SCS) has been established as an effective therapy for neuropathic pain. However, the analgesic mechanisms involved in HF-SCS remain to be clarified. In our study, adult rat neuropathic pain was induced by spinal nerve ligation. Two days after modeling, the rats were subjected to 4 hours of HF-SCS(motor threshold 50%, frequency 10,000 Hz, and pulse width 0.024 ms) in the dorsal horn of the spinal cord. The results revealed that the tactile allodynia of spinal nerve-injured rats was markedly alleviated by HFSCS, and the effects were sustained for 3 hours after the stimulation had ceased. HF-SCS restored lysosomal function, increased the levels of lysosome-associated membrane protein 2(LAMP2) and the mature form of cathepsin D(matu-CTSD), and alleviated the abnormally elevated levels of microtubule-associated protein 1 A/B-light chain 3(LC3)-II and sequestosome 1(P62) in spinal nerve-injured rats. HF-SCS also mostly restored the immunoreactivity of LAMP2, which was localized in neurons in the superficial layers of the spinal dorsal horn in spinal nerve-injured rats. In addition, intraperitoneal administration of 15 mg/kg chloroquine for 60 minutes reversed the expression of the aforementioned proteins and shortened the timing of the analgesic effects of HF-SCS. These findings suggest that HF-SCS may exhibit longlasting analgesic effects on neuropathic pain in rats through improving lysosomal dysfunction and alleviating autophagic flux. This study was approved by the Laboratory Animal Ethics Committee of China Medical University, Shenyang, China(approval No. 2017 PS196 K) on March 1, 2017.

Effect of pre-electroacupuncture on p38 and c-Fos expression in the spinal dorsal horn of rats suffering from visceral pain

Background Acupuncture is an effective way to relieve pain, but the mechanism by which electroacupuncture （EA） decreases the visceral pain state still remains unclear. This study aimed to evaluate the effects of pre-electroacupuncture on pain behaviors, p38 phosphorylation, and c-Fos protein and mRNA expression in both the colonic wall and spinal dorsal horn of rats suffering from visceral pain. This study also investigated the probable signaling regulatory mechanism of the analgesic effect induced by electroacupuncture. Methods All rats were randomized into the control （Con） group, the Con＋EA group, the visceral pain （VP） group, and VP＋EA group （n=8 for all groups）. The visceral pain model was established using 40 ul of 5% formalin solution injected into the colon of rats. EA was applied to the bilateral Jiaji acupoints for 20 minutes before application of visceral pain. Parameters for EA were set at a continuous wave （20 Hz） and intensity where the rats shook their whiskers but did not scrabble （≤1 mA）. The visceral pain score was recorded and the expressions of p38 and c-Fos protein were detected using Western blotting. Real-time quantitative PCR was also used to determine the expression of c-Fos mRNA. Results Rats in the VP group immediately presented with obvious visceral pain behaviors after being injected with formalin. p38 activity and c-Fos protein and mRNA expression in both the colonic wall and spinal dorsal horn were higher in the VP group than in the Con group （P 〈0.05）. By contrast, visceral pain behaviors were delayed in rats from the VP＋EA group. p38 activity and c-Fos protein and mRNA expression were lower in the VP＋EA group than that in the VP group （P〈0.01）. Conclusions Pre-electroacupuncture of the Jiaji acupoint has prophylactic analgesic effects on rats suffering from visceral pain. The p38 signal transduction pathway may be partly involved in the regulatory mechanism of this analgesic effect.

