Effect of Y-27632 on the cultured retinal neurocytes of rats

AIM:To investigate the effect of Y-27632 on the survival and neurite outgrowth of the cultured retinal neurocytes. METHODS:After the postnatal day 2-3, Sprague-Dawley retinal neurocytes were cultured for 48 hours, the culture media was replaced with serum-free media (control group) and serum-free media contained 30μmol/L Y-27632 (Y-27632 group), and the cells were continually cultured another 48 hours. The cultured retinal neurocytes were identified with anti-neuron specific enolase (NSE) immunocytochemistry. The survival state of those cells was estimated by MTT assay, and the neurite outgrowth of those cells was evaluated by the computerized image-analysis system. RESULTS:Compared with the control group, the absorbance values of cells survival in Y -27632 group increased 12.90% and 33.33% respectively after 72 and 96 hours culture. Y-27632 had no significant effect on the diameter of cultured retinal neurocytes. Compared with the control group, Y-27632 induced a stable improvement of neurite outgrowth of retinal neurocytes after 72 and 96 hours culture (P =0.001). CONCLUSION:Y-27632 could promote the survival and neurite outgrowth of the early postnatal cultured retinal neurocytes.

Spinal cord injury regeneration using autologous bone marrowderived neurocytes and rat embryonic stem cells:A comparative study in rats

BACKGROUND Spinal cord injury(SCI)is an important cause of traumatic paralysis and is mainly due to motor vehicle accidents.However,there is no definite treatment for spinal cord damage.AIM To assess the outcome of rat embryonic stem cells(rESC)and autologous bone marrow-derived neurocytes(ABMDN)treatment in iatrogenic SCI created in rats,and to compare the efficacy of the two different cell types.METHODS The study comprised 45 male Wistar rats weighing between 250 and 300 g,which were divided into three groups,the control,rESC and ABMDN groups.The anesthetized animals underwent exposure of the thoracic 8th to lumbar 1st vertebrae.A T10-thoracic 12th vertebrae laminectomy was performed to expose the spinal cord.A drop-weight injury using a 10 g weight from a height of 25 cm onto the exposed spinal cord was conducted.The wound was closed in layers.The urinary bladder was manually evacuated twice daily and after each evacuation Ringer lactate 5 mL/100 g was administered,twice daily after each bladder evacuation for the first 7 postoperative days.On the 10th day,the rats underwent nerve conduction studies and behavioral assessment[Basso,Beattie,Brenham(BBB)]to confirm paraplegia.Rat embryonic stem cells,ABMDN and saline were injected on the 10th day.The animals were euthanized after 8 wk and the spinal cord was isolated,removed and placed in 2%formalin for histopathological analysis to assess the healing of neural tissues at the axonal level.RESULTS All the animals tolerated the procedure well.The BBB scale scoring showed that at the end of the first week no recovery was observed in the groups.Post-injection,there was a strong and significant improvement in rats receiving rESC and ABMDN as compared to the control group based on the BBB scale,and the Trainof-four-Watch SX acceleromyography device exhibited statistically significant(P<0.0001)regeneration of neural tissue after SCI.Histological evaluation of the spinal cord showed maximum vacuolization and least gliosis in the control group compared to the rESC and ABMDN treated animals.In the ABMDN group,limited vacuolization and more prominent gliosis were observed in all specimens as compared to the control and rESC groups.CONCLUSION This study provided strong evidence to support that transplantation of rESC and ABMDN can improve functional recovery after iatrogenic SCI.The transplanted cells showed a beneficial therapeutic effect when compared to the control group.

Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neuroeyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass （inner cell mass, ICM） which were isolated by mechanical method on the mouse embryonic fibroblaste cell （MEF） feeder layer or 0.1% gelatin coated dishes. The stem ceils were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunoehemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 （ stage specific embryonic antigen 1 ）. Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated ceils presented the characters of ESCs. Then the isolated cells were able to differentiate into neuroeytes in vitro. Conclusion Mouse embryonic stem ceils isolation, culture and differentiation system has been established.