| Spinal cord injury can be divided into the primary damage and the second damage. The primary damages are unpredictable and uncontrollable, and so targets of the research on SCI focus on the pathophysiology of secondary injury in the injured spinal cord and the therapeutic interventions. Based on the comprehending of the pathophysiology of SCI, it's known now that there are several factors attributed to the failure of CNS regeneration: exists of growth-inhibitory molecules, lack of appropriate trophic support, and reactions of the immune system et al. Building a 'bridge'is one of the ways to solve these problems: cellular scaffolds across the injury not only provided a growth-permissive substratum environment, abundant trophic factors, but also enhance the regenerative capacities of axotomized neurons. There are several types of cells including olfactory ensheathing cells, Schwann cells, fibroblasts, embryo stem cells, nerve stem cells, and Bone marrow stromal cells having been used as graft seed cells in treating SCI. It's regretted that, up to date, none of those kinds of the cells can restore the functional deficit after spinal cord transection. On the contrary, they are confronted with problems of the deficit of cell donors and problems of the immune rejection. In addition, there are also legal or ethical problems, differentiation problems and tumorrized growth problems associated with embryonic stem cell graft and neural stem cell. Most of all, no kinds of those cells can be commercially provided to meet a single therapy demand for an accident or a large-scale therapy demands for a disaster. So we perform a research on HAECs and explore its utility on neuro-transplantation. Our study has been divided into four sections: Section one: Isolation of human amniotic epithelial cells and the primary exploration on its biologic characterizations HAECs were isolated from a piece of fresh amnion. Using immunocytochemistry methods, we investigated the expression of neuronal phenotype (MAP-2, GFAP, Nestin) in HAECS. We also investigated the effect of the conditioned medium of HAECS on the rat glial cells in in vitro. At last, HAECS were transplanted into the Corpus triatum of healthy adult rats; the survival and migration of the graft cells were observed. In this study, we successfully isolated HEACs. HAECs displayed positive immunoreactivity to MAP-2, GFAP, and Nestin and could secret the neurotrophic factors. When transplanted into the Corpus triatum of healthy adult rats, HAECs survived well for a long time, migrated for a distance and did not induce immune rejection Our data indicate that HAECs may be new seed cells in neurograft. Section two: Investigating the histological changes, the processes and outcomes of spontaneous functional recovery after spinal cord hemisection or transection in adult rats. The different degrees of spinal cord injury in adult rats were produced by hemisection or transection at T10 thoracic level. The behavioral function recovery was assessed by using the BBB open field locomotion score and the narrow beam test. The bladder function recovery was assessed by using several parameters included the day at which autonomic function of the bladder was recovered, the residual volume obtained by measure the urine voided by expressing the bladders constantly,the bladder weight, the thickness of the bladder wall. The histology of the lesions and the myelin sheath fibers around the lesions were also detected by staining with Cresyl Fast Violet or by Pal-Weigert method. At 5 weeks after spinal cord injury, the BBB scores of the transection group(3.67±1.03)were significantly different from the hemisection group(19.4±0.89)or the sham-operation group(21)(P﹤0.01), there was no significant difference in behavioral recovery between the hemisection group and the sham-operation group(P>0.05). The rats in the transection group could not complete the narrow beam test and the scores of the narrow beam tests between the hemisection group(0.8±0.27) and the sham-operation group(2) were significantly different(P﹤0.01). The time of the autonomic bladder function recovering of the transection group(7.3±1.0) were longer than the hemisection group(1.0), the rats in the sham-operation group recovered the bladder function soon after the anesthesia. The rats in the hemisection group and the sham-operation group recovered the normal voiding after the operations and no residual urine was detected. The mean residual volumes of the transection group raised at the first week and then descended, at last reached at 4.25±0.84ml. The bladder weight of the transection group(0.79±0.18g)were significantly different from the hemisection group(0.22±0.09g)or the sham-operation group(0.14±0.02g)(P﹤0.01), there was no significant difference in the bladder weight between the hemisection group and the sham-operation group(P, 0.289). the thickness of the bladder wall of the transection group(1375±228μm)were significantly different from the hemisection group(1065±210μm)or the sham-operation group(930±113μm)(P﹤0.01), there was no significant difference in the thickness of the bladder wall between the hemisection group and the sham-operation group(P, 0.271). The lesions of the hemisection group were infiltrated with hyperplastic glial cells and imflammation cells. There were a few cysts or cystic cavities present at the lesions.The cut ends of spinal cord of the transection group were connected with