Low-Frequency Electroacupuncture Alleviates Chronic Constrictive Injury-Induced Mechanical Allodynia by Inhibiting NR2B Upregulation in Ipsilateral Spinal Dorsal Horn in Rats

Objective: To study the effects of electroacupuncture(EA) in chronic constrictive injury(CCI) rat model and the expression of N-methyl-D-aspartate receptor type 2B(NR2B) in ipsilateral spinal dorsal horn in rats to explore the analgesic mechanisms of EA. Methods: According to the random number table, totally 180 rats were evenly divided into a sham group, a CCI group, and an EA group. CCI model was conducted with four4–0 chromic gut ligatures loosely ligated around the left sciatic nerve 1 cm above the trifurcation. Rats in the EA group received 2 Hz EA therapy bilaterally at acupoints of Zusanli(ST 36) and Sanyinjiao(SP 6) once daily(30 min/d) for 30 days after surgery. Paw withdrawal thresholds(PWTs) were measured on 0(baseline), 1, 3, 7, 15,30 days after surgery. Rats were sacri?ced on 0, 1, 3, 7, 15 and 30 days after surgery, and the L4–5 segments of spinal cord were removed to detect the expression of NR2B by immunohistochemistry and quantitative polymerase chain reaction. Results: PWTs in the CCI group were signi?cantly lower than the sham group at Day1–30 after surgery, and reached its lowest at Day 1(P<0.01). After EA treatment, the PWTs recovered rapidly and were signi?cantly higher than those in the CCI group on 3, 7, 15 and 30 days after surgery(P<0.01). The numbers of NR2B-immunoreactive cells of the CCI group signi?cantly increased after CCI surgery compared with the sham group(P<0.01). Compared with the CCI group, stimulation of EA markedly decreased the numbers of NR2B-immunoreactive cells at Day 3, 7, 15 and 30(P<0.05). In the sham group, NR2B mRNA was expressed at a low level. It increased after CCI surgery, which increased rapidly at Day 7(P<0.01) and reached its peak value at Day 15(P<0.01). After EA stimulation, relative quantity of NR2B mRNA expression was less than that in the CCI group at Day 15 and 30(P<0.05). Conclusions: Low frequency of EA had antinociceptive effect in CCI rat model. The analgesic effects of EA might be through the inhibition of NR2B.

THE PROJECTION LINKAGE BETWEEN THE SPINAL DORSAL HORN NEURONS AND BOTH THE SOLITARY TRACT AND DORSAL COLUMN NUCLEI

Electrical stimulation of the solitary tract nucleus (SN) and dorsal column nuclei (DCN)as well as microelectrode recording from the lumbal spinal dorsal horn have been used tofind and identify the axonal projection of and the afferent innervation on the spinal neuronsof pentobarbital- anesthetized rats. A total of 92 neurons was recorded and identified mainly in laminae Ⅲ-Ⅴ of the lumbarspinal dorsal horn. Of them, 38 neurons were activated antidromically from stimulation ofboth the SN and DCN. The other 54 neurons responded synaptically to both the SN andDCN stimulations. The initial antidromic responses of 8 neurons in the first group werefollowed by one or more responses synaptically driven from the SN and/or DCN stimulation.Conduction velocities were in the range of A delta fibers, but faster in the antidromicresponses and slower in the synaptic responses. These results indicate that (i) some spinal neurons issue branched axons of larger-sizedA delta fibers and double project to both the SN and DCN; (ii) some of these doubleprojection neurons receive in turn smaller A delta fiber innervation from the SN and/orDCN; and (iii) some other neurons in the spinal cord are dually innervated by smaller Adelta fibers originating from both nuclei.

The Functional Linkage Among the "ZSL"-Spinal Dorsal Horn-SN

Electrical stimulation of the Zusanli point (ZSL) and solitary tract nucleus (SN) as well as microelectrode recording from the laminae Ⅲ-Ⅴ of the lumbar spinal dorsal horn have been used on the pentobarbital anesthetized rats, finding and identifying 57 spinal neurons responding to the stimulations of both ZSL and SN. Among them, 34 responded antidromically to SN; the others responded orthodromically to SN. Among them, the lowthreshold mechano-receptive(LTM) neurons and wide-dynamic-range (WDR) neurons were 50 percent respectively. The results indicate that (ⅰ) a single spinal dorsal horn neuron receives somatic afferent input and then conveys it to the visceral sensory nucleus-SN; (ⅱ) some spinal dorsal horn neurons receive, in turn, innervation from the SN; (ⅲ) the convergence and integration between somatic and visceral sensory inputs might occur in the spinal dorsal horn neurons and/or SN.

