Effect of elevated pressure on astrocytes on an in vitro Optic Nerve Head chip

Introduction: Glaucoma is the leading cause of irreversible blindness worldwide affecting close to 80 million people by 2020. Elevated intraocular pressure (IOP) is the main risk factor for developing glaucoma. The human lamina cribrosa (LC) region, which is the first site of injury in glaucoma, contains two major cell types that are critical to disease progression - astrocytes and LC cells. Changes in protein expression and cellular morphology of these cells in response to increased IOP are thought to trigger degeneration of neuronal axons leading to gradual vision loss. Most glaucoma research is conducted using conventional 2D cell cultures and in vivo mice models. However, the absence of LC cells and significantly different biomechanics in mice, relative to humans, make it challenging to study the interactions between the different cell types and their respective contributions to glaucoma progression. This complicates the translation of findings from animal studies to the human condition. Therefore, we developed an optic nerve head (ONH) chip that captures the effects of both major cell types and mimics the biomechanics of the human LC. We investigated the effect of elevated hydrostatic pressure of physiological relevance on astrocyte monoculture and astrocyte-LC cell co-culture encapsulated in a 3D collagenous hydrogel. The results showed that changes in morphology, reactivity, and cell interactions in response to elevated pressure play a vital role in glaucoma pathophysiology.

Materials and

Methods: A rectangular PDMS (Sylgard 184) structure with a hollow cylindrical chamber of 15 mm in diameter was fabricated by casting on a 3D printed mold. A cover slip was attached to the bottom of the chamber using an adhesive. Two metal tubes channel through the PDMS into the chamber (Figure). Human ONH astrocytes and LC cells were isolated from human donor eyes. Two studies were conducted - astrocyte monoculture and astrocyte-LC cell co-culture. In both studies, the cells were encapsulated in collagen I hydrogel and seeded on the cover slip. The chamber was sealed from above using a glass slide and filled with growth media. The lower and upper metal tubes were connected to a syringe and pressure gauge respectively. Different masses were placed on the syringe to produce three different pressures - normal (15 mmHg), moderate (30 mmHg) and high (40 mmHg) ocular pressure. Cells were subjected to the pressure for 48 hours. Immunofluorescence staining and Quantitative PCR were carried out to image and quantify the changes in cell reactivity and morphology.

Results, Conclusions, and Discussions: Immunofluorescence staining was used to assess the changes in cell morphology and reactivity of astrocytes through the expression of F-actin, glial fibrillary acidic protein (GFAP), transforming growth factor (TGF) β2, collagen IV, matrix metalloproteinase 1(MMP-1). This assay demonstrated that astrocytes subjected to normal pressure had thinner, longer, and laterally grown processes while those exposed to higher pressures had shorter and thicker processes with increased processes growth parallel to the pressure axis. Immunofluorescence images of astrocytes under elevated pressure showed higher expression of GFAP, TGF- β2, Collagen IV and MMP-1. Results from quantitative PCR showed an upregulation of reactivity associated genes. These results agree with in vivo animal studies validating the functioning of our ONH chip.

Our results indicate that exposure to elevated pressure leads to significant changes in the cell morphology and reactivity in the LC. High cell reactivity could lead to an increase in LC matrix stiffness which is a common observation in glaucoma patients. Thus, in subsequent studies, we plan to investigate how elevated pressure affects cell morphology and disease progression at different matrix stiffnesses corresponding to different stages of glaucoma. Current and future work with our ONH chip could be pivotal to understanding the mechanisms of human glaucoma progression at the cellular level and aid in developing therapeutics that could prevent or reverse degenerative changes associated with glaucoma and similar neurological conditions.

Acknowledgements (Optional): This work is supported by National Institute of Health (no. R15EY033967)