The Effects Of Fingolimod On Neurofunctional After Acute Spinal Cord Injury And Neural Stem Cells In Vitro

Objective:To observe the effects of a novel immunemodulator FTY720 on acute spinal cord injury and blood-spinal cord barrier in rats and analysise its underly mechanism. Investigate the influences of FTY720-P on proliferation, differentiation and the migration ability of NSCs in vitro. This study aims to provide evidences for the following studies and new treatment strategy for SCI.Main methods and techniques:1.140 adult Sprague-Dawley (SD) rats were randomly divided into four groups:Normal control group (NG Group):rats without any treatment. Sham-operated group (SO Group):rat spinal cords were exposed by laminectomy without injury. Hemisection group (HS Group): rats received spinal cord hemisection injury and intraperitoneal injection with saline solution. FTY720 treated group (FTY720 Group):rats received spinal cord hemisection injury and intraperitoneal injection with FTY720 (1mg/kg/day) for 7 days.The neurofunctional was assessed by Basso Beatlie Bresnahan (BBB) scores, grid walking, record N1 and PI delays of motor evoked potential (MEP) and somatosensory evoked potentials (SEP), histological analysis by light microscopy with HE staining, and determination of blood-spinal cord barrier permeability with fluorescence microscope at different times after injury.2. Isolate NSCs from E14.5-16.5 Sprague-Dawley (SD) rat fetal brains. The isolated cells were cultured in serum-free medium and identified by immunofluorescence with Nestin and BrdU staining. Their potential to generate neurons and glial cells were examined through their special markers’expressions respectively with immuocytochemisty. Investigate the effects of different concentrations FTY720-P on NSCs’ biological characteristics including their ability of proliferating, the potential to generate neurons and glial cells and the ability of migration in vitro.Main results:1. The neurofunctional of HS group and FTY720 group were impaired after hemisection injury. They were still incurable at Day 28 after damage compared with NG group or SO group. The recovery rate of motor function in FTY720 group was higher than it in HS group. The BBB scores and the potential period of SEP-P1 showed statistical significance from Day 7 to 28 after injury between FTY720 group and HS group(P<0.05).The grid walking test results and the potential period of MEP-N1 showed statistical difference from Day 14 to 28 after injury between FTY720 group and HS group(P<0.05). Compared the HE staining slices at Day 14 and Day 28 after injury, the number of chronic inflammatory cells, the reaction of glial cells and the size of syringomyelia cavities in gray matter in FTY720 group were significantly less than HS group. In addition, the leakage of Evans Blue from the damaged BSCB in FTY720 group were increased than NG group and SO group in 7 days after injury(P<0.01), but reduced compared to HS group(P<0.05), the biggest difference was observed at Day 3 after injury.2. NSCs were successfully isolated and identified from fetus rats. Culture media containing OnM, 1nM, 10nM and 100nM FTY720-P was applied for NSCs to research. lOnM and 100nM FTY720-P stimulated proliferation of NSCs coordinated with bFGF. There was no significant alteration of proliferation between controlled group and FTY720-P treated group alone. With the concentration increased, the ratio of NSCs migration was increased, which suggested that FTY720-P has a chemoattractant effect on NSCs. After NSCs differentiation for 7days, the number of neurons showed no significant attenuation in different concentrations of FTY720-P groups but the proportion of protoplasmic astrocytes increased.Conclusion:FTY720 can decrease the permeability of blood-spinal barrier at acute phase of acute spinal cord injury. And it mainly promotes the neural functional recovery after acute phase. We demonstrate that FTY720 has some potential neuroprotective effects. In addition, FTY720-P mediate NSCs biological characteristics including their ability of proliferating, the potential to generate neurons and glial cells and the ability of migration in vitro.