Experimental Study On The Use Of Human Amniotic Cells In Traumatic Brain Injury

【Objectives】:Traumatic brain injury(TBI) continues to be an important cause of human mortality and morbidity, however the effect of current treatment is unsatisfactory. Human amniotic cells(HACs) not only express the markers for both neuronal and glial cells but also synthesize and release neurotrophic factors ,which suggests that HACs may have multipotential neuronal properties.So we explored the differentation and proliferation of human amniotic cells which culture conditioned by traumatic brain tissue extracts to simulate the microenvironment of injured brain,and evaluated the effects after transplanting HACs and GDNF-expressing HACs on the motor function and hippocampal neuron following TBI in rats. 【Method】: A human amniotic sheet obtained from an uncomplicated elective cesarean section,then the separated amnion was treated with 0.25%trypsin three times to collect HACs within an hour.A rat model of traumatic brain injury was established by Feeney techniques,and the traumatic brain tissue extracts was made 24 hours post-TBI. HACs were cultured in RPMI1640 medium(1640), RPMI1640 and normal brain tissue extracts(1640+NB), RPMI1640 and traumatic brain tissue extracts(1640+TBI).The cell morphology of each group was observed under phase-contrast microscope at 7 days after co-culture.The immunohistochemical analysis for Nestin and MAP2 was performed before and after co-culture. HACs were cultured in three different medium by the cell density of 10000/ml and 100000/ml,then the cellular viability of each group was investigated at 1,4,7 days after co-culture.HACs were resuspended 105/μl after HACs were separated and labeled with Hoechst33342. Hindlimb sensorimotor cortex area in rats was impacted by Feeney weight drop techniques.10 μl Hoechst33342-labeled HACs were injected into the center and the margin of contusion 24h after injured by microsyringe and stereotactic frame. Groups consisted of treatment group(TBI+HACs), control group(TBI+PBS) and sham TBI group.The motor function was evaluated by pegged beam walking test during 28 days after injured.After then, the rats were anesthetized and perfused transcardially with saline followed by 4% paraformaldehyde. The brains were stored in 25% sucrose-phosphate buffer overnight and cut (coronal sections,40μm) into three series on a cryostat for fluorescence microscopy, hematoxylin and eosin staining for morphological analysis under light microscopy and for immunohistochemical.The model of hippocampal neuronal death following TBI was built up by Feeney weight drop techniques.5μl GDNF-expressing HACs were injected into the margin of contusion 24h after injured by microsyringe and stereotactic frame.Groups consisted of GDNF-expressing HACs group(TBI+GDNF), eGFP-expressing HACs group(TBI+eGFP),PBS group (TBI+PBS)and sham TBI group.The spatial memory performance was evaluated on 5 consecutive days by Morris water maze on 7 days after transplantation.After the probe trial, the rats were sacrificed for hippocampal neuron morphological analysis by cresyl violet staining under light microscopy.Expression of GDNF was detected by PCR in mRNA level. 【Results】: HACs in group 1640 showed round and oval in shape,however in group 1640+NB and group 1640+TBI some cells were transformed pyramidal cells like neuron. Cells were immunostained by anti-nestin before culture,and displayed MAP2-positive in group 1640+NB and group 1640+TBI. The cell proliferation of each group was not significant.The HACs cultured by the cell density of 100000/ml decreased little by little in group 1640+NB and group 1640+TBI.The rats of treatment group had less footslips than those of control group,but had more footslips than those of sham TBI group. Under fluorescence microscopy, Hoechst33342-labeled HACs were observed as a blue area around the injection site and the transplantation trajectory. Hematoxylin and eosin staining showed the majority of HACs were visualized as either oval or round around the injection site and the transplantation trajectory. Some HACs were stained with antibody to MAP2. The rats of TBI+GDNF group exhibited less escape latencies than those of TBI+eGFP group and TBI+PBS group,but had longer escape latencies than those of sham TBI group.Cresyl violet staining showed hippocampal neuronal loss,shrinkage and dark staining of neurons in TBI+eGFP group and TBI+PBS group.Compared with other groups,GDNF expression of TBI+GDNF group obviously increased in mRNA level. 【Conclusions】: HACs can transform the neuron-like cell by co-culture with traumaticbrain tissue extracts;the proliferation of HACs is not significant; the HACs cultured by low cell density survives more easily than those by high cell density in group 1640+NB and group 1640+TBI.HACs can not only survive in the cerebrum of TBI rats up to 4 weeks after transplantation but also express the specific neuronal antigen-MAP2 and improve the motor deficits of TBI rats. Transplanted GDNF-expressing HACs can protect against hippocampal neuronal death following TBI; HACs can be used as a transgene carrier in gene therapy. So the HACs can be exploited as an alternative graft source in cellalur transplantation in TBI.