The Research About The Protective Effect Of Tacrolimus On Spinal Motor Neurons And Its’ Related Mechanism

Acute Spinal Cord Injury(Acute Spinal Cord Injury, ASCI) is a serious nervous system injury, often cause nerve damage below the injury level and part or all loss of sensory and motor functions. In the developed world, the patients with spinal cord injury costs average of $750000 or more for treatment, it bring great economic burden to individuals, families and society. Therefore, the treatment of acute spinal cord injury and make the patient back to normal social life has the vital significance.Tacrolimus(FK506), the commodity name is Prograf,has passed the FDA certification.It was isolated from the soil of fermentation product by Fujisawa pharmaceutical company, it belong to the macrolide antibiotics, have stro ng immunosuppressive effects. FK506 receptor protein-FKBP(FK506 Binding Protein) not only exists in the immune system,but also in the nervous system, it about 10 to 50 times of the former. The present study showed that tacrolimus as an immunosuppressive drug has neurotrophic and neuroprotective effects,but the drug’s neurotoxicity was related to the concentration, avoid to produce neurotoxic effect is difficult. At present the mechanism of nerve nutrition effects in FK506 is not clear, and there is no study talk about the protective effect of tacrolimus on hypoxia-ischemia spinal motor neurons. On the basis of training system of neonatal rats’ spinal motor neurons established, then we select the optimal concentration of tacrolimus to protect neurons’ growth. After optimal concentration of tacrolimus has been finded,we set up the hypoxia-ischemia’s model, observe the nerve nutrition and protection effects of tacrolimus and investigate its related mechanism.This research fills the blank of the research about tacrolimus affect for hypoxia-ischemia spinal motor neurons.At the same time, it provides support for the nutritional function of tacrolimus, which provides a new theoretical basis for clinical treatment of acute spinal cord injury.Part 1:Spinal motor neurons from neonatal rats: purification, culture and identificationObjective:To establish the culture system of spinal motor neurons from neonatal rats.Classification, identification and survival rates determination of spinal cord neuron.Method: Ventral spinal cord tissues from neonatal rats were made into cell suspension that was subjected to density gradient centrifugation and differential adherence followed by purification culture. Then, the cells on cover plates were identified and classified using immunocytochemical double staining method, and the content of cell components was calculated in combination with fluorescent Hoechst nuclear staining.Continue to develop 5 days, randomly selected 50 fields from each time point to calculate the number of living cells and did the statistical analysis.Results: The cells adhered the wall and grows well, the neurons accounted for 85.8%, motor neurons reached 71.6%, astrocytes accounted for 7.8%, cells negative for neurofilament 200 and glial fibrillary acidic protein were 6.4%.The number of the neurons were more,and the survival rate remained 57.8% after 5d.Conclusions:These findings indicate that the ventral spinal cord tissues from neonatal rats combined with density gradient centrifugation and differential adherence can develop high-purity spinal motor neurons. At the same time, the high survival rate of neurons, ensure the smooth progress of the follow-up experiments and successfully established a neonatal rats’ spinal motor neurons culture system.Part 2:Experimental study of the protective effect of tacrolimus on spinal motor neurons and its’ mechanismObjective:To find the optimal concentration of tacrolimus(FK506) for the growth of spinal cord motor neurons, observe the neuroprotective effect of the hypoxia-ischemia spinal motor neurons, and to explore the possible mechanism.Method: After the motor neuron cells were cultured for three days, we added different concentration of FK506 to classified the motor neurons into: blank control group(Group A), 100 pmol FK506 group(Group B), 1nmol/L FK506 group(Group C) and 10nmol/L FK506 group(Group D). Continue to culture two days, we use immunofluorescence staining to observe the growth of spinal motor neurons,MTT colorimetric assay to detect the vitality of spinal motor neurons,RT-PCR to detect the growth associated protein-43 m RNA expression of spinal motor neurons. After obtained the optimal concentration of FK506, we divided the spinal cord motor neurons into control group, hypoxia-ischemia group, FK506 treatment group.On the third day, the culture medium was aspirated and replaced serum-free medium in hypoxia-ischemia group and FK506 treatment group,close the hole with paraffin oil cause hypoxia condition. The control group without any treatment.In the hypoxia-ischemia group, we added 800μL 0.01 mol PBS per pore.In FK506 treatment group,we added 800μL optimal concentration of FK506 per pore,continue to culture two days.Then we use immunofluorescence staining to observe the growth of spinal motor neurons,MTT colorimetric assay to detect the vitality of spinal motor neurons,RT-PCR to detect the growth associated protein-43 m RNA expression of spinal motor neurons. Western blot to detect the motor neuron cell p-ERK protein expression after FK506 treatment.Results: The number and the length of the spinal cord motor neurons in group C were significantly better than group A, group B and group D. The OD value of MTT and m RNA relative expression level in group C was higher than group A, group B and group D, the difference has statistically significant(P<0.01). With optimal concentrations of FK506 treatment of spinal motor neurons immunofluorescence results showed that, the motor neuron number and neurite length in FK506 treatment group were significantly better than hypoxia-ischemia group, the OD value of MTT and GAP-43 m RNA relative expression levels in FK506 treatment group were significantly higher than hypoxia-ischemia group, has significant difference(P<0.01). According to the results of Western blot, the spinal motor neurons’ p-ERK expression were significantly increased, the peak time were 4 hours and the GAP-43 expression peak time were 8 hours.Conclusions:The optimal concentration of FK506 for neuroprotective effect is 1nmol/L. FK506 can improve the cell activity of hypoxia-ischemia spinal motor neurons in neonatal rats.Hypoxia-ischemia can increase p- ERK and GAP-43 protein expression in spinal motor neurons, the peak time of p-ERK protein expression were earlier than the GAP-43, support the view that GAP43 is downstream molecular of p-ERK,which have the function of nerve regeneration and repair.Part 3:Experimental study of tacrolimus in the treatment of acute spinal cord injury in ratsObjective:To observe the tacrolimus(FK506) in the improvement of nerve function after acute spinal cord injury in rats and explore the possible mechanism.Method:Randomly divided 72 healthy SD rats into three groups: control group,injury model group, FK506 treatment group.The injury model group and FK506 treatment group’s T10 spinal cord wounded by Allen’s blow device to make the acute spinal cord injury model, the control group only do laminectomy. 10 minutes after injury,the FK506 treatment group did the intraperitoneal injection with 0.3mg/kg FK506,once a day, during 2 weeks.The rest of the two groups were given equal volume of saline solution.According to the time after injury(1,2,4,6h) the rats in their group were divided into four teams, each team has 6 rat,measure the MDA, SOD and NO levels of spinal cord tissue. Using toluidine blue staining to observe spinal cord tissue pathology change,BBB score and inclined plate experiment to detect the spinal cord function at 1,3,7,14,21 days post- injury.Results: At 1, 3, 7,14,21 days post-injury,the situation of hemorrhage and necrosis in the spinal cord in FK506 treatment group were better than injury model group.The inclined plate test score and the BBB score in FK506 treatment group were higher than the injury model group, the difference between the two groups is significant(P<0.05).The content of MDA, SOD and NO in the injury model group and the FK506 treatment group had no significant difference at postoperative 1h, but at postoperative 2,4,6h,the FK506 can inhibited the contents of MDA and NO increase obviously, improved the content of SOD decreased significantly, has significant difference(P<0.01).Conclusions: Early application of tacrolimus can reduce the apoptosis of nerve cells, relieve secondary spinal cord injury, promote the recovery of spinal cord function. The protective effect may be related to inhibit the inflammatory reaction and enhance the cell viability of spinal cord.