Relationship Between Neuropathic Pain And Protein Nitrosation In Rats

Objective1.A rat model of neuropathic pain(SNI)was established,which provides a good basis for the study of the physical and chemical factors leading to the neuropathic pain.2.To study the changes in the degree of mercapto-nitroso occurring at the global level in the corresponding region of neuropathic pain.3.Normal SD rats were given intrathecal administration of different concentrations of No donor(GSNO)or precursor to observe the changes of behavior and protein nitrite level in rats.4.Whether the use of reagents that can reverse the occurrence of mercaptoso nitrite can significantly reduce the hyperalgesia in rats with neuropathic pain.Methods1.The male SD rats weighing 300-350 g were selected to establish SNI animal model,that is,the common peroneal nerve and tibial nerve,which are the branches of the right sciatic nerve after ligation,were preserved and the sural nerve was preserved so as to avoid traction or injury.The sham-operation group as the control group,protected all nerves and branches from pulling or injury after nerve exposure,while the rest were the same as the SNI group.After observing the pain behavior of rats,dynamic plantar tactile was used to quantify the degree of mechanical pain in right foot of rats in each group every day,and the threshold of 50% foot contraction response was compared.The PWT=10log(x)+kδ,PWT was less than 4g as the standard of mechanical pain sensitivity.The model failure will exclude the experiment and Western blot will be used to detect the nitrosation level of all proteins.2.Male SD rats weighing 300-350 g were placed in subarachnoid space.After anaesthesia,the rats were treated with a side approach.Connect the line parallel to the point to the beginning of the caudal vertebra in the iliac spine connection on both sides,side open 0.5cm,the tip of the catheter enters the subarachnoid space about 2cm,one end of the catheter is fixed to the fascia layer,the other end of the catheter is led to the back neck of the rat to be fixed on the skin,and the heat seal is closed for standby.Rats with no abnormal hindlimb movement were selected after waking and were given 2% lidocaine by catheter for 10 μl.If the paralysis of hind limbs proved the success of intrathecal catheterization,it could be used for experiment one week after recovery.3.Thirty-six male SD rats weighing 300-350 g were selected to make SNI animal model.After observing the pain behavior of the rats,they were randomly divided into 6 groups with 6 rats in each group,and each experimental group was established: Group N(normal group);Group S(sham-operation group):right lower extremity nerve was not ligated or transected in rats;In group SNI,they were divided into D1 group according to the number of days,group D4,group D7 and group D14.On the 14 th day,6 rats were killed in each group.Take out the lumbar enlargement of spinal cord tissue and store it.4.Male SD rats weighing 300-350 g were placed intrathecal tube and recovered for 7 days.Twenty-four rats were randomly divided into 4 groups according to intrathecal administration of different concentrations of no donor GSNO,with 6 rats in each group.Each experimental group was established:Control group O(DMSO 10 μl);Group A(GSNO 10μg/10μl);Group B(GSNO30μg/10μl);Group C(GSNO 90μg/10μl).Intrathecal injection of the above drugs was carried out every day.On the 7th day after continuous administration,therats in each group were killed.Take out the lumbar enlargement of spinal cord tissue and store it.5.Male SD rats weighing 300-350 g were placed intrathecal tube and recovered for 7 days.SNI animal models were made in 18 rats.After observing the pain behavior of the rats,they were randomly divided into 3 groups,6 rats in each group,and each experimental group was established: Group O(control group): Intrathecal administration of 100% DMSO 10μl;NEM group: intrathecal administration of NEM 100μg/10μl,nitroso blocker;ODQ group: intrathecal administration of ODQ 30μg/10μl,PKG pathway blocker;Intrathecal injection of the above drugs was carried out every day.On the 7th day after continuous administration,the rats in each group were killed.Take out the lumbar enlargement of spinal cord tissue and store it.6.The degree of mechanical pain of rats in each group was quantified daily by dynamic plantar tactile meter and compared with the level of nitrosation of all proteins detected by Western blot.7.Detection of total protein in spinal dorsal horn: Under the condition of shading,the spinal cord of rat L4-L6(lumbar enlargement)was lysated and centrifugated to obtain supernatant.Biotinylated protein was denatured by biotin transformation.The biotin protein was extracted from the lytic solution by streptavidin-agarose,and the samples were prepared.Results1.SNI model SD rats showed spontaneous pain and hyperalgesia after operation with mild lateral foot valgus.The model SD rats developed obvious pain on the 4th day after operation.The threshold value of 50% foot contraction was significantly decreased,and there was significant difference between the two groups(P < 0.05).This is clear the model of neuropathic pain wasestablished successfully.The threshold value of 50% foot contraction decreased to a peak on the 7th day after operation and lasted until 28 days after operation.2.SD rats were given different concentrations of no donor GSNO.The threshold of 50% contraction of foot began to increase at the concentration of GSNO 30μg/10μl in each group.The decrease trend of protein nitrosation level on the whole level is obvious,significant statistical significance(P < 0.001).3.SNI model SD rats were given intrathecal tube with DMSO,blocker NEM and ODQ respectively.The mechanical pain threshold and the level of nitroso in all proteins in each group were compared: The mechanical pain value of rats treated with blocker NEM was significantly increased,and the total protein nitrosation was significantly decreased,which was statistically significant(P < 0.001).The content of PKG pathway protein in ODQ was also decreased,which was statistically significant(P < 0.05).Conclusions1.The degree of protein nitrosation at the global level is positively correlated with neuropathic pain.2.Starting from a certain concentration of no,the protein can be nitrosated.3.Nitroso blocking agent NEM and PKG pathway blocker ODQ have therapeutic effects on neuropathic pain,and NEM is more effective.