Exploring minocycline's effect on retinal degeneration following N-methyl- N-nitrosourea exposure in rats

Abstract

Retinal degeneration (RD) is often associated with deficiencies or the inaccurate production of photoreceptor-specific proteins, which are encoded by various genes and characterised by the apoptotic and ongoing death of photoreceptor cells. This study involved administering a single intraperitoneal (i.p.) dose of 50 mg/kg of N-methyl-N-nitrosourea (MNU) to rats to induce RD. Some of these rats also received intraperitoneal minocycline at varying doses to prevent RD. Euthanasia was conducted at five intervals: at 12, 24, 48, and 72 h, and on the 7^th day; and eye samples were taken. These samples were analysed using histopathology, immunohistochemistry, and electron microscopy. Significant RD was observed in the MNU-treated groups, with photoreceptor cell apoptosis demonstrated by the TUNEL method. Compared with those in the control group, there was a progressive thinning of the photoreceptor layer and outer nuclear layer, along with increased levels of glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA), and reduced levels of rhodopsin and red/green opsin starting from the 12^th hour in the experimental groups. Electron microscopy revealed that amacrine and bipolar cells, in addition to photoreceptors, were also affected. The minocycline treatment did not show significant differences in retinal layer thickness or the staining levels of PCNA, GFAP, and opsins in the MNU-induced RD model.

Keywords: minocycline; retinal degeneration; retinitis pigmentosa.

Figure 1. Photoreceptor layer and outer nuclear layer measurement points (μm) in the central retina (A) and peripheral retina (B–C)

(B) Superior quarter (P1 and P2); (C) Inferior quarter (P3 and P4) S1, S2, S3 = superior quarter; I1, I2, I3 = inferior quarter

Figure 2. Time-dependent change of the retinal layers

Increased thinning in the ONL and PL in all the groups receiving MNU MNU = N-methyl-N-nitrosourea; ONL = outer nuclear layer; PL = photoreceptor layer

Figure 3. (A) Severe karyorrhexis in all the experimental groups, characterised by the disruption of the general histological structures of the ONL (region between arrows) and PL (region between the arrow heads). (B) Severe thinning and loss of borders of the ONL and PL (region between the red arrows), cellular infiltrations of macrophages (black arrows), and a mitotic figure in a Müller cell (arrowhead). (C–D) General view of the retina in the optic head at the 72^nd hour, normal retina (C) and thinned ONL (D) (arrows). (E–F) Normal peripheral retina (E) and thinned peripheral retina (D), ONL (regions between arrows) and PL (regions between the arrow heads)

MNU = N-methyl-N-nitrosourea; ONL = outer nuclear layer; PL = photoreceptor layer

Figure 4. TUNEL positivity in the ONL in all the MNU given groups (brown stained cells)

MNU = N-methyl-N-nitrosourea; ONL = outer nuclear layer; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling

Figure 5. Rhodopsin (A–C) and red/green opsin (D–F) positivity (arrows) in the ONL and PL (72^nd hour and 7^th day)

ONL = outer nuclear layer; PL = photoreceptor layer

Figure 6. GFAP and PCNA positivity in the retinal layers (arrows) (24^th, 72^nd hours and 7^th day)

GFAP = glial fibrillary acidic protein; MNU = N-methyl-N-nitrosourea; PCNA = proliferating cell nuclear antigen

Figure 7. Electron microscopic view of the retina

(1) Normal view of photoreceptor nuclei (N). (2) Apoptotic nuclei of the photoreceptors (A), chromatolysis (C), cell degeneration (asterisk), and nuclear fragmentation (arrows) (MNU group, 24^th hour). (3) Normal view of the photoreceptor body parts, outer (O) and inner (I) segments. (4) Pycnotic mitochondria (arrows) in the inner segments, condensation in the outer segment discs (stars) of the photoreceptors (MNU group, 24^th hour). (5) Normal view bipolar cell (B) Müller cell (M) and amacrine cell (A). (6) Degeneration and intracytoplasmic oedema (stars), myelin figures (thick arrows), dilatation and loss of crystals in mitochondria (thin arrows) in the bipolar cell (B) and amacrine cells (A), normal Müller cell (M) (MNU group, 48^th hour) INL = internal nuclear layer; MNU = N-methyl-N-nitrosourea; ONL = outer nuclear layer; PL = photoreceptor layer