Differential Expression of Alpha-Adrenoceptor Subtypes in Rat Dorsal Root Ganglion after Chronic Constriction Injury

Summary： mRNAs of alpha-adrenoceptor （α-AR） subtypes are found in neurons in dorsal root ganglion （DRG） and change after peripheral nerve injury. In this study, the distribution of α-AR subtype proteins was studied in L5 DRG of normal rats and rats with chronic constriction injury of sciatic nerve （CCI）. Using immunofluorescence technique, it was found that α1A-, α1B-, and α2A-AR proteins were expressed in large, medium, and small size neurons in normal DRG, and significantly increased in all size neurons 14 days after CCI. α1D- and α2C-AR was also expressed in all size neurons in normal DRG. However, α1D-AR was significantly increased and α2C-AR was decreased in small size neurons 14 days post CCI. α2B-AR neurons were not detectable in normal and CCI DRG. Co-expression of α1A- and α2A-AR in the same neuron was observed in normal DRG and increased post CCI. Collectively, these results indicated that there is distinct distribution of α-AR subtypes in DRG neurons, and the distribution and levels of expression of α-AR subtypes change differently after CCI. The up-regulation of α-AR subtypes in DRG neurons may play an important role in the process of generating and transmitting neuropathic pain.

Effect of Touch stimulus on the Expression of C-fos and TrkA in Spinal Cord Following Chronic Constriction Injury of the Sciatic Nerve in Rats

Summary: To study the mechanism of the innoxious touch-stimulus on the modulation of hyperalgesia and the expression of the C-fos and the nerve growth factor (NGF) receptor-TrkA in the spinal dorsal horn neurons following the chronic constriction injury (CCI) of the sciatic nerve in rats, 60 female Sprague-Dawley rats were randomly divided into sham-operation group and CCI group, with each group being further divided into 3 subgroups on the 7th,14th and 28th day after operation (n=10). The mechanical and the thermal withdrawal threshold were assessed following the touch stiumulation after the CCI, immunohistochemical methods were employed to observe the expression of the C-fos and TrkA in spinal dorsal horn. Our results showed that the hyperalgesia appeared on the 4th day and reached the maximal level on the 14th day after operation. The expression of the C-fos also increased significantly and reached its maximal level on the 14th day after the touch-stimulus. Meanwhile, the TrkA expression was elevated significantly in both groups, as compared with basic data, and the difference was statistically significant (P<0.05). It is concluded that the level of the C-fos expression changed with the paw withdrawal threshold variation and increased markedly following the innoxious touch-stimulus. The expression of the TrkA receptors also increased gradually following the development of the neuropathic pain. The results suggest that C-fos may play a crucial role in the development of the hyperalgesia in the earlier-time of the neuropathic pain, but TrkA receptors may be involved in the long-lasting adaptive changes of the central pathway in neuropathic pain.

Aloin attenuates chronic constriction injury-induced neuropathic pain in rats by inhibiting inflammatory cytokines and oxidative stress

Objective:To investigate the effect of aloin against chronic constriction injury(CCI)-induced neuropathic pain in rats.Methods:Rats were randomly divided into 7 groups:GroupⅠ(normal control),GroupⅡ(sham-operated),GroupⅢ(CCI control)and GroupⅣ,Ⅴ,Ⅵ,andⅦ,which underwent CCI surgery and then were administered with aloin(5 mg/kg,p.o.;25 mg/kg,p.o.;125 mg/kg,p.o.)and gabapentin(50 mg/kg,p.o.),respectively for 14 days.Peripheral neuropathy was induced by silk ligatures(4-0)loosely placed around the sciatic nerve.Nociceptive thresholds against mechanical stimuli(Von-Frey filaments)and thermal stimuli(12℃and 40℃)were measured at midplantar paw region ipsilateral to the compressed nerve on day-3,7,11,and 14.The concentration of cytokines including tumor necrosis factor-α(TNF-α),interleukin-6,and interleukin-1βwas estimated at day-7.At day 14,motor nerve conduction velocity was determined under urethane anesthesia(1.25 g/kg).Oxidative stress parameters(malondiadehyde,glutathione,catalase,and superoxide dismutase)were estimated in sciatic nerve homogenates at day 14.Representative nerve samples were processed for histological investigations.Results:Aloin significantly reduced CCI-induced mechanical and thermal allodynia.It also improved motor nerve conduction velocity and decreased oxidative stress in nerve tissues.In addition,it decreased pro-inflammatory cytokine levels and restored the histoarchitecture of compressed sciatic nerve.Conclusions:Aloin mitigates CCI-induced neuropathic pain in rats by inhibiting oxidative stress and pro-inflammatory cytokines in the afflicted sciatic nerve.

