| Objective:Stem cells from human exfoliated deciduous teeth(SHED)are derived from the embryonic neural crest,are homologous to nerve cells,and have strong proliferative and multi-differentiation abilities.Human deciduous teeth are ideal stem cell sources for neurodegenerative disease therapies.In the nervous system,synapses serve as the foundation for forming neural circuits and transmitting neural signals.Chronic cerebral ischemia(CCI)causes synaptic dysfunction,which is linked to cognitive impairment.This study aimed to investigate the effect of SHED transplantation on synaptic plasticity in CCI rats,providing an experimental and theoretical foundation for the mechanism of SHED on recovering cognitive impairment and the application of dental stem cells in treating neurological diseases.Methods:1.SHED were isolated and extracted from human exfoliated deciduous teeth using enzyme digestion and the tissue block method.Flow cytometry was used to detect surface markers CD73,CD90,and CD105 of stem cells,and adipogenic and osteogenic inductions were used to detect their multidirectional differentiation ability.2.The CCI rat model was created by permanently ligating the bilateral common carotid arteries.Further,2,3,5-triphenyl tetrazolium chloride(TTC)staining was used to detect brain ischemia.In addition,the cognitive function of rats was evaluated.Morris water maze experiment was used to detect the number of rat crossing the platform within 60 s and Y-maze experiment to detect the autonomic alternation rate of rats,so as to identify the CCI rat animal model.3.The rats were divided into three groups,Sham,CCI,and SHED transplantation groups.In the sham group,only the median neck opening was performed,and the common carotid artery on both sides was separated without ligation.In the CCI group,the common carotid arteries were separated on both sides,ligated and cut at both proximal and distal ends to establish the CCI rat model,and 5μL PBS per side was transplanted into the hippocampus 10 days after CCI.In the SHED transplantation group,SHED(cell number:1×10~5/side,5μL/side)were transplanted into the hippocampus on both sides10 days after CCI.Four weeks after SHED transplantation,Morris water maze and Y maze experiments were used to detect the cognitive function of rats.4.Four weeks after SHED transplantation,the density and morphological changes in dendritic spines in the hippocampus were detected by Golgi staining,and changes in synaptic density and synaptic structure in the hippocampus were detected by transmission electron microscopy.5.Synaptophysin(SYN)expression in the hippocampal CA1 region of rats was detected using immunofluorescence.6.The expression of synaptophysis-related proteins SYN,postsynaptic density protein 95(PSD95),and growth-associated protein 43(GAP43)was detected using western blotting.7.The experimental data were statistically analyzed using SPSS v22.0.One-way analysis of variance was used for intra-group comparison.LSD-t test was used for inter-group comparison between the two samples.The Pearson test was used to evaluate the correlation between the density of dendritic spines in the hippocampus and the times of rats across the platform.P-values less than 0.05 were considered statistically significant.Results:1.SHED extracted from human exfoliated deciduous teeth could differentiate into osteogenic and adipogenic cells.The surface markers CD73,CD90,and CD105 of mesenchymal stem cells were positive,whereas the surface markers CD34 and CD45 of hematopoietic stem cells were negative in SHED.2.A CCI rat model was successfully established.TTC staining revealed extensive ischemia in CCI rat brain tissue.Morris water maze and Y maze behavioral tests showed that the cognitive function of rats in CCI group decreased compared with Sham group(P<0.001).3.After 4 weeks of SHED transplantation,behavioral tests showed that the cognitive function of rats in the SHED transplantation group was improved compared with that in CCI group.Morris water maze space exploration experiment showed that the number of rats in CCI group traversed the platform area was 1.58±0.79 times in 60 s,which was significantly less than that in sham group(3.33±0.65 times)(P<0.001).After SHED transplantation,the number of rats crossing the platform area increased to 2.50±0.67times within 60 s,which had a significant difference compared with CCI group(P<0.01).Y maze experiment showed that the alternation rate of rats in CCI group was(59.39±5.27)%,which was significantly lower than that in sham group(73.78±5.61)%(P<0.001).After SHED transplantation,the alternation rate of rats was(70.54±6.83)%.It was significantly higher than CCI group(P<0.001).4.Golgi staining revealed that the density of dendritic spines in the hippocampal region of CCI rats was decreased compared to that of sham rats(P<0.05),but the density increased and tended to normalize in the hippocampal region of SHED transplantation rats(P<0.05),and the density of dendritic spines in hippocampus was positively correlated with the times of rats across the platform within 60 s(r=0.767,P=0.016).Electron microscopy results showed that the number of synapses and synaptic vesicles in the hippocampus of CCI rats was decreased,and the structure of postsynaptic density and synaptic cleft was sparse and unclear.The number of synaptic structures and the synaptic vesicles increased,and the structure of postsynaptic density and synaptic cleft was clear after SHED transplantation.5.Immunohistochemical results showed that the intensity of SYN positive staining in the hippocampal CA1 region of CCI rats was significantly decreased compared with that of Sham group(P<0.01),while the intensity was significantly increased after SHED transplantation compared with that of CCI group(P<0.01),indicating that the density of synapses became more intense after SHED transplantation.6.Western blot results showed that the expression levels of synaptophysis-related proteins in the hippocampus of CCI rats were significantly decreased(P<0.05),and the expression levels of SYN,PSD95,and GAP43 proteins were upregulated in SHED transplantation rats(P<0.05).Conclusions:SHED transplantation could upregulate the expression level of synaptic related proteins in the hippocampus of CCI rats,restore the density of dendritic spines in the hippocampus of CCI rats,improve the synaptic plasticity of CCI rats,and alleviate the cognitive dysfunction of CCI rats. |
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