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Tasneem Putliwala Sharma, Abbot F. Clark, Colleen M McDowell; Novel Ex-Vivo Translaminar Autonomous System to study effects of Intraocular and Intracranial pressure differential on Human Retinal Ganglion Cells.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1214.
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© ARVO (1962-2015); The Authors (2016-present)
Elevated IOP is a prominent 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 within and across the laminar region, where RGC axons exit the eye. Here, we generated a novel ex-vivo model of translaminar pressure to determine if ICP has a role in glaucoma pathogenesis.
To recapitulate human in vivo translaminar pressure, we created the translaminar autonomous system (TAS). Dissected human posterior cups (N=3) were decellularized and stained for cellular and ECM markers (COL4A1/laminin). The optic nerve head (ONH) region was plugged with Matrigel to ensure a translaminar barrier. Posterior cups were seeded with human iPSC-derived RGCs and Matrigel to form a thin layer of cells on the cup. A cell culture roller was utilized to align cells towards the ONH. These iPSC-derived RGCs (N=3) were cultured for 6 days before ex-vivo pressurized organ culture. Recellularized posterior cups were stained for ECM component (COL4A1), RGCs (BRN3A), and glial cells (GFAP). The posterior cup with human iPSC-RGCs was cultured in the TAS model (Figure 1) which contained two independent pressure chambers. The pressure within the posterior region of the eye (IOP) and around the ON (ICP) was autonomously modulated. We generated translaminar pressure 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).
Decellularized posterior cups stained positive for COL4A1 and laminin, but not BRN3A or GFAP. Recellularized posterior cups stained positive for COL4A1, BRN3A and GFAP. Using the TAS model we maintained and autonomously regulated pressure in both chambers (IOP,20-40 mmHg/ ICP,5-10 mmHg). We also maintained translaminar pressure differentials (IOP: ICP; elevated IOP, 40:10; normal, 20:10; decreased ICP, 20:5) for 7 days (N=3). No significant changes were observed between all three experiments for each pressure differential.
Our TAS model will allow the study of IOP and ICP glaucoma insults especially TGFB2 and TLR4 mediated pathogenesis. Through this model we hope to provide a unique biomechanical insult to study and target glaucoma.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Figure 1: Schematic of translaminar autonomous system.
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