| Spinal cord injury(SCI)is a familiar disease with a high disability rate and mortality,which seriously threatens human health.With the continuous development of the economy and transportation industry,the incidence of SCI tends to increase.Approximately 70% of patients with SCI will have spasm.Spasticity is a motor dysfunction characterized by speed-dependent tonic stretch reflex(muscle tone)enhancement and tendon hyperreflexia.Spasticity can cause many problems such as muscle fibrosis,pain,joint stiffness.Severe cramps will increase nursing expenditures and adversely affect patients’ lives and functions.Spasticity is a medical problem that needs to be solved urgently.Nowadays,there are many treatment methods for spasm after SCI,which have a certain effect on the relief of spasm,but there are some problems.In order to achieve a better treatment of spasticity,the research on the mechanism of spasticity and the search for new drug targets are particularly critical.The mechanism of spasticity after SCI is complicated and unclear.Some studies believe that one of the important mechanisms of spasticity is that the monoamine neurotransmitter serotonin after SCI generates a continuous inward current by activating its receptors,so that motor neurons continuously discharge and cause spasticity.Literature reports and previous studies of this group have found that SCI can induce phenotypic changes in aromatic amino acid decarboxylase cells and use exogenous 5-hydroxytryptophan to synthesize 5-HT.Synthetic 5-HT can increase the excitability of motor neurons and cause spasm.It is found that the mechanism of 5-HT production by AADC cells after SCI is related to 5-HT1’s auto-receptor 5-HT1 B receptor,which plays a negative feedback regulation effect(auto-receptor 5-HT1 A receptor does not play a role).But activation of 5-HT1 B receptor only inhibit the activity of AADC cells by29%.We speculate that other 5-HT1 receptors such as 5-HT1 D receptor may also participate in this process.At present,there is no literature report on the characteristics of 5-HT1 D receptor expression at different time points after SCI.The correlation between the development of spasticity and 5-HT1 D receptor expression at different time points after SCI has not been reported in the literature.And Whether 5-HT1 D receptors are involved in the changes in AADC cell function after spinal cord injury is still unclear.In this study,a rat model of spinal cord sacral 2 full transection tail spasticity was established.Focus on observation of rat tail spasm score and 5-HT1 D receptor expression characteristics at different time points 2d,5d,14 d,28d and 60 d after SCI;Correlation was analyzed between the spasm score and5-HT1 D expression at different time points after SCI;explore whether the 5-HT1 D receptor is involved in regulating the spasm mechanism of 5-HT synthesis by AADC cells after SCI.Through this research,we hope to lay a foundation for the in-depth study of the mechanism of spasticity after SCI and provide new ideas for the clinical targeted treatment of spasticity after SCI.Objective:Through the establishment of a rat spinal cord S2 complete transectioninduced spasticity model,the characteristic behavioral changes of rat tail spasticity and 5-HT1 D receptor expression in spinal cord at different time points after SCI were studied at different time points after SCI were studied,and analyze the relationship between the development of spasticity and the expression of 5-HT1 D receptors.Explore whether the 5-HT1 D receptor is involved in the changes in AADC cell function to produce 5-HT.The purpose is for the further study of the mechanism of spasticity after SCI and provide new ideas for the clinical targeted treatment of spasticity after SCI.Methods:There were 164 healthy male Wistar rats with a body weight of(200±20)g.Among them,84 rats were randomly selected for spinal cord S2 total transection(SCI),66 rats were subjected to sham surgery(Sham),and 14 normal rats.1.2,5,14,28,60 days after SCI,12 rats from both sham and SCI group were selected randomly.First,the rat tail spasm score was performed,extract all rats S3～tail 2(Caudal 2,Ca2)fresh spinal cord tissue below S2.Then 5-HT1 D receptor expression was tested by Western blotting(n=6)and q RT-PCR(n=6).2.Correlation between rat tail spasm score and 5-HT1 D receptor expression were analyzed.3.60 days after SCI,SCI rats were scored for tail spasm.6 SCI rats with scores of 4～5 and 6 rats in the Sham group were selected randomly.After paraformaldehyde perfusion,the spinal cord below the S2 injury plane was taken for 5-HT1 D receptor Immunofluorescence detection.Another 2 healthy rats were used only for 5-HT1 D receptor immunofluorescence control staining.4.60 days after the operation,the rats were scored for tail spasm,and 12 SCI rats with scores of 4～5 were randomly selected.Among them,6 SCI rats served as the 5-HT1 D receptor agonist group,and 6 SCI rats served as the 5-HT1 D receptor agonist control group.Then 12 healthy rats were selected randomly.Among them,6 were used as 5-HT1 D antagonist group and 6 were used as 5-HT1 D antagonist control group.The 5-HT1 D receptor agonist group and the control group were injected subcutaneously with the agonist L694247 and normal saline for 8 consecutive days.The 5-HT1 D antagonist group and the control group were injected subcutaneously with the antagonist LY310762 and normal saline for 8consecutive days.Subsequently,each rat received intraperitoneal injection of 5-HTP.The immunofluorescence double-labeling method was used to detect the expression