Reduction of GABA-and substance P-immunoreactivities in spinal dorsal horn by capsaicin

γ-aminobutyric acid (GABA)-containing neurons are densely located in laminae Ⅰ—Ⅱ of the spinal cord where substance P (SP)-containing primary afferent C fibers terminate. By the antibody microprobe technique, we found that capsaicin, a chemical irritant selectively activating C fibers, significantly produced the release of SP in lamina Ⅱ.

“Three Methods and Three Points” regulates p38 mitogen-activated protein kinase in the dorsal horn of the spinal cord in a rat model of sciatic nerve injury

Tuina is a traditional Chinese treatment for sensory disturbances caused by peripheral nerve injury and related diseases. Our previous studies showed that tuina regulates relevant regions and indices of the spinal dorsal horn using the Dian, Bo, and Rou method in Yinmen（BL37）, Yanglingquan（GB34）, and Weizhong（BL40）. Treatment prevents muscle atrophy, protects spinal cord neurons, and promotes sciatic nerve repair. The mechanisms of action of tuina for treating peripheral nerve injury remain poorly understood. This study established rat models of sciatic nerve injury using the crushing method. Rats received Chinese tuina in accordance with the principle of ＂Three Methods and Three Points,＂ once daily for 20 days. Tuina intervention reduced paw withdrawal latency and improved wet weight of the gastrocnemius muscle, as well as promoting morphological recovery of sciatic nerve fibers, Schwann cells, and axons. The protein expression levels of phospho-p38 mitogen-activated protein kinase, tumor necrosis factor-α, and interleukin-1β also decreased. These findings indicate that ＂Three Methods and Three Points＂ promoted morphological recovery and improved behavior of rats with peripheral nerve injury.

Effect of propofol on glutamate-induced activation and related inflammatory cytokines of astrocytes from spinal cord dorsal horn

BACKGROUND： Astrocytes participate in central nervous system-mediated physiological or pathological processes, such as pain. Activated dorsal horn astrocytes from the spinal cord produce nerve active substances and proinflammatory cytokines, such as interleukin-lbeta （IL-1 β ）, IL-6, and tumor necrosis factor- α （TNF-α ）, which play important roles in pain transduction and regulation. OBJECTIVE： To investigate the effects of different doses of propofol on activation of cultured spinal cord dorsal horn astrocytes induced by glutamate, as well as changes in IL-1β, IL-6, and TNF- α, and 1L-10 （anti-inflammatory cytokine） expression in rats, and to explore the dose relationship of propofol. DESIGN, TIME AND SETTING： The cellular and molecular biology experiment was performed at the Central Laboratory of Yunyang Medical College between March 2006 and December 2007. MATERIALS： Forty healthy, Wistar rats, aged 2-3 days, were selected. Propofol was provided by Zeneca, UK; glutamate by Sigma, USA; EPICS XL flow cytometry by Beckman culture, USA; rabbit-anti-mouse glial fibrillary acidic protein （GFAP） antibody kit and inflammatory cytokine detection kit were provided by Zhongshan Biotechnology Company Ltd., Beijing; multimedia color pathologic image analysis system was a product of Nikon, Japan. METHODS： Astrocytes were harvested from T11- L6 spinal cord dorsal horn of Wistar rats and incubated for 3 weeks. The cells were divided into seven groups, according to various treatment conditions： control group was cells cultured in Hank＇s buffered saline solution; intralipid group was cells cultured in intralipid （0.2 mL/L）; glutamate group was cells cultured with 100 u mol/L glutamate; propofol group was cells cultured with 250 u mol/L propofol; three glutamate plus propofol groups were cultured in 100 11 mol/L of glutamate, followed by 5, 25, and 250 u mol/L of propofol 10 minutes later. MAIN OUTCOME MEASURES： GFAP-labeled astrocytes were analyzed using a multimedia pathology imaging analysis system to detect area density （AD） and average optical density （AOD） of positive cells. The supernatant concentrations of IL-1β, TNF- α, IL-6, and IL-10 were determined using radioimmune assays. RESULTS： Compared with the control group, cells in the glutamate plus low-dose propofol group were activated and hypertrophic, and AD and AOD were significantly increased （P 〈 0.01 ）. Concentrations of IL-1β, TNF- α, and IL-6 were also significantly increased （P 〈 0.01）, while IL-10 levels remained unchanged （P 〉 0.05）, but still higher than the control and glutamate groups （P 〉 0.05）. Compared with the glutamate group, astrocyte activation was inhibited by moderate and high-dose propotol. In addition, with moderate and high-dose propofol, AD, AOD, IL-1β, TNF- α, and IL-6 concentrations were significantly decreased （P 〈 0.05-0.01）, and IL-10 levels were increased （P 〈 0.01 ）. CONCLUSION： Propofol can effectively inhibit glutamate-induced astrocyte activation in the spinal cord dorsal horn, significantly inhibit production of IL-1 β, TNF- α, and IL-6, and increase IL-10 synthesis and release in a dose-dependent manner.

