June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Role of apicobasal polarity in retinal pigmented epithelial cells evaluated by ablation of Pard3
Author Affiliations & Notes
  • Peri Sohnen
    ophthalmology, University of South Florida Morsani College of Medicine, Tampa, Florida, United States
  • Peng Shang
    ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Satinder Kaur
    ophthalmology, University of South Florida Morsani College of Medicine, Tampa, Florida, United States
  • Sudha Swamynathan
    ophthalmology, University of South Florida Morsani College of Medicine, Tampa, Florida, United States
  • Tomonori Hirose
    ophthalmology, Yokohama Shiritsu Daigaku Igakubu Daigakuin Igaku Kenkyuka, Yokohama, Kanagawa, Japan
  • Debasish Sinha
    ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Shivalingappa Swamynathan
    ophthalmology, University of South Florida Morsani College of Medicine, Tampa, Florida, United States
  • Footnotes
    Commercial Relationships   Peri Sohnen None; Peng Shang None; Satinder Kaur None; Sudha Swamynathan None; Tomonori Hirose None; Debasish Sinha None; Shivalingappa Swamynathan None
  • Footnotes
    Support  NIH EY 026533, P30 EY008098
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 2978. doi:
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      Peri Sohnen, Peng Shang, Satinder Kaur, Sudha Swamynathan, Tomonori Hirose, Debasish Sinha, Shivalingappa Swamynathan; Role of apicobasal polarity in retinal pigmented epithelial cells evaluated by ablation of Pard3. Invest. Ophthalmol. Vis. Sci. 2023;64(8):2978.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The retinal pigmented epithelium (RPE) is a highly polarized monolayer that performs many functions critical to the maintenance of the retina. This study employed a binary transgenic Pard3-conditional knockout (Pard3-cKO) mouse model to investigate the effects of RPE-specific ablation of Pard3, a component of the tight junction complex and a crucial determinant of apicobasal polarity (ABP).

Methods : Pard3-cKO (Pard3-LoxP/LoxP; Best1-Cre/+) mice were maintained by breeding Pard3-cKO females with control (Pard3-LoxP/LoxP; +/+) males, which resulted in roughly equal numbers of Pard3-cKO and control littermates. RPE-specific ablation of Pard3 in Pard3-cKO mice was confirmed by immunofluorescent staining. Control and Pard3-cKO retinal histology was compared by brightfield microscopy of hematoxylin-eosin (H&E) stained 5 mm-thick sections from paraformaldehyde-fixed paraffin embedded eyes. 40x magnification images of the H&E-stained central sections from at least 4 eyes were examined qualitatively, and the number of photoreceptors was quantified by TensorFlow based AI software that counts the density of photoreceptor nuclei through the retina, mapped relative to the optic nerve. Whole-mount RPE staining with fluorescently labeled phalloidin was employed to evaluate the organization of actin cytoskeleton.

Results : Immunofluorescent staining of cryosections from 10-week-old Pard3-cKO and control mouse eyes confirmed the RPE-specific ablation of Pard3. Immunofluorescent staining also revealed that the tight junctions are disrupted in Pard3-cKO RPE. Qualitative assessment of the H&E images showed only subtle differences between control and Pard3-cKO retinae, with a slight decrease in stain intensity of the outer segments of the photoreceptors in Pard3-cKO mice. Quantitation (n=4) showed a slight decrease (statistically non-significant; p>0.05) in photoreceptor nuclei in the Pard3-cKO mice. Whole-mount RPE staining revealed that the actin cytoskeleton organization was disrupted in Pard3-cKO compared with the control RPE.

Conclusions : We have successfully developed a mouse model to directly evaluate the role of ABP in RPE. Detailed examination of the aged Pard3-cKO mouse RPE is anticipated to shed light on how ABP plays a key role in RPE functions and overall retinal health.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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