An in vitro pressure model towards studying the response of primary retinal ganglion cells to elevated hydrostatic pressures

Abstract

Glaucoma is a leading cause of blindness characterized by progressive degeneration of retinal ganglion cells (RGCs). A well-established risk factor for the development and progression of glaucoma is elevation of intraocular pressure (IOP). However, how elevated IOP leads to RGC degeneration remains poorly understood. Here, we fabricate a facile, tunable hydrostatic pressure platform to study the effect of increased hydrostatic pressure on RGC axon and total neurite length, cell body area, dendritic branching, and cell survival. The hydrostatic pressure can be adjusted by varying the height of a liquid reservoir attached to a three-dimensional (3D)-printed adapter. The proposed platform enables long-term monitoring of primary RGCs in response to various pressure levels. Our results showed pressure-dependent changes in the axon length, and the total neurite length. The proportion of RGCs with neurite extensions significantly decreased by an average of 38 ± 2% (mean ± SEM) at pressures 30 mmHg and above (p < 0.05). The axon length and total neurite length decreased at a rate of 1.65 ± 0.18 μm and 4.07 ± 0.34 μm, respectively (p < 0.001), for each mmHg increase in pressure after 72 hours pressure treatment. Dendritic branching increased by 0.20 ± 0.05 intersections/day at pressures below 25 mmHg, and decreased by 0.07 ± 0.01 intersections/day at pressures above 25 mmHg (p < 0.001). There were no significant changes in cell body area under different levels of hydrostatic pressure (p ≥ 0.05). Application of this model will facilitate studies on the biophysical mechanisms that contribute to the pathophysiology of glaucoma and provide a channel for the screening of potential pharmacological agents for neuroprotection.

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“…On the other hand, the relationship between IOP increases and RGC degeneration remains to be clarified. For instance, pressure levels of 25 mmHg have been recently reported as the threshold above which RGCs begin to degenerate [68], while IOP higher than 21/22 mmHg has been generally reported as a critical level.…”

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“…Although a number of in vitro 3D/engineered models of glaucoma pathophysiology have emerged in recent years, most address the biology of the trabecular meshwork and regulation of intra-ocular pressure, or how this affects retinal ganglion cells [ 38 – 40 ]. Nevertheless, such models have demonstrated that 3D cultures are better at reproducing the pathophysiology of the disease, and better mimic in vivo behaviour, for example in terms of the cell responses to stress [ 40 ].…”

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“…Elevated IOP is the main risk factor of glaucoma and, together with other factors, it has been implicated in RGC degeneration and death [71]. Several in vitro models have been developed [72] and allowed the demonstration that there are pressure-dependent changes in the length of axons and neurites of RGCs [73]. When cultures of RGCs are challenged with elevated pressure there is a severe impact in axon length and in the total neurite length, with a weakened neurite extension (Figure 3), without interfering with cell body area [73].…”

“…Several in vitro models have been developed [72] and allowed the demonstration that there are pressure-dependent changes in the length of axons and neurites of RGCs [73]. When cultures of RGCs are challenged with elevated pressure there is a severe impact in axon length and in the total neurite length, with a weakened neurite extension (Figure 3), without interfering with cell body area [73]. In glaucoma, the increased IOP perturbs anterograde and retrograde axonal transports that lead to deprivation of RGCs of neurotrophic factors produced by brain targets [74].…”