Knocking Down Retinal Pigment Epithelium Hemoglobin Expression Results in Accelerated Death of Photoreceptors with Aging

Abstract

Purpose : To investigate the physiological importance of retinal pigment epithelium hemoglobin (RPE-Hb) expression using an inducible Cre/loxP system for generating tissue-specific Hb knockout mouse model.

Methods : An inducible Cre/loxP system was used to create a conditional Hb knock-out mouse. The temporal impact of silencing the RPE-Hb on retinal cell integrity was studied in 8 eyes of 4 aged animals using light microscopy and image analysis. Animals divided into two groups and conditional Hb knock-out group results were compared with control animals where the Cre/loxP system was not induced. The eyes of the sacrificed animals were embedded in paraffin and sectioned. DAPI-stained sections were used to analyze outer nuclear layer (ONL) cell integrity utilizing the ImageJ/FIJI program (version 1.52b). Retinal cells were counted using CellProfiler (version 4.2.6) (Figure 1A-F). Photoreceptor layer (ONL) and ganglion cell layer (GCL) cell densities were compared between the RPE-Hb-knockout and control groups. Statistical analysis has been performed via SPSS (V28.0, IBM) and p-value less than 0.05 was accepted as significant.

Results : The mean age of the RPE-Hb knock-out group is 552 ± 132 days, and the control group is 416 ± 45 days (p>0.05). Image analysis was conducted on 192 sections. A significant decrease in the number of photoreceptors was noted in RPE-Hb knockout animals (49,791 ± 7,405 cells/mm² vs. 54,752 ± 9,078 cells/mm², p<0.001) (Figure 2). No difference was noted in the number of GCL densities (5,326 ± 999 cells/mm² vs. 5,525 ± 879 cells/mm², p=0.172).

Conclusions : Tissue-specific silencing of the RPE-Hb expression results in an accelerated loss of photoreceptors in aged mice, a condition mimicking early stages of age-related macular degeneration. Hb production defects of the aged RPE and, thus, the failure of oxygen delivery to photoreceptors may be the underlying pathology leading to dysfunction of the aged retina and age-related macular degeneration.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Figure 1: A. 40x DAPI stained image B. Segmenting outer nuclear layer (ONL) C. Cropping ONL D. Thresholding ONL E. Identifying ONL Cell Nucleus F: Counting ONL Cell Number

 

Figure 2: Outer nuclear layer (ONL) cell count boxplot between groups