Radial shock wave therapy in the treatment of chronic constriction injury model in rats： a preliminary study

Background Pain physicians pay close attention to neuropathic pain （NP）,since there is currently no ideal treatment.Radial shock wave therapy （RSWT） is a noninvasive treatment to chronic pain of soft tissue disorders.So far,there is no information on the use of RSWT for the treatment of NP.Therefore we observe the effects of RSWT on a NP model induced by chronic constriction injury （CCI） in rats.Methods Four different energy densities （1.0,1.5,2.0 and 2.5 bar） RSWT administered as a single session or repeated sessions in rats with NP induced by CCI of the sciatic nerve.The analgesic effect was assessed by measuring mechanical withdrawal threshold （MWT） and thermal withdrawal latency （TWL）.The safety was assessed through calculating sciatic functional index （SFI）.Results MWT and TWL increased after a single session of RSWT from day 1 to day 5 but retumed to baseline levels by day 10.Following repeated sessions of RSWT,both the MWT and TWL were significantly higher than NP group （P ＜ 0.01）for at least 4 weeks.In addition,no significant changes of SFI were observed in any groups after repeated sessions of RSWT and no increased pain or other side effects in any animals.Conclusions A single session of RSWT is rapidly effective in the treatment of CCI,but the efficacy maintained in a short period.However,repeated sessions of RSWT have prolonged efficacy.

Effect of Pulsed Radiofrequency on Rat Sciatic Nerve Chronic Constriction Injury： A Preliminary Study

Background：Pulsed radiofrequency （PRF） application to the dorsal root ganglia can reduce neuropathic pain （NP） in animal models,but the effect of PRF on damaged peripheral nerves has not been examined.We investigated the effect of PRF to the rat sciatic nerve （SN） on pain-related behavior and SN ultrastructure following chronic constriction injury （CCI）.Methods：The analgesic effect was measured by hindpaw mechanical withdrawal threshold （MWT） and thermal withdrawal latency （TWL）.Twenty rats with NP induced by ligating the common SN were then randomly divided into a PRF treatment group and a sham group.The contralateral SN served as a control.The MWT and TWL were determined again 2,4,6,8,10,12,and 14 days after the PRF or sham treatment.On day 14,ipsilateral and contralateral common SNs were excised and examined by electron microscopy.Results：Ipsilateral MWT was significantly reduced and TWL significantly shorter compared to the contralateral side 14 days after CCI （both P =0.000）.In the PRF group,MWT was significantly higher and TWL significantly longer 14 days after the PRF treatment compared to before PRF treatment （both P =0.000）,while no such difference was observed in the sham group （P ＞ 0.05）.Electron microscopy revealed extensive demyelination and collagen fiber formation in the ipsilateral SN of sham-treated rats but sparse demyelination and some nerve fiber regrowth in the PRF treatment group.Conclusions：Hyperalgesia is relieved,and ultrastructural damage ameliorated after direct PRF treatment to the SN in the CCI rat model of NP.

Microencapsulation improves inhibitory effects of transplanted olfactory ensheathing cells on pain after sciatic nerve injury

Olfactory bulb tissue transplantation inhibits P2X2/3 receptor-mediated neuropathic pain. However, the olfactory bulb has a complex cellular composition, and the mechanism underlying the action of purified transplanted olfactory ensheathing cells（OECs） remains unclear. In the present study, we microencapsulated OECs in alginic acid, and transplanted free and microencapsulated OECs into the region surrounding the injured sciatic nerve in rat models of chronic constriction injury. We assessed mechanical nociception in the rat models 7 and 14 days after surgery by measuring paw withdrawal threshold, and examined P2X2/3 receptor expression in L4–5 dorsal root ganglia using immunohistochemistry. Rats that received free and microencapsulated OEC transplants showed greater withdrawal thresholds than untreated model rats, and weaker P2X2/3 receptor immunoreactivity in dorsal root ganglia. At 14 days, paw withdrawal threshold was much higher in the microencapsulated OEC-treated animals. Our results confirm that microencapsulated OEC transplantation suppresses P2X2/3 receptor expression in L4–5 dorsal root ganglia in rat models of neuropathic pain and reduces allodynia, and also suggest that transplantation of microencapsulated OECs is more effective than transplantation of free OECs for the treatment of neuropathic pain.

