Abstract
Purpose :
To develop a short-term intraocular pressure (IOP) elevation model in enucleated porcine eyes
Methods :
Pig eyes (N=48) were incubated in Ames media at room temperature, cannulated at the anterior chamber and maintained at 10 or 50mmHg for 4, 16 or 24hours (hrs). Samples were microdissected into 4 regions- retina, pre-lamina (PL), lamina cribrosa (LC), and myelinated optic nerve (MON) for protein expression analysis via western blot, or cryopreserved for immunohistochemistry. Quantitative amyloid precursor protein (APP) fluorescence was used to compare axonal transport[i].
[i] Korneva A, Schaub J, Jefferys J, Kimball E, Pease ME, Nawathe M, Johnson TV, Pitha I, Quigley H. A method to quantify regional axonal transport blockade at the optic nerve head after short term intraocular pressure elevation in mice. Exp Eye Res. 2020 Jul;196:108035. doi: 10.1016/j.exer.2020.108035. Epub 2020 Apr 27. PMID: 32353427; PMCID: PMC7335019.
Results :
With high pressure elevation- axonal transport block was detected only at the LC (Fig 1A), as in human glaucoma[ii], after 4hrs (p= 0.000018) and 16hrs (p = 0.00050, Mann Whitney). In western blot analysis, 4hr pressure elevation led to a 70-80% reduction at the LC for vinculin, a key cell matrix linking protein in astrocytes. Cofilin, an actin binding protein downstream from Rho kinase, reduced by 50-75% at the PL, LC and MON (Fig 1B), while phosphorylated-cofilin, stimulated by cell membrane response to physical stress, increased by 2-3x at the PL, LC and MON (Fig 1B).
Figure 1. A: Distribution in APP shows transport block by brightness increase in LC at 50 mmHg (orange). B: Protein expression for cofilin reduced and p-cofilin increased sequentially from retina to myelinated nerve (ratios of 50 vs 10 mmHg values, adjusted for GAPDH).
[ii] Quigley HA, Addicks EM, Green WR, Maumenee AE. Optic nerve damage in human glaucoma. II. The site of injury and susceptibility to damage. Arch Ophthalmol. 1981 Apr;99(4):635-49. doi: 10.1001/archopht.1981.03930010635009. PMID: 6164357.
Conclusions :
Inexpensive pig eyes, with features similar to human, can be studied for relevant changes in axonal and glial structure and function for 24 hours post mortem. Axonal transport block localizes to the LC with IOP elevation, and integrin-linked signaling of extracellular mechanical stress can be studied.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.