The Neural Pathway Innervating Diaphragm After End-to-side Anastomosis Of Phrenic Nerve And Vagus Nerve To Restore Respiratory Function In Rats With Cervical Spinal Cord Injury

Objective: Cervical spinal cord injuries can result in paralysis of the diaphragm and impaired breathing.These patients often have a poor quality of life and a high mortality rate but with no effective treatments.In our previous study,the function of diaphragm in rats with cervical spinal cord injury was reconstructed by end-to-side anastomosis of phrenic nerve(PN)and vagus nerve(VN),but we knew nothing about the mechanism.The purpose of this study is to compare the neural pathways innervating the diaphragm between normal rats and nerve-transferred rats so as to provide a basis for future researches and clinical treatments.Method: The rat model was constructed by end-to-side anastomosis from phrenic nerve to vagus nerve.The effectiveness of respiratory function after nerve transfer was evaluated by C2 transection test.The vagus nerve and phrenic nerve of normal rats as well as the phrenic nerve of nerve-transfered rats were injected with adeno-associated virus(AAVRetro-GFP)to identify the primary motor neurons innervating the diaphragm.Similarly,pseudorabies virus(PRV-CMV-EGFP)which could retrograde through multiple synapses was injected into these nerves to label the nerve centers of cerebral cortex and anterior motor neurons.These nerve centers labeled by AAV and PRV were identified by immunohistochemical staining and immunohistochemical double labeling.By comparing the neural pathways innervating diaphragms between mormal rats and nerve-transferred rats,this study could preliminarily clarify the mechanism of nerve transfer to reconstruct respiratory function.Results:(1)The rat model suffering end-to-side anastomosis from phrenic nerve to vagus nerve was established.After C2 spinal cord transection,the survival time of rats in the anastomosis group was significantly longer than that in the control group,which proved that nerve transfer was effective to reconstruct respiratory function.(2)The primary motoneurons innervating the vagus nerve of normal rats were located in the dorsal vagal nucleus and nucleus ambiguus.The premotoneurons were located in contralateral dorsal vagal nucleus,nucleus tractus solitarius,nucleus spinalis nervi trigemini,intermediate reticular nucleus,the ventral portion of gigantocellular intermediate reticular nucleus,nucleus raphe obscurus,nucleus raphes pallidus,area postrema and adrenergic/noradrenergic neurons(A1/C1,A2/C2,C3,A5,A6).The advanced center of cerebral cortex is located in infralimbic cortex,lateral septal nucleus-intermediate part,agranular insular cortex,olfactory tubercle,piriform cortex.(3)The primary motoneurons innervating the diaphragm of normal rats were located in the ventral horn of C4-6 spinal cord.The premotoneurons were located in rostral ventral respiratory group,pre-Botzinger complex,parafacial respiratory group,nucleus tractus solitarius,nucleus spinalis nervi trigemini,Intermediate reticular nucleus,the ventral portion of gigantocellular intermediate reticular nucleus,nucleus raphe obscurus,nucleus raphes pallidus,area postrema,Adrenergic/noradrenergic neurons(A1/C1,A2,C3,A5,A6).The advanced center of cerebral cortex is located in primary motor cortex,secondary motor cortex,prelimbic cortex,agranular insular cortex,lateral septal nucleus-intermediate part,substantia innominate,granular insular cortex.(4)The primary motoneurons innervating the diaphragm in rats suffering PN-VN anastomosis were located in the dorsal vagal nucleus and nucleus ambiguus.The premotoneurons were located in contralateral dorsal vagal nucleus,nucleus tractus solitarius,nucleus raphe obscurus,nucleus raphes pallidus,area postrema and adrenergic/noradrenergic neurons(A1/C1,A2/C2,C3,A5,A6).The advanced center of cerebral cortex is located in infralimbic cortex,amygdalopirform transiton area/piriform cortex,primary motor cortex,secondary motor cortex,prelimbic cortex,agranular insular cortex,lateral septal nucleus-intermediate part,granular insular cortex and cingulate cortex-area1.Conclusion: The diaphragm function in rats with cervical spinal cord injury was reconstructed successfully by end-to-side anastomosis of phrenic nerve(PN)and vagus nerve(VN).Nerve transfer led to neural plasticity in cerebral cortex and reconstructed the connection between diaphragm and original area in cerebral cortex innervating diaphragm.Eventually,the new neural pathway “Original diaphragm cortex area—vagus cortex area—vagus premotor neurons—vagus motoneuron—vagus nerve—phrenic nerve—diaphragm ” was formed.