The Study Of Cortical Calcium Signal Changes After Spinal Cord Injury Based On In Vivo Calcium Imaging

Objective Spinal cord injury(SCI)is a severe traumatic condition of central nervous system,which results in interruption of the intact sensorimotor pathway between brain and spinal cord.Due to the high plasticity of the nervous system,neuropathic neuralgia,hyperalgesia,abnormal sensation below the injury and other neurological complications related to brain occur after spinal cord injury.However,the mechanism of reorganization after injury has not been fully understood.Calcium ions are important signaling molecules in the nervous system,whose activity can indirectly reflect the activity of neurons.We use the genetically encoded calcium indicator and in vivo calcium imaging technology to observe the calcium signal dynamics and the change of cortical plasticity of the primary sensory and motor cortex neurons after spinal cord injury in order to investigate reorganization mechanism after spinal cord injury.It can provide theoretical guidance for prevention of pathological neural plasticity after spinal cord injury.Methods 1.Craniotomy and injection of GCa MP6 f virus: 8-10 weeks female C57BL/6J mice,performing stereotypical craniotomy and injecting AAV2/1-syn-GCaMP6f-WPRESV40 virus.The virus was injected into primary sensory cortex or primary motor cortex.2.Experimental group and spinal cord hemisection: mice with GCa MP6 f expressed in the primary somatosensory cortex of the right primary sensory cortex were divided into three groups randomly: sham group,left spinal cord hemisection group(contralateral injury)and right spinal cord hemisection group(ipsilateral injury).Laminectomy was performed in the sham group and left or right spinal cord hemisection was performed in the injured group.The mice with GCa MP6 f expressed in the right primary motor cortex were left spinal cord hemisection group(contralateral injury).3.Primary sensory cortex two-photon in vivo calcium imaging: the changes of calcium signals in the primary sensory cortex of mice before and after spinal cord injury at 3,7,14 and 28 days were observed in vivo by two-photon microscopy.4.In vivo calcium signal recording in the primary motor cortex by photometry in free moving mice: the changes of calcium signal in the primary motor cortex neurons before and after spinal cord injury at 3,7,14 and 28 days after spinal cord injury by photometry.5.Data analysis: a correction software Mosaic and analysis software Fluoro SNNAP based on MATLAB was used,and calcium signal frequency,rise time and decay time were analyzed,at the same time neurons synchronized coefficient and functional connectivity coefficient were also analyzed;The Op Signal analysis software based on MATLAB was used to analyze the calcium signal recorded by optical fiber,and the frequency and amplitude of calcium signal at each time point before and after spinal cord injury were analyzed.Results 1.In ipsilateral primary sensory cortex,there was no significant difference in calcium signal frequency at day 3,14 and 28 after injury but increased at day 7 after spinal cord injury.For rise time and decay time at 3,7,14 and 28 days after spinal cord injury,there was no significant difference.In contralateral primary sensory cortex and sham group,there was no significant difference in calcium signal frequency.2.In ipsilateral primary sensory cortex,synchronization coefficient had no significant difference at day 3 after spinal cord injury but it was increased at day 7 after injury with significant difference,and it was no significant difference at day 14 and 28 after spinal cord injury.Functional connectivity coefficient had no significant difference at day 3 after injury but it increased at day 7,14 and 28 after injury with significant difference.In contralateral primary sensory cortex and sham group,there was no significant difference in the synchronization coefficient and functional connectivity coefficient.3.In free moving mice,the frequency and amplitude in the contralateral primary motor cortex of the brain after spinal cord injury had no significant difference at 3,7,14 and 28 days after spinal cord injury.Conclusion 1.Calcium signal frequency and synchrony of primary sensory cortex neurons increased on day 7 after spinal cord injury,suggesting that frequency and synchrony are involved in early abnormal sensory formation and early cortical reorganization.2.Functional connectivity of calcium signals in primary sensory cortical neurons increased at 7,14,and 28 days after spinal cord injury,with the most significant increase at 28 days,suggesting that functional connectivity of neurons is involved in long-term chronic cortical reorganization.3.No changes were founded in the frequency and amplitude of calcium signals from primary motor cortex neurons after spinal cord injury,indicating that excitability and synchrony of calcium signals from neurons were not involved in motor cortex reorganization.