July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Validating animal models of Limbal Stem Cell Deficiency: A histopathological and immunohistochemical study
Author Affiliations & Notes
  • Abhinav Reddy Kethiri
    SSR-Stem cell biology Laboratory, Prof. Brien Holden Eye Research Center, L V Prasad Eye Institute, Hyderabad, Telangana, India
    Manipal Academy of Higher Education, Manipal, Karnataka, India
  • Mukesh Damala
    SSR-Stem cell biology Laboratory, Prof. Brien Holden Eye Research Center, L V Prasad Eye Institute, Hyderabad, Telangana, India
    University of Hyderabad, Hyderabad, Telangana, India
  • Kiran Kumar Bokara
    Center for Cellular and Molecular Biology, Medical Biotechnology Complex, Uppal Road, Hyderabad, Telangana, India
  • Dilip Mishra
    Ophthalmic Pathology Laboratory, L V Prasad Eye Institute, Hyderabad, Telangana, India
  • Sayan Basu
    SSR-Stem cell biology Laboratory, Prof. Brien Holden Eye Research Center, L V Prasad Eye Institute, Hyderabad, Telangana, India
    Center for Ocular Regeneration (CORE), L V Prasad Eye Institute, Hyderabad, Telangana, India
  • Mohan Rao Ch
    Center for Cellular and Molecular Biology, Medical Biotechnology Complex, Uppal Road, Hyderabad, Telangana, India
  • Virender Singh Sangwan
    SSR-Stem cell biology Laboratory, Prof. Brien Holden Eye Research Center, L V Prasad Eye Institute, Hyderabad, Telangana, India
    Center for Ocular Regeneration (CORE), L V Prasad Eye Institute, Hyderabad, Telangana, India
  • Vivek Singh
    SSR-Stem cell biology Laboratory, Prof. Brien Holden Eye Research Center, L V Prasad Eye Institute, Hyderabad, Telangana, India
    Center for Ocular Regeneration (CORE), L V Prasad Eye Institute, Hyderabad, Telangana, India
  • Footnotes
    Commercial Relationships   Abhinav Kethiri, None; Mukesh Damala, None; Kiran Bokara, None; Dilip Mishra, None; Sayan Basu, None; Mohan Ch, None; Virender Sangwan, None; Vivek Singh, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4715. doi:https://doi.org/
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      Abhinav Reddy Kethiri, Mukesh Damala, Kiran Kumar Bokara, Dilip Mishra, Sayan Basu, Mohan Rao Ch, Virender Singh Sangwan, Vivek Singh; Validating animal models of Limbal Stem Cell Deficiency: A histopathological and immunohistochemical study. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4715. doi: https://doi.org/.

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

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Abstract

Purpose : Loss of corneal clarity due to limbal stem cell deficiency (LSCD) is a serious cause of visual impairment and blindness across the globe. Reliable animal models of LSCD are useful for understanding the pathophysiology and developing novel therapeutic approaches. The purpose of this study was to validate LSCD creation in a small and large animal model by histopathological and immuno-histochemical comparison with human specimens.

Methods : Human corneal specimens (h, n=12) were obtained from patients with chemical burn-induced LSCD undergoing limbal transplantation surgery. The animal models of LSCD were created by topical administration of sodium hydroxide on the ocular surface of C57BL/6 mice (m, n=12) and New Zealand white rabbits (r, n=12) and the animals were sacrificed after at least six weeks. The excised corneal specimens were either paraffin embedded or frozen in cryogenic medium and the sections were stained with hematoxylin-eosin and Periodic acid–Schiff to analyze the morphology and histopathological features of the corneal surface such as vascularization, inflammation, presence of goblet cells, epithelial hyperplasia and keratinization. Immunofluorescence was performed to distinguish between corneal (CK3+), conjunctival (CK19+) epithelial and epidermal (CK10+) phenotype.

Results : Histological analysis of corneal specimens from the three groups showed the presence of goblet cells (h:83%, m:50%, r:50%, p=0.014), epithelial hypertrophy (h:92%, m:50%, r:66.6%, p=0.04), epithelial hyperplasia (h:50%, m:17%, r:17%, p=0.18), intra epithelial edema (h:42%, m:33%, r:100%, p=0.02), stromal inflammation (h:100%, m:67%, r:67%, p=0.01)and stromal vascularization (h:100%, m:50%, r:67%), in varying proportions. Immunostaining showed presence of total LSCD (CK19+ and/or CK10+, CK3) in 92% of human and 50% of animal specimens. While partial LSCD (CK19+ and/or CK10+, CK3+) was seen in 8% of human and 50% of animal specimens.

Conclusions : Our study shows the significant differences in the extent of vascularization, inflammation, epithelial thickness and goblet cell formation in mice and rabbit models of LSCD when compared to post-chemical burn LSCD in human corneas. In both mice and rabbit models complete LSCD developed in only 50% of cases and this important fact needs to be considered when working with animal models of LSCD.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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