Proteomic analysis of the dorsal spinal cord in the mouse model of spared nerve injury-induced neuropathic pain

Peripheral nerve injury often causes neuropathic pain and is associated with changes in the expression of numerous proteins in the dorsal horn of the spinal cord. To date, proteomic analysis method has been used to simultaneously analyze hundreds or thousands of proteins differentially expressed in the dorsal horn of the spinal cord in rats or dorsal root ganglion of rats with certain type of peripheral nerve injury. However, a proteomic study using a mouse model of neuropathic pain could be attempted because of abundant protein database and the availability of transgenic mice. In this study, whole proteins were extracted from the ipsilateral dorsal half of the 4th-6th lumbar spinal cord in a mouse model of spared nerve injury（SNI）-induced neuropathic pain. In-gel digests of the proteins size-separated on a polyacrylamide gel were subjected to reverse-phase liquid-chromatography coupled with electrospray ionization ion trap tandem mass spectrometry（MS/MS）. After identifying proteins, the data were analyzed with subtractive proteomics using ProtAn, an in-house analytic program. Consequently, 15 downregulated and 35 upregulated proteins were identified in SNI mice. The identified proteins may contribute to the maintenance of neuropathic pain,and may provide new or valuable information in the discovery of new therapeutic targets for neuropathic pain.

基于SP/NK1R/βARRS通路电针缓解神经性疼痛的实验研究

目的观察针刺对坐骨神经慢性缩窄模型(chronic constriction injury,CCI)大鼠疼痛行为及脊髓背角P物质(substance P,SP)含量和神经激肽1受体(neurokinin 1 receptor,NK1R)、β-抑制蛋白1抗体(β-arrestin 1,βARR1)蛋白水平的影响。方法将36只雄性Sprague-Dawley(SD)大鼠随机分为假手术组、模型组、电针组和手针组,每组9只。暴露模型组、电针组和手针组大鼠左侧股骨中段坐骨神经并进行结扎,建立坐骨神经慢性缩窄模型;假手术组暴露坐骨神经但不结扎。造模后第8天开始,电针组和手针组分别对环跳和阳陵泉穴进行干预。测定4组大鼠造模后第0、7、13、21、29天的机械痛阈、热痛阈及后肢负重分布。采用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测4组大鼠L4~L6腰膨大脊髓背角组织中SP含量;用蛋白质印迹法(Western blotting)检测4组大鼠L4~L6腰膨大脊髓背角组织中NK1R和βARR1蛋白表达。结果造模后第7天,模型组、电针组和手针组大鼠机械痛阈和热痛阈较假手术组降低(P<0.05),两后肢负重分布差异较假手术组增加(P<0.05)。针刺干预后,造模后第13、21、29天,手针组和电针组大鼠机械痛阈和热痛阈高于模型组(P<0.05),两后肢负重分布差异小于模型组(P<0.05);造模后第29天,电针组机械痛阈和热痛阈均高于手针组(P<0.05);造模后第21天,电针组两后肢负重分布差异小于手针组(P<0.05)。针刺干预全部结束后,模型组脊髓背角组织SP含量低于假手术组(P<0.05),手针组和电针组SP含量高于模型组(P<0.05)。针刺干预全部结束后,模型组脊髓背角组织NK1R和βARR1蛋白表达高于假手术组(P<0.05),电针组和手针组NK1R和βARR1蛋白表达低于模型组(P<0.05)。结论针刺可能通过调控脊髓背角SP水平及NK1R和βARR1蛋白表达对神经病理性疼痛起到镇痛效应。

