Study On 5-HT Produced By AADC Cells At Different Time Points Following Spinal Cord Injury In Rats

With the rapid development of China's social economy and transportation industry,the incidence of spinal cord injury?SCI?has been increasing year by year.Due to the high rate of disability and mortality,it causes a huge econom-ic burden on patients and society.Most patients with SCI may present with obvious spastic symptoms in later stages,which seriously affect the quality of life of patients.The mechanism of spasticity is more complicated,one of the important mechanisms is that motor neurons have increased sensitivity to monoamine neurotransmitter such as 5-hydroxytryptamine?5-HT?.5-HT con-stitutively active receptors which in concert increase the excitability of motor neurons by facilitating the PICs?persistent inward currents,PICs?.PICs pro-duce involuntary muscle contractions by maintaining motor neuron discharge?spasticity?.In the spinal cord of mammals,almost all monoamine neuro-transmitters such as 5-HT,noradrenalin?NA?and dopamine?DA?are mainly derived from brain descending fibers.The study found that after a complete spinal cord injury,the spinal cord below the injury level still contains a small amount of monoamine neurotransmitters such as 5-HT?about 2-15%of the normal level?.It has been reported that the sensitivity of motoneurons to 5-HT is increased after complete spinal cord injury,so these small amounts of 5-HT are sufficient to maintain the normal activity of motoneurons and even in-crease their excitability to produce spasticity.After the spinal cord is com-pletely transected,the descending brain-derived monoamine neurotransmitters in the spinal cord have been completely removed.The experimental study found that these small amounts of neurotransmitters in the spinal cord below the lesion level originate from the Aromatic L-amino acid decarboxylase?AADC?cells,AADC cells produce 5-HT after phenotypic changes and are an important source of 5-HT.AADC is widely distributed in peripheral tissues and brain tissues of the central nervous system.The study found that AADC plays an important role in the development and motor function of the brain.Children with AADC deficiency may experience symptoms such as develop-mental delay,abnormal motor function,and autonomic dysfunction.Since AADC?also known as dopamine decarboxylase?decarboxylates levodopa?L-dopa?to dopamine?DA?in the brain,increasing AADC enzyme activity is a potential target for L-dopa replacement therapy for Parkinson's disease and in the Parkinson animal model AADC gene therapy has a significant effect,it has now entered the preclinical phase of the trial.Therefore,in this study,the expression characteristics of AADC cells and the expression characteristics of 5-HT were studied at different time points af-ter thoracic T10-11 spinal cord injury.Objectives:By studying the distribution of AADC cells before and after SCI and the characteristics of AADC cells expressing 5-HT cells at different time points after SCI,the role of AADC in SCI was revealed,which laid the foundation for further research on the mechanism of spasticity after SCI.Methods:In this study,90 healthy male Wistar rats[SPF level,body weight?200±20?g]adapted to feed for 1 week,randomly selected 52 rats were sub-jected to detach spinal cord of thoracic T10-11?1-3mm?,ensuring that the transverse transection was complete and the spinal dorsal vein without damage.After ensuring it,suture the muscle layer and skin layer.Rats were randomly divided into normal control group?12 rats?,spinal cord injury 2d group 24 rats?12 rats in 2d Sham,12 rats in SCI 2d?,SCI 5d group?12 rats?,SCI 15d group?12 rats?and spinal cord injury 60d group 24 rats?12 rats in Sham 60d,12 rats in SCI 60d?.Another with 2 normal rats?2 SCI2d rats and 2 SCI60d rats were used for control study.Postoperative nursing of SCI rats and assisted urination by people were performed until the rats resumed spontaneous urination.The BBB scores of SCI rats at different time points after surgery were used to evaluate the hindlimb motor function of SCI rats.At the corresponding time points,perfusion was performed and exogenous 5-HTP was given 30 minutes before perfusion.After tissue processed,the spinal cord was divided into lumbar segment?L1-L6?and sacrocaudal segment?S1-Ca3?to slice.Rats in the control study were not given exogenous 5-HTP.The other methods were the same as above.Immunofluorescence was used to detect the expression of AADC and 5-HT in the spinal cord below the injured segment.Experimental data were expressed as mean±standard deviation?