| Partâ… An Experimental Study on Model Foundation of Choroidal Neovascularization Induced by Krypton Laser in RatsObjectiveTo study the efficacy and safety of the model foundation of choroidal neovascularization(CNV)induced by krypton laser in Brown Norway(BN)rats, observe the trend of the formation and changes of CNV,and ascertain the initial and peak time of CNV formation after laser photocoagulation.MethodsTwenty-five male BN rats with weight from 180 to 200g were used in this study. One rat(2 eyes)was randomly selected as control group and another 24 rats(48 eyes) were as experimental group.The experimental group(24 rats)was randomly divided into 6 sub-groups with 4 rats in each sub-group according to the different follow-up time.The rats were anesthetized with intraperitoneal injection of 846 combined anesthetic(0.5mg/kg).The pupils were dilated with 0.5%tropicamide 5 minutes before laser photocoagulation.Eight laser photocoagulation spots emitted by krypton laser,with the spot size of 50μm,duration of 0.05 second,intensity of 350mW and wave length of 647.1nm,were applied to each eye in the area of 2 PD away from and around the optic disc.Four rats were randomly selected to receive the examinations of fundus fluorescein angiography(FFA),histopathology and transmission electron microscopy 3,7,14,21,28 and 56 days after the photocoagulation.Results1.After laser photocoagulation,the leakage appeared in burns on day 7 (190/16×20,59%),increased on day 14(172/16×16,67%),reached the peak on day 21(162/16×12,84%)(P＜0.01),and kept stable after day 21(P＞0.05).2.Three days after laser photocoagulation,cellular tissue extended from the choroid through the rupture of Bruch's membrane.In association with this tissue,there were many pigment-containing macrophage-like cells,and many retinal pigmented epithelial(RPE)cells were at the boundary of the burns.Seven days after laser photocoagulation,newly formed vessels with wide lumens extended from the choroid to the subretinal space where they became partially enveloped by RPE cells.Fourteen days after laser photocoagulation,neovascularization increased and RPE decreased. Twenty-one days after laser photocoagulation,CNV kept fibrovascular proliferation (FVP),and large collections of vascular channels were seen.Fifty-six days after laser photocoagulation,excavations were seen from outer retina to inner,and there were a large amount of collagen fibers and elastic fibers in CNV.3.The thickness of CNV increased from day 7 to day 21(P＜0.01),and no significant progress occurred after day 21(P＞0.05).4.Complication:Except little hemorrhage in few photocoagulation spots(8/384), none of other complications were observed.ConclusionThe experimental model of choroidal neovascularization can be successfully founded by krypton laser in rats with a stable,long lasting and a highly successful probability.The leakage appears on day 7,increases on day 14,reaches the peak on day 21,and keeps stable on day 21 after laser photocoagulation.Partâ…¡:Experiment Study on Construction of Ad-PEDF and It's Eukaryotic ExpressionObjectiveTo construct recombinant adenovirus carrying pigment epithelium-derived factor (PEDF)gene for gene therapy research on choroidal neovascularization(CNV).Methods1.The PEDF cDNA was prepared by nest RT-PCR technique using the template mRNA isolated from the tissue of the retina of BN rat.The PEDF coding sequence was cloned into the vector pGEM-Teasy and then sequenced.A pair of primers for PEDF which contained EcoRâ… and Hindâ…¢specific sites on each end was designed. The PEDF coding sequence was subcloned into adenovirus expression plasmid pDC316,the recombinant vector pDC316/PEDF was analysised by restricted endonuclease Sacâ… ,and sequenced.The 293 cells were cotransfected with pDC316/PEDF and pBHGloxΔE1,3Cre by Lipofectamine 2000,thus Ad-PEDF was constructed by Cre/loxp site intracellular homologous recombination.After the recombinant adenovirus was identified by PCR,the recombinant adenovirus stock reproduced in 293 cells and was concentrated by Cscl ultracentrifugation,and detected the titer of the recombined virus.2.Detecting the expression of PEDF protein in eukaryotic cell.HepG2 cells were infected with Ad-PEDF in vitro.The medium was changed into culture medium without FBS 1 hours later.The supernatant was collected 48 hours