September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Quantitative Immunohistochemistry (IHC) in the Non-Human Primate (NHP) Early Experimental Glaucoma (EG) Model – Method and Initial Results
Author Affiliations & Notes
  • Cheri Stowell
    Optic Nerve Head Research Laoratory, Devers Eye Institute, Portland, Oregon, United States
    Discoveries in Sight Research Laboratories, Devers Eye Institute, Portland, Oregon, United States
  • Juan Reynaud
    Optic Nerve Head Research Laoratory, Devers Eye Institute, Portland, Oregon, United States
    Discoveries in Sight Research Laboratories, Devers Eye Institute, Portland, Oregon, United States
  • Stuart Keith Gardiner
    Discoveries in Sight Research Laboratories, Devers Eye Institute, Portland, Oregon, United States
  • Nicholas Marsh-Armstrong
    Department of Ophthalmology, University of Califonia, Davis, Davis, California, United States
  • John W Crabb
    Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
    Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States
  • Claude F Burgoyne
    Optic Nerve Head Research Laoratory, Devers Eye Institute, Portland, Oregon, United States
    Discoveries in Sight Research Laboratories, Devers Eye Institute, Portland, Oregon, United States
  • Footnotes
    Commercial Relationships   Cheri Stowell, None; Juan Reynaud, None; Stuart Gardiner, None; Nicholas Marsh-Armstrong, None; John Crabb, None; Claude Burgoyne, None
  • Footnotes
    Support  NIH Grants R01-EY011610 (CFB), EY021840 (JWC), EY022134 (JWC); Legacy Good Samaritan Foundation (CFB); AGS Mid-Career Award (CFB); Alcon Research Institute (CFB); Sears Medical Trust (CFB); Research to Prevent Blindness Challenge Grant (JWC), RPB Senior Investigator Award (JWC).
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 6017. doi:
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    • Get Citation

      Cheri Stowell, Juan Reynaud, Stuart Keith Gardiner, Nicholas Marsh-Armstrong, John W Crabb, Claude F Burgoyne; Quantitative Immunohistochemistry (IHC) in the Non-Human Primate (NHP) Early Experimental Glaucoma (EG) Model – Method and Initial Results. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6017.

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

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Abstract

Purpose : To regionalize and quantify multiple IHC signals in paraffin optic nerve head (ONH) sections to study the cellular mechanisms of connective tissue remodeling and axonal insult in unilateral EG animals and corroborate quantitative proteomic findings.

Methods : After sacrifice by paraformaldehyde perfusion, a 10 mm ONH trephine was excised from the EG and control eyes of n=4 animals, embedded in paraffin and serial sectioned (sagittal) at 5 um thickness. Four sections from each eye were stained with a combination of 3 primary antibodies and corresponding secondary antibodies. Negative controls (n=4 sections) were prepared without primary antibodies. For each section, 10x polarized images were acquired and composited. 40x fluorescent images were acquired at each wavelength using an epifluorescent scope (Leica DMIRB) with an LED light source (Lumen Dynamics X-Cite 120) and an automatic focusing algorithm, then composited (Fig 1). For each section, ONH anatomy was delineated within the polarized composite allowing a standard vessel mask and prelaminar, laminar (3 bands), retrolaminar (15 bands), scleral and retinal regions to be consistently imposed on each wavelength image (Fig 2). For each eye, the average fluorescence intensity for each sampling area within the 4 sections was recorded. After subtracting the negative control, overall (n=4 animals) EG vs control eye differences were assessed with a linear mixed effects model accounting for intra- and inter-eye correlations.

Results : Image acquisition is complete for 4 combinations of 3 antibodies. Within the first combination, Septin 8 and 2’,3’-cyclicnucleotide 3’-phosphodiesterase (CNP) are altered within the EG eyes as follows: septin 8 is significantly decreased in the nerve fiber layer (p = 0.031), retina (p < 0.005), prelamina (p = 0.034), lamina (p < 0.0001, all bands) and retrolaminar optic nerve (p < 0.03, 5 bands nearest the lamina). CNP is significantly decreased in the retrolaminar optic nerve (p < 0.02, all bands) – confirming previously reported CNP IHC findings.

Conclusions : We have built a method to perform regionalized quantitative IHC in the NHP ONH. A total of 16 proteins previously identified as changed in early NHP EG by proteomic analysis are under study using these techniques. EG eye IHC reductions in Septin-8 and CNP support the proteomic findings for these proteins in early EG.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

 

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