Molecular mechanisms underlying the effects of acupuncture on neuropathic pain

Acupuncture has been used to treat neuropathic pain for a long time, but its mechanisms of action remain unknown. In this study, we observed the effects of electroacupuncture and manual acu- puncture on neuropathic pain and on ephrin-B/EphB signaling in rats models of chronic constriction injury-induced neuropathic pain. The results showed that manual acupuncture and electroacu- puncture significantly reduced mechanical hypersensitivity following chronic constriction injury, es- pecially electroacupuncture treatment. Real-time PCR results revealed that ephrin-B1/B3 and EphB1/B2 mRNA expression levels were significantly increased in the spinal dorsal horns of chronic constriction injury rats. Electroacupuncture and manual acupuncture suppressed the high expres- sion of ephrin-B1 mRNA, and elevated EphB3/B4 mRNA expression. Electroacupuncture signifi- cantly enhanced the mRNA expression of ephrin-B3 and EphB3/B6 in the dorsal horns of neuro- pathic pain rats. Western blot results revealed that electroacupuncture in particular, and manual acupuncture, significantly up-regulated ephrin-B3 protein levels in rat spinal dorsal horns. The re- sults of this study suggest that acupuncture could activate ephrin-B/EphB signaling in neuropathic pain rats and improve neurological function.

Estrogen affects neuropathic pain through upregulating N-methyl-D-aspartate acid receptor 1 expression in the dorsal root ganglion of rats

Estrogen affects the generation and transmission of neuropathic pain,but the specific regulatory mechanism is still unclear.Activation of the N-methyl-D-aspartate acid receptor 1（NMDAR1） plays an important role in the production and maintenance of hyperalgesia and allodynia.The present study was conducted to determine whether a relationship exists between estrogen and NMDAR1 in peripheral nerve pain.A chronic sciatic nerve constriction injury model of chronic neuropathic pain was established in rats.These rats were then subcutaneously injected with 17β-estradiol,the NMDAR1 antagonist D（-）-2-amino-5-phosphonopentanoic acid（AP-5）,or both once daily for 15 days.Compared with injured drug na？ve rats,rats with chronic sciatic nerve injury that were administered estradiol showed a lower paw withdrawal mechanical threshold and a shorter paw withdrawal thermal latency,indicating increased sensitivity to mechanical and thermal pain.Estrogen administration was also associated with increased expression of NMDAR1 immunoreactivity（as assessed by immunohistochemistry） and protein（as determined by western blot assay） in spinal dorsal root ganglia.This 17β-estradiol-induced increase in NMDAR1 expression was blocked by co-administration with AP-5,whereas AP-5 alone did not affect NMDAR1 expression.These results suggest that 17β-estradiol administration significantly reduced mechanical and thermal pain thresholds in rats with chronic constriction of the sciatic nerve,and that the mechanism for this increased sensitivity may be related to the upregulation of NMDAR1 expression in dorsal root ganglia.

Microencapsulated Schwann cell transplantation inhibits P2X3 receptor expression in dorsal root ganglia and neuropathic pain

Schwann cell transplantation is a promising method to promote neural repair, and can be used for peripheral nerve protection and myelination. Microcapsule technology largely mitigates immune rejection of transplanted cells. We previously showed that microencapsulated olfactory ensheathing cells can reduce neuropathic pain and we hypothesized that microencapsulated Schwann cells can also inhibit neuropathic pain. Rat Schwann cells were cultured by subculture and then microencapsulated and were tested using a rat chronic constriction injury（CCI） neuropathic pain model. CCI rats were treated with Schwann cells or microencapsulated Schwann cells and were compared with sham and CCI groups. Mechanical withdrawal threshold and thermal withdrawal latency were assessed preoperatively and at 1, 3, 5, 7, 9, 11 and 14 days postoperatively. The expression of P2X3 receptors in L4-5 dorsal root ganglia of the different groups was detected by double-label immunofluorescence on day 14 after surgery. Compared with the chronic constriction injury group, mechanical withdrawal threshold and thermal withdrawal latency were higher, but the expression of P2X3 receptors was remarkably decreased in rats treated with Schwann cells and microencapsulated Schwann cells, especially in the rats transplanted with microencapsulated Schwann cells. The above data show that microencapsulated Schwann cell transplantation inhibits P2X3 receptor expression in L4-5 dorsal root ganglia and neuropathic pain.