later,and detected the expression of PEDF by Western blot.Results1.Compared with the sequence of the rat PEDF mRNA published in NCBI Sequence Viewer(NM-177927),the PEDF cDNA fragment contained all the sequences of open reading frame of the rat PEDF gene.The plasmids pDC316/PEDF was constructed successfully,and the nucleotide sequences of pDC316/PEDF were confirmed by sequencing.The 293 cells were cotransfected with pDC316/PEDF and pBHGloxΔE1,3Cre.Ad-PEDF were constructed by Cre/loxp site intracellular homologous recombination.The recombinant adenovirus titer increased to 3.08×1010 pfu/mL after amplification and CsCl gradient purification,which was qualified for experiments in vivo and in vitro.2.Western blot confirmed that the PEDF protein was significantly expressed in infection group,while it was not happened in control group.It was demonstrated that Ad-PEDF could transfected eukaryotic cells,and the PEDF protein could be detected in supernatant cells.ConclusionThe successful construction of Ad-PEDF sets an important foundation for Ad-PEDF therapy of CNV. Partâ…¢An Experimental Study on Choroidal Neovascularization Inhibited by Ad-PEDFObjectiveTo study the inhibiting effect of adenoviral vectored pigment epithelium-derived factor(Ad-PEDF)gene on choroidal neovascularization(CNV)in BN rat model, observe the trend of the regression and changes of CNV,and ascertain a better method between intravitreal injection and periocular injection for further Ad-PEDF therapy.MethodsSixty-eight female BN rats with weights from 160 to 180g were used in this study.Four rats(8 eyes)were randomly selected as normal control group and 64 rats (128 eyes)were as experimental group.The experimental group(64 rats)was randomly divided into 4 groups with 16 rats in each group.Group A:intravitreal injection with Ad-PEDF 1μl;Group B:intravitreal injection with control vector (AdNull)1μl;Group C:periocular injection with Ad-PEDF 1μl;Group D:periocular injection with AdNull 1μl.Then each group was randomly divided into 4 sub-groups with 4 rats in each sub-group according to different follow-up time.All the rats were anesthetized and mydriasis.Eight laser photocoagulation spots emitted by krypton laser,with the spot size of 50μm,duration of 0.05 second,intensity of 350～380mW and wave length of 647.1nm,were applied to each eye in the area of 2 PD away from and around the optic disc.After 14 days,the normal control group were tested by fundus fluorescein angiography(FFA)and sacrificed without any operations just for comparing with the experimental groups.At the same time,the experimental groups were tested by FFA,then they were received intraviteal or periocular injection of Ad-PEDF or AdNull.Finally,the rats were sacrificed 3,7,14,28 days after injection. Histopathology and TUNEL staining were tested and observed 3,7,14,28 days after injection.Results1.The leakages appeared decrease after treatment in group A(54.7%)and C (56.3%)(t=2.75;t=3.15,P＜0.01).2.CNV decreased in group A(57.3%)and C(57.8%)7 days after injection,and kept fibrovascular proliferation in group B and D.3.The thickness of CNV in group A(44.51±0.53μm)and C(44.37±0.48μm) was significantly less than that in normal control group(46.35±0.93μm)after the treatment(F=7.57,8.85;P＜0.01),and it diminished with the time prolongation (F=4.31,5.25;P＜0.05).The thickness of CNV in group A(46.35±0.62μm)was greater than that in group C(44.90±0.44μm)3 days after treatment(F=3.55,P＜0.05), and it was less in group A than that in group C 14 and 28 days after treatment (F=6.54,P＜0.01;F=4.41,P＜0.05).4.There were positive TUNEL stainings in choroidal neovascular endothelium in group A and C.5.Postoperative complication included cataract(5 eyes)after intravitreal injection and no complication occurred after periocular injection.Conclusion1.AdPEDF is effective in inhibiting CNV in an animal model.2.The effect appears 7 days after treatment,reaches the peak on day 14,and keeps stable on day 28.3.The inhibition effect on CNV appears slowly in eyes with intravitreal injection than that with periocular injection,and it is stronger in eyes with intravitreal injection.4.The method of periocular injection is easy and can be repeated.The method of Intraviteal injection is complex and not repeated easily and can induce cataract.
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