| Object:Cerebral palsy（CP）is the most common cause of physical disability in children,which brings a heavy economic and spirituaburdensen to families and society.Recent animal experiments and clinical studies suggest that stem cell transplantation may be a potential new method for the treatment of CP,but its efficacy is still unclear due to the heterogeneity of transplantation route,dose,and time.In particular,it is very important to determine the intravenous（IV）,intrathecal（IT）,lateral ventricle（LV）,and other drug delivery routes and clarify their role and mechanism for optimizing stem cell therapy for CP.Therefore,in this study,the hemiplegic CP（HCP）rat model with hypoxic-ischemic injury was constructed,and human umbilical cord-derived mesenchymal stem cells（h UC-MSCs）were infused through an intravenous,intrathecal,l and lateral ventricle to explore the effects of three administration routes on improving motor impairment in an HCP rat model,and by comparing their cell homing Explore the mechanism of immune inflammation regulation,neurotrophic and endogenous regeneration.Method:174 5-day-old Wistar rats were divided into control group（control,n=35）and hypoxic-ischemic injury group（n=139）.The control group was not treated.The hypoxic-ischemic injury group was ligated in the left common carotid artery and exposed to hypoxia at 37℃with 6-8%O2for 3 hours.On a postnatal day 18（P18）,136 HCP rats were selected through behavioral experiments for follow-up experiments.The rats in the hemiplegic cerebral palsy group were not treated（HCP,n=35）,and the other HCP rats were infused with 1×106Di R labeled h UC-MSCs through three routes on P21.The intravenous group were infused from the intravenous（HCP+IV+MSCs,n=33）,the intrathecal group from the lumbar 5-6space（HCP+IT+MSCs,n=33）,and the lateral ventricles group were infused in the brain with brain stereotactic instrument（HCP+LV+MSCs,n=33）.The brain pathological sections of HCP rats on P18 were observed by Nissl staining and transmission electron microscope;In vivo imaging was used to track the homing of h UC-MSCs caused by three infusion routes in HCP rats for 3 hours,4 days,7days,and 2 weeks;The changes of P32,P46 and P60 motor function in five groups were recorded by rotation and suspension experiment;The expressions of Iba-1,BDNF,MBP,Olig-2 and NG2 in brain tissue were detected by immunofluorescence and Western blot（WB）,the inflammatory-related factor TNF-α,IL-6,and IL-10 was detected by ELISA,the expression levels BDNF and the density of dendritic spines were analyzed by Golgi staining,above pathological changes of P32,P46 and P60brains of five groups were recorded.ANOVA and nonparametric rank-subtests were used to compare the differences between groups.The Spearman correlation coefficient is used to detect the correlation between the indicatorsResult:（1）Validation of the model revealed a low number of nuclei in the motor cortex of the damaged rats,with nuclei solidified and sparsely arranged,and a sparse and disorganized myelin structure;13 days after surgery,the rotation and front-limb suspension times of CP rats were reduced to 54.03±2.73s and 25.60±2.58s,respectively,compared with normal rats（101.17±3.24,70.94±2.27）（P<0.05）,and the HCP rat model was successfully established.（2）In vivo imaging tracing revealed that:IV infusion did not detect cellular signals in the brain,IT infusion detected cellular signals in the brain and the signals lasted for 7 days,LV infusion concentrated cellular signals in the brain and the signals lasted for at least 2 weeks.（3）In terms of motor function,11 days after infusion,the rotation times in the IT,LV,and IV groups were 137.05±6.02,119.10±7.40,and 106.50±3.51 seconds,respectively,which were significantly more than those in the HCP group（66.27±3.07 seconds）（P<0.05）;the rotation and suspension times in the IT group were significantly more than those in the IV group（P<0.05）.Twenty-five days after infusion,the rotation（138.93±9.31 s,136.90±6.84 s,115.57±6.89 s）and suspension times were significantly more in the IT,LV,and IV groups than in the HCP group（72.07±5.2 s）（P<0.05）;the rotation times were significantly more in the IT and LV groups than in the IV group（P<0.05）.At 39 days post-infusion,the rotation time（178.30±11.46 seconds,144.41±16.35 seconds）and suspension time was significantly more in the LV and IT groups than in the HCP group（97.14±8.32 seconds）（P<0.05）;the rotation and suspension time were significantly more in the LV group than in the IV group,and the rotation time was significantly more in the LV group than in the IT group,all with statistically significant differences（P<0.05）.（4）In terms of immune inflammation regulation,11 days after infusion,the expression of TNF-αin the IT group was significantly less than that in the LV group,and the expression of IL-10 in the LV group was significantly more than that in the IV group,and the differences were statistically significant（P<0.05）,suggesting that the inflammation suppression effect of intrathecal infusion was better than that of lateral ventricular infusion,and the anti-inflammatory effect of lateral ventricular infusion was better than that of intravenous infusion;25 and 39 days after infusion,the expression of Iba-1 and TNF-αexpression in the LV group was significantly less than that in the IV group,and TNF-αexpression in the LV group was significantly less than that in the IT group,and the differences were statistically significant（P<0.05）,suggesting that the inflammatory inhibitory effect of lateral ventricular infusion was superior to that of the intravenous and intrathecal infusion.（5）In terms of neurotrophy,the expression of BDNF in the IT group was significantly more than that in the IV and LV groups at 11days post-infusion（P<0.05）;at 25 days post-infusion,the density of neuronal dendritic spines in the IT group was significantly greater than that in the IV and LV groups（P<0.05）,suggesting that the neurotrophic effect of intrathecal infusion was superior to that of the intravenous and lateral ventricular infusion at earlier time points post-infusion;at 39 days post-infusion,the expression of BDNF in the IV group,IT group and LV group showed no significant difference（P>0.05）.（6）In terms of endogenous regeneration,the expression of MBP and Olig-2 in the IT and LV groups was significantly more than that in the IV group at 11 days post-infusion（P<0.05）,suggesting that intrathecal and lateral ventricular infusions were superior to intravenous infusions in terms of myelin formation;at 25 days post-infusion,the expression of MBP in the LV group was significantly more than that in the IV group（P<0.05）;at 39 days post-infusion,the expression of MBP in the LV group was significantly more than that in IV group;the expression of NG2 in the IV group was significantly more than that in the LV group（P<0.05）,suggesting that the myelin formation effect of lateral ventricular infusion was superior to that of intravenous infusion at longer time points after infusion.（7）Iba-1 was negatively correlated with MBP（r=-0.492,P<0.05）;motor function was positively correlated with MBP（r=0.239,P<0.05）and negatively correlated with Iba-1（r=-0.422,P<0.05）.Conclusion:Intravenous,intrathecal,and lateral ventricular infusions of h UC-MSCs all improved motor function in HCP rats.Intrathecal infusions were superior to intravenous infusions at 11 and 25 days,probably due to a significant neurotrophic effect;lateral ventricular infusions were superior to intravenous infusions at 25 days and superior to both intravenous and intrathecal infusions at 39days,probably due to a significant immunosuppressive effect.In conclusion,intrathecal and lateral ventricular infusions have similar therapeutic effects at early time points,and the recommended repeated intrathecal infusion approach may ultimately provide greater benefit. |
