An experimental pig model with outer retinal degeneration induced by temporary intravitreal loading of N-methyl-N-nitrosourea during vitrectomy

Abstract

We aimed to develop an outer retinal degeneration pig model induced by temporary intravitreal loading of N-methyl-N-nitrosourea (MNU) during vitrectomy. In a preliminary experiment involving 5 mini-pig cases to determine the appropriate concentration of MNU, the vitreous cavity of each eye was filled with 4, 8, 10, 12, or 16 mg/mL MNU for 10 min, which was then replaced with a balanced salt solution. Multimodal examinations including spectral-domain optical coherence tomography (OCT) images and full-field electroretinography (ffERG) were obtained at baseline and week 2, week 6, and week 12. The retinal degeneration was classified according to the amplitudes of a dark adaptive (DA) 10.0 a-wave amplitude. The degree of moderate retinal degeneration was defined as DA 10.0 a-wave amplitude ≥ 10% and < 60% of baseline amplitude. The degree of severe degeneration was defined as DA 10.0 a-wave amplitude < 10% of baseline amplitude, noise, or flat signal. Hematoxylin and eosin staining and immunohistochemistry were performed at week 12. The main experiments were conducted first with 10 cases of 5 mg/mL and later with 13 cases of 10 mg/mL. In the preliminary experiment, degree of outer retinal degeneration increased with MNU concentration. Use of 4, 8, 10, 12, and 16 mg/mL MNU showed no, moderate, severe, severe, and atrophic changes, respectively. In the main experiments, there were 9 cases of moderate retinal degeneration and 1 case of severe degeneration in 5 mg/mL MNU group. Two cases of moderate degeneration and 11 of severe degeneration were recorded in 10 mg/mL group. Mean thickness of total retina, inner nuclear layer, and outer nuclear layer decreased at week 2 in both groups. The mean amplitudes on ffERG decreased at week 2. The ffERG and OCT findings did not change from week 2 to week 6 or week 12. The results of staining supported those of ffERG and OCT. Temporal MNU loading in a vitrectomized pig-eye model induced customized outer retinal degeneration with changing the concentration of MNU.

Electrical response of retinal ganglion cells in an N-methyl-N-nitrosourea-induced retinal degeneration porcine model

“…We previously showed that our new RD model could be successfully induced with MNU in a larger animal 28 . In that study, the results were evaluated by multimodal examinations, including spectral-domain optical coherence tomography (OCT) imaging, ffERG recording, and immunohistochemistry.…”

“…Although the degeneration process observed in the MNU-treated model was similar to that of genetically induced ones, there were differences in the distribution of degeneration in the retina. Unlike genetically induced RD, which causes global homogeneous expression of RD throughout the retina, MNU-treated RD exhibited local heterogeneities in RD expression even when MNU was spread evenly throughout the retina 28 , 34 .…”

“…In our previous study, we reported the reduced light responsiveness in MNU-induced porcine RD model using the full-field electroretinogram (ffERG) recording 28 , wherein ERG recording represented the overall average responses of photoreceptors (a-wave) and BCs (b-wave). In this study, we focused on the responses of individual RGCs using an in-vitro multi-electrode array (MEA) recording setup to identify changes in light responsiveness in local areas (2 mm × 2 mm) of the retinal tissue, not the whole retina (Fig.…”

“…We previously found that even after the photoreceptor layer degenerated, RGCs remained intact 28 . Therefore, in the current study, we speculated that RGCs would be differentially responsive to electrical stimuli (ES) with varied current pulses (duration: 0.5, 1, and 2 ms; amplitude: 10, 15, 20, 25, and 30 μA).…”

“…We chose the porcine model because of several similarities in the eyes of pigs and humans, not only in the size of the eyeball but also in its composition and the shape of the retina 27 . Previously, we developed an N -methyl- N -nitrosourea (MNU)-induced porcine RD model, and showed that the porcine model is a good animal model for patients with RP 28 . In this study, we investigated the correlation between the severity of RD and electrically elicited RGC responses using the same porcine model.…”