The Experimental Study Of Bone Marrow Stromal Cells Transplantation In Spinal Cord Injury

PrefaceExperimental evidences have shown that bone marrow stromal cells ( BMSCs ) , when transplanted into the injured spinal cord of rats, promote functional recovery. Bone marrow stromal cells survived after transplantation and appeared to differentiate into oligodendrocytes, astrocytes and neurons. BMSCs may reduce functional deficits and have a therapeutic role after spinal cord injury, but further neurobiologic understanding and mechanisms that promote recovery with bone marrow cell transplantation after spinal cord injury remain incompletely understood up to now.In the present study, we tested the ability of survival, migration and integration of BMSCs, as well as the expression of glial fibrillary acidic protein (GFAP) and neurofliament ( NF) after spinal cord injury by immunohistochem-istry. We also investigated the expression of brain - derived neurotrophic factor ( BDNF) , glial cell line - derived neurotrophic factor( GDNF) and proteolipid protein ( PLP) using RT - PCR method, and performed functional tests after BMSCs translpantion, to discuss further mechanisms that BMSCs transplantation promote regeneration and recovery of the injured spinal cord.Materials and MethodsIsolation of BMSC and cell cultureBone marrow stromal cells were obtained from donor Wistar rats. Freshcomplete bone marrow was harvested aseptically from tibias and femurs. Bone marrow was mechanically dissociated to obtain a homogeneous cell suspension. The cells were incubated with containing 10% fetal bovine serum (FBS) at 37 C in 5% CO2. After tightly adhered to plastic, cells were resuspended in fresh DulbeccoS modified Eagle medium (DMEM -LG) , and BMSCs were grown for three to fifteen passages. To identify cells derived from bone marrow, cells were trypsinized and subcultured in chambered slides. BMSCs were examined with microscope and identified by surface markers, CD45 and CD90, after Immuno-histochemical staining. BrdU labelling of BMSCsWhen cells were cultured for 3 passages, BMSCs were marked in 20 mol/L of the bromodeoxyuridine (BrdU) 72 hours before transplantation. After several washes in PBS, cells were collected for transplantation. Spinal cord injury model and transplantationWistar rats were anesthetized with chloral hydrate (30mg/kg) and a lami-nectomy was performed was performed to expose the dorsal surface of the T7_u segment, followed by a hemi - transection at T9 level. Rats were assigned, without bias, to control and experimental groups. Experimental groups had 6 1 containing 6 105 BMSCs injected into the cross site of white and grey matter around the injury. The control group had 6 1 of PBS injected within the same site as those of experimental groups. ImmunohistochemitryOn 1, 3, 7, 14, 28 days after transplantation, immunohistostaining of BrdU and the survival of BMSCs were observed respectively. NF and GFAP expression was quantitatively determined. Slides were viewed and photographed with a photo microscope. The photographs were digitized with a video image a-nalysis system ( Metamorph Image System 4.6) in conjunction with a computer. After background correction, the grey levels of each slide were automatically detected. Then the mean of grey levels for all slides from each animal were obtained and statistical analysis was performed. RT-PCR analysisT9 spinal cord tissue of 80 - lOOmg was collected 24h, 72h and 7 days aftercell transplantation. Total RNA was isolated from the tissue using TRIzol (GIB-CO BRL) and the RNA concentration was measured photometrically. For PCR, specific primers were designed to amplify GDNF, BDNF and PLP cDNA. After 35 cycles of PCR, expression GDNF, BDNF and PLP mRNA of samples was analyzed by image analysis system. Functional evaluationFunctional tests were performed before surgery and 1 day and 1, 2, 3, and 4weeks after surgery. The locomotor functions of animals were evaluated using the modified Tarlov scale and inclined plane method of Rivlin.ResultsCharacterization of BMSCsThe BMSCs in cultures, which formed clone after 10-14 days, had a spindle - like morphology, a large flat, or a small granular appearance, and displayed CD90 positive and CD45 negative immunoreactivity. The positive marker remained during 2—15 passages, it was indicated that the cultured cells measure up to the characterization of BMSCs.BrdU - positive BMSCs cells survivals were observed in spinal cord after transplantation, with cell migration more than 2mm rostrally and caudally from the injection sites. Histological and molecular examinationHistological examination of the spinal cord at different time after BMSCs transplantation showed that immunostaining for GFAP and NF was much stronger in BMSCs transplantation groups than in control groups.After surgery and BMSCs transplantation, RT - PCR was used to measure the mRNA level of GDNF, BDNF and PLP in the injured spinal cord. We examined the similar time - dependent change in mRNA expression of GDNF, BDNF and PLP. The expression of GDNF, BDNF and PLP mRNA began to increase at 24 hours, peaked at 72 hours at levels 2 - fold higher than controls, began to decline at 3 days, and did not returned to basal levels by 7 days after transplantation. In injured spinal cord injected with BMSCs, significantly higher levels ofGDNF, BDNF and PUP mRNA expression were detected compared with that in the spinal cord of rats injected with PBS. Functional evaluationUsing the modified Tarlov scale and inclined plane method of Rivlin, we demonstrated a progressive recovery over time on experimental groups. The functional recovery in the BMSCs transplantation group showed statistically significant improvements compared with the control group. This result is in accordance with the morphological experiments.DiscussionMany exciting and promising experimental therapeutic strategies have emer-genced to promote regeneration 'of the injured spinal cord in laboratory animals in the past several years. Transplantation is perhaps one of the most powerful strategies to promote spinal cord repair.A greater understanding of the pathophysiologic