Treatment with acetyl-L-carnitine exerts a neuroprotective effect in the sciatic nerve following loose ligation:a functional and microanatomical study

Peripheral neuropathies are chronic painful syndromes characterized by allodynia,hyperalgesia and altered nerve functionality.Nerve tissue degeneration represents the microanatomical correlate of peripheral neuropathies.Aimed to improve the therapeutic possibilities,this study investigated the hypersensitivity and the neuromorphological alterations related to the loose ligation of the sciatic nerve in rats.Effects elicited by treatment with acetyl-L-carnitine（ALCAR） in comparison to gabapentin were assessed.Axonal injury,reduction of myelin deposition and accumulation of inflammatory cells were detected in damaged nerve.A decrease of phosphorylated 200-k Da neurofilament（NFP） immunoreactivity and a redistribution in small clusters of myelin basic like-protein（MBP） were observed in ipsilateral nerves.Treatment with ALCAR（100 mg/kg intraperitoneally-i.p.） and gabapentin（70 mg/kg i.p.） administered bis in die for 14 days induced a significant pain relieving effect.ALCAR,but not gabapentin,significantly countered neuromorphological changes and increased axonal NFP immunoreactivity.These findings indicate that both ALCAR and gabapentin significantly decreased the hypersensitivity related to neuropathic lesions.The observation of the positive ALCAR effect on axonal and myelin sheath alterations in damaged nerve supports its use as neurorestorative agent against neuropathies through mechanism（s） consistent to those focused in this study.

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.

Screening of differentially expressed genes in the hypothalamus of a rat neuropathic pain model following sciatic nerve injury

Background Neuropathic pain is induced by injury or disease of the nervous system. Most studies have so far focused only on a few known molecules and signaling pathways among neurons. However, all signal transmissions involved in neuropathic pain appear to be an integral system at different molecular levels. This study was designed to screen the differentially expressed genes of the hypothalamus in chronic constriction injury （CCI） rats and analyze their functions in developing neuropathic pain. Methods Ten adult female Sprague-Dawley rats （（200±10） g） were used in experimental group and sham group （n=-5 in each group）. Mechanical allodynia tests were performed to ensure that the CCI rat model was constructed successfully. Total hypothalamus RNAs were isolated from each group. Forward suppression subtractive hybridization （SSH） library of rat hypothalamus was constructed and up-regulated cDNA clones at neuropathic pain states were obtained via suppressed subtractive hybridization technique and the functions of these genes were analyzed bioinformatically. Results Mechanical allodynia tests showed that the experimental rats had a significantly reduced mechanical allodynia threshold 3 to 13 days after CCI vs sham surgery rats （P 〈0.01）, indicating that the model was successful. Forward SSH library of the rat hypothalamus was constructed successfully and 26 over-expressed expression sequence tags （ESTs） were obtained from these up-regulated cDNA clones. Conclusion Twenty-six up-regulated genes, involved in the regulation of cell cycle and apoptosis, signal transduction, and neuroprotection, may play key roles in decreasing mechanical withdraw thresholds in CCI rats, which implicates a multidimensional and integrated molecular mechanism at gene level in developing neuropathic pain with the supraspinal contributions.

Compounds of traditional Chinese medicine and neuropathic pain

Neuropathic pain(NP)has become a serious global health issue and a huge clinical challenge without available effective treatment.P2 receptors family is involved in pain transmission and represents a promising target for pharmacological intervention.Traditional Chinese medicine(TCM)contains multiple components which are effective in targeting different pathological mechanisms involved in NP.Different from traditional analgesics,which target a single pathway,TCMs take the advantage of multiple components and multiple targets,and can significantly improve the efficacy of treatment and contribute to the prediction of the risks of NP.Compounds of TCM acting at nucleotide P2 receptors in neurons and glial cells could be considered as a potential research direction for moderating neuropathic pain.This review summarized the recently published data and highlighted several TCMs that relieved NP by acting at P2 receptors.

Antinociceptive effects of novel epibatidine analogs through activation of α4β2 nicotinic receptors

The study of α4β2 nicotinic receptors has provided new indications in the treatment of pain. Efforts have been made to explore new α4β2 nicotinic receptor agonists, including TC-2559, as antinociceptive drugs. In this study, we discovered a set of novel epibatidine analogs with strong binding affinities to the α4β2 nicotinic receptors. Among these compounds, C-159, C-163, and C-9515 attenuated formalin-induced nociceptive responses in mice; C-9515 caused the most potent analgesic effect, which was blocked by mecamylamine, a non-selective nicotinic receptor antagonist. Furthermore, C-9515 potently inhibited chronic constriction injury(CCI)-induced neuropathic pain in rats, which was sensitive to DHβE, a selective α4β2 subtype antagonist,indicating that its analgesic effect was mediated by the activation of the α4β2 nicotinic receptors. In conclusion, the epibatidine analog C-9515 was found to be a potent α4β2 nicotinic receptor agonist with potent analgesic function, which demonstrated potential for the further exploration of its druggability.