| Part I Reconstruction of Respiratory Function by Nerve Transfer in Rats with High Cervical Spinal Cord InjuryObjective:High cervical spinal cord injury often leads to diaphragmatic paralysis and respiratory dysfunction,resulting in a high mortality rate.Our research team focused on the respiratory function reconstruction in patients with high cervical spinal cord injury,and successfully reconstructed the diaphragmatic movement in rats with high cervical spinal cord injury through the phrenic-vagus nerve transfer technique.This part aims to demonstrate the feasibility of nerve transfer to restore diaphragmatic function after high cervical spinal cord injury.Method:A rat model of end-to-side anastomosis between vagus nerve and phrenic nerve was constructed.After 6 months of nerve transfer,spinal cord injury model was established at C3 level.We compared the survival time after high cervical spinal cord injury in the sham group and the nerve transfer group;Compound muscle action potential(CMAP)was used to assess the recovery of the diaphragm;The respiratory motility was observed by X-ray fluoroscopy;Toluidine blue staining quantified nerve regeneration by counting myelinated nerve fibers.Results:(1)End-to-side anastomosis of the vagus nerve-phrenic nerve was performed with a 12-0 suture under a 10 x surgical microscope.Six months after surgery,the rats were treated with high cervical spinal cord injury at C3 level.The survival time of rats in the nerve transfer group was significantly higher than that in the control group(6.87 ± 3.36 min vs.6.63 ± 3.11 h);(2)The compound muscle action potential(CMAP)of the diaphragm showed that the latency(Lat)gradually shortened and the amplitude gradually increased after nerve transfer;(3)The respiratory motility of rats was observed by chest X-ray.The results showed that the rats in the control group could only survive a few minutes after high cervical spinal cord injury,as well as rats in the nerve transfer group could breathe without abnormality after high cervical spinal cord injury(respiratory mobility 0.54±0.06cm);(4)Toluidine blue staining was used to observe the regeneration of myelinated nerve fibers.The results showed that the regeneration of myelinated nerve fibers was basically complete 6 months after nerve transfer.However,compared with the normal group,the myelin sheath in the transfer group was thinner and different in size,and arranged disorderly.At the same time,there was no statistical difference in the number of myelinated nerve fibers between the proximal and distal end of the donor nerve(vagus nerve),indicating that endto-side anastomosis had little disturbance to the donor nerve.Conclusion:The respiratory function of rats with high cervical spinal cord injury can be effectively reconstructed by phrenic nerve-vagus nerve end-to-side transfer.End-to-side anastomosis has a good nerve regeneration effect without damaging the important function of donor nerve.Part II Cortical Neuronal Circuit Remodeling During Peripheral Nerve TransferObjective:Numerous studies have shown that the cerebral cortex needs to be readapted or relearned after peripheral nerve injury and nerve transfer.At present,most of the studies on the reconstruction of respiratory function by phrenic nerve transfer focus on the selection of donor nerve and the functional recovery of target organs.It is not clear whether the reconstruction of respiratory function by phrenic nerve transfer can trigger extensive cortical functional reorganization.In this part,we compare the nerve centers at all levels innervating the diaphragm before and after nerve transfer to elucidate the changes in neural circuits after nerve transfer.Method:Pseudorabies virus(PRV)was used to infect the phrenic nerve and vagus nerve of rats in control group and nerve transfer group,respectively,to locate the cortical centers innervating the diaphragm and clarify the remodeling of neural circuits;resting-state functional magnetic resonance imaging(rs-f MRI)was performed to discover the differences in the excitatory areas of the cortical center after nerve transfer.Results:(1)Retrograde tracing of PRV showed that the virus was retrogradely infected through the phrenic nerve to the vagus nerve,the lower center of vagus nerve,and the cortical center of vagus nerve,eventually reaching the cortical center innervating the phrenic nerve(primary motor cortex).In other words,after nerve transfer,a new neural circuit is formed between the originally unconnected phrenic cortical center and the vagal cortical center;(2)fMRI of the rats in the nerve transfer group showed that the ALFF value and ReHo value of the phrenic cortical center(primary motor cortex)were significantly increased,suggesting that the spontaneous activity level of neurons in the phrenic cortical center was increased.Conclusion:After end-to-side anastomosis between the phrenic nerve and the vagus nerve,cerebral cortex regions were remodeled.A new neural circuit is formed between the otherwise unconnected phrenic cortical center(primary motor cortex)and the vagal cortical center,which can re-innervate the movement of the diaphragm through the neural network of the vagus nerve.Part III Mechanism of IGFBP-6/IGFs/IGF-1R Axis in Neuronal PlasticityObjective:In the process of neural circuit remodeling,neurons regulate the morphology and function through a large number of genes and molecules and complex intracellular and intercellular signal transduction.Therefore,understanding the molecular mechanisms of synaptic plasticity is one of the prerequisites for dissecting neuronal circuit remodeling.Method:10 × Genomics spatial transcriptome sequencing was performed on the phrenic cortical center(primary motor cortex),a key site for neural circuit remodeling,to quantitatively analyze the related gene expression differences and cellular behavioral changes in the phrenic cortical center during neural circuit remodeling,and to excavate and verify the potential downstream signaling pathways.Results:(1)Spatial transcriptomics sequencing was performed in the phrenic neurocortical center.It was found that cluster1 and cluster4 were significantly changed in the same spatial location after nerve transfer,and cluster1 accounted for an increased proportion and cluster4 accounted for a decreased proportion after nerve transfer.Quasi-chronological analysis for cluster1 and 4 showed that they had a significant evolution process on the time axis.Further analysis of all hypervariable genes during the temporal sequence revealed that IGFBP-6 gene expression gradually increased with the differentiation of cluster4 to cluster1 on the time axis;(2)Spatial transcriptomics sequencing identified a total of 76 differentially expressed genes in the phrenic cortical center,including 40 up-regulated genes and 36 down-regulated genes in the nerve transfer group.Among them,IGFBP-6 was up-regulated in the phrenic cortex of nerve transfer group;(3)Co-IP confirmed that IGFBP-6 regulates the competitive binding of IGFs(IGF-1 and IGF-2)to IGF-1R.By binding IGF-2 with high affinity,IGFBP-6 inhibits the binding of IGF-1R with IGF-2,so that more IGF-1R sites bind to free IGF-1 and enhance the biological effect of IGF-1;(4)IGFBP-6 is overexpressed or knocked down in neurons,and immunofluorescence showed that the IGFBP-6OE group had increased in volume,axonal length,branch complexity,and dendritic spine density.When IGFBP-6OE cells were added with PI3K/Akt signaling pathway inhibitor(Wortmannin),the above effects of IGFBP-6 overexpression were reversed.The results showed that IGFBP-6 promoted the neuronal structural plasticity through PI3K/Akt signaling pathway;(5)Western-blot showed that overexpression of IGFBP-6 significantly increased the expression levels of synaptic plasticity-related proteins SYN,NF-20,and PSD-95 and increased the phosphorylation levels of Akt downstream molecules Girdin and NR2 B.When Wortmannin was added into IGFBP-6OE cells,the increase of above protein expression levels were reversed.These results suggest that IGFBP-6 promotes the neuronal functional plasticity through PI3K/Akt signaling pathway.Conclusion:By up-regulating the expression of IGFBP-6 gene,the phrenic cortical center mediates IGFs/IGF-1R axis to promote dendrite spines,axon lengthening and synaptic transmission efficiency,and achieve the remodeling of neural circuits. |
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