| Traumatic brain injury is the most common form of trauma in the contemporary war injuries,with the incidence of more than 20%.The mortality and disability rates is the top of all kinds of injuries.Up to now,there is not timely and effective treatment and prognosis measures for TBI.In addition to the war trauma,the traumatic brain injury caused by accident in normal life is also a thorny problem in the general public.In the new era,TBI has some new characteristics with the development of new technology.Some recent Statistics show that the incidence of nerve trauma,the rate of mortality and disability in high-tech war have not decreased.At present,more than 200 clinical multicenter randomized double-blind studies have been carried out on the factors of primary injury and secondary injury after craniocerebral trauma.The results show that there is no definite clinical effect of any drug.The beneficial method is the surgical intervention for TBI treatment in the acute phase.With the development of surgical technology,the surgical treatment in the acute stage of traumatic brain injury has significantly improved the survival rate of patients.But by the limit of medical technology,the hemiplegia,cognitive and other neurological dysfunction,which cannot avoid after TBI surgery,can still be the most important problem affecting the quality of the patients'life.it is also a worldwide problem that scientists are trying their best to find the solution.Besides the medical effort for TBI patients,some researchers of other fields also focus on this thorny problem.For the deepening of nervous system research,the molecular mechanism and physiological characteristics of brain injury are constantly revealed.Some innovative ideas for the treatment of traumatic brain injury have been noted.The drug research and the cell therapy have been launched in succession.Neural stem cells are a kind of cells with self-renewal ability which have found in the brain tissues of many kinds of animals.NSCs can be differentiated into other subtypes of neural cells,such as neurons,astrocytes and oligodendrocytes.NSCs are most active in the embryonic stage and larval stage.With the continuous development of organisms,they will gradually disappear or stay in a dormant state.Current researches shows that neural stem cells can be activated under the condition of lesions and participate in the neural repair of the focus area.Therefore,the possibility of using neural stem cells as a repair treatment in brain injury seems to deserve further attention and exploration.However,there is one thing need to solve that it is difficult for NSCs to survive in the focus area for a long time after migrating to the focus area.The dramatic change of local microenvironment could be one of the hardest conditions,which is difficult to provide a good living environment for NSCs.Some studies have shown that the number of local proliferation and differentiation of neural stem cells is too limited to repair the neural loss.So transplantation of exogenous neural stem cells may be an appropriate way to break through this problem.Melatonin is a hormone secreted by pineal gland at night under the influence of normal circadian rhythm.Melatonin or its metabolites can be detected in plasma,saliva and urine.Melatonin get involved in the regulation of many physiological functions of organism,mainly through light perception to regulate the circadian rhythm and seasonal rhythm.Melatonin also plays a key role in the regulation of core temperature and sleep awakening.Melatonin can precisely regulate the physiological activities and life cycle of cells by regulating the intracellular ion channels(such as calcium channels),affecting the molecular signal channels(such as notch,MAPK,etc.),regulating mitochondrial respiration,etc.In the nervous system,it has been shown that melatonin can affect the survival of neural stem cells under oxidative stress,hypoxia and other experimental pathological conditions.In addition,melatonin can also affect the proliferation and differentiation of neural stem cells.To explore the possibility of NSCs as cell resource for cell therapy,we combine neural stem cells,melatonin and Matrix Gel as 3D culture medium and transplant mixed culture medium which combine with neural stem cells,melatonin and Matrix Gel into lesion of traumatic brain injury.1.The protection of melatonin for neural stem cellsIn order to study the effect of melatonin on neural stem cells,we add 0?6.25?12.5?25?50?100?mol/L six concentration melatonin into each experimental group in vitro,and then determine the ability of cell survival through morphological observation.In order to study the effect of melatonin on neural stem cell treatment,we found that melatonin has a certain neuroprotective effect under the condition of oxygen and glucose deprivation using CoCl2+EBSS sugar free medium in vitro.Under the condition of oxygen and glucose deprivation,the