电针调控脊髓背角NRG1及小胶质细胞活化缓解癌性骨痛研究

目的观察电针腰(L)3~L5“夹脊”对癌性骨痛(CIBP)大鼠疼痛行为及脊髓背角神经调节蛋白1(NRG1)表达和小胶质细胞活化的影响,探讨电针缓解CIBP的作用机制。方法30只雌性SD大鼠随机分为假手术组、模型组和电针组,每组10只。向模型组和电针组大鼠右侧胫骨骨髓腔内注射10μL Walker 256乳腺癌细胞,假手术组注射10μL Hank's液。于术后15 d开始,选取双侧L3~L5“夹脊”进行电针治疗30 min,每日1次,共6次。于术前(0 d)和术后3、7、10、14、16、18、20 d进行机械性痛觉敏化测试。Western blot检测L4~L6脊髓背角NRG1的蛋白表达;免疫荧光组织化学法检测脊髓背角离子钙结合适配器分子1(Iba-1)和NRG1的表达;ELISA检测L4~L6脊髓背角肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和IL-6的含量。结果与假手术组相比,模型组大鼠自术后7 d开始右后肢机械缩足反射阈值(MWT)显著下降(P<0.001),L4~L6脊髓背角NRG1蛋白表达明显增加(P<0.05),且Iba-1与NRG1的共表达显著增加(P<0.01),TNF-α、IL-1β、IL-6含量明显升高(P<0.001)。与模型组相比,术后15 d开始电针治疗后,电针组大鼠右后肢MWT显著上升(P<0.001),L4~L6脊髓背角NRG1蛋白表达明显降低(P<0.05),且Iba-1与NRG1的共表达显著减少(P<0.01),TNF-α、IL-1β、IL-6含量明显降低(P<0.001)。结论电针双侧“夹脊”可有效缓解CIBP大鼠的疼痛行为,其机制可能与抑制脊髓背角NRG1的上调及小胶质细胞的活化有关。

腹部推拿对溃疡性结肠炎大鼠脊髓背角PI3K、NR1表达及神经元形态的影响

目的观察腹部推拿对溃疡性结肠炎(UC)大鼠脊髓背角磷脂酰肌醇-3-激酶(PI3K)、N-甲基-D-天冬氨酸受体(NMDAR)亚基NR1表达及神经元形态的影响,探讨其治疗UC的作用机制。方法36只SD大鼠随机分为正常组、模型组、腹部推拿组、美沙拉嗪组、PI3K激动组和PI3K激动+腹部推拿组,每组6只。采用5%葡聚糖硫酸钠溶液自由饮用建立UC大鼠模型,腹部推拿组和PI3K激动+腹部推拿组予腹部推拿干预,美沙拉嗪组予美沙拉嗪溶液灌胃,PI3K激动组和PI3K激动+腹部推拿组鞘内注射PI3K激动剂,均1次/d,连续15 d。腹壁撤退反射(AWR)评分及乙酸扭体反应观察大鼠腹痛症状,免疫荧光染色和Western blot检测大鼠脊髓背角PI3K、NR1表达,尼氏染色观察脊髓背角神经元形态。结果与正常组比较,模型组大鼠AWR评分及扭体次数显著增加(P<0.01),脊髓背角PI3K、NR1蛋白表达显著升高(P<0.05,P<0.01),脊髓背角神经元形态紊乱、形成大量空泡样结构,尼氏体结构模糊、不完整。与模型组比较,腹部推拿组和美沙拉嗪组大鼠AWR评分及扭体次数显著减少(P<0.05,P<0.01),脊髓背角PI3K、NR1蛋白表达显著降低(P<0.05,P<0.01),脊髓背角神经元水肿较轻、空泡样改变较少,尼氏体数量增加;PI3K激动组和PI3K激动+腹部推拿组AWR评分及扭体次数显著增加(P<0.05,P<0.01),脊髓背角PI3K、NR1蛋白表达显著升高(P<0.05,P<0.01),大量神经元固缩坏死,尼氏体数量减少,甚至溶解消失。结论腹部推拿能有效改善UC模型大鼠腹痛症状,其作用机制可能与抑制脊髓背角PI3K、NR1表达,改善脊髓背角神经元形态有关。

