Transmembrane member 16 A(TMEM16 A) is involved in many physiological functions, such as epithelial secretion, sensory conduction, nociception, control of neuronal excitability, and regulation of smooth muscle contrac...Transmembrane member 16 A(TMEM16 A) is involved in many physiological functions, such as epithelial secretion, sensory conduction, nociception, control of neuronal excitability, and regulation of smooth muscle contraction, and may be important in peripheral pain transmission. To explore the role of TMEM16 A in the persistent hyperalgesia that results from chronic constriction injury-induced neuropathic pain, a rat model of the condition was established by ligating the left sciatic nerve. A TMEM16 A selective antagonist(10 μg T16 Ainh-A01) was intrathecally injected at L5–6. For measurement of thermal hyperalgesia, the drug was administered once at 14 days and thermal withdrawal latency was recorded with an analgesia meter. For measurement of other indexes, the drug was administered at 12 days,once every 6 hours, totally five times. The measurements were performed at 14 days. Western blot assay was conducted to analyze TMEM16 A expression in the L4–6 dorsal root ganglion. Immunofluorescence staining was used to detect the immunoreactivity of TMEM16 A in the L4–6 dorsal root ganglion on the injured side. Patch clamp was used to detect electrophysiological changes in the neurons in the L4–6 dorsal root ganglion. Our results demonstrated that thermal withdrawal latency was shortened in the model rats compared with control rats.Additionally, TMEM16 A expression and the number of TMEM16 A positive cells in the L4–6 dorsal root ganglion were higher in the model rats, which induced excitation of the neurons in the L4–6 dorsal root ganglion. These findings were inhibited by T16 Ainh-A01 and confirm that TMEM16 A plays a key role in persistent chronic constriction injury-induced hyperalgesia. Thus, inhibiting TMEM16 A might be a novel pharmacological intervention for neuropathic pain. All experimental protocols were approved by the Animal Ethics Committee at the First Affiliated Hospital of Shihezi University School of Medicine, China(approval No. A2017-170-01) on February 27, 2017.展开更多
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 stu...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.展开更多
Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain.We investigated whether the increase of NKCC1 and KCC2 is associa...Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain.We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons.To this aim,rats with persistent hyperalgesia were randomly divided into four groups.Rats in the control group received no treatment,and the rat sciatic nerve was only exposed in the sham group.Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide,an inhibitor of NKCC1,based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group.In the experiment measuring thermal withdrawal latency,bumetanide (15 mg/kg) was intravenously administered.In the patch clamp experiment,bumetanide (10 μg/μL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour,or bumetanide (5 μg/μL) was intrathecally injected.The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats.We found that the thermal withdrawal latency of rats was significantly decreased on days 7,14,and 21 after model establishment.After intravenous injection of bumetanide,the reduction in thermal retraction latency caused by model establishment was significantly inhibited.Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7,14,and 21 after model establishment.No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment.The Cl^– (chloride ion) fluorescent probe technique was used to evaluate the change of Cl^– concentration in dorsal root ganglion neurons of chronic constriction injury model rats.We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl^– fluorescent probe whose fluorescence Cenintensity decreases as Cl– concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment.The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased,and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment.After bumetanide administration,the above indicators were significantly suppressed.These results confirm that CCI can induce abnormal overexpression of NKCC1,thereby increasing the Cl^– concentration in dorsal root ganglion neurons;this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia.In addition,bumetanide can achieve analgesic effects.All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital,College of Medicine,Shihezi University,China on February 22,2017 (approval No.A2017-169-01).展开更多
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 follo...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.展开更多
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),Gr...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.展开更多
基金supported by the National Natural Science Foundation of China,No.30160026(to JQS)the High Level Talent Research Project of Shihezi University of China,No.RCSX201705(to YW)
文摘Transmembrane member 16 A(TMEM16 A) is involved in many physiological functions, such as epithelial secretion, sensory conduction, nociception, control of neuronal excitability, and regulation of smooth muscle contraction, and may be important in peripheral pain transmission. To explore the role of TMEM16 A in the persistent hyperalgesia that results from chronic constriction injury-induced neuropathic pain, a rat model of the condition was established by ligating the left sciatic nerve. A TMEM16 A selective antagonist(10 μg T16 Ainh-A01) was intrathecally injected at L5–6. For measurement of thermal hyperalgesia, the drug was administered once at 14 days and thermal withdrawal latency was recorded with an analgesia meter. For measurement of other indexes, the drug was administered at 12 days,once every 6 hours, totally five times. The measurements were performed at 14 days. Western blot assay was conducted to analyze TMEM16 A expression in the L4–6 dorsal root ganglion. Immunofluorescence staining was used to detect the immunoreactivity of TMEM16 A in the L4–6 dorsal root ganglion on the injured side. Patch clamp was used to detect electrophysiological changes in the neurons in the L4–6 dorsal root ganglion. Our results demonstrated that thermal withdrawal latency was shortened in the model rats compared with control rats.Additionally, TMEM16 A expression and the number of TMEM16 A positive cells in the L4–6 dorsal root ganglion were higher in the model rats, which induced excitation of the neurons in the L4–6 dorsal root ganglion. These findings were inhibited by T16 Ainh-A01 and confirm that TMEM16 A plays a key role in persistent chronic constriction injury-induced hyperalgesia. Thus, inhibiting TMEM16 A might be a novel pharmacological intervention for neuropathic pain. All experimental protocols were approved by the Animal Ethics Committee at the First Affiliated Hospital of Shihezi University School of Medicine, China(approval No. A2017-170-01) on February 27, 2017.
基金supported by grants from the National Natural Science Foundation of China(No.30160026)the Youth Science and Technology Innovation Special Foundation of Xinjiang Production and Construction Corps,China(No.2010JC33)
文摘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.
基金supported by the National Natural Science Foundation of China,No.30160026(to JQS)the High Level Talent Research Project of Shihezi University of China,No.RCSX201705(to YW)
文摘Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain.We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons.To this aim,rats with persistent hyperalgesia were randomly divided into four groups.Rats in the control group received no treatment,and the rat sciatic nerve was only exposed in the sham group.Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide,an inhibitor of NKCC1,based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group.In the experiment measuring thermal withdrawal latency,bumetanide (15 mg/kg) was intravenously administered.In the patch clamp experiment,bumetanide (10 μg/μL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour,or bumetanide (5 μg/μL) was intrathecally injected.The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats.We found that the thermal withdrawal latency of rats was significantly decreased on days 7,14,and 21 after model establishment.After intravenous injection of bumetanide,the reduction in thermal retraction latency caused by model establishment was significantly inhibited.Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7,14,and 21 after model establishment.No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment.The Cl^– (chloride ion) fluorescent probe technique was used to evaluate the change of Cl^– concentration in dorsal root ganglion neurons of chronic constriction injury model rats.We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl^– fluorescent probe whose fluorescence Cenintensity decreases as Cl– concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment.The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased,and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment.After bumetanide administration,the above indicators were significantly suppressed.These results confirm that CCI can induce abnormal overexpression of NKCC1,thereby increasing the Cl^– concentration in dorsal root ganglion neurons;this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia.In addition,bumetanide can achieve analgesic effects.All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital,College of Medicine,Shihezi University,China on February 22,2017 (approval No.A2017-169-01).
文摘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.
文摘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.
