| Objective:The syringomyelia(SM)is the most common intraspinal malformation associated with scoliosis.However,the relationship between them is still unclear.Abnormal hydrodynamics of cerebrospinal fluid(CSF)and apoptosis of spinal cord neurons are important causes of SM formation,and further studies about their roles in the formation and progression of scoliosis with SM are still on the road.This study is based on a kaolin-induced rabbit model of scoliosis with SM.It is proposed to explore the effects of CSF hydrodynamics and spinal cord neuronal apoptosis in this pathological process from both positive and negative aspects.Through studying the pathogenesis of scoliosis with SM and looking into the deep relationship between scoliosis and SM,a scientific basis would be built for exploring novel strategies and methods to achieve etiological treatment to scoliosis.Methods:1.To research the correlation between scoliosis with SM and CSF hydrodynamics.Fifty Japanese rabbits(2-3 months old)were randomly selected,in 40 of them,kaolin was injected from the subarachnoid cavity to the spinal cord layer by layer at the level of C7 for inducing and establishing a rabbit model of scoliosis with SM.The remaining 10 were injected by saline as the control.Preoperative and postoperative 4-week,6-week,8-week,10-week,and 12-week,the scoliosis and SM formation were observed dynamically through radiography and magnetic resonance imaging(MRI)scanning of the spinal cord,and the earliest time of scoliosis/SM occurrence were observed and recorded.Meanwhile,the peak flow velocity of CSF in the pathological process was detected dynamically by phase contrast magnetic resonance imaging(PC-cine MRI).According to the final formation of scoliosis at postoperative 12-week,the experimental rabbits were divided into two groups and the differences of CSF peak flow velocity were compared among them to analyze the relationship between CSF hydrodynamics and the formation/progression of scoliosis.2.To research the neuronal apoptosis in spinal cord and denervation of bilateral paraspinal muscles in the pre-occurrence of scoliosis with SM.Twenty-five Japanese rabbits(2-3 months old)were randomly selected,20 were modeled by kaolin-induction,and the remaining as the control.Preoperative and postoperative 3-day,2-week,4-week,6-week,the cervical-thoracic spinal cord tissue and the bilateral thoracic paraspinal muscle tissue were respectively taken for the HE staining histological observation,and the TUNEL examination of the spinal cord tissue for detecting neuronal apoptosis through apoptotic index(AI)changes.The Masson's staining histological observation of the paravertebral muscle was performed to compare the fibrotic degree of bilateral paravertebral muscle.Moreover,the mRNA and protein expressional differences of AChR-y in bilateral paraspinal muscle were detected and compared.3.To research the effect of bone marrow stromal cells(BMSCs)to improve the spinal cord neuronal apoptosis in vitro.On the basis of isolation and primary culture of rabbit spinal cord neurons,serum deprivation method was used to construct a spinal cord neuronal apoptosis model in vitro.The rabbit BMSCs served as assistant cells were cocultured with apoptotic neurons.After coculture,the neuronal apoptosis rate was detected by the flow cytometry,and the cell proliferation was detected by CCK-8 kit.4.To research the effect from BMSCs transplantation to inhibit the formation of scoliosis with SM.Thirty-four Japanese rabbits(2-3 months old)were randomly selected for kaolin-induction.In which,17 experimental rabbits were treated by intratracheal injection with rabbit BMSCs in the original area for modeling at week 2 and 4 after kaolin-induction.The others were treated with saline injection as the control.After the transplantation intervention being completed,the neuronal AI was detected and calculated by TUNEL method at week 6 after kaolin-induction.Moreover,at week 6 and 12 after kaolin-induction,radiography and MRI were also used to observe the formation of scoliosis/SM after BMSCs transplantation,and PC-cine MRI was used to detect the hydrodynamic changes of