ABSTRACT
Post-amputation pain causes great suffering to amputees, but still no effective drugs are available due to its elusive mechanisms. Our previous clinical studies found that surgical removal or radiofrequency treatment of the neuroma at the axotomized nerve stump effectively relieves the phantom pain afflicting patients after amputation. This indicated an essential role of the residual nerve stump in the formation of chronic post-amputation pain (CPAP). However, the molecular mechanism by which the residual nerve stump or neuroma is involved and regulates CPAP is still a mystery. In this study, we found that nociceptors expressed the mechanosensitive ion channel TMEM63A and macrophages infiltrated into the dorsal root ganglion (DRG) neurons worked synergistically to promote CPAP. Histology and qRT-PCR showed that TMEM63A was mainly expressed in mechanical pain-producing non-peptidergic nociceptors in the DRG, and the expression of TMEM63A increased significantly both in the neuroma from amputated patients and the DRG in a mouse model of tibial nerve transfer (TNT). Behavioral tests showed that the mechanical, heat, and cold sensitivity were not affected in the Tmem63a-/- mice in the naïve state, suggesting the basal pain was not affected. In the inflammatory and post-amputation state, the mechanical allodynia but not the heat hyperalgesia or cold allodynia was significantly decreased in Tmem63a-/- mice. Further study showed that there was severe neuronal injury and macrophage infiltration in the DRG, tibial nerve, residual stump, and the neuroma-like structure of the TNT mouse model, Consistent with this, expression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β all increased dramatically in the DRG. Interestingly, the deletion of Tmem63a significantly reduced the macrophage infiltration in the DRG but not in the tibial nerve stump. Furthermore, the ablation of macrophages significantly reduced both the expression of Tmem63a and the mechanical allodynia in the TNT mouse model, indicating an interaction between nociceptors and macrophages, and that these two factors gang up together to regulate the formation of CPAP. This provides a new insight into the mechanisms underlying CPAP and potential drug targets its treatment.
Subject(s)
Animals , Mice , Amputation, Surgical , Chronic Pain/pathology , Disease Models, Animal , Ganglia, Spinal/pathology , Hyperalgesia/etiology , Ion Channels/metabolism , Macrophages , Neuroma/pathologyABSTRACT
Objective To investigate the effects of minocycline,a selective microglia inhibitor on dorsal GABAB receptor expression in the spinal cord in a rat model of neuropathic pain and the possible mechanism.Methods Forty-eight male SD rats weighing 220-260 g were used in this study.Neuropathic pain was induced by ligation of L5 spinal nerve(SNL).The animals were randomly divided into 4 groups(n=12 each):Ⅰ sham operation;Ⅱ SNL;Ⅲ sham operation+intrathecal(IT)minocycline 50 μg;IV SNL+IT minecycline 50 μg.Paw withdrawal threshold to von Frey hair stimulation was measured before operation(baseline)and at post-surgery days 1,2,4,6,8,10,12,14,16,18 in 6 animals in each group.The GABABR2 expression in dorsal horn of spinal cord was detected by Western blot at the time point when paw withdrawal threshold to mechanical stimulation was the lowest in another six animals which did not undergo yon Frey hair stimulation test.Results The paw withdrawal threshold to mechanical stimuli and the GABABR2 expression in the dorsal horn of spinal cord were significantly lower in SNL group than in sham operation group.The decreased paw withdrawal threshold to von Frey hair stimulation and the down-regulated GABABR2 expression in the spinal cord induced by SNL were ameliorated by intrathecal administration of micmglia inhibitor- minocycline in group Ⅳ.Conclusion The mechanism of neuropathic pain mediated by microglia activation may be related to the inhibition of GABAB receptor activation.