推拿通过NF-κB/GT通路对minor CCI大鼠的镇痛启动机制研究

目的:探讨推拿治疗神经病理性疼痛的镇痛启动机制。方法:将32只雄性大鼠随机分为假手术组、模型组、推拿组和推拿+PDTC组,模型组、推拿组和推拿+PDTC组建立minor CCI模型。造模后7 d推拿组进行三法三穴干预1次。通过Von Frey法检测大鼠的机械疼痛和热痛阈值,双重免疫荧光标记法、Western blot和RT-PCR技术检测大鼠SDH中NF-κB/GT通路相关分子变化。结果:推拿干预1次后,与模型组相比,推拿组和推拿+PDTC组的MWT和TWL值、EAAT2细胞数目、蛋白及mRNA表达显著升高(均P<0.05),GFAP细胞数目、蛋白、mRNA表达和NF-κB mRNA表达显著降低(均P<0.05);与推拿组相比,推拿+PDTC组的MWT和TWL值、EAAT2细胞数目、蛋白及mRNA表达升高(均P<0.05),GFAP细胞数目、蛋白、mRNA表达和NF-κB mRNA表达降低(均P<0.05)。结论:推拿可通过调节NF-κB/GT信号通路发挥即刻镇痛作用。

“三法三穴”推拿手法干预早期对Minor CCI模型大鼠脊髓背角IL-17A/RA、C/EBPβ信号通路影响的研究

目的 通过观察“三法三穴”推拿手法干预早期对轻度坐骨神经慢性压迫性损伤(Minor chronic compression injury of sciatic nerve, Minor CCI)模型大鼠脊髓背角中白介素-17A(interleukin-17A,IL-17A)、白介素-17A受体(interleukin-17A receptor, IL-17RA)、转录因子CCAAT增强子结合蛋白β(transcription factor CCAAT/enhancer binding protein β, C/EBPβ)表达的影响,明确推拿对周围神经病理性疼痛(peripherally neuropathic pain, pNP)的即刻镇痛机制。方法 SD大鼠随机分为假手术组、模型组、推拿组,8只/组,模型组、推拿组复制Minor CCI模型。使用按摩推拿手法模拟仪模拟点法、拨法、揉法,对推拿组大鼠患侧殷门、承山、阳陵泉进行定时、定性、定量干预1次,通过机械缩足反射阈值(paw mechanical withdraw threshold, PMWT)与冷敏阈值(cold sensitivity threshold, CST)评价干预后即刻对大鼠机械痛觉、温度觉敏化情况变化;通过蛋白免疫印迹法(Western Bolt, WB)检测脊髓背角中IL-17A、C/EBPβ表达;通过荧光定量聚合酶链式反应(real time-polymerase chain reaction, RT-PCR)检测脊髓背角中IL-17A、IL-17RA、C/EBPβ表达。结果 与假手术组、模型组相比,推拿干预1次后可提高Minor CCI模型大鼠PMWT与CST(P<0.01);WB结果显示,与模型组相比,推拿可降低IL-17A、C/EBPβ在脊髓背角中蛋白的表达(P<0.05);RT-PCR结果显示,与模型组相比,推拿可降低IL-17A、IL-17RA、C/EBPβ在脊髓背角中mRNA的表达(P<0.05)。结论 推拿干预1次后,可通过抑制脊髓背角中IL-17A、IL-17RA、C/EBPβ信号通路的表达,改善机械痛觉及冷刺激敏化情况,从而实现即刻镇痛的作用。

