June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Immunohistochemical grading of human glaucomatous eye globes to ascertain disease severity and correlation with glial reactivity
Author Affiliations & Notes
  • Chuanxi Xiang
    Ophthalmology, Novartis Institutes for BioMedical Research Inc, Cambridge, Massachusetts, United States
  • Manisha Mehta
    Ophthalmology, Novartis Institutes for BioMedical Research Inc, Cambridge, Massachusetts, United States
  • Yubin Qiu
    Ophthalmology, Novartis Institutes for BioMedical Research Inc, Cambridge, Massachusetts, United States
  • Christopher W. Wilson
    Ophthalmology, Novartis Institutes for BioMedical Research Inc, Cambridge, Massachusetts, United States
  • Ganesh Prasanna
    Ophthalmology, Novartis Institutes for BioMedical Research Inc, Cambridge, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Chuanxi Xiang None; Manisha Mehta None; Yubin Qiu None; Christopher Wilson None; Ganesh Prasanna None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 1014. doi:
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      Chuanxi Xiang, Manisha Mehta, Yubin Qiu, Christopher W. Wilson, Ganesh Prasanna; Immunohistochemical grading of human glaucomatous eye globes to ascertain disease severity and correlation with glial reactivity. Invest. Ophthalmol. Vis. Sci. 2023;64(8):1014.

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

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Abstract

Purpose : Glaucoma is an optic neuropathy characterized by progressive loss of RGCs and nerve fibers, and cupping of ONH. A general challenge when procuring donor human eye globes (HEG) is related to inaccuracies in clinical diagnoses and paucity of clinical information on disease severity for glaucoma. The present study was to assess and grade disease severity of human glaucoma through pathological examination and immunohistochemical (IHC) staining of HEGs.

Methods : 19 HEGs with glaucoma were collected from the Lion’s Eye Institute (Tampa, FL) between 2017 and 2019. All samples were fixed in MDF for 48 hours, and embedded in paraffin. 5 um transverse sections with macula and ONH were cut and stained with H&E. IHC was performed with antibodies against RBPMS, NF-L (RGC markers), GFAP and IBA1. IHC staining was quantified by Halo software. BMO was used as a landmark. IHC signals in the GCC (including NFL, GCL and IPL) were collected from the central region (1 mm range from temporal BMO), peripheral retina (7 mm from temporal BMO), and ONH (1 mm area below BMO). 16 Non-glaucoma controls were selected to match by age and procurement time. Data were normalized as % positive area of total area measured. Statistical analyses were performed by t-test, or one-way ANOVA with P<0.05 as being significant.

Results : RBPMS+ RGCs and NF-L were significantly reduced in glaucomatous temporal retina and ONH. RBPMS was 43% in the central and 58% in peripheral retina; NF-L was 40% in central, 51% in peripheral retina, and 45% in ONH respectively. NF-L reduction had a linear correlation with RBPMS RGCs (r=0.73 and 0.77 in central and peripheral region). GFAP and IBA1 were significantly upregulated in glaucoma. Glaucoma disease severity was graded as mild, moderate and severe based on NF-L and GFAP positivity findings. HEGs with glaucoma history, but without significant NF-L reduction (<30%) as compared to normal, were graded as mild; those with significant NF-L reduction (30% to 65%) and strongest upregulation of GFAP as moderate; Samples with NF-L reduction >65% were graded as severe glaucoma.

Conclusions : The study developed an objective assessment of glaucoma severity based on IHC changes of RGCs and nerve fibers and provided correlations of IHC changes with glaucoma severity, which may contribute to a better understanding of the pathological mechanisms underlying disease progression.

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

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