Padronização de retinopatia em larvas de zebrafish (Danio rerio)

Abstract

Retinopathy is an illness that causes the progressive loss of eyesight. It can lead to total vision loss which currently presents a public health problem. Beyond the challenge of developing new therapies for this disease, ocular researchers cannot count on an ideal model to further their studies. Therefore, Danio rerio larvae has been appointed as a potential substitute for the investigation of ophthalmological problems due to advantages, such as: visual homology; early eye development; early visual response and diurnal behavior, similar to those of human’s. Thus, the goal of this thesis was to standardize different methods of retinopathy induction in zebrafish larvae in order to test new ocular therapies. At first, zebrafish eggs were generated from crossing adult fish. These were incubated in E2A 0.5x or PTU 0.001% environment and stored in an oven at 28 ºC for up to 72 hpf. The retinopathy induction occurred by: (1) exposure of larvae for 72h in a CoCl2 0.5 mM solution; and (2) retention of larvae in total darkness for 24h (LIRD group), followed by intense light exposition for 24h. The control group included larvae cultivated in the E2A or PTU environment under normal photoperiod, and the survival of the groups was observed daily. Following the treatments, the Visual Motor Response (VMR) Test was performed, which consisted of one period of acclimatization for 15 min under light, followed by 6 alternating clear-dark cycles. The individual displacement during the test was measured and registered in order to be used as a behavioral parameter. After the VMR, euthanasia of the larvae was conducted by exposing them to 4% tricaine. Afterwards, the process included the fixation in 4% formalin, dehydration, rehydration, and incubation in 30% sucrose for the preparation of histological slides, then the sectioned larvae were tinted with Hematoxylin-eosin. To identify the changes in the zebrafish retina, the thickness of retinal layers was measured and statistical analysis was performed, considering the p<0.05 value as significant. The larvae in the incubating solutions (E2A e PTU) and treatment (CoCl2) showed low mortality. The VMR demonstrated that all treated groups, except for the PTU-incubated LIRD, showed a smaller displacement of the controls, suggesting alterations in visual behavior of treated larvae. In the LIRD group, the difference between the retinal layers showed to be significant: in the Photoreceptor Layer, where the LIRD group thickness was smaller than the control’s; and in the Inner Plexiform and Ganglion Layers, where the LIRD group thickness was higher than control group. However, concerning the larvae on which the retinopathy was induced by CoCl2, the statistical difference was observed only in the Outer Plexiform Layer, where the layer thickness in the treated group was higher than the control’s. Therefore, the results suggest that the protocols performed seem to be effective for the induction of retinopathy in zebrafish larvae, corroborating with the use of this model as alternative for therapeutic studies of ophthalmological diseases. Thus, zebrafish in your larval stage can be canonical as a model to study of ocular diseases.