Effect of subconjuctival and intraocular bevacizumab injection on angiogenic gene expression levels in a mouse model of corneal neovascularization

Olga Dratviman-Storobinsky, Bat-Chen R Avraham-Lubin, Murat Hasanreisoglu, Nitza Goldenberg-Cohen, Olga Dratviman-Storobinsky, Bat-Chen R Avraham-Lubin, Murat Hasanreisoglu, Nitza Goldenberg-Cohen

Abstract

Purpose: This study sought to characterize the expression of angiogenesis-related genes in a mouse model of corneal neovascularization, either untreated or after treatment with a single injection of bevacizumab by three different routes. In addition, the effectiveness of the treatment was compared to a rabbit model.

Methods: A chemical burn was induced in the mid-cornea of the right eye in 119 mice; 56 of them were untreated and 63 were bevacizumab-treated. Neovascularization was evaluated 2, 4, 8, 10, and 14 days later using digital photos, angiography and India ink perfusion. The relative area of new blood vessels was analyzed using slit-lamp examination in vivo and on histological and flat-mount sections. The levels of gene expression involved in the angiogenic process vascular endothelial growth factor [VEGF], insulin-like growth factor-1 [IGF-1], pigment epithelium derived factor [PEDF], and macrophage-inflammatory protein-2 [MIP-2]) were measured by a real-time polymerase chain reaction. Six rabbits underwent the same injury and treatment, and the response was compared to the mouse model.

Results: Neovascularization was first observed two days after injury. The affected section increased from 11.24% (+/-7.0) of the corneal area to 47.42% (+/-25.4) on day 8 and 50.62% (+/-24.7) on day 10. In the mice treated with bevacizumab, the relative area of neovascularization was significantly lower at the peak time points (p<0.005): 24.90% (+/-21.8) on day 8 and 28.29% (+/-20.9) on day 10. Spontaneous regression was observed on day 14 in both groups, to 26.98% (+/-19.9) in the untreated mice and 10.97% (+/-10.8) in the bevacizumab-treated mice (p<0.005). Rabbits also showed peak corneal neovascularization on days 8-10, with significant regression of the vessels following intracameral bevacizumab injection. In the mice, intraocular (intravitreal, intracameral) injection was more effective than subconjuctival injection. VEGF gene expression was upregulated in both the untreated and treated mice, but was slightly less in the treated mice. PEDF gene expression decreased in both the treated and untreated mice. In the untreated group, gene expression peaked (above baseline) at 14 days, and in the untreated mice, it had already peaked by day 8. IGF-1 was upregulated early in the model; at 8 days, there was only a slight change in the untreated group compared to a significant increase in the treated group. MIP-2 was upregulated in both groups in the early stage and returned to baseline on day 14.

Conclusions: Bevacizumab treatment partially inhibits the progressive corneal neovascularization induced by chemical damage in a mouse model. Treatment is more effective when administered via the intraocular than the subconjunctival route. The clinical findings are compatible with the angiographic and histologic data and are supported by molecular analysis showing a partial change in expression of proangiogenic genes. The molecular mechanisms involved in corneal neovascularization and inflammation warrant further exploration. These findings may have important therapeutic implications in the clinical setting.

Figures

Figure 1
Figure 1
Corneal neovascularization in rabbits. A-B: Clinical appearance of neovascularization in a rabbit cornea 10 days (peak) after chemical burn induction. Note the central scar (yellow) covering approximately 10% of the corneal area. C-D: Intracameral bevacizumab-treated cornea at 10 days. Note the reduced neovascularization.
Figure 2
Figure 2
Clinical appearance of neovascularization in a mouse cornea. Comparison between normal eye, without blood vessels on the cornea (A) and 8 days following cauterization without treatment, with high level of neovascularization (B). Less blood vessels observed after bevacizumab treatment by (H) intravitreal injection, (I) intracameral injection or (J) subconjunctival injection at the same time point. Fluorescein angiography 8 days after chemical burn induction showing (C) early leakage and (D-E) increased late leakage. Flat mount cornea of the control mouse showing transparent cornea without blood vessels (F). In the neovascular corneal model, studies with fluorescein dye (G) and India ink (K, black) reveal new vessels 8 days after induction of chemical burn, compared to the control avascular cornea (K), and a reduced level of NV detected following bevacizumab treatment (M-O).
Figure 3
Figure 3
Histology analysis using hematoxylin-eosin staining. No blood vessels detected in the normal cornea (A). Following cauterization, development of the scar (arrow) was observed in the epithelial and anterior stroma of the center cornea (B). New pathological blood vessels (C) were located in the superficial stroma, filled with erythrocytes (*).
Figure 4
Figure 4
Corneal flat-mount immunostaining for endothelial marker. Flat-mount corneas were stained with endothelial marker using anti-CD31 antibody. No positive staining detected in a control mouse (avascular cornea; A) compared to blood vessels detected in the flat mount cornea  8 days after cauterization (red staining; B).
Figure 5
Figure 5
Immunostaining for intraocular bevacizumab. No staining was detected using Anti-human antibody labeled with CY3 dye in an untreated mouse after chemical burn injury (A) without bevacizumab injection. Bevacizumab staining identified in the anterior chamber, filtrating into the cornea, following intraocular injection one day after injury (B).
Figure 6
Figure 6
Molecular analysis of gene expression levels after chemical cauterization in the untreated and bevacizumab treated mice. VEGF expression up-regulated in all time points, with a slight decrease following bevacizumab treatment (A). PEDF showed an inverse pattern of expression to VEGF (B). IGF-1 expression increased between days 8 and 10 following injury, with a higher level in the bevacizumab treated group (C). MIP-2 expression significantly increased in both the untreated and treated mice on day 2 and then dropped to near-normal levels by day 14, with higher level in the bevacizumab treated eyes (D).

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