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.

Spinal processing of bee venom-induced pain and hyperalgesia

Subcutaneous injection of bee venom causes long-term neural activation and hypersensitization in the dorsal horn of the spinal cord,which contributes to the development and maintenance of various pain-related behaviors.The unique behavioral 'phenotypes' of nociception and hypersensitivity identified in the rodent bee venom test are believed to reflect a complex pathological state of inflammatory pain and might be appropriate to the study of phenotype-based mechanisms of pain and hyperalgesia.In this review,the spinal processing of the bee venom-induced different 'phenotypes' of pain and hyperalgesia will be described.The accumulative electrophysiological,pharmacological,and behavioral data strongly suggest that different 'phenotypes' of pain and hyperalgesia are mediated by different spinal signaling pathways.Unraveling the phenotype-based mechanisms of pain might be useful in development of novel therapeutic drugs against complex clinic pathological pain.

Up-regulation and time course of protein kinase C immunoreactivity during persistent inflammation of the rat spinal cord

BACKGROUND： It has been reported that activation and/or translocation of protein kinase C （PKC） is related to hyperalgesia, and changes in PKC expression in the dorsal horn of spinal cord take place during inflammatory pain. OBJECTIVE： To observe PKC changes in the dorsal horn of spinal cord using immunohistochemistry and to measure the time-course during persistent pain produced by chemical stimulation with a right hind-paw injection of formalin. DESIGN： Randomized controlled animal experiment. SETTING： Institute of Basic Medical Science, Hebei Medical University MATERIALS： The present experiment was performed at the Department of Pathophysiology, Institute of Basic Medical Science, Hebei Medical University between September 2000 and June 2002. Forty-two Sprague-Dawley rats, weighing 260-280 g, irrespective of gender, were provided by the Center of Animal Experimentation at Hebei Medical University. PKC antibody was provided by Sigma, USA. Immunohistochemistry kits were purchased from Zhongshan Biotechnology Company, Beijing. HPIAS-1000 definition multicolor system was provided by Qianping Wuxiang Project Company of Tongji Medical University. Animal use during experimentation was consistent with the standards of Animal Ethics Committee. METHODS： Sprague-Dawley rats were divided randomly into control （n = 6） and experimental groups （n = 36）. Experimental rats were given an intracutaneous injection of 5% formalin into the planta surface of the right hind-paw. Animals with inflammatory pain were anesthetized and sacrificed to obtain the L5 spinal region at 1, 3, 12 hours, 1, 3, and 7 days after formalin treatment, with 6 rats in each time group. The spinal cords at the L5 region were collected from the control group following sodium chloride injections into the planta surface of the right hind-paw, identical to the experimental group. MAIN OUTCOME MEASURES： Pain reaction of experimental rats after formalin treatment. PKC-positive neurons, and distribution of PKC-immunoreactive particles, in the ipsi- and contralateral dorsal horn were investigated during different stages of inflammatory pain using immunohistochemistry. RESULTS： All 42 rats were included in the final analysis, without any loss. Pain reaction： consistent with previous findings, it was determined that a unilateral injection of formalin into the hind-paw resulted in significant edema and induced a series of nociceptive responses, such as licking, biting, or shaking the injected paw. The maximal inflammation change was observed 1 day after formalin injection and changes did not disappear until the day 7. Number of the PKC positive neurons： results demonstrated that the number of PKC immunoreactive neurons in the dorsal horn increased slightly after formalin injection at 1 hour, compared with the control group. PKC immunoreactivity was up-regulated at day 1, reduced at day 3, and appeared to recover at day 7. The number of PKC-positive neurons in the contralateral side was less than the ipsilateral side at each time sampled. Distribution of PKC immunoparticles over the neurons： PKC immunoreactivity was observed in the nucleus and cytoplasm, as well as on or near the membrane of neurons and synaptosomes in the spinal cord of the control group. PKC activated and translocated from nucleus to the membrane-associated site following formalin treatment. Significant changes were observed at 1 hour and 1 day. The intensity of staining was stronger in the ipsilateral side than the contralateral side at all time points following formalin injection （P 〈 0.01）, whereas the expression patterns of PKC immunoreactivity in the nuclei were very similar in the right and left hemispheres. CONCLUSION： PKC expression in the dorsal horn of the spinal cord peaked at 1 hour and 24 hours, and was very obvious at 24 hours. Protein kinase C expression in the spinal cord increased bilaterally, although it was greater in the ipsilateral hemisphere. In addition, PKC expression at the neuronal membrane and synaptosome was significantly increased. These results indicate that PKC expression is activated in the dorsal horn of the spinal cord during hyperalgesia.

