Co-transplantation Of Neural Stem Cells And Olfactory Ensheathing Cells Promotes Neurological Function Repair And Its Mechanism In PD Rats

Parkinson’s disease（PD）is a progressive neurodegenerative disorder affecting millions of predominantly elderly individuals worldwide.Clinically,patients with PD are characterized with both motor and non-motor symptoms.The cardinal motor symptoms of PD include resting tremor,bradykinesia,muscle rigidity,posture reflex disorder,etc.Currently,the reliable identification of non-motor symptoms,such as anxiety,rapid eye movement sleep disorders and cognitive disorder,are attracted more attention.In recent years,the achievements of stem cell research have brought hopes for the treatment of PD.The typical pathological feature of PD is the loss of dopamine（DA）neurons in substantia nigra（SN）.However,the pathogenesis of PD is still unclear,a large number of studies have shown that the neuroinflammation plays a critical role in the process of dopaminergic neuronal degeneration.Many studies have reported that the transplantation of neural stem cells（NSCs）plays an important role in improving the neuroinflammation microenvironment in PD models.Olfactory ensheathing cells（OECs）were often used in the co-transplantation with other stem cells for their excellent neurotrophic function,and were called"nutrient pumps"for the co-transplanted cells with them.So far,more and more evidences indicate that OECs play a critical role in improving the microenvironment of the co-transplanted cells.we will construct the PD rat models by injecting 6-hydroxydopamine（6-OHDA）into the right medial forebrain bundle and substantia nigra in adult male rats,to investigate the effects of co-grafting NSCs with OECs on neurological function of rats with 6-OHDA lesions and explore the underlying mechanism associated with the neuroinflammation.PD model was prepared by injecting 6-hydroxydopamine（6-OHDA）into the right medial forebrain bundle and substantia nigra in adult male Wistar rats.The NSCs derived from midbrain of E11¹2d fetal rats were cultured,differentiated,and identified.The OECs derived from olfactory bulbs of 1³d new born rats were cultured and identified.NSCs were infected by GFP-HIV,and OECs were labeled by Hoechst 33342 before transplantation surgery,then NSCs and OECs engrafted into the right striatum of PD rats through the microinjection needle of a stereotaxic instrument.the injection was made,containing 2μl of suspended cells（1×10⁵ cells/μl）.For co-grafting group,NSCs（1μl）and OECs（1μl）were mixed and injected,suggesting the total number of cells for each treatment was the same.At the same time,6-OHDA group animals received only 2μl DMEM medium,except for the control group animals without any treatment.The transplanted cells were observed at 7 days after the transplantation.The spontaneous exercise ability,anxiety level,muscle rigidity and motor coordination ability of PD rats were measured by open field test（OFT）,balance beam test and grip strength test at 7d after the transplantation.The expression of tyrosine hydroxylase（TH）in the striatum（Str）and SN of PD rats were detected by Western blot（WB）.Similarly,the expression of tumor necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）and interleukin 4（IL-4）in the right Str of PD rats were also detected by WB.The results are as follows:1.The third passages of NSCs with suspension growth characteristics formed neurospheres.Most NSCs were stained by nestin,which confirming as NSCs.Differentiated NSCs have TH immunoreactivity typical of dopaminergic neurons and GFAP immunoreactivity typical of astrocytes.A great deal of OECs appeared with bipolar and multipolar shapes,were positive P^75NTR which were located in cytoplasm and processes.2.NSCs were infected by GFP-HIV,and OECs were labeled by Hoechst 33342.Most of NSCs and OECs were successfully labeled.At 7 days after cell transplantation,no fluorescence was observed in the Control group and the 6-OHDA group,Blue and green fluorescence was observed,respectively,suggesting grafted NSCs and OECs can survive in each cell