±s?,SPSS19.0 software was used for statistical analysis.One-way analysis of variance was used for comparison between groups.P<0.05 was statistically significant.Results:1.Number of assisted urination of rats at different time points after SCI:Within 14 days after surgery,the number of assisted urination was maintained at 1-3times/day,and urination was generally no longer supported after 15 days after surgery.2.On the 1st,3rd,7th,14th,28th,and 60th day after operation,the SCI rats were evaluated as 0.00±0.00,1.00±0.58,3.30±0.95,6.30±0.10,7.50±0.36,7.87±0.08 respectively.3.Expression of AADC cells before and after SCI and changes in the ex-pression of AADC cells.AADC cells are mainly expressed in the spinal dorsal horn,intermediate zone and the surrounding of the central canal;Furthermore,there was no sig-nificant change in the distribution of AADC cells before and after spinal cord injury.In the spinal cord lumbar segment?L?in the normal spinal cord,SCI 2d group,Sham 2d group,SCI 5d group,SCI 15d group,SCI 60d group and Sham 60d group,the number of AADC cells was 46.75±5.50,49.50±4.87,46.17±4.59,48.33±5.55,47.17±5.46,48.50±6.37 and 49.17±3.95.In rat sac-rocaudal?S+C?segment,the number of AADC cells was 1026.50±66.59,1066.75±80.56,1030.25±57.48,1041.25±63.24,1037.50±60.38,1046.25±67.79 and 1051.42±54.32,there was no significant difference between the groups?P>0.05?.4.Characteristics of AADC cells expressing 5-HT cells after different time points after SCI.4.1 Normal rats,2d injured rats and 60d injured rats did not express5-HT in AADC cells of spinal cord below the injured level when exogenous5-HTP was not administered.4.2 When exogenous 5-HTP was administered,there was no expression of 5-HT in AADC cells in the L and S+C segments of normal control rats.4.3 When exogenous 5-HTP was administered,there was no expression of 5-HT in AADC cells in L segment and S+C segment in Sham 2d and Sham60d rats.4.4 When exogenous 5-HTP was administered,the positive expression rates of 5-HT in L segment and S+C segments of SCI 2d rats were?44.43±6.03?%and?44.45±5.71?%respectively.4.5 When exogenous 5-HTP was administered,the positive expression rates of 5-HT in L segment and S+C segments of SCI 5d rats were?97.22±2.03?%and?95.05±2.78?%respectively.4.6 When exogenous 5-HTP was administered,the positive expression rates of 5-HT in L segment and S+C segments of SCI 15d rats were?97.61±1.56?%and?98.57±1.61?%respectively.4.7 When exogenous 5-HTP was administered,the positive expression rates of 5-HT in L segment and S+C segments of SCI 60d rats were?96.20±1.53?%and?95.14±3.02?%respectively.Conclusions:1.The number and location of AADC cells were similar before or after spinal cord thoracic T10-11 injury,the number of AADC cells in L segment was less than that of S+C segments.2.After injury of rat thoracic spinal cord T10-11,AADC cells in the spi-nal cord below the lesion increased their ability to produce 5-HT from exoge-nous 5-HTP.3.After injury of rat thoracic spinal cord T10-11 at different time points,the trend is that increased AADC cell ability to produce 5-HT,which was sim-ilar to the change trend of spinal cord S₂ after injury.4.The expression of 5-HT in AADC cells in the spinal core below the le-sion could be observed at 2 days post-SCI,the increased peak reached at 5days post-SCI and entered the plateau phase.The plateau period lasted for 60days after the longest period of the study.