Alcohol and nicotine consumption exacerbates choroidal neovascularization by modulating the regulation of complement system

Abstract

The objective of the present study was to investigate the effect of alcohol and nicotine consumption on the pathogenesis of choroidal neovascularization (CNV) in rats after laser-photocoagulation. Confocal microscopic analysis demonstrated an increase in CNV complex size in rats fed with alcohol (2.3-fold), nicotine (1.9-fold), and the combination of alcohol and nicotine (2.7-fold) compared with the control groups. Immunohistochemical analysis revealed that alcohol and nicotine consumption increased MAC deposition and VEGF expression in laser spots. Expression of CD59 by RT-PCR and Western blot was drastically reduced in the animals that were fed with alcohol, nicotine and alcohol and nicotine compared to those fed with water alone and this was associated with exacerbation of CNV.

Fig. 1

Effect of alcohol and nicotine consumption on laser-induced CNV. (A–E) Confocal microscopic images of flat mounts (RPE–choroid– sclera) from rats perfused with fluorescein-dextran (green color) and stained with elastin (red color). Elastin stains Bruchs membrane (red), the CNV complex is green. There was a significant increase in the size of the CNV complex in alcohol-fed rats (C), nicotine-fed rats (D), combination of alcohol and nicotine-fed rats (E) compared with control rats (A and B). There was no difference in the CNV complex size between controls fed with only water-fed (A) and pair-fed (glucose and water fed) controls (B) (2000×). Comparison of CNV complex size (i.e. area) in only water-fed control, pair-fed control, alcohol, nicotine, and combination of alcohol and nicotine-fed rats (F). CNV complex size was measured using Image Pro-Plus software in micron unit. Note the increase in the CNV complex size in alcohol-fed rats, nicotine-fed rats and alcohol and nicotine-fed rats compared with water-fed and pair-fed controls. In panel F, “W” represents only water-fed animals, “G” represents glucose in water-fed animals, “A” represents animals fed with alcohol, “N” represents nicotine-fed animals while “AN” represents combination of alcohol and nicotine-fed animals.

Fig. 2

Choroidal vessel density in laser spots. Representative microphotographs of formalin fixed paraffin embedded sections (4 μm) stained with hematoxylin and eosin. The laser-injured tissues were obtained from rats fed with water (A), alcohol (B), nicotine (C) and combination of alcohol and nicotine (D). The choroidal vessels are shown by arrow head (Objective magnification: 40×). The severity of CNV was measured by measuring the density of vessels in laser-injured areas in the above mentioned groups (E). The number of vessels and area was measured using Image Pro-Plus program and density of vessels (number of vessels/mm² area) was calculated. Note the increase in density of choroidal vessels in rats fed with alcohol, nicotine and combined alcohol and nicotine compared to water fed control animals. In panel E, “W” represents only water-fed animals, “A” represents alcohol-fed animals, “N” represents nicotine-fed animals while “AN” represents combination of alcohol and nicotine-fed animals. * P < 0.05

Fig. 3

Effect of alcohol and nicotine on MAC deposition during laser-induced CNV. Paraffin sections of choroid (Ch) in the laser-injured area obtained from water-fed animals (control; A); alcohol-fed (B); nicotine-fed (C); and combination of alcohol and nicotine-fed (D) were stained for MAC as described in Section 2. The red fluorescence indicates MAC deposition (objective magnification: 20×).

Fig. 4

Effect of alcohol and nicotine consumption on VEGF expression in CNV complex. Paraffin sections of laser-injured area obtained from water-fed animals (control; B); alcohol-fed (C); nicotine-fed (D); and combination of alcohol and nicotine-fed (E) were stained for VEGF using of rabbit polyclonal anti-rat VEGF₁₆₄ antibody. Nuclei were counterstained with Vector Methyl Green and appeared as greenish blue color. Purple color indicates VEGF staining in the choroidal vessels (V). Note the higher intensity of positive staining for VEGF in the choroidal vessels of alcohol (C), nicotine (D) and combination of alcohol and nicotine (E) fed groups compared to the control (B). (A) Is negative control (NC) (with out primary antibody) which shows no staining for VEGF (Objective magnification: 100×). (F) Shows the histograph of this figure.

Fig. 5

Effect of alcohol and nicotine on CD59 expression. (A) RT-PCR products for CD59 in RPE–choroid–sclera of water-fed rats (control) (lane 1), alcohol-fed (lane 2), nicotine-fed (lane 3) and combination of alcohol and nicotine-fed (lane 4). (B) represents densitometric analysis of PCR products. Intensity of PCR products was quantitated using Quantity One 4.2.0 image analyzer, and the relative intensity was expressed as ratio of the intensity of the CD59 transcripts to those of β-actin transcripts (B). Semiquantitative Western blot (C) and densitometric analysis of CD59 protein (D) in RPE–choroid–sclera obtained from the water-fed rats (control) (lane 1), alcohol-fed (lane 2), nicotine-fed (lane 3) and combination of alcohol and nicotine-fed (lane 4). The results of densitometric analysis are expressed as ratio of the intensity of CD59 protein (C) bands to those of β-actin protein bands.