Programmed pressure platform fabrication for ophthalmologic study

Abstract

Glaucoma, the second leading cause of blindness worldwide, is a neurodegenerative disease characterized by progressive degeneration of retinal ganglion cells (RGCs). Intraocular pressure (IOP) is a well-established risk factor for the development and progression of glaucoma. However, it is still not clear how IOP elevation leads to degeneration of RGCs in glaucoma. Due to the circadian rhythm and pulsatile variation with time, the IOP level is not static throughout the day and is difficult to be monitored and regulated. Therefore, a new platform that has ability to simulate pathological IOP controllably is critically important. Compared to in vivo models, the in vitro models possess higher reproducibility, as well as greater flexibility and more precise control over cell culture environments. In vitro pressure model provides a versatile tool for investigating cellular response and biomolecular changes on glaucomatous problems. In this thesis, we propose new platforms with tunable hydrostatic pressure and programmable dynamic pressure for in vitro ophthalmologic study.

In Chapter 4, we devise a hydrostatic pressure platform to simulate IOP in the eye. By connecting with a liquid column, a monitored hydrostatic pressure is generated inside the cell culture chamber. This platform enables the direct testing of hypotheses related to the role of IOP in primary RGCs degeneration. The morphological changes are recorded under the microscope and then quantified by measuring the total neurite length, axon length, cell body area, and branching complexity. We find that a critical pressure threshold is present at 25 mmHg, beyond which the RGCs become vulnerable. In particular, at pressure above 25 mmHg, the RGCs do not show any neurites extension and begin to degenerate.

In Chapter 5, a new facile programmed pressure platform, that uses the electronic valve to simulate the dynamic pressure, is further designed. Dynamic pressure with convertible profile, programmable amplitude, and frequency can be generated inside a microfluidic PDMS chamber. The output pressure can be calculated by the magnitude of air bubble shrinkage via the ideal gas law, which is identical to that measured by the pressure sensor. Moreover, the ability to generating pulsatile pressures with different amplitude provides great potential to study the mechanism of neurodegeneration under normal IOP. In particular, we find that the expression of ZO-1 between ARPE-19 is increased under dynamic pressure, when compared to that of control group (no pressure).

In summary, we propose a hydrostatic pressure platform and a programmable dynamic pressure platform for in vitro ophthalmologic study in this thesis. The hydrostatic pressure platform can simulate a wide range of physiologically relevant intraocular pressures, and the programmable dynamic pressure platform can generate cyclic and diverse pressure profiles, with tunable amplitude and frequency. Successful establishment of hydrostatic and dynamic pressure platforms will facilitate further studies on the IOP related problems in the eye. We believe that the advancement in this thesis will be beneficial to a variety of research areas, such as drug screening and biomaterials screening in eye care.