Development of a Workflow to study Neuronal Injury In Vivo in the Retina and In Vitro in Collagen Hydrogels

Abstract

Ocular trauma affects 1.5-2 million individuals and is the fourth leading cause of blindness in the United States (Bourne et al., 2021a; Hashemi et al., 2023). In the military, the frequency of blasts has increased during recent military conflicts where 186,555 active-duty military personnel are now diagnosed with ocular injuries (Hilber, 2011). This study used in vitro and in vivo models to determine the cellular responses to mechanical trauma. A whole-body blast injury model in rats was used to observe changes in retinal structure and function in Aim 1. To study the mechanical mechanisms of injury in isolation, an in vitro model of compression to neuronal cells was developed in Aim 2. The first aim of the study employed an established preclinical blast model to expose the entire body of rats to three blast waves, each one hour apart. Immunohistochemistry was performed on retinas isolated twenty-nine days after blast exposure. Major differences in immunolabeling were found between retinas from the blast-exposed group and the sham group. Immunolabeling against RNA Binding Protein with Multiple Splicing (RBPMS), revealed significantly fewer retinal ganglion cell (RGC) somas in the blast-exposed group than the sham group (P < 0.001). Nitrotyrosine, an indicator of oxidative stress, was elevated in the ganglion cell layer of the blast-exposed group. Müller cells of the retina express glial fibrillary acidic protein (GFAP). GFAP expression was similar between the two groups. The whole-body blast model resulted in degeneration of RGCs and heightened oxidative stress in the ganglion cell layer, but no activation of Müller cells 29 days after exposure to blast. Therefore, this rat model of traumatic injury results in pathology of RGCs in the retina and needs to be further studied to determine the mechanisms underlying blast-induced retinal injury. An in vitro compression model was developed to study the effects of mechanical stress on neuronal cells using a 3D platform. Additionally, a protocol was established to differentiate the SH-SY5Y neuroblastoma cell line into neurons in 3D collagen hydrogels. The protocol determined the optimal collagen concentration and seeding density. SH-SY5Y differentiation was effective in a 0.5 mg/mL and 1.0 mg/mL collagen hydrogels seeded at a density of 6x105 cells/mL. Next, a method to statically compress collagen hydrogels between 0-18% was developed. This compression model can be used to study the mechanical response of neurons, such as differentiated SH-SY5Y cells, or retinal ganglion cells, in a 3D environment. Together, the two aims present the opportunity to better understand the mechanisms underlying neuronal injury caused by mechanical stress both in vivo in the retina and in vitro in a 3D environment.