Evaluating Gene Therapy Effects on Corneal Anatomy

To study the effect of gene delivery on corneal anatomy, rabbit corneal tissue sections from the naïve and AAV5-naked, and AAV5–Dcn groups were subjected to hematoxylin and eosin (H&E) and Mason trichome staining following our published protocols.²⁵ (link)^,²⁶ (link) The H&E and Mason trichome stained corneal sections were imaged with a bright-field microscope (Leica DM 4000B, Leica Microsystems Inc.) equipped with a digital camera and imaging software (SpotCamRT KE; Diagnostic Instruments, Sterling Heights, MI). The effect of gene therapy on cellular density was studied by mounting corneal sections with an antifade Vectashield mounting medium with propidium iodide (PI) (Vector Laboratories, Inc., CA). The PI-stained nuclei in corneal sections were recorded with a fluorescence microscope (Leica DM 4000B, Leica Microsystems Inc.) equipped with a digital camera (SpotCam RT KE, Diagnostic Instruments Inc.). The cellular density in the corneas of the naïve, AAV5 naked, and AAV5–Dcn groups was quantified by counting PI-stained nuclei²⁷ (link) in ten randomly selected areas in corneal sections at 200 and/or 400 magnification field following the method reported by our laboratory earlier.²¹ (link)

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Mohan R.R., Balne P.K., Muayad M.S., Tripathi R., Sinha N.R., Gupta S., An J.A., Sinha P.R, & Hesemann N.P. (2021). Six-Month In Vivo Safety Profiling of Topical Ocular AVV5–Decorin Gene Transfer. Translational Vision Science & Technology, 10(10), 5.

Publication 2021

Leica DM 4000B

Camera Corneal Delivery Diagnostic Eosin Fluorescence microscope Gene Gene therapy Hematoxylin Microscope Nuclei Propidium iodide Rabbit Tissue Trichome Vector

Corresponding Organization : University of Missouri

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Variable analysis

independent variables

Gene delivery (naïve, AAV5-naked, and AAV5–Dcn groups)

dependent variables

Corneal anatomy (cell density, histological changes)

control variables

Rabbit corneal tissue sections
H&E and Mason trichome staining protocols
Microscopy equipment (bright-field and fluorescence microscopes)

controls

Naïve group (no gene delivery)

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