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Tasneem Putliwala Sharma, Abbot F Clark, Husain Lohawala, Colleen M McDowell; Modulating human intraocular and intracranial pressure using the ex-vivo Translaminar Autonomous System. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2247.
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© ARVO (1962-2015); The Authors (2016-present)
Elevated intraocular pressure (IOP) is the main risk factor for high tension open angle glaucoma, while IOP-independent factors play a role in normal tension glaucoma. Increased IOP and decreased intracranial pressure (ICP) could increase the strain in the laminar region, where retinal ganglion cell (RGCs) axons exit the eye. Here, we utilized multiple strategies to mimic the ex-vivo model of human translaminar pressure (TLP) and determine if ICP has a role in glaucoma pathogenesis.
To recapitulate human in vivo TLP, we developed the translaminar autonomous system (TAS) (Figure 1) and adjusted the model design and hydrostatic pressure reservoirs for long-term ICP analysis. Dissected human posterior cups (PCs) (N=3) were decellularized and the optic nerve head (ONH) region plugged with Matrigel to ensure a TLP barrier. PCs were seeded with Matrigel infused human induced pluripotent stem cells (iPSC)-derived RGCs and aligned (cell roller) towards the ONH. These iPSC-derived RGCs (N=3) were cultured on the PC for 6 days to ensure attachment, alignment and growth. Recellularized PCs were stained for ECM component (COL4A1, LAM), RGCs (BRN3A), and glial cells (GFAP). These recellularized PCs were cultured in the TAS model (Figure 1). We then autonomously generated TLP differentials by raising the media reservoir connected to IOP and ICP chambers to different heights (hydrostatic pressures; IOP: ICP; 40:10, 20:10, 20:5 mmHg) for 5, 7 and 10 days. Viability of pressurized PCs seeded with iPSC-RGCs (DAPI and BRN3A) was assessed at all timepoints.
Decellularized PCs stained positive for COL4A1 and LAM, but not BRN3A or GFAP. Recellularized PCs stained positive for COL4A1 and BRN3A but not GFAP. Using the modified TAS model we autonomously regulated pressure in both chambers (IOP,20-40 mmHg/ ICP,5-10 mmHg). We maintained TLP differentials (IOP: ICP; elevated IOP, 40:10; normal, 20:10; decreased ICP, 20:5) for 5,7 and 10 days (N=3). PCs seeded with iPSC-RGCs showed different BRN3A marker expression through various pressure gradients and timepoints (N=3).
Our novel TAS model will allow the study of IOP and ICP associated glaucoma insults. It will be a unique tool to test new therapeutics that can target both pressures in glaucoma pathogenesis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
Figure 1: Schematic of translaminar autonomous system. A) Model, B) transparent and C) transverse view. Patent: UNTH.P0006US.P1
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