Correlation Study Of Nicotinic Acetylcholine Receptors(nAChR) And Neuregulin1(NRG1) Expression In The Skeletal Muscle Sample Of Rat With Spinal Cord Injury

Objective:The purpose of this study is on the basis of animal spasm due to the spinal cord injury(SCI) models has established, to improve behavior and electrophysiological evaluation system.The study was to detect nAChR distribution and expression of change in neuromuscular junction of rat skeletal muscle with spasms from spinal cord injury(SCI), while detecting the key signaling molecules-Neuregulin1(NRG1) and its receptor ErbB3, ErbB4expression in Neuregulin1/ErbB signal transduction pathways, from the tissue and the molecular level to explore the correlation between skeletal muscle nAChR. NRG1and its receptor ErbB3, ErbB4expression regulation and spasms of SCI in different spastic periods, Further research on the inner molecular mechanisms of spasm due to the spinal cord injury.Methods:60male SD adult rats, weight270-350g were chosen, randomly divided into4groups, each group contain15samples, the control group received no intervention, the SCI group applying Allen’s method to produce spasms were divided into3groups, the14day group, the21day group and the35day group after surgery. After SCI, make modified Ashworth, BBB score, as well as the associated with spasm nerve electrophysiology index test for bilateral hind limbs, to ensure that the spasm model is successful.The gastrocnemius was selected and tetramethyl rhodamine labelled α-BTX solution (TMR-α-BTX) and nAChR of neuromuscular junction specific binding were used, distribution and change of nAChR in endplate were observed under fluorescence microscope. Using double-immunofluorescence labeling assay, western blot analysis method (western blotting)and reverse transcription polymerase chain reaction (RT-PCR) to detect protein level and mRNA expression change of Neuregulin1(NRG1) and its receptor ErbB3, ErbB4.Results:1.The experimental group and control group nAChR distribution and expression change in skeletal muscle neuromuscular junction:After spinal cord injury (SCI), spasm of skeletal muscle of segment below injury section began to appear after a week (day-7), continuous progress after a week(day-14) to two weeks (day-21) reaches a steady state,during the steady spasm, nAChR increasingly expressed and gathered in the neuromuscular junction of skeletal muscle, compared with the control group was statistically significant difference (p<0.05), during the steady spasm(day-14, day-21and day-35after SCI), the expression of nAChR in skeletal muscle neuromuscular joint has no obvious change, no statistical differences between two groups (p>0.05).2.The key signaling molecules-Neuregulin1(NRG1) and its receptor ErbB3, ErbB4expression change in Neuregulinl/ErbB signal transduction pathways in skeletal muscle:NRG1mRNA and protein expressed increasingly in the skeletal muscle compared with control group (p<0.05),NRG1mRNA and protein expressed increasingly in the skeletal muscle of segment below injury section progressively increased reaching a peak at day-21after the SCI then significantly decreased (p<0.05)within day-35after the SCI,compared with the control group was statistically different (p<0.05).ErbB3, ErbB4mRNA and protein expression were upregulated after SCI compared with control muscles(day-O,baseline control), but the peak of expression of mRNA and protein were not synchronized, ErbB3mRNA expression progressively increased and is still upregulated over the further time points(day-35) after the SCI(p<0.05), protein expression showed a faster upregulation,that reached the maximum already at day-14after the SCI,then decreased within day-35after the SCI.ErbB4mRNA expression showed a faster upregulation,that reached the maximum already at day-14after the SCI,then decreased within day-35after the SCI. Protein expression progressively increased reaching a peak at day-21after the SCI and is still higher compared with baseline.Conclusion:1.Production of spasm after SCI may be associated with the expression and aggregation increase of nAChR in skeletal muscle neuromuscular junction.2.NRG1and its receptor ErbB3, ErbB4expressed increasingly in the spastic skeletal muscle after SCI, which may promote nAChR expressing and gathering in neuromuscular junction.