Effects of oxidized phospholipids on formation of laser induced choroidal neovascularization in mice

Abstract

Purpose: The wet form of age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV). Drusen is highly enriched with polyunsaturated fatty acid, which can be modified to oxidized phospholipids (Ox-PLs) under the high oxidative condition. Our former experiments shown that Ox-PLs on oxidized low density lipoprotein (Ox-LDL) could induce CNV in mice when injected subretinally. However, due to the unevenness and difficulties of subretinal injection, this model is not ideal for a quantitative research. As laser induced CNV model is now the most commonly used one in quantitative research, the purpose of this experiment is to test whether Ox-PLs injection intravitreally can stimulate CNV formation in this model.

Methods: Male C57Bl/6 mice of 6 to 8 weeks old were anesthetized by intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Laser burns were performed two disk diameters from the optic disk in each quadrant with the following parameters: 120 mW power, 75 μm spot size, and 100 ms duration. For Ox-LDL effects on CNV formation experiments, mice were given an intravitreal injection of 1 μl of Ox-LDL (100 μg/ml in PBS) right after laser treatment. An equal volume of native LDL (Nat-LDL) (100 μg/ml in PBS) or PBS was given by intravitreal injection as control. Each group had 4 mice (4*4 laser spots, n=16). Seven days after laser treatment, mice were sacrificed and choroidal flat mounts were generated. FITC-conjugated isolectin was used to perform immunolabelling of CNV. Images were taken and areas of CNV were calculated by using a Zeiss AX10 fluorescence microscope (Carl Zeiss, Inc.). Two-tailed Student’s t-test was used for statistical analysis.

Results: At day 7, the mean CNV areas per lesion were 54596.25±9516.09 μm², 33635.94±6234.57μm², and 36598.50±10553.75 μm² in the Ox-LDL, PBS and Nat-LDL injected eyes respectively. The mean CNV area of Ox-LDL injected eyes was larger than that of PBS (p=8.19E-8) or Nat-LDL injected eyes (p=1.93E-5). There had no significant difference between PBS and Nat-LDL injected eyes (p=0.34).

Conclusions: As we estimated, Ox-PLs intravitreal injection can stimulate laser induced CNV formation in mice. This Laser&Ox-PLs Intravitreal Injection model may be an ideal model to quantitatively study the influence of Ox-PLs on AMD development, and how impaired lipid metabolism and intense oxidative stress influence such activity.