电针对紫杉醇诱导大鼠神经病理性疼痛的影响

目的观察电针(EA)对紫杉醇诱导神经病理性疼痛大鼠脊髓背角NKCC1、KCC2表达和小胶质细胞活化的影响及其可能机制。方法将48只雄性SD大鼠随机分为溶媒组(Vehicle组)、紫杉醇组(PTX组)、紫杉醇+电针组(PTX+EA组)、紫杉醇+假电针组(PTX+Sham EA组),每组12只。采用腹腔注射PTX的方法建立PTX诱导的神经病理性疼痛大鼠动物模型,建模完成后,PTX+EA组给予“足三里”、“阳陵泉”电针刺激,连续7 d。于紫杉醇注射前2 d和注射后第1、3、5、7、14、21天进行机械撤足阈值和热缩足潜伏期痛行为学测试。利用免疫荧光染色技术和Western blot技术检测脊髓背角组织中钠钾氯联合转运蛋白1(NKCC1)、钾氯联合转运蛋白2(KCC2)和小胶质细胞标志物离子钙结合衔接分子1(Iba1)表达的变化。结果与Vehicle组比较,PTX组大鼠出现双后足机械和热痛觉过敏,脊髓背角组织中NKCC1表达升高和活化的小胶质细胞数增加。与PTX组比较,PTX+EA组大鼠在第14、21天的机械和热痛觉过敏得到显著改善,脊髓背角组织中NKCC1和Iba1表达量降低,4组间KCC2表达差异无统计学意义。结论电针可有效缓解紫杉醇诱导的神经病理性疼痛,其机制可能与抑制大鼠脊髓背角组织中NKCC1表达和小胶质细胞活化有关。

Effects of Electroacupuncture on Expression of COX-2 in the Dorsal Horn of Spinal Cord of Arthritic Rats

Objective： The present study was to observe the expression of COX-2 protein in the dorsal horn of the spinal cord in rats with chronic inflammatory pain to investigate whether COX-2 is involved in the mechanism of electroacupuncture （EA） analgesia. Methods： After EA treatment, the pain score and expression of COX-2 protein in the dorsal horn of spinal cord in rats with complete Freund＇s adjuvant （CFA）-induced arthritis were measured by the behavioral test and immunohistochemistry technique, and compared with those of the normal group, model group and medicine group. Results： EA could decrease the pain score significantly and repeated EA treatments had the remarkable cumulative analgesic effect; and with the increase in EA treatments, the number of COX-2 immunoreactive cells was obviously decreased. Conclusion： EA at acupoints Yanglingquan （GB 34） and Kunlun （BL 60） had remarkable analgesic effect on adjuvant arthritis rats; and in the meantime, could inhibit the expression of COX-2 protein in the dorsal horn of spinal cord.

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。

人参皂苷Rd通过中枢γ-氨基丁酸能神经对神经病理性疼痛的镇痛作用机制

目的探讨人参皂苷Rd(ginsenoside Rd,GSRd)对小鼠坐骨神经分支选择性损伤(spared nerve injury,SNI)所致神经病理性疼痛(neuropathic pain,NP)的镇痛作用及机制。方法建立SNI模型,检测行为学指标验证模型稳定性、GSRd对SNI所致神经痛的镇痛作用。GSRd与γ-氨基丁酸(gamma aminobutyric acid,GABA)系统工具药联用,分析GSRd治疗SNI所致神经痛与GABA能神经的关系。免疫荧光染色观察SNI所致神经痛小鼠腹外侧视前核(ventrolateral preoptic nucleus,VLPO)、中脑导水管周围灰质腹外侧区(ventrolateral periaqueductal gray,VLPAG)和脊髓背角(spinal dorsal horn,SDH)中c-Fos、c-Fos与GAT-1免疫阳性细胞共表达情况,分析GSRd镇痛作用与痛觉传导通路核团及核团神经元的关系。结果SNI神经痛小鼠术后第3天开始痛阈变化、产生痛敏,可持续至少14 d;GSRd 500、1000 mg·kg^(-1)使SNI所致神经痛小鼠痛阈升高。GABA系统工具药能协同或拮抗GSRd对SNI所致神经痛小鼠的镇痛作用;SNI所致神经痛小鼠VLPO、VLPAG和SDH的c-Fos免疫阳性细胞增加,GSRd 500 mg·kg^(-1)可减少SNI所致神经痛小鼠VLPO、VLPAG和SDH的c-Fos与GAT-1共表达免疫阳性细胞。结论SNI所致神经痛模型稳定,GSRd具有明显的镇痛作用,机制与下调VLPO、VLPAG、SDH中的GAT-1,减少其对突触间隙GABA的重摄取,增强中枢GABA能神经的抑制作用相关。

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。

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。