rate of 5-HT positive cells in AADC cells in the spinal cord below the S2 injury plane.Results:1.The test results of the rat tail spasticity score at different time points after SCIAt 2d,5d,14 d,28d,and 60 d postoperatively,the tail spasm scores of rats in the Sham group were 0.17±0.02,0.19±0.02,0.22±0.02,0.22±0.01,0.21±0.01;tail spasm of rats in the SCI group was evaluated as 0.00±0.00,0.59±0.05,1.62±0.05,3.09±0.22,4.42±0.14.2.Results of 5-HT1 D receptor protein and 5-HT1 D receptor m RNA at different time points after SCIAt different time points 2d,5d,14 d,28d,60 d,the relative levels of protein in the spinal cord of the Sham group were 0.63±0.06,0.59±0.04,0.59±0.04,0.64±0.13,0.60±0.05;the relative levels of protein in the spinal cord of the SCI group were 0.52±0.06,0.33±0.09,0.37±0.05,0.50±0.09,0.52±0.05.At different time points,compared with the Sham group,the relative levels of 5-HT1 D receptor protein in the spinal cord of the SCI group decreased(P<0.05).At different time points 2d,5d,14 d,28d,60 d,the relative levels of 5-HT1 D receptor m RNA in the spinal cord of the Sham group were 1.02±0.09,1.04±0.07,1.05±0.06,1.01±0.07,1.07±0.11;the relative levels of 5-HT1 D receptor m RNA in the spinal cord of the SCI group were 0.28±0.04,0.50±0.06,0.56±0.04,0.69±0.03,0.79±0.06.At different time points,compared with the Sham group,the relative level of 5-HT1 D receptor m RNA in the spinal cord of the SCI group decreased(P<0.05).3.Correlation analysis resultsWith the prolonged operation time,Score of tail spasm was correlated with5-HT1 D receptor protein level positively(r=0.97,P<0.05),Rat tail spasm score was correlated with 5-HT1 D receptor m RNA positively(r=0.95,P<0.05).4.Detection of 5-HT1 D receptor expression in rat spinal cord by immunofluorescence staining(1)Distribution of 5-HT1 D receptors: 5-HT1 D receptors are in the spinal cord S3～Ca2 of normal rats,and are widely distributed in spinal cord DH,IMZ and VH.The 5-HT1 D receptors in superficial DH are mainly in the fibers,and5-HT1 D receptors can be seen in neurons in the DH,IMZ and VH regions.The distribution of 5-HT1 D receptors in the spinal cord S3～Ca2 of rats in the Sham and SCI groups was similar to that of the normal rats.(2)Observation and measurement results of 5-HT1 D receptor in the spinal cord of rats 60 days after SCI: The measurement result of 5-HT1 D receptor in spinal cord gray matter in Sham group was 22.55±1.13.The measurement result of 5-HT1 D receptor in spinal cord gray matter of rats in SCI group was13.33±0.50.Compared with the Sham group,the 5-HT1 D measurement value in the spinal cord gray matter of the SCI group decreased(P<0.05).Measurement results of 5-HT1 D receptors in three different areas of DH,IMZ and VH in the gray matter of the spinal cord of rats 60 days after SCI: The measured values of 5-HT1 D receptor in the DH,IMZ and VH regions of spinal cord gray matter in the Sham group were 21.91±1.70,23.72±1.31,22.91±1.51.The measured values of 5-HT1 D receptor in the DH,IMZ and VH regions of spinal cord gray matter in the SCI group were 13.15±0.53,13.83±0.83,13.66±0.62.Compared with the Sham group,the 5-HT1 D measurement values of DH,IMZ and VH in the spinal cord gray matter of the SCI group were reduced(P<0.05).5.5-HT expression in AADC cells after 5-HT1 D receptor agonist and antagonist intervention(1)Distribution and expression of AADC cells:Distribution style of AADC cells in spinal cord below S2 segment were similar among 5-HT1 D receptor antagonist group,5-HT1 D receptor antagonist control group,5-HT1 D agonist group and 5-HT1 D agonist control group.They are mainly distributed in the DH,IMZ,and CC areas around the gray matter of the spinal cord.The AADC cells in the DH area are the most dense.There was no statistical difference in the total number of AADC positive cells in the four groups(P>0.05).(2)After 5-HT1 D receptor antagonist interferes with normal rats,AADC cells in the spinal cord below the S2 segment express 5-HT positive cells:5-HT1 D receptor antagonist group and control group: In the S3～Ca2segment of the spinal cord of rats in the 5-HT1 D receptor antagonist group,it can be seen that the co-existence rate of AADC cells and 5-HT was(1.31±0.15)%.No 5-HT positive cells were seen in the spinal cord of the control group,only 5-HT fibers were seen.It can be seen that the co-existence rate of AADC cells and 5-HT was(0.00±0.00)%,and there was a significant statistical difference between the two groups(P<0.05).(3)After 5-HT1 D receptor stimulants interfere with chronic spinal cord injury in rats,AADC cells in the spinal cord below the S2 segment express 5-HT positive cells:5-HT1 D receptor stimulant group and control group: In the spinal cord below the S2 segment of the spinal cord,the 5-HT1 D receptor stimulant group and the control group had 5-HT positive expression rates in AADC cells,respectively(12.97±1.60)% And(46.06±7.70)%.There are statistical differences between the two groups(P<0.05).Conclusion:1.At different time points(2d,5d,14 d,28d,60d)after complete spinal cord S2 injury in rats,5-HT1 D receptors were reduced.2.The expression changes of 5-HT1 D receptors are positively correlated with the development of rat tail spasticity after SCI.3.Below the S2 segment of the spinal cord of normal,Sham and SCI rats,5-HT1 D receptors are not only in the superficial DH fibers of the spinal cord,but also in the neurons of DH,IMZ and VH.4.The 5-HT1 D receptor regulates the 5-HT production mechanism of AADC cells after SCI through negative feedback. |
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