Histological and Immunohistochemical Retinal Changes Following the Intravitreal Injection of Aflibercept, Bevacizumab and Ranibizumab in Newborn Rabbits

Purpose: To analyze the retinal effects of the intravitreally administered vascular endothelial growth factor (VEGF) inhibitors (aflibercept, bevacizumab and ranibizumab) in newborn rabbits.Methods: Right eyes of 28 two-week-old New Zealand albino rabbits comprised the study population. Four eyes received intravitreal injection of 0.025cc balanced salt solution (BSS) (group I, control group); six 0.25 mg ranibizumab (group II), six 0.3125 mg bevacizumab (group III), six 0.625 mg bevacizumab (group IV), and six 1 mg aflibercept (group V) intravitreally. Blood samples were obtained to evaluate serum VEGF levels. Retinal tissues were examined by light microscopy and immunohistochemical examination (TUNEL and caspase-3 staining) to evaluate the level of apoptosis at the end of the third week.Results: Light microscopic evaluation did not show any retinal abnormality in all study and control eyes. Positive TUNEL staining was present in 16.75 1.25%, 23.6 +/- 1.36%, 33.1 +/- 5.03%, 49.3 +/- 9.3%, and 32.33 +/- 8.06% of the eyes recruited in groups I, II, III, IV, and V, respectively. Mean percentage of apoptotic cell counts detected by caspase-3 staining was as follows: 6.75 +/- 2.06% in Group I, 12.6 +/- 13.44% in Group II, 15.5 +/- 1.37% in Group III, 24.0 +/- 2.7% in Group IV, and 17.33 +/- 1.96% in Group V. TUNEL and caspase-3 staining ratio was found to be statistically higher in all anti-VEGF drug groups compared to the controls (TUNEL stain; p = 0.01, p = 0.01, p = 0.01, p = 0.01; caspase-3 stain; p = 0.024, p = 0.009, p = 0.01, p = 0.01, respectively). Serum VEGF levels were 82.16 +/- 1.72 pg/mL, 54.53 +/- 12.69 pg/mL, 33.09 +/- 17.26 pg/mL, 39.66 +/- 5.77 pg/mL, and 36.90 +/- 28.14 pg/mL for the control groups II, III, IV, and V, respectively. Serum VEGF concentrations were found to be statistically lower in the anti-VEGF groups compared to the control eyes (p = 0.011, p = 0.011, p = 0.011, p = 0.014, respectively).Conclusion: This study demonstrates that apoptosis was induced in the retina of newborn rabbits by intravitreal administration of anti-VEGF agents together with reduction in serum VEGF levels. Among the three anti-VEGF agents, ranibizumab caused the least apoptotic activity in the retina and reduction in serum VEGF levels. In light of our study, we believe that intravitreal anti-VEGF agents should be used with caution as a first line treatment for the treatment of retinopathy of prematurity.