Objectives To observe the changes of axonal transport in the rabbit optic nerve under acute ocular hypertensions. Methods 24 adult healthy New Zealand white rabbits were divided into 4 groups according to the intraocular pressure (IOP), with 6 rabbits in each group. There are 3 experimental groups with an IOP of 20, 30, 40 mm Hg (1 mm Hg=0.133 kPa). respectively, and 1 control group with an IOP of 10 -15 mm Hg.Two 25-gauge cannulas were inserted into each rabbitprime;s anterior chamber to create the model of acute ocular hypertension. At the beginning of the experiment, rhodamine-beta;-isothiocyanate (RITC) was injected into the vitreous of each eye to label axonal transport. After 3 hours of high intraocular pressure, rabbits were sacrificed with anesthetic overdose. The retina and the optic nerve were then carefully exposed. Fluorescent microscopy was used for quantitative measurements of the changes of optic nerve axonal transport. Statistically analyze the average grey level in different groups and sites by Leica RITC Q500IW image analysis software. Results RITC, a fluorescent tracer, was transported in the anterograde direction by axonal transport. With the increasing of the intraocular pressure, the distance of the axonal transport was declined (F=159.3, P＜0.05). The difference of the grey level in the pre-laminar region of 20, 30, 40 mm Hg group was not statistically significant (F=0.2545,P＞0.05 ). Compared the grey level of 40 mm Hg group with control group, the differences in lamina cribrosa（t=5.684）and the proximal 350 mu;m of the post-laminar (t=5.124) were statistically significant (P＜0.05). Compared the grey level of 20, 30 mm Hg group with control group, the difference was not statistically significant(t=1.747, P＞0.05 ).Conclusion 40 mmHg intraocular pressure lasts for 3 hours can reduce axonal transport in the lamina cribrosa and post laminar of optic nerve.
Objective To investigate the feasibility of gene transfection into retinal pigment epithelial (RPE) cells and photoreceptors (PRs) in vivo electroporation. Methods A total of 147 Sprague-Dawley (SD) rats were divided into 5, 10, 15, 20, 25, 30 and 35 V group according to different voltage. The right eyes of rats underwent the injection of eukaryotic expressive plasmid of enhanced green fluorescent protein (EGFP) pEGFP-N1 into subretinal space as experimental eyes; the left eyes were injected with TE buffer as control eyes. Each group was divided into RPE and RP subgroups according to different transfection direction. There were same parameters of 99 ms pulse width, 0.5 s pulse interval and 5 consecutive pulses except different voltage in groups. With a negative charge in the electric field was transfected into RPE cell layer, reverse electrode set to be transfected into PR cell layer. Retina mounts were made on seven days after transfection and the fluorescence of EGFP was photographed by fluorescent microscope. The expression of EGFP mRNA and protein were detected by reverse transcription polymerase chain reaction technique (RT-PCR) and Western blot.Results On seven days after transfection, in RPE subgroups, there were no specific fluorescence expressions in RPE cell layer and retina mounts of control eyes, while there were fluorescence expressions in experimental eyes. Western blot showed that the grayscale ratio of EGFP protein and beta;actin protein bands rose with the increased voltage. RT-PCR showed that each group produced positive amplification bands, and the relative ratio of gray level of EGFP mRNA and GADPH mRNA amplified bands gradually increased with the increased voltage.Conclusion Electroporation is an effective method for gene delivery into RPE cells in vivo.
Objective To construct expression plasmid of the fusion protein of brainderived neurotrophic factor (BDNF)green fluorescent protein (GFP), and observe its characteristics.Methods BDNF cDNA segment was inserted into plasmid pcDNA3.1/ NT-GFP-TOPO and in the same reading frame with GFP. After verified by sequencing, the BDNFGFP plasmid was transferred into cultured Schwann cells by electroporation. And the expression of BDNFGFP fusion protein was observed by immunohistochemistry and Western blotting. The neuralprotective function of the fusion protein was evaluated by transferring the plasmid into adult rat retinas with transected optic nerve.Results The sequence of BDNFGFP plasmid was verified correctly by autosequencing. The results of Western blotting showed that the BDNF-GFP fusion protein expressed a brand with the relative molecular mass of 41times;103. Seven days after the optic nerve was transected, the number of survival retinal ganglion cells (RGC) in BDNF-GFP group and GFP group was （1201plusmn;286） and（482plusmn;151）cells/mm2, respectively; and the survival rate was （51.39plusmn;12.24）% and （20.62plusmn;6.46）% , respectively. Twentyeight days after the optic nerve was transected, the number of survival RGC in the two groups was （715plusmn;71） and （112plusmn;24）cells/mm2, respectively; the survival rate was（30.59plusmn;3.04）% and (4.79plusmn;1.03）% respectively. The differences of the survival rate of RGC between the two groups were significant (t=3.144，11.378；Plt;0.01).Conclusion BDNF-GFP fusion plasmid can express a fusion protein which emit green fluorescence and has the biological activity of BDNF.