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Objective:To observe the protective effect of etomidate (ET) on cultured retinal ganglion cells (RGC) with mechanical injury in vitro.Methods:New Sprague-Dawley rat RGC was cultured in vitro and identified by double immunofluorescent labeling of Thy1.1 and microtubule associated protein 2. The cultured primary cells were randomly divided into control group, RGC scratch group, ET low dose group (1 μmol/L), ET medium dose group (5 μmol/L) and ET high dose group (10 μmol/L). The RGC mechanical injury model was established by using iris knife to culture cells in RGC scratch group and ET group with different concentration. Seven days after modeling, the RGC survival rate of each group was detected by cell count Kit 8 proliferation assay. The apoptosis rate of RGC was detected by Annexin Ⅴ/propyl iodide double staining. Single factor analysis of variance was used to compare the groups. The pairwise comparison between groups was tested by the least significant difference method.Results:The survival rates of RGC in RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (72.60±2.97)%, (73.73±1.14)%, (79.19±1.79)% and (83.88±0.94)%, respectively. The RGC apoptosis rates of control group, RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (5.08±0.17)%, (18.67±1.24)%, (17.96±0.74)%, (15.11±0.56)% and (11.67±1.32)%, respectively. Comparison of RGC survival rate between groups: compared with RGC scratch group, the cell survival rate of ET low-dose group, ET medium-dose group and ET high-dose group was increased, and the difference between RGC scratch group and ET low-dose group was not statistically significant ( P=0.728); the differences between RGC scratch group, ET medium dose group and ET high dose group were statistically significant ( P<0.001); the difference between ET medium dose group and ET high dose group was statistically significant ( P=0.002). Comparison of apoptosis rate of RGC among groups: the apoptosis rate of RGC scratch group was significantly higher than that of control group, the difference was statistically significant ( P<0.001). Compared with RGC scratch group, the apoptosis rate of ET low-dose group, ET medium-dose group and ET high-dose group was decreased, and there was no statistical significance between RGC scratch group and ET low-dose group ( P=0.869). The differences of apoptosis rate between RGC scratch group, ET medium dose group and ET high dose group were statistically significant ( P<0.05). The difference of apoptosis rate between ET medium dose group and ET high dose group was statistically significant ( P=0.007). Conclusion:ET has neuroprotective effect on RGC cultured in vitro with mechanical injury, and the protective effect increases with the increase of ET dose in a certain range.
ABSTRACT
The optic nerve belongs to the central nervous system (CNS).Because of the lack of neurotrophic factors in the microenvironment of the CNS and the presence of myelin and glial scar-related inhibitory molecules,and the inherent low renewal potentials of CNS neurons comparing to the peripheral nerve system,it is difficult to spontaneously regenerate the optic nerve after injury.Protecting damaged retinal ganglion cells (RGCs),supplementing neurotrophic factor,antagonizing axon regeneration inhibitory factor,and regulating the inherent regeneration potential of RGCs can effectively promote the regeneration and repair of optic nerve.Basic research has made important progress,including the restoration of visual function,but there are still a lot of unsolved problems in clinical translation of these achievements,so far there is no ideal method of treatment of optic nerve injury.Therefore,it is rather urgent to strengthen the cooperation between basic and clinical research,to promote the transformation of basic research to the clinical applications as soon as possible,which will change the unsatisfactory clinical application status.
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Objective cAMP promotes neurite outgrowth in vitro. The study is aimed to understand whether cAMP can repair spinal cord injury of rats. Methods 56 rats models of spinal cord hemisection were adopted and randomly distributed into six groups. Dibutyryl-cAMP or physiological saline was injected either once in the motor cortex with an amount of 6 ml of 50 mmol/L cAMP, or continuously infused through a polyethylene tube connecting with a micro-pump in the spinal lesion area or in the subarachnoid space with a total amount of 72 ml of 10 mmol/L cAMP for 72 h. The distribution of neurofilament (NF) in the lesion area was observed by immunohistochemistry. Corticospinal tracts (CST) and spinal axons regeneration were investigated by CST and spinal axons anterograde tracing with biotinylated dextran amine (BDA). The function of hindlimb movements were evaluated by BBB scales and as a reference to assess the repairing effect of treating strategy. Results NF were densely distributed and extended in the lesion area in the cAMP groups, but no connection could be found with the NF in the caudal. No axonal regeneration could be achieved when cAMP was input into the subarachnoid space. Many regenerated axons, including some CST axonal regeneration were presented in the lesion areas in cAMP groups though no continuous long regenerated axons traversed the lesion area, while there was no regenerated axon in the lesion areas in the control groups. All the rats restored to normally walk 4 to 5 weeks after operations, BBB scale exceed 20, and no significant difference between cAMP groups and control groups by comparing the BBB scales of hindlimb movements. Conclusion cAMP injected in the brain cortex or continuously infused in the spinal lesion area can induce the axonal regeneration and is beneficial to repair the spinal cord injury, but could not directly promote hindlimb movements recovering.
