Title: An advanced 3D human based in vitro model to identify disease specific molecular markers of glaucoma neurodegeneration
Abstract:
Several neurodegenerative diseases, including glaucoma, diabetic retinopathy, and age-related macular degeneration all involve retinal cell death. Reliable experimental models of such diseases are important for developing novel therapeutic strategies and for assessing the efficacy and adverse effects of symptomatic treatments. Primary Open Angle Glaucoma, the most common type of Glaucoma, the second leading cause of irreversible blindness worldwide, is usually characterized by an elevated Intraocular Pressure (IOP) associated with the progressive loss of retinal ganglion cells and the injury of the optic nerve.
However, the underlying mechanisms of glaucoma are not entirely understood, and IOP is only one component of a more intricate puzzle. Reliable experimental models of such diseases are important for developing novel therapeutic strategies and assessing the efficacy and adverse effects of symptomatic treatments. The obvious anatomical and morphological differences between animal and human eyes, together with the inflammation pathway used in vivo to induce prolonged or transient elevation of IOP in animals, indicate that instead human-specific models of glaucoma are highly desirable. These species-specific differences may lead to an over estimation of effects or the oversight of fundamental issues, resulting in a no translatability of the results from animal experiments to humans. So, there is the pressing need for translationally-relevant human biology-based advanced models to better understand the disease in humans at multiple biological scales, by mimicking the micro-environment involvement in glaucoma, to identify biomarkers of early/advanced stages; and finally, to assess the potential of active compounds to improve therapeutic approaches.
3D in vitro Human Trabecular Meshwork cells (HTMC) could well represent a good starting point to study several molecular features of glaucoma. Millifluidic platforms are a useful support to better represent in vivo conditions, thanks to the small dimension bioreactor scenario, the connecting cell-culture chambers and the use of a peristaltic pump to cultured cells with a monitored flow medium, comparable with the human blood circulation. In this way, cells can interact with each other in a physiological manner for prolonged time.
Recently, we developed an innovative in vitro human-based reliable HTMC, using Matrigel® as scaffold, and bioreactor technologies in order to explore, in a more physiological way, the first molecular changes in the HTMC during prolonged exposure to H2O2 as Oxidative Stress (OS), for more than 96 hrs, and increase In Pressure flow (IP) by adding a small plunger at outlet pipe of the perfusion chamber. Analysis of F-actin expression, cellular metabolic activity, proinflammatory cytokine and pro-/anti-apoptotic proteins, PARP-1 cleavage, NFκB activation, mitochondrial activity, evidenced that HTMCs are less sensitive to IP than OS and when both stimuli were merged, as in an advanced glaucoma stage, OS-related damage was exacerbated by IP. Furthermore, our platform revealed to be a useful tool for assessing the different action of two patented formulation eye drops as anti-oxidant/inflammatory and antiapoptotic approaches. Moreover, exposure of human 3D-neuron-like cells to stressed HTMC-conditioned medium, evidenced an impairment of neural cell behavior, suggesting that neuronal damage had occurred as consequence of the adaptive response of HTMC towards stressors.
