Establishment of multielectrode array-based retinal ex vivo stress models : hypoxia and pressure

Ingensiep, Claudia; Kampa, Björn M. (Thesis advisor); Johnen, Sandra (Thesis advisor); Müller, Frank (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022


Hypoxia and pressure are prominent stress factors in diverse eye diseases. Hypoxia (lack of oxygen) and pressure, in the form of elevated intraocular pressure (IOP), can occur in combination within glaucoma disease. Glaucoma is a heterogeneous eye disease causing atrophy of the optic nerve head (ONH). The optic nerve is formed by the axons of the retinal ganglion cells (RGCs) that transmit visual input to the brain as electrical signals. The progressive RGC loss during glaucoma leads to irreversible vision loss and blindness of the patient. In this study, two multielectrode array (MEA)-based ex vivo stress models were established to investigate the effect of hypoxic conditions and hydrostatic pressure on the retinal electrical activity of adult wild-type (WT) mice (C57BL/6). In the hypoxia model, hypoxic stress was induced by the exchange of carbogenated medium by nitrogen (N2)-saturated medium. The hypoxic period lasted for 30 min. In the pressure model, a lid was added to the MEA setup and hydrostatic pressure was applied to simulate increased IOP. Pressure levels of 10, 30, 60, and 90 mmHg were tested. Spontaneous activity, response rate to electrical and light stimulation, and bursting behavior of RGCs was analyzed before, during, and after stress conditions. RGC activity completely vanished during hypoxia, but it conditionally returned after reestablishment of the conventional test conditions. After 30 min of hypoxia and 30 min of recovery, electrical activity returned on 27 ± 16% of the initially active recording channels. Addition of the amino sulfonic acid taurine (1 mM) increased the returning activity to 40 ± 21%. Whereas RGCs entered an inactive stress mode during hypoxia, the RGC activity persisted under pressure. Even a high pressure level of 90 mmHg for 2 h did not disturb the RGC functionality. However, the cells’ bursting behavior significantly changed under 90 mmHg: the number of spikes in bursts doubled during pressure application and stayed on a high level afterwards. Addition of taurine (1 mM) showed a counteracting effect. OFF RGCs did not reveal an increase in bursts under elevated pressure. In the hypoxia model, a gene expression analysis showed only small differences between the expression profiles of the single groups. However, live/dead staining revealed an immense cell loss in the ganglion cell layer (GCL) of 85 ± 13% under hypoxic conditions which was lowered by taurine (73 ± 12%). Live/dead staining after pressure stress showed no significant differences in the number of dead cells. The findings generated in this work suggest that RGCs react very sensitively to hypoxic stress, but are tolerant to high, short-time pressure stress. The models established here therefore help to better understand the role of hypoxia and pressure in glaucoma. Moreover, the neuroprotective and neuromodulatory effect of taurine was confirmed.