Estudio de la degeneración de los fotorreceptores, epitelio pigmentario de la retina y de la reacción microglial en un modelo de fototoxicidad focal con led y neuroprotección en roedor

Abstract

Objectives The general objectives of this thesis are as follows: 1. To study "in vivo" in the adult albino rat the time course of focal phototoxic retinal injury and to characterize "ex vivo" the microglial reaction in the photoreceptor outer segment layer (FES). 2. To characterize in the adult pigmented mouse retina the degeneration of photoreceptors in a new model of focal LED phototoxicity (FFL). 3. To study cone survival, associated microglial reaction and retinal pigment epithelium after focal LED photoexposure in pigmented mice treated with bFGF and minocycline administered in isolation or in combination. Material and Methods. Adult female albino rats and adult female pigmented mice were used for the experiments. Animal manipulations were performed following the current European (Directive 2010/63/EU) and national (RD 53/2013) regulations on the protection of animals used for experimental and other scientific purposes. For the "in vivo" study of the time course of the phototoxic focal lesion the retinas were examined by spectral domain optical coherence tomography (SD-OCT; Spectralis®, Heidelberg Engineering, Heidelberg, Germany) at 24 hours, 3, 5, 7, 14, 30 days after focal LED photoexposure (FFL) To characterize "ex vivo" the microglial reaction in the photoreceptor outer segment layer (FES) in the adult albino rat, the retinas of the animals were immunodetected with two antibodies, anti-OPN1SW for detection of S cones and anti-Iba-1 for detection of microglia cells. For the study of the quantification of arrestin+ cones and their distribution in the pigmented mouse retina, flat retinas and transverse sections were immunodetected with an anti-arrestin, anti-OPN1SW and anti-opsin-L antibody. For in vivo study of FFL injury, pigmented mice were studied by SD-OCT at 1, 3, 5 and 7 days after FFL. For the ex vivo analysis of cone degeneration after FFL, 4 groups of 6 mice each were established and analyzed at 1, 3, 5 and 7 days after FFL and cones were immunodetected with an antibody against arrestin. For the study of cone survival, associated microglial reaction and retinal pigment epithelium (RPE) after focal LED photoexposure in pigmented mice treated with bFGF and minocycline administered alone or in combination, the retinas of the animals were studied by SD-OCT and immunohistochemistry at 3 and 7 days after FFL. For the study by immunohistochemistry, anti-arrestin antibodies were used for the study of cones, anti-Iba1 for the labeling of microglia and anti-ZO-1 for the study of the EPR. For statistical analysis, One Way ANOVA or Kruskal Wallis was used for comparison of more than two groups and t-test for comparison of two groups. Results The "in vivo" study by SD-OCT of the evolution of retinal damage after FFL in the albino rat retina showed a rounded lesion located in the superotemporal quadrant of the retina in which progressive retinal atrophy circumscribed to this lesion was observed. The mean total thickness of the right eyes was 192.3 ± 3.8 µm while the thickness of the outer retina was 104.7 ± 3.9 µm. At 30 days after FFL, the total retinal thickness decreased to 102.8 ± 17.2 µm and the outer retinal thickness decreased to 10.2 ± 5.7 µm Analysis of the survival of S-cone outer segments (SES+) in the lesion area in the adult albino rat retina showed a total number of SES+ of 2398 ± 388. At day FFL, the number of SES+ in the ACC decreased significantly to 1369 ± 364. At 3 days the SES+ number was maintained and then decreased progressively until 14 days FFL, when the number of SES+ declined by 85% in leison area and remained constant until 30 days FFL. After FFL, the appearance of Iba-1+ cells circumscribed to the center of the lesioned area was observed at 24 hours after FFL and was maintained until 30 days after FFL. The morphology of these cells changed at different study intervals. The study of the quantification and distribution of arrestin+ cones (SEa+) in the pigmented mouse retina showed that the total number of SEa+ was 141,842 ± 11,288. These showed an increasing dorso-ventral gradient and high density in the central retina versus the periphery. The number of L (SEL+) and S (SES+) cones was lower (131,172 ± 8,945 and 111,158 ± 9,805). The study of retinal cross-sections showed that 99% of L cones also expressed arrestin, while 92% of arrestin+ cones also expressed S opsin. The "in vivo" study by SD-OCT after FFL in the pigmented mouse retina showed a lesion in the superior temporal quadrant of the retina. The mean total retinal thickness of the pigmented mouse retina in control right eyes was 205.05 ± 4.2 µm (n=6), while the mean outer retinal thickness was 116.5 ± 2.88 µm. In the lesion area, progressive total retinal atrophy was observed until 5 days FFL, when the mean total retinal thickness was 127.15 ± 4.47 µm and remained constant until 7 days FFL. Ex vivo analysis of arrestin+ cone degeneration in the lesion area after FFL showed a circular lesion in the superior temporal quadrant whose distance to the optic nerve was approximately 1 mm. The number of SEa+ of control right eyes in the lesion area was 6218 ± 341. Analysis of SEa+ degeneration in the lesion area revealed a progressive degeneration of SEa+ that was significant at 3 days after FFL (5516 ± 183) and continued until 7 days after FFL when the number of SEa+ was 3966 ± 311. The in vivo study of retinal thickness in the lesion area after FFL and administration of bFGF and minocycline alone or in combination showed that those retinas treated with bFGF alone or in combination had a slowing of retinal atrophy, at the expense of the outer layers, at 5 days after FFL relative to vehicle-treated retinas). This effect disappeared 7 days after FFL. Immunofluorescence study of the survival of arrestin+ cones in retinas treated with bFGF and minocycline alone or in combination revealed a neuroprotective effect on the number of SEa+ in the bFGF- and bFGF+minocycline-treated group at 7 days after FFL versus their vehicles. In the lesion area microglia located in the outer plexiform layer (EPC) revealed that those retinas treated with intravitreal therapies had 22-40% more microglia cells in the lesion area than those treated with intraperitoneal therapies or control right retinas. However, retinas treated with bFGF+minocycline had a lower number of microglia cells in the SPC at 7 days after FFL. Qualitative analysis of Iba-1+ cells in the photoreceptor outer segment layer (PES) located in the lesion area after FFL revealed that in control right retinas the presence of these cells is anecdotal. However, after FFL we observed the appearance of Iba-1+ cells in the lesion area whose morphology was different at 3 days after FFL, presenting a more rounded or amoeboid morphotype than at 7 days after FFL when the morphology was more elongated. In retinas treated with minocycline the presence of these cells in the SEF layer was lower than in those treated with other therapies. The study of the RPE showed that after FFL there is a progressive degeneration of the RPE cells, with a decrease in the number of cells contained in the lesion area and alterations in their morphology. The right control epithelia showed a number of RPE cells in the 10,000 µm2 study area of 24.3 ± 3 cells with a mean cell area of 353.4 ± 124.4 µm2 (n=6). At 7 days, the EPR cell number declined by about 47% and there was an increase in the mean cell area of the EPRs. At 3 days after FFL, retinas treated with bFGF and bFGF+minocycline preserved the mean cell area, However, bFGF and bFGF+minocycline therapies did not produce significant differences in the number of RPE cells at 3 and 7 days after FFL