Selecting An Appropriate Time Window For Transplanting Neural Stem Cells Into The Injured Spinal Cords

Back Grounds: Spinal cord injury (SCI) represents one of the most devastating injuries to afflict the human body, the functional recovery of which is restricted mainly due to the limited regeneration and plasticity of injured spinal cord in adult mammalians. Making the paralyzed walk has been standard of medical miracle and magic for much of recorded history, and it is not surprising that it remains us as potent cultural image. However, with breakthroughs in the molecular understanding of neural injury and recent neuroscience advances, new promising strategies for neural preservation and regeneration are on the horizon. Successful isolation and expansion in vitro of the neural stem cells form fetal and adult mammalian neural tissues have opened the door for hope toward prevention and cure of the devastating effects of SCI.Neural stem cells (NSCs) are a potential expandable source of graft material for transplantation aimed at repairing the injured spinal cord. The main rationale for NSCs-based therapies following SCI are: (i) replacement of degenerated spinal cord parenchyma by an axon growth supporting matrix; (ii) generation of neurons to replenish and replace those lost in and after injury; (iii) generation of oligodendrocytes to remyelinate of regenerating axons; and (iv), local delivery of growth promoting molecules. So it was disappointed to find out that the NSCs could not generate new neurons and oligodendrocytes after implanted into the intact or injured spinal cord in some published reports.However, there were also some surprising findings that the NSCs could differentiate into neurons in the injured spinal cord, although they could only generate glial cells after transplanted into the adult mammalian non-neurogenic areas such as new cortex and intact spinal cord. So in this study, we investigated that whether the transplanting time could contribute to NSCs' differentiation and consequent functional recovery of the treated SCI model. The possible mechanisms of influencing NSCs' differentiation and therapeutic results were also investigated.Objectives: (1) to isolate NSCs form embryonic (E) 13 days fetal brains and spinal cords and expand them in vitro; (2) to investigate NSCs' biological characteristics including their dependency on mitogen, proliferation, cryoperservation, and expression of some specific proteins, etc. in vitro; (3) to investigate NSCs' therapeutic effect on SCI repair; (4) to seek for a time window for transplanting in vitro-expanded fetus-derived neurosphere cells into injured spinal cords that could gain the best results and NSCs transplanted in which could generate neurons in vivo; (5) to investigate the possible mechanisms underline the existing therapeutic time window after SCI.Methods: El3 Sprague Dawley (S-D) rat were used as the source for isolating NSCs. The isolated cells were cultured for long period in vitro in serum-free defined medium in the existence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Their abiJity of self-renew was tested through single clone experiment. Their potential to generate neurons, astrocytes, and oligodendrocytes in vitro were examined through their special markers' expressions respectively with immuocytochemistry. Their proliferating abillity and expressions of some specific proteins such as nestin and c-Met, an acceptor for hepatocyte growth factor (HGF), were also examined with single-clone test, fluorescence- activated cell sorting (FACS), and immunocytochemistry respectively.The transected SCI animal models were produced using the methods reported everywhere. The animals were divided into 6 groups according to the time of their acceptance of NSCs transplantation, which were selected as 1 day, 4, 7, 10, 14, and 21 days after injury. The animals accepted medium transplantation were assigned into the control group. NSCs derived from the fetal spinal cord were labeled with BrdU and DAPI before transplantation. 8 weeks after transplantation, the therapeutic effects were manifested with the combine behavioral score (CBS), electrophysiology (motion evoked potential, MEP), and histological analysis. The survival and differentiation of transplanted NSCs were also examined through histology and immunohistochemistry. The results were further compared and analyzed between different groups respectively to find out the best time window for transplantation.In order to investigate the mechanisms underline the existence of such a time window, the pathological changes after transected SCI were analyzed under light microscope. Furthermore, the mRNAs' expressions of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), bFGF, EGF, glial cell line-derived neurotrophic factor (GDNF), nervegrowth factor (NGF), Idl and Id2 in different days after SCI corresponding to the therapeutic time point were detected through reverse transcription-polymerase chain reaction (RT-PCR).Results: NSCs were isolated successfully from El3 rat fetal brains and spinal cords. These cells could proliferate as "neurospheres" in defined serum-free medium under the stimulation of basic fibroblast growth factor and epidermal growth factor. They were confirmed as NSCs by their ability of self-renew and potential to differentiate into neurons, astrocytes, and oligodendrocytes, the three main cell types in CNS, as identified through their specific markers' expression respectivly. They also expressed c-Met and the specific NSCs' marker of nestin. And they could be cryopereserved for long period of time in liquid nitrogen without losing their self-renewal ability and multipotency.CBS: CBS of the rats in control group and the groups received cell transplantation 1 day, 4, 7, 10, 14, and 21days after injury were 84.00±3.14, 65.00 + 8.67, 60.50 + 6.60, 43.63± 10.24, 49.21 ±9.92, 61.71 ±5.41, and 60.50 ± 11.24 respectively. All CBS obtained from the therapeutic groups were lower than that of control group (PO.01). CBS of the 7d and lOd group were lower than those of the other groups.MEP: The MEPs of the treated rats in 7, 10, and 14days were better than those in other groups.Histological analysis and neural fibers staining: The rats in 7days and 10 days group had better results than those in other groups did: there were more neural fibers paralleled to the longitude direction of the spinal cord crossing the transected areas and less cavity formation.Survival, migration and integration with the host spinal cord of the transplanted NSCs: There were numerous NSCs survived in and integrated with the host spinal cord. The survived NSCs could migrate for long distance from transplanting area in the host spinal cord.Transplanted NSCs' differentiaon: Over 60% transplanted NSCs maintained as undifferentiated in 8 weeks after transplantation. NSCs transplanted in 7, 10, 14, and 21 days after injury could generate some neurons.After comparing the treated rats' CBS, MEPs, histological analysis, and the survival and differentiation of the transplanted NSCs between different groups, we confired that in vitro-expanded fetal spinal cord-derived NSCs cells were able to improve the motor function and found out that there was a time window for transplanting the NSCs into the transpected spinal cord, which was a time course between 7 days and 14 days after SCI. The NSCstransplanted in this period could acquire best neurological functional recovery and generate neurons in vivo.All the checked factors' mRNA expression fluctuated greatly. They were upregulated in different time course after SCI. The upregulation of some factors, including BDNF, NT-3, bFGF, GDNF, and NGF, might account for the existence of such a time window.In this study, we also found out that the spinous process of the 11th thoracic vertebrae could be served as an anatomical landmark in the lower thoracic spine fro SCI research.Conclusion: (1) In vitro-expanded fetal spinal cord-derived NSCs cells are able to improve the motor function after transplanted into the adult rat spinal-cord-transection injury model. (2) Combining NSCs transplantation with other intervening strategies may be necessary for repairing the injured spinal cord. (3) There is a time window for transplanting the NSCs into the transpected spinal cord, NSCs transplanted in which can acquire best neurological functional recovery and generate neurons in vivo. (4) The up-regulation of some factors, including BDNF, NT-3, bFGF, GDNF, and NGF, might account for the existence of such a time window. (5) The mRNAs' up-regulated expression of Idl and Id2 may regulate the inherent proliferating NSCs to generate glial cells only.