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AIM: To study the interactions of propofol and midazolam on the whole cell sodium channel currents in rat sympathetic ganglion neurons. METHODS: Whole cell patch clamp recordings were made from enzymatically isolated rat (7- 10 d ) superior cervical sympathetic ganglion neurons. Isobolographic analysis was applied to evaluate the potency of combinations of propofol and midazolam on Na + channel currents. RESULTS: Under V h= 80 mV and V t= 0 mV . Propofol and midazolam dose dependently blocked Na + currents with a mean drug concentration required to produce 50% current inhibition (IC 50 ): 33.12 ?mol?L -1 and 18.35 ?mol?L -1 ; clinically relevant concentrations of propofol and midazolam reduced Na + peak currents by 27.66 % (P
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Aim The effects of diazepam on the whole cell sodium currents in rat sympathetic ganglion neurons were studied to investigate the mechanisms by diazepam mediates hypotension. Methods Whole cell patch clamp recordings were performed on enzymatically isolated rat superior cervical sympathetic ganglion neurons. Results Diazepam dose dependently blocked the whole cell sodium currents. Under a V t of 0 mV and a V h of 80 mV 0.3 ?mol?L -1 diazepam reduced sodium peak currents by 14.76 %(P
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Objective To investigate the effects of ropivacaine on the whole cell sodium currents in rat sympathetic ganglion neurons in order to determine whether sympathetic ganglion is involved in the ropivacaine cardiotoxity. Methods The sympathetic neurons were enzymatically isolated from the superior cervical ganglion. The effects of ropivacaine on the whole cell sodium channel currents were assessed using patch clamp technique. Results Ropivacaine blocked the whole cell sodium channel currents dose dependently. When the holding potential (Vh) was -80mV and Vt Omv , 0.01?mol/L ropivacaine reduced peak currents by 30.02% with a mean IC50 of 2.68?mol/L. The blockade was also potential dependent. When Vh was -60mv, the mean IC50 was 1.55?mol/l. 1.0?mol/L ropivacaine reduced the peak value of I V curve by 30.66% but did not affect the shape of I V curve and caused 2.56mv shift of voltage dependence activation curve to depolarized potentials and 3.56mv shift of steady state inactivation curve to more hyperdepolarized potentials.The conditioning pulse potential at which half maximal channels were activated (V1/2), changed from -52.99mv to -56.44mv and the test potential at which half maximal channels were activated (V1/2), changed from -25.2mv to -22.64mv. Conclusions Subconvulsive concentration of ropivacaine significantly inhibits sympathetic ganglion neurons in a dose dependent and potential dependent way through the inactivation of sodium channel,indicating that sympathetic ganglion neurons may contribute to the cardiotoxity of ropivacaine.
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Objective To investigate the effect of GABAB receptor in spinal cord by detecting semi-quantitatively the mRNA for isoforms of GABAB receptor in a chronic neuropathic pain model. Methods Twenty-four male SD rats weighing 200-250g were anesthetized with intraperitoneal pentobarbital. Lamina of L5 vertebra and superior articular process of L6 were removed and posterior root of left L5 spinal nerve was exposed and tightly ligated with 7/0 atraumatic suture. The animals were randomly divided into 4 groups of 6 animals in each group: group A in which animals were observed for 7 days after sham operation; group B in which animals were observed for 7 days after ligation of posterior root of L5 spinal nerve; in group C animals were observed for 14 days after sham operation and in group D animals were observed for 14 days after ligation. Pain threshold was measured by heat stimulation of bilateral hindlimbs before and on the 1st , 3rd , 7th , 10th and 14th day after operation. Lumbar spinal cord was removed on the 7th or 14th day after operation and mRNA for isoforms of GABAB receptor in the spinal cord was detected semi-quantitatively by digoxin hybridization in situ.Results Pain threshold of left hindlimb (ligated side) was significantly lower than that of right hindlimb on the 7th, 10th and 14th day after operation as measured by heat stimulation. There were less mRNA for three isoforms of GABAB receptor in group B and D as compared with group A and C respectively. Conclusion The gene expression of three isoforms of GABAB receptor in the spinal cord has plasticity and is reduced during chronic neuropathic pain.
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To compare the clinical effect and side-effect of intravenous PCA(PCIA) with epidural PCA (PCEA). Method: One hundred and eighty postoperative patients, who were randomly divided into three groups: group PCIA, group PCEA and control, were observed for 3 days after operation. Result: Overall patients in two PCA groups were satisfied with the postoperative analgesia. The incidence of urinary retention in group PCIA were significantly lower than that in group PCEA (P0.05). 10% in group PCIA were in medium sedation whereas no eases in group PCEA were in sedation. At 6th hour after operation,serum cortisol level of control patients was much higher than that of PCA patients. Conclusion=Both PCIA and PCEA have excellent analgesia and reduce stress response after operation. PClA has lower incidence of urinary retention and is performed easily,but inhibits gastrointestinal motility much more and has higher sedative incidence compared with PCEA.