the connective tissue and glial scar. Stained by Pal-Weigert method, there were little myelinated axons existed within the lesions of the transection group, but in the hemisection group the myelinated axons existed at one side and was absent at the other side. Our data indicate that (1) Experimental modelsof acute spinal cord injury can be produced consistently and reduplicatedly by hemisection or transection at T10 thoracic level. (2) There were no obvious locomotor or bladder dysfunctions in spinal cord hemisection rats. The histological and functional changes after spinal cord transection in adult rat were distinct. (3) The rat spinal cord transection model can be used to evaluate the effects of therapeutic interventions. Section three: Grafting HEACs(Human amniotic epithelial cells) into the rat's injury spinal cord to improve the rat's hindlimb motor function and ameliorate the bladder function. Labeled with Hoechst33342, HAECs, obtained from a piece of fresh amnion, were transplanted into the site of complete midthoracic spinal transections in adult rats. Hindlimb motor function was assessed by using the open-field BBB scoring system and the morphology and sizes of the axotomized red nucleus neurons were observed. The bladder function recovery was assessed by using several parameters included the day at which autonomic function of the bladder was recovered, the residual volume obtained by measure the urine voided by expressing the bladders constantly,the bladder weight, the thickness of the bladder wall. Survival of the graft cells was observed at two weeks and two months after transplantation. We also detected the histology of the lesions and the myelin sheath fibers around the lesions. The amounts of HAECs are easily obtained for one graft. From a piece of human amniotic epithelial tissue(15×15cm2), about 1×106 cells can be harvested. When transplanted into the injured spinal cord, HAECs survive well for a long time (8 weeks) and integrate well with the host. HAECs do not induce immune rejection after transplantation. Comparing with the control group, HAECs can promote the regeneration and sprouting of the axons and improve the hindlimb motor function (cells-graft group vs PBS group, 9.0±0.89 vs 3.7±1.03,P<0.01). They also rescue the atrophy of axotimized red nucleus (cells-graft group vs PBS group, 526.47±148.42μm2 vs. 473.69±164.73μm2, P<0.01). The time of the autonomic bladder function recovering of cells-graft group (2.67±1.63d) were obviously shorter than PBS group (7.33±1.03d)(P<0.01).The mean residual volumes of the cells-graft group (2.27±1.23ml)was obviously smaller than the PBS group(4.28±0.97ml()P<0.01).There was no significant difference in the bladder weight or the thickness of the bladder wall between the cells-graft group (bladder weight 1.01±0.21g, bladder wall thickness 1161±280μm) and the PBS group (bladder weight 0.84±0.16g, bladder wall thickness 1386±236μm)(P, 0.067and 0.121). Our study indicated that HAECs transplants do obviously improve the rat's hindlimb motor function and ameliorate the bladder function after spinal cord transection. Section one:Graft HEACs genetically modified to overexpress GDNF into the rat's injury spinal cord to rescue the axotomized rubrospinal neurons and improve the rat's hindlimb motor function. We transferred the GFP or GDNF into HEACs by lentiviral vectors. The expression of GFP byHEACs was detected by using Leica fluorescent microscope. Expression of GDNF or GFP was also detected by RT-PCR in mRNA level. HEACs genetically modified were transplanted into the site of complete midthoracic spinal transections in adult rats. Hindlimb motor function was assessed by using the open-field BBB scoring system and the morphology and sizes of the axotomized red nucleus neurons were observed. GFP proteins expressed by HEACs could be seen with fluorescent microscope and GFP mRNA expression could also be confirmed by RT-PCR. In our study, we first proved that GDNF mRNA was naturally expressed by HEACs at low level. When genetically modified to overexpress GDNF by lentiviral vectors, HEACs did significantly increase the expressins of GDNF mRNA. Compared with PBS group, HAECs transferred with GDNF or GFP can promote the hindlimb motor function (PBS group vs GDNF group or GFP group, 5.3±1.37 vs 9.7±1.63 or 10±2.28,P<0.01). The BBB scores between GDNF group and GFP group were not significantly different(P>0.05), but the time of GDNF group when the rats reached a plateau of BBB score curve was shorter than GFP group. Graft of HEACs transferred with GDNF could resume the morphology of the injured neurons in cresyl violet staining. Compared with PBS group, cell transplantations (HAECs transferred with GDNF or GFP) rescue the atrophy of axotimized red nucleus (PBS group vs GDNF group or GFP group, 473.69±164.73μm2 vs 585.27±159.92μm2 or 526.47±148.42μm2, P<0.01). The sizes of the axotomized red nucleus neurons of GDNF group were larger than GFP group but still smaller than sham-operation group (679.25±256.04μm2)(P<0.01). Our study indicated that HAECs could express the GDNF effectively, chronically and stably by lentiviral vectors. Compared wtih GFP-HAECs graft, graft of HEACs transferred with GDNF could resume the morphology of the injured neurons, do obviously improve the rat's hindlimb motor function and shorter the time when the rats reached a plateau of BBB score curve. |
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