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Objective To explore the effects of expressing human ciliary neurotrophic factor (hCNTF) mediated by retroviral vector in olfactory ensheathing cells(OECs) on the survival and neurite outgrowth of cultured neurons. Methods S\|hCNTF fragment was digested with endonucleases(Kpn I and Xba I) from pcDNA\-3\|S\|hCNTF plasmid and cloned into pRev\|TRE vector.The harvested pRev\|TRE\|hCNTF was identified and transfected with pRev\|Tet\|On into ecotropic Ecopack\|293 cells,resulting in 2 retroviral supernatants(pRev\|TRE\|hCNTF and pRev\|Tet\|On).Primarily cultured rat olfactory ensheathing cells(OECs) were co\|infected with the 2 retroviruses,and induced to secrete hCNTF with different concentrations of doxycline.The secreted hCNTF in OEC culture supernatant was detected with Western\|blot.Dorsal root ganglion (DRG) from a postnatal rat of 2 days was co\|cultured with CNTF\|modified OECs,and the supernatant was used to culture retinal ganglion cells(RGCs).Following ?\|tubulin immunocytochemical staining,the length of DRG neurites were measured,while the numbers of surviving RGCs were counted. Results 1.Individual 630bp and 400bp fragments were digested from pRev\|TRE\|S\|hCNTF expression vector with endonucleases(Hind Ⅲ and BamH Ⅰ),and respected direction and integration of hCNTF cDNA which inserted pRev\|TRE vector were identified; 2.The expression of 24kD CNTF proteins in CNTF\|modified OEC culture supernatant was positively\|correlated with the concentration of doxycline,while no such protein expression was detected in the control groups; 3.The number of surviving RGCs in CNTF\|modified OECs group(41^34?5^4) was significantly higher than those in unmodified OEC(23^15?4^7),OECs(24^55?5^8) and blank(16^8?6^5) groups;and 4^The neurites of DRG were longer (660?67?m) and denser in CNTF\|modified OECs group,as compared with unmodified OECs(418?45?m),Mock+OECs(400?65?m) and blank (0?m) control groups.No process migrated and grew from the tissue mass in blank group.Conclusion\ hCNTF can be expressed in OECs with a doxycline concentration\|dependent manner after transfected via pRev\|TRE\|S\|hCNTF vector,and possesses a marked enhancing effect on the survival and neurite outgrowth of cultured neurons.[
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Cho and So studied, with horseradish peroxidase retrograde tracing technique, the initial delay time and the rate of regrowth of damaged retinal ganglion cell axons regenerating into the autologous sciatic nerve implanted into the retinae in adult hamsters. This is the only report, to our knowledge, on the rate of regeneration of damaged central neuron axons. The present experiment tackles this issue using autologous sciatic nerve transplantation into the dorsal horn of the damaged spinal cord in adult rats, a model introduced by David and Aguayo, and visualized the regenerating axons with anti-neurofilament monoclonal antibody immunohistochemical method. Our results are as follows: the minimum initial delay time of the regenerating spinal axons in peripheral nerve grafts is 4 days. After which axons continue to regrow into the grafts within a definite period, suggesting different initial delay time for different regenerating axons. The regenerating spinal axons differ in their rate of regrowth, the fastest rate being 2.14 mm/d.
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Objective\ The present study was carried out to examine the relationship between the distance of axotomy and axonal regeneration systematically,and the effect of a pre\|degenerated peripheral nerve(PN) graft on axonal regeneration of retinal ganglion cells(RGCs) axotomized at different distances. Methods\ The optic nerve(ON) was transected at 0^5,1,1^5,2,3 or 7mm from the optic disc and a normal (the normal group) or pre\|degenerated(the pre\|degeneration group) PN graft was transplanted onto the ocular ON stump in adult hamsters. Results\ In both groups,the number of regenerating RGCs 28 days after grafting decreased significantly when the distance of axotomy increased from 0^5 to 7mm with a sharp decline between 0^5 and 3mm.When the corresponding distance points between the two groups were compared,enhanced regeneration was observed at 2 and 3mm in the pre\|degeneration group. Conclusion\ These results show that the distance of axotomy on the ON of adult hamsters is critical in determining the number of regenerating RGCs.The beneficial effect of a pre\|degenerated PN graft on axonal regeneration of axotomized RGCs is conditional on the distance of axotomy.\;