Set-up of a 3D Human Trabecular Meshwork Cells in vitro model for the study of the pathophysiology of the aqueous humor conventional outflow pathway, through the use of oxidative and pressors stimuli and medical compounds

Glaucoma is a progressive optic neuropathy that leads to irreversible blindness. From a molecular point of view it is possible to define glaucoma as a syndrome that develops pro-apoptotic signals towards the optic nerve head. In the pathogenesis of glaucoma a central role is played by the trabecular meshwork (TM) that, together with the juxtacanalicular connective tissue and the inner wall of the Schlemm’s canal, forms the conventional way of outflow of the aqueous humor. Although, the etiology of the disease is partly still unknown, the main features are an elevated intraocular pressure (IOP) related to an alteration of the trabecular outflow resistance, ocular vascular alteration, extracellular matrix (ECM) changes. There are many other conditions that contribute to the development of glaucomatous disease, such as oxidative stress and cellular responses to damage that lead to autophagy or senescence [1–5]. Up-to date the IOP is the only target of therapy, without counteracting blindness. Unfortunately, actually there is not reliable experimental model for analyzing and identifying crucial factors for prevention and therapy towards this multifactorial disease [6–8]. Taking account of those issues, it is crucial to develop in vitro human-based model. During my PhD research activity of these three years, I set up three different in vitro TM models, to check which of those could better mimic the glaucoma onset: 2D conventional and 3D static and innovative biodynamic models. Since in glaucoma TM seems to be the main ocular tissue that is affected by oxidative stress, as first approach, all TM models were subjected to prolonged oxidative stress and at each selected check point I analyzed some markers related to cellular responses to damage. Afterwards, has been added a pressure modulator to 3D dynamic chamber, to examine the role of increased pressure, to better mimic glaucoma onset. Lastly, I evaluated the performance of our dynamic 3D-HTMC model as platform to test the effects of therapeutic compounds for glaucoma disease. I analyzed the effect of iTRAB ®, a concentrate mixture of polyphenols ≥ 2.5%. Preliminary experiments were performed by the analysis of collagens, ECM glycoproteins and regulators, as well as several cytokines by qPCR. The results are reported as two independent sets: 1) response to OS plus iTRAB ®, and 2) response to increased pressure flow plus iTRAB®. The selected genes were found to be differentially expressed in response to the OS and increased pressure flow. However, under both experimental conditions, the administering of the PM down-regulated the expression of crucial genes involved in TM dysfunction, confirming the potential usefulness of our model References: 1. Saccà SC, Gandolfi S, Bagnis A, Manni G, Damonte G, Traverso CE, et al. From DNA damage to functional changes of the trabecular meshwork in aging and glaucoma. Ageing Res Rev. 2016;29: 26–41. doi:10.1016/j.arr.2016.05.012 2. Izzotti A, La Maestra S, Micale RT, Longobardi MG, Saccà SC. Genomic and post-genomic effects of anti-glaucoma drugs preservatives in trabecular meshwork. Mutat Res. 2015;772: 1–9. doi:10.1016/j.mrfmmm.2014.11.006 3. Quigley HA. Number of people with glaucoma worldwide. Br J Ophthalmol. 1996;80: 389–393. doi:10.1136/bjo.80.5.389 4. Acott TS, Kelley MJ. Extracellular matrix in the trabecular meshwork. Exp Eye Res. 2008;86: 543–561. doi:10.1016/j.exer.2008.01.013 5. Awai-Kasaoka N, Inoue T, Kameda T, Fujimoto T, Inoue-Mochita M, Tanihara H. Oxidative stress response signaling pathways in trabecular meshwork cells and their effects on cell viability. Mol Vis. 2013;19: 1332–1340. 6. A. Bouhenni R, Dunmire J, Sewell A, Edward DP. Animal Models of Glaucoma. J Biomed Biotechnol. 2012;2012. doi:10.1155/2012/692609 7. Johnson TV, Tomarev SI. Rodent models of glaucoma. Brain Res Bull. 2010;81: 349–358. doi:10.1016/j.brainresbull.2009.04.004 8. Aires ID, Ambrósio AF, Santiago AR. Modeling Human Glaucoma: Lessons from the in vitro Models. Ophthalmic Res. 2017;57: 77–86. doi:10.1159/000448480