CSF.Results:1.In the 50 experimental rabbits,47 survived to postoperative 12 weeks:of the 40 experimental rabbits being modeled by kaolin-induction,37(92.5%)survived to postoperative 12 weeks.At postoperative 6-week after kaolin-induction,the occurrences of scoliosis and/or SM were observed in some experimental rabbits.At postoperative 12-week,the incidence of scoliosis was 73.0%,and SM was 70.3%.The rate of scoliosis associated with SM was 54.1%.In the control,neither scoliosis nor SM was detected.In the rabbits with scoliosis,the peak flow velocity of CSF decreased gradually after kaolin-induction:being compared with rabbits without scoliosis,the peak flow velocity of CSF became more obvious from postoperative 4-week.And there was a significant positive correlation between the scoliotic Cobb angle and the decreased peak flow velocity of CSF at postoperative 12-week after kaolin-induction(P<0.001).2.Through the HE staining of spinal cord tissue at different phases after kaolin-induction,pathological changes of SM was characterized by early inflammation,perivascular space expansion,ependymal cell shedding,and central canal dilation.In the surgical region of spinal cord and its adjacent region,the neuronal apoptosis index was increased progressively from postoperative 3-day to 6-week.The HE staining of bilateral paraspinal muscles showed pathological changes were characterized by early inflammatory response and progressive fibrosis.The Masson's staining showed a progressive but symmetric fibrosis in the bilateral paravertebral muscle(P>0.05).There was no difference in the changes in AChR-y mRNA and protein expressions between bilateral paraspinal muscle(P>0.05).3.The AI of spinal cord neuron was increased gradually from 24h to 48h after serum deprivation,and which was increased greatly at 72h from excessive injury.Therefore,rabbit spinal cord neurons cultured by serum deprivation 48h were chosen as the apoptotic cell model in vitro for subsequent research.Coculture of apoptotic rabbit spinal cord neurons by serum deprivation 48h with BMSCs effectively decreased the apoptotic index of neurons(8.60%±0.30%vs 6.13%±0.12%,P<0.01)and improved the proliferation of neurons(1.52±0.27 vs 0.73±0.20,P<0.05).4.After BMSCs were transplanted in vivo,the neuronal apoptotic index was decreased than the control at week 6 after kaolin-induction in the surgical area of the spinal cord(13.04%±1.39%vs 26.57%±5.72%,P<0.05).A similar trend was found at the adjacent region(17.79%±1.57%vs 26.08%±3.04%,P<0.05).At week 6 after kaolin-induction,the incidences of scoliosis(25.0%vs 33.3%),SM(16.7%vs 33.3%),the concomitant rate of scoliosis with SM(8.3%vs 25.0%)were much lower than the control.At week 12 after kaolin-induction,the incidences of scoliosis(66.7%vs 75.0%),SM(50.0%vs 66.7%),the concomitant rate of scoliosis with SM(41.7%vs 50.0)were also decreased by BMSCs transplantation,but the downward trend was not so obvious as the week 6 after kaolin-induction.The peak flow rate of CSF in the experimental rabbits was also improved more obviously than the control,especially at week 6 after kaolin-induction.But from the week 6 to 12,the peak flow velocity of CSF was being much stable without obvious changes.Conclusions:1.A stable and reliable rabbit model of scoliosis with SM can be successfully established by injecting kaolin into the subarachnoid cavity and spinal cord layer by layer,which can provide a key technique and serve as an experimental platform for the studies about the pathogenesis of scoliosis2.There is not a single causal relationship between scoliosis and SM.The continuous changes of lower CSF hydrodynamics play an important role in the formation and progression of scoliosis with SM.3.Apoptosis of spinal neurons and hydrodynamics changes of CSF causing the scoliosis formation is not through inducing the asymmetric denervation of bilateral paraspinal muscles.4.BMSCs transplantation can protect spinal cord neurons and inhibit their excessive apoptosis,which could decrease the occurrence of scoliosis with SM by improving the CSF hydrodynamics status. |
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