survival ability of neural stem cells in the untreated group decreased significantly.After melatonin treatment,we found that under the condition of 25?mol/L melatonin treatment,the survival number of neural stem cells increased,and the survival rates of neural stem cells was improved.2.Establishment of traumatic brain injury model in ratsTo estimate the potential of this effect for clinical using,we choose traumatic brain injury model for research.After many literature learning and discussing,we design a new method to make the traumatic brain injury model.We used open surgery to open a bone window at the top of the skull of the rats and locate the injury in M1 and M2 motor cortex of the rats,which was 1?4 mm to the right and 1?4mm to the front of the bregma.Then we take out 3 mm~3 cortical tissue to cause the neural tissue lost.Finally,the skin tissue was sutured and the traumatic brain injury model was complete.Compared with the traditional impact model,the cavity in the lesion area can effectively simulate the structural damage caused by the loss of brain tissue after traumatic brain injury.Seven days after the model was made,the rats were scored with NSS behavior.Compared with the normal rats,the scores showed that the rats after surgery had obvious neurological dysfunction,which means that the performance of new model was stable and feasible.3.Combined transplantation of melatonin,neural stem cell and Matrix Gel to repair traumatic brain injury in ratsIn the above study,melatonin has a neuroprotective effect on neural stem cells under the condition of25?mol/L melatonin treatment in OGD model.Therefore,we co-cultured melatonin and neural stem cells,and filled them with matrix gel to the focal area of the open TBI model of rats.Compared with the previous methods of cell injection transplantation,we add matrix gel to build the neural stem cell transplantation and repair system,transplant it to the focus cavity in the trauma area which can effectively offer 3D structure support for the tissue cavity,and provide a basic platform for cell growth.Seven days after cell transplantation into the rat trauma model,was evaluated the recovery of neural function by by means of NSS scored,MRI,immunohistochemistry,immunofluorescence,laser confocal detection and others.The results showed that the neural function of the injured rats in each group decreased to the sham.Compared with the cell-free group,the recovery of neural function of the rats after the transplant with stem cell was significantly improved.The melatonin treated group also had a better recovery effect of neural function compared with the non-treated group.The brain tissues of rats were perfused,sectioned and stained with immunohistochemistry and immunofluorescence.Immunohistochemically results showed that in the experimental group of melatonin,neural stem cells and Matrix Gel co-transplantation,the number of cells around the focal area significantly increased and the edge of the focal area was more complete and smooth.The results of immunofluorescence showed that the rate of MAP2 positive cells increased significantly in the brain tissue of traumatic rats,which were transplanted with melatonin and neural stem cells.The result suggested that melatonin and NSCs co-transplantation could provide better structural support,cell protection.It also confirmed co-transplantation promoted the survival of cells in the focus area and repaired the neural function.In conclusion,this experiment simulated local microenvironment of brain tissue after trauma in vitro using Oxygen and glucose deprivation model.Under this condition,neural stem cells cannot have good viability.So we treat neural stem cells with melatonin to confirm neuroprotective effect of melatonin.Finally,we proved that melatonin can improve the survival ability of neural stem cells in Oxygen and glucose deprivation model.And then we mixed matrix gel with melatonin and neural stem cells.The cell culture mixture was transplanted into a traumatic brain injury model.The results show that the transplantation of melatonin and neural stem cells can also promote the recovery of neural function in the model rats.The addition of matrix gel can provide structural support in three-dimensional level to the brain focal cavity and enhance the effect of cell repair.All above research shows that the combined transplantation of melatonin,neural stem cell and Matrix Gel promotes the function recovery of traumatic brain injury rats.Melatonin can protect neural stem cell against the microenvironment of traumatic lesion.Matrix Gel can provide the Structural support for the tissue cavity after traumatic brain injury,building a tridimensional condition for neural stem cell living,enhance the effect of neural stem cell repair.Based on the Basic research,our study may provide a new method for the clinic treatment of traumatic brain injury. |
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