mechanisms that contribute to the initial and secondary cord injury may facilitate the development of neuro-protective strategies that preserve axonal function and prevent apoptotic cell death, thus optimizing neurologic function. There are at least seven overlapping principal approaches to SCI repair: bridging the gap, building new circuits, re-myelinating demyelinated axons, providing trophic support, overcoming myelin - associated growth inhibition, overcoming glial - scar - associated growth inhibition, and protecting neurons and glia from secondary cell death.Recent studies have shown that transplantation of BMSCs appears to be one of the most promising strategies to promote recovery in the injured spinal cord. BMSCs were proved survival, growth and differentiation in the spinal cord with transplantion. It is conceivable that the transplanted BMSCs have several beneficial effect on the recovery of spinal cord injury. BMSCs can bridge the injury site, provide the mechanical or chemical induction, stimulate nerve regenreation and facilitate axonal growth across the lesion. On the other hand, BMSCs may provide nerve cells to compensate loss cellular structure. Moreover, BMSCs may also provide trophic support which can promote nerve regeneration.Glial fibrillary acidic protein (GFAP) , which is regulated dynamically by astrocytes, is used to mark the activity of injured astrocytes. Astrocytes respond to central nerve system (CNS) injury by hyperplasia and hypertrophy. Astrocytes play an important role in neurons survival and synapses formation after spinal cord injury. These cells can secrete a number of molecules that are neurotrophic to the recovery of spinal cord injury, such as NGF, bFGF, IL - 1, IL -6, IL - 3 and TNF and so on. Data presented in this study demonstrated significant increase of the GFAP immunostaining 7 days after BMSCs transplantation, and the sustained high - level expression has remained for 28 days in transplantation groups. However, the expression of GFAP in control groups began to decrease at 14 days, and returned to basal levels by day 28. This result was in accordance with the functional evaluation. It was indicated that BMSCs transplantation can increase GFAP immunoactivity of injured spinal cord and promote the recovery of locomotor functions of rats.Neurofilament ( NF) plays a critical role in neuronal function both as a slc€:letal protein maintaining neuronal shape and caliber and as a facilitator of ax-onal transport. Neurofilament consists of three subunits called the neurofilament triplet proteins NF - L {NF68; 68 kDa) , NF - M ( NF160; 160 kDa) , and NF - H ( NF200; 200 kDa) that assembled together build filaments. Previous stud-y proved that the number of NF200 positive neurons and the degree of neuron immunostaining were significantly related to functional recovery of incomplete spinal cord injury. The current findings showed that NF200 positive neurons were observed disciplinary with time and distribution. NF200 positive in motor neurons of anterior horn were determined earlier than those of posterior horn. This difference indicated that neurons of different types responded to injury differently , which may correlate to the direction of signal conduction. In this study it was indicated that BMSCs transplantation can increase NF immunoactivity of injured spinal cord and promote the recovery of locomotor functions of rats.Neurotrophic factors were originally identified as critical mediators of neuronal survival and nerve fibre outgrowth during development. The beneficial effects of neurotrophic factors on neuronal protection and repair in the CNS have been well documented. The glial cell line -derived neurotrophic factor {GDNF) , o-riginally identified as a trophic factor for midbrain dopaminergic neurons, have been found to be the most potent trophic factor for motoneurons. GDNF may have stimulated the repair or survival of spinal motor neurons, which may have contributed to the functional recovery. GDNF may have a more potent effect in stimulating axonal growth. Furthermore, GDNF was able to induce motor axon outgrowth across the surrounding white matter. When applied into the spinal cord, GDNF was able to exert a trophic effect on corticospinal neurons and promote long - term survival after axotomy. Moreover, GDNF has recently been shown to exert behavioural and anatomical neuroprotection following SCI. In the present study, high levels of GDNF mRNA could be detected even 1 week after BMSCs transplantation. The sustained high - level expression of GDNF indicated the possibility of neuron self - protection and more need of GDNF to maintain physiological activity of nerve cells following spinal cord injury. We also demonstrated that GDNF mRNA expression in injured spinal cord injected with BMSCs was significantly higher than that in the spinal cord of control rats. Brain - derived neurotrophic factor ( BDNF) plays a major role in maintaining and promoting development, differentiation, growth and regeneration of sensory neurons, cholinergic neurons and dopaminergic neurons. BDNF has been studied extensively to find whether they have a role in promoting regeneration of spinal motor pathways. Brain - derived neurotrophic factor was reported to promote rubrospi-nal tract ( RST) regeneration, but has limited effects on corticospinal tract { CST) regeneration. BDNF can prevent neuron atrophy after spinal cord injury and promote synapse growth. Moreover, BDNF promote regeneration of axons and myelin sheath continuously. In the current study, it was the same as the expression of GDNF expression that BDNF mRNA expression increased significantly after BMSCs transplantation.In normally myelinated nerve, voltage - dependent sodium channels, responsible for action - potential generation, are concentrated at the nodes of Ran-vier and are scarce along myelin — covered internodal segments. Loss of myelin causes resistive and capacitative shunting of current across unexcitable demye-linated segments, and reduced current density at excitable segments. This causes either delayed saltatory conduction, as more time is required for excitable...