Histological and Immunohistochemical Retinal Changes Following the Intravitreal Injection of Aflibercept, Bevacizumab and Ranibizumab in Newborn Rabbits.
To analyze the retinal effects of the intravitreally administered vascular endothelial growth factor (VEGF) inhibitors (aflibercept, bevacizumab and ranibizumab) in newborn rabbits.



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.



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).



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.
a Department of Ophthalmology , State Hospital , Tunceli , Turkey.
Duygu
D
Cam
b Department of Ophthalmology , Dokuz Eylul University, School of Medicine , Izmır , Turkey.
Ayse Tulin
AT
Berk
c Department of Histology and Embryology , Dokuz Eylul University, School of Medicine , Izmır , Turkey.
Serap Cilaker
SC
Micili
d Department of Biochemistry , Dokuz Eylul University, School of Medicine , Izmır , Turkey.
Tuncay
T
Kume
c Department of Histology and Embryology , Dokuz Eylul University, School of Medicine , Izmır , Turkey.
Bekir Ugur
BU
Ergur
e Multidisciplinary Laboratory , Dokuz Eylul University, School of Medicine , Izmır , Turkey.
Osman
O
Yilmaz
D020533:Angiogenesis Inhibitors / Q000008:administration & dosage; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D000068258:Bevacizumab / Q000008:administration & dosage*; D004195:Disease Models, Animal; D007150:Immunohistochemistry / Q000379:methods*; D020287:In Situ Nick-End Labeling; D058449:Intravitreal Injections; D011817:Rabbits; D000069579:Ranibizumab / Q000008:administration & dosage*; D040262:Receptors, Vascular Endothelial Growth Factor / Q000008:administration & dosage*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage*; D012160:Retina / Q000378:metabolism / Q000473:pathology*; D012178:Retinopathy of Prematurity / Q000097:blood / Q000175:diagnosis / Q000188:drug therapy*; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000097:blood
D020533:Angiogenesis Inhibitors; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; C533178:aflibercept; D000068258:Bevacizumab; D040262:Receptors, Vascular Endothelial Growth Factor; D000069579:Ranibizumab
Aflibercept; VEGF inhibition; apoptosis; bevacizumab; ranibizumab; retinopathy of prematurity