推拿按揉法对慢性坐骨神经挤压伤大鼠脊髓背角星形胶质细胞和神经元的影响

目的:观察推拿按揉法对慢性坐骨神经挤压伤(CCI)大鼠星形胶质细胞和神经元的影响,探讨其治疗神经病理性疼痛的潜在作用机制。方法:将入组的40只SD大鼠随机平均分成5组(每组8只),分别为空白组、模型组、推拿组、星形胶质细胞抑制剂组和星形胶质细胞抑制剂+推拿组。除空白组外的组别均建立CCI模型,造模后第4天起,推拿组接受按揉法干预,10分钟/次/天,星形胶质细胞抑制剂组鞘内注射氟代柠檬酸(fluorocitrate,FCA),10ul/只/天,星形胶质细胞抑制剂+推拿组进行鞘内注射FCA和按揉委中穴联合干预,连续14天。观察各组大鼠各时间点的机械痛阈值情况,使用免疫荧光检测脊髓背角星形胶质细胞标志物(glialfibrillaryacidicprotein,GFAP)蛋白的表达,尼式染色法观测脊髓背角内神经元的形态、排列和数量的变化,并分析GFAP表达水平与神经元存活数量的相关性。结果:相较空白组,模型组机械痛阈值显著下降(P<0.01);推拿干预后,推拿组阈值较模型组逐渐上升,累计治疗14天后具有显著差异(P<0.01),注射FCA第7天起,星形胶质细胞抑制剂组和星形胶质细胞抑制剂+推拿组疼痛阈值明显上升(P<0.01)。相较空白组,模型组脊髓背角GFAP表达显著升高(P<0.01),神经元形态不规则、排列松散,数量减少(P<0.01),推拿治疗后,GFAP表达下调(P<0.01),神经元形态趋于规则、排列趋于整齐,数量增加(P<0.01)。注射FCA后,星形胶质细胞抑制剂组和星形胶质细胞抑制剂+推拿组的GFAP表达水平较模型组显著降低(P<0.01),神经元形态、排列较模型组更加规整、数量增加(P<0.01)。GFAP平均荧光强度与神经元存活数量在相关性分析中提示负(r=-0.4649,P<0.05)相关。结论:推拿按揉法可以缓解CCI大鼠的疼痛行为,减轻脊髓背角神经元的受损程度,其机制可能与其抑制星形胶质细胞活化相关。

Chinese Tuina remodels the synaptic structure in neuropathic pain rats by downregulating the expression of N-methyl D-aspartate receptor subtype 2B and postsynaptic density protein-95 in the spinal cord dorsal horn

OBJECTIVE:To investigate whether the Chinese massage system,Tuina,exerts analgesic effects in a rat model of chronic constriction injury(CCI)by remodeling the synaptic structure in the spinal cord dorsal horn(SCDH).METHODS:Sixty-nine male Sprague–Dawley rats were randomly and evenly divided into the normal group,sham group,CCI group,CCI+Tuina group,CCI+MK-801[an N-methyl D-aspartate receptor subtype 2B(NR2B)antagonist]group,and CCI+MK-801+Tuina group.The neuropathic pain model was established using CCI with right sciatic nerve ligation.Tuina was administered 4 d after CCI surgery,using pressing manipulation for 10 min,once daily.Motor function was observed with the inclined plate test,and pain behaviors were observed by the Von Frey test and acetone spray test.At 19 d after surgery,the L3-L5 spinal cord segments were removed.Glutamate,interleukin 1β(IL-1β),and tumor necrosis factor-α(TNF-α)levels were detected by enzyme-linked immunosorbent assay.The protein expression levels of NR2B and postsynaptic density protein-95(PSD-95)were detected by Western blot,and the synaptic structure was observed by transmission electron microscopy(TEM).RESULTS:CCI reduced motor function and caused mechanical and cold allodynia in rats,increased glutamate concentration and TNF-αand IL-1βlevels,and increased expression of synapse-related proteins NR2B and PSD-95 in the SCDH.TEM revealed that the synaptic structure of SCDH neurons was altered.Most of these disease-induced changes were reversed by Tuina and intrathecal injection of MK-801(P<0.05 or<0.01).For the majority of experiments,no significant differences were found between the CCI+MK-801 and CCI+MK-801+Tuina groups.CONCLUSIONS:Chinese Tuina can alleviate pain by remodeling the synaptic structure,and NR2B and PSD-95 receptors in the SCDH may be among its targets.

