Study of of Complement 9 expression in the laser-induced horoidal neovascularization and cobra venom factor intervention in mice

1. Dr Song Chen
2. Dr Huaxiang Yu

Tianjin Eye Hospital

Objective
Age-related macular degeneration£¨AMD£©is the most common cause of low vision and blindness in the elderly worldwide persons,choroidal neovascularization (CNV£©is the leading cause of blindness .The aim of theses experiments is to observe the components of Drusen- membrane attack complex(MAC)expression in the laser-induced choroidal neovascularization in mice and to study the formation of MAC playing the role in the development of CNV. In this study we prewent CNV development with cobra venom factor (CVF) by intraperitoneal injection.Further explain the immunological mechanisms of AMD and explore a effective methord to inhibite complement activity,provide a theoretical basis and new methods.

Method
1. Thirty six 6-8 weeks C57BL/6J mice,male and female unlimited,they are all healthy and no eye disease checked by ophthalmoscope and slit lamp.The mice divide into three groups by random, group one as control group(n= 12)£¬In group two, CVF treated :C57BL/6 mice (n= 12) were treated i.p. with 0.1¦Ìg/g of CVF 2 days before laser photocoagulation and every day after laser treatment. In group three, C57BL/6 mice (n= 12) were only treated by laser photocoagulation.
2. Induction of CNV in mice: the mice are anesthetized by the 10% chloral hydrate (0.3ml/100g), mydriasis with Tropine amide compound,surface anesthesia, drops 1% methyl cellulose amount in Conjunctival sac of experimental eye, place the slide as a contact lens ,the krypton red laser (50¦Ìm spot size; 0.1s duration; 120 mW, Wavelength 647nm),four laser spots are placed in each eye close to the optic nerve. The bubbles generated after photocoagulation can be regarded as the symbol of breakage of bruch¡®s membrane,checked the laser spots by ophthalmoscope.
3. At the1,3,5,7day,the mice are anesthetized by the 10% chloral hydrate. Put out the eyes taking the Frozen section to the SABC-FITC histochemical stain and observe the positive stain under Fluorescence microscope .Each eye select two laser regional integrity retina structure by SABC-FITC histochemical stain and HE stain.Each section selects 5 fields by random(¡Á200 times),figure the positive stain in each field.The HE stain is to observate the morphological changes in retina under the light microscope.

Results:
1. HE stain display that in the control group,the mice retina has the regular layers, the photoreceptor cells are regular,the nuclear stains deep violet,the boundary is sharp.retinal pigment epithelium£¨RPE£©cell nuclear is light violet,ellipse,clear and stain uniformity. Two group mice:One day after retinal photocoagulation,we can find that retinal tissue have edema,Bruch membrane and RPE layer are significantly damaged,the outer and inner nuclear layers structure of retina are floc chaos;three days after retinal photocoagulation, choriocapillaries is obviously congestive, RPE cell are hyperplasia,but don¡¯t find neovascularization;Seven days after retinal photocoagulation,a small amount of CNV are formed in facula area,mild retinal detachment and subretinal scarring lesions,cells arrange irregularly in scar lesions, injuried RPE decompose and break,photoreceptors fracture and some disappear, outer nuclear layer disappeare,inner nuclear layer disorder.Three group of mice:one day after retinal photocoagulation, retinal edema is lighter than group two, three days after retinal photocoagulation retinal tissue remains edema,congestion of choriocapillaries are lighter than guoup two, seven days after retinal photocoagulation don¡¯t find CNV.
2. SABC-FITC histochemical stain appears that under different treatment groups and at different time point groups,with the two way ANOVA.The mice C9 IOD(integrated optical density)in SABC-FITC histochemical stain are differernts,and these difference between different treatment groups are significant at the 0.05 level analyzed by statistics software spss11.5.(p£½0.000).So do the different time points.We compare each group with SNK test, except the group one and group three, the difference among other groups all has significant statistic meaning(P£½0.000). Under the only treatment group, we use one way ANOVA£¬besides the group one and group three(P1=1.000£¬P2=1.000), then we use SNK test shows besides group two between one day,three day and five day (P£½0.993,P=0.924,P=0.917 ),the mice C9 IOD in SABC-FITC histochemical stain are differents among other groups, and these difference between different treatment groups are significant at the 0.05 level analyzed by statistics software (P<0.01). Under the only time factor, we use one way ANOVA, at every time period, the mice C9 IOD in SABC-FITC histochemical stain are significant differents at the 0.05 level(P=0.000). then we use SNK test shows that besides every time period between the group one and group three£¨P£½0.995,P=0.983,P=1.000£¬P=0.977£©,the mice C9 IOD in SABC-FITC histochemical stain between other groups are significant differents at the 0.05 level(P<0.01).

Conclusion:
Our reseach sets up the model of laser-induced choroidal neovascularization in mice. We then focused our attention on the formation and deposition of the MAC in CNV lesions. we observed that the neovascular complex stained very strongly for C9 in complement-sufficient C57BL/6 mice with SABC-FITC histochemical stain.In contrast, C57BL/6 mice depleted of complement with CVF ,laser spots did not stain for C9 and did not develop CNV ,we demonstrated that complement was essential for the development of CNV in C57BL/6 mice after laser photocoagulation .These results showed a correlation between the presence of complement, MAC deposition, and choroidal new vessel formation following laser photocoagulation.
Keywords£ºlaser photocoagulation, choroidal neovascularization, cobra venom factor, complement 9, animal model