The Effects Of Intra-articular Administration Of Botulinum Toxin Type A On The Rats With Osteoarthritis Pain And The Underlying Mechanisms

Objective:To observe the analgesic effect of botulinum toxin type A（BoNT/A）on MIA（Monoiodoacetate）induced osteoarthritis in rats,and to compare it with compound betamethasone injection,which is commonly used in clinical practice.In addition,the effects of intraarticular injection of botulinum toxin A on P2X4R and BDNF protein levels in spinal dorsal horn of rats with MIA-induced osteoarthritis were observed to explore the possible analgesic mechanism of botulinum toxin A on osteoarthritis.Methods:Sixty-nine SD rats were selected as the experimental subjects,6 rats were selected as the control group and the rest as the model group according to random number table method.The left ankle joint of the two groups of rats was intra-articular injected with 50μL normal saline and 8%MIA,respectively.After 21 days of modeling,rats with osteoarthritis pain were selected and divided into saline group,betamethasone group and BoNT/A group,with 15 rats in each group.25μL of normal saline,25μL of compound betamethasone,5IU botulinum toxin A was injected into the joint cavity,respectively.The body weigh,mechanical allodynia and weight bearing of the hind limbs of rats in both groups were conducted at 1 days prior to induction of osteoarthritis,3,7,14,21 days after induction of osteoarthritis,and 3,7,14,21 days following drug treatments.The protein levels of P2X4R and BDNF in L4-L6 spinal cord of rats in model group and each group were detected 1 day before modeling,3,7,14,21 days after modeling,and 7,14 days after administration.The expression of P2X4R protein in the left spinal dorsal horn of each group was observed by immunohistochemical staining 14 days after administration.Results:（1）Compared with the control group,the paw withdrawal threshold of of hind limbs and the percentage of weight bearing in the model group were decreased at the injection side（P<0.05）,and the difference lasted until 42 days after administration（P<0.05）.Compared with before the experiment,there were no significant changes in the paw withdrawal threshold and weight bearing percentage of the hind limbs of rats in the control group（P>0.05）.（2）Compared with the control group,the protein levels of P2X4R and BDNF in the spinal cord of rats in the model group were increased,and the differences began to show statistically on the 7th day after modeling（P<0.05）.（3）Compared with the saline group,the ipsilateral hindlimb PWT gradually increased after intraarticular betamethasone injection,with a statistically significant difference on day 7（P<0.001）,and then gradually decreased to the pre-injection level.The PWT of the ipsilateral hindlimb improved significantly 14 days after intraarticular injection of BoNT/A,the difference was statistically significant（P<0.001）,and the difference remained statistically significant 21 days after injection（P<0.001）.On day 14 and 21,the withdrawal threshold of the BoNT/A group was significantly higher than that of the betamethasone group(P₁₄<0.01,P₂₁<0.001).Compared with the saline group,there was no significant improvement in ipsilateral hindlimb loading in the betamethasone group,but loading in the BoNT/A group was significantly increased 7 days after intraarticular injection（P<0.001）,and this improvement in hindlimb loading was observed 21 days after administration（P<0.001）.On days 7,14,and 21,the percentage of weight bearing in the left hind limb of the BoNT/A group was significantly higher than that of the betamethasone group(P₇<0.05,P_14,21<0.001).（4）Compared with the saline group,the protein expression levels of P2X4R and BDNF in the spinal cord of betamethasone group were not significantly different on days 7 and 14,while the protein expression levels of P2X4R and BDNF in the spinal cord of the BoNT/A group were significantly decreased on days 7 and 14（P<0.01）.（5）Compared with the control group,the number of P2X4R positive cells in the spinal dorsal horn of the ipsilateral spinal cord was significantly increased in the saline group of betamethasone group of BoNT/A group（P<0.05）,Compared with saline group,the number of P2X4R positive cells in betamethasone group was not significantly different,while the number of P2X4R positive cells in BoNT/A group was decreased,and the difference was statistically significant（P<0.01）.Conclusion:（1）In MIA-induced osteoarthritis of rats,the production of pain may be related to the increased level of P2X4R receptor on microglia in spinal dorsal horn and the increased release of BDNF by microglia after the injury of the endings of intraosseous neurons in articular subchondral injection.（2）BoNT/A can significantly improve the mechanical hyperalgesia of rats and increase the percentage of weight bearing of affected limbs.In addition,this study found that the analgesic effect of BoNT/A was related to the protein expression of P2X4R in spinal dorsal horn and the down-regulation of BDNF protein level.（3）We compared BoNT/A with compound betamethasone and found that in this model,BoNT/A showed better analgesic effect,which may be related to this model may be more biased towards neuropathic pain model.