推拿对坐骨神经慢性压迫损伤大鼠脊髓背角小胶质细胞P2Y12/RhoA/ROCK2通路及c-Fos蛋白表达的影响

目的观察推拿对坐骨神经慢性压迫损伤大鼠脊髓背角小胶质细胞P2Y12/RhoA/ROCK2通路及c-Fos蛋白表达的影响,探讨推拿治疗腰椎间盘突出症的作用机制。方法采用右侧坐骨神经慢性压迫损伤模拟腰椎间盘突出症神经性疼痛。将24只雄性SD大鼠随机分为空白组、模型组和推拿组,每组8只。造模后第4日,推拿组以按揉法干预,连续14 d。测量造模前及造模后第4、10、17 d大鼠机械缩足阈值(PWT)、热痛阈值(PWL),免疫荧光染色检测大鼠右侧脊髓背角Iba1、P2Y12蛋白表达,Western blot检测右侧脊髓背角RhoA、ROCK2蛋白表达,免疫组化检染色测右侧脊髓背角c-Fos阳性细胞数量。结果与空白组比较,模型组大鼠造模后第4、10、17日PWT、PWL明显降低(P<0.001),右侧脊髓背角Iba1、P2Y12、RhoA、ROCK2蛋白表达明显升高(P<0.001,P<0.05),c-Fos阳性细胞数明显增加(P<0.001);与模型组比较,推拿组大鼠造模后第10、17日PWT、PWL明显升高(P<0.05,P<0.01,P<0.001),右侧脊髓背角Iba1、P2Y12、RhoA、ROCK2蛋白表达明显降低(P<0.001,P<0.01,P<0.05),c-Fos阳性细胞数明显减少(P<0.001)。结论推拿可能通过调控脊髓背角P2Y12/RhoA/ROCK2通路及c-Fos表达抑制小胶质细胞激活,降低神经元兴奋性,对腰椎间盘突出症发挥镇痛作用。

褪黑素在小鼠脊髓背角神经元发挥镇痛效应的受体机制研究

目的研究神经病理性痛状态下,褪黑素(MLT)是否在脊髓水平参与镇痛效应的调节及其可能的受体机制。方法采用雄性C57BL/6小鼠,进行坐骨神经部分损伤(SNI)模型及假手术(Sham)造模,7 d后鞘内分别给予MLT以及MLT不同受体激动剂和/或拮抗剂,运用von Frey丝测试比较SNI组及Sham组给药前后小鼠机械刺激缩足反应阈值,即机械痛阈值的变化。以雄性C57BL/6和转基因小鼠为研究对象,采用免疫荧光染色技术明确不同类型脊髓背角神经元上MLT受体的表达情况。通过膜片钳技术比较SNI造模及Sham术药物干预前后,脊髓背角神经元兴奋性的改变。结果与Sham组相比,SNI组小鼠的机械痛阈值明显降低(P<0.05);鞘内给予MLT和Ramelteon(MT1/MT2受体激动剂)均能显著恢复SNI组小鼠的机械痛阈值(P<0.05),但同时给予Ramelteon+4PP(MT2受体拮抗剂)后SNI组小鼠的机械痛阈值未恢复。免疫荧光结果表明,MT2广泛分布于小鼠脊髓背角的兴奋性神经元。膜片钳实验发现,与Sham组相比,SNI组脊髓背角兴奋性神经元的动作电位基强度明显降低(P<0.05),静息电位绝对值减小(P<0.05),并且MT2受体激动剂(8MP)可以逆转上述变化(P<0.01)。结论神经病理性痛状态下,MLT在脊髓水平显著缓解SNI小鼠的机械性痛敏,且其镇痛效应是通过MT2介导的。