Testing the Biocompatibility of a Glutathione-containing Intraocular Irrigation Solution by Using an Isolated Perfused Bovine Retina Organ Culture Model — an Alternative to Animal Testing

Abstract

The effects of a glutathione-containing intraocular irrigation solution, BSS Plus®, on retinal function and on the survival of ganglion cells in whole-mount retinal explants were studied. Evidence is provided that the perfused ex vivo bovine retina can serve as an alternative to in vivo animal testing. Isolated bovine retinas were prepared and perfused with an oxygen-saturated standard irrigation solution, and an electroretinogram was recorded to assess retinal function. After stable b-waves were detected, the isolated retinas were perfused with BSS Plus for 45 minutes. To investigate the effects of BSS Plus on photoreceptor function, 1mM aspartate was added to the irrigation solution in order to obtain a-waves, and the ERG trace was monitored for 75 minutes. For histological analysis, isolated whole retinal mounts were stored for 24 hours at 4°C, in the dark. The percentages of cell death in the retinal ganglion cell layer and in the outer and inner nuclear layers were estimated by using an ethidium homodimer-1 stain and the TUNEL assay. General swelling of the retina was examined with high-resolution optical coherence tomography. During perfusion with BSS Plus, no significant changes in a-wave and b-wave amplitudes were recorded. Retinas stored for 24 hours in BSS Plus showed a statistically significant smaller percentage (52.6%, standard deviation [SD] = 16.1%) of cell death in the retinal ganglion cell layer compared to the control group (69.6%, SD = 3.9, p = 0.0031). BSS Plus did not seem to affect short-term retinal function, and had a beneficial effect on the survival of retinal ganglion cells. This method for analysing the isolated perfused retina represents a valuable alternative for testing substances for their retinal biocompatibility and toxicity.

Ex vivo biophysical characterization of a hydrogel-based artificial vitreous substitute

“…The isolated bovine retina, which shows significant analogies to the human retina, is a sensitive and well-evaluated tool for short term pharmacological testing and, in this respect, has shown comparable results to human isolated organ cultures [16,24]. A possible toxic effect of a tested substance on retinal neurons can be detected as a reduced electrical signal.…”

“…The superperfusion with standard solution was resumed for another 70 min to monitor b-wave recovery. The b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave [16].…”

“…A series of 5 independent experiments with different retinas was conducted. The recovery of a- and b- waves was compared to the corresponding amplitudes after application of VBS and at the end of the washout [16].…”