transplantation group.3.Behavioral experiments showed that compared with the Control group,the6-OHDA group had a shorter distance in the OFT（P<0.01,n=9-11）,and which suggested that 6-OHDA can reduce the spontaneous exercise capacity of rats;Compared with the6-OHDA group,the distance of 6-OHDA+NSCs+OECs group was longer（P<0.05,n=9-11）,and the distance of the 6-OHDA+NSCs and 6-OHDA+OECs group was slightly longer,but there was no statistical difference,and the above indicating that the co-transplantation of NSCs and OECs can improve the spontaneous exercise ability of rats in a short period of time.Compared with the Control group,the balance beam score of 6-OHDA group was significantly lower（P<0.001,n=9-11）,and which suggested that6-OHDA can reduces the motor coordination of rats;Compared with the 6-OHDA group,the balance beam score of 6-OHDA+NSCs,6-OHDA+OECs group and 6-OHDA+NSCs+OECs group was slightly increased,but there was no statistical difference（P>0.05）,and the above indicating that the single transplantation or co-transplantation of NSCs and OECs have no effect on the motor coordination ability of rats in a short-time.Compared with the Control group,the hang time of 6-OHDA group was significantly longer（P<0.05,n=9-11）,and which suggested that 6-OHDA can casue muscle rigidity in of rats;Compared with the 6-OHDA group,the hang time of 6-OHDA+NSCs,6-OHDA+OECs and 6-OHDA+NSCs+OECs group was slightly shorter,but there was no statistical difference（P>0.05）,and the above indicating that the single transplantation or co-transplantation of NSCs and OECs have no obvious relief effect on muscle rigidity ofrats in a short-time.4.The results of the OFT showed that compared with the Control group,peripheral to central activity time ratio（Pt/Ct）of 6-OHDA group was increased,which suggested that the anxiety of the 6-OHDA group was higher than the Control group（P<0.05,n=9-11）;Compared with the 6-OHDA group,the anxiety level of the OECs group and the 6-OHDA+NSCs+OECs group were lower（P<0.05,n=9-11）.5.There was no change in the expression of TH in the left Str and SN of PD rats（P>0.05）.The expression of TH in right Str was significantly changed.Compared with Control group,the expression of TH in the other four groups was significantly lower.（P<0.001,n=5-7）.Compared with the 6-OHDA group,the expression of TH in the6-OHDA+NSCs group and the 6-OHDA+NSCs+OECs group was slightly up-regulated,but the difference was not statistically significant（P>0.05）;The expression of TH protein in right SN was significantly changed.Compared with the Control group,the expression of TH in the other four groups was significantly decreased（P<0.001,n=5-7）.Compared with the 6-OHDA group,the TH expression in the 6-OHDA+NSCs+OECs group was slightly up-regulated but the difference was not statistically significant（P>0.05）.6.The inflammatory factors in right Str of PD rats was significantly changed.Compared with Control group,the expression of TNF-α,IL-1βand IL-4 in 6-OHDA group significantly increased（P<0.05,n=5-7）.Compared with 6-OHDA group,TNF-αand IL-1βwas significantly decreased in the 6-OHDA+NSCs+OECs group（P<0.05,n=5-7）,and the expression of IL-4 in the 6-OHDA+NSCs+OECs group was slightly up-regulated,but the difference was not statistically significant（P>0.05）,and the expression of IL-1βof 6-OHDA+NSCs and 6-OHDA+OECs group was significantly decreased（P<0.05,n=5-7）,and the expression of TNF-αand IL-4 of 6-OHDA+NSCs and 6-OHDA+OECs group was slightly decreased,but the difference was not statistically significant（P>0.05）.The above results shown that the spontaneous motor ability of PD rats was improved,while the anxiety of PD rats was alleviated,at the same time,the expression of pro-inflammatory factors TNF-αand IL-1βwas decreased in right Str of PD rats at 7 days after co-grafting NSCs with OECs.In summary,the co-transplantation of NSCs and OECs promoted neurological restoration in PD rats,and its mechanism may be related to the expression of pro-inflammatory factors TNF-αand IL-1β.These results will provide new thoughts and new targets to develop drugs for PD treatment.