Abstract
Purpose :
Organ culture is a potential avenue to reduce burden on animal testing and complementary to animal testing in pre-clinical investigations such as in studying pathophysiology of glaucoma, evaluation of diagnostic instruments, and assessing ocular drug delivery. In this study, we evaluate the structural integrity (by imaging) and functional viability (by electroretinography) of saline-suspended porcine eyes with and without orbital tissues after 4 days of harvesting.
Methods :
Porcine eyes were obtained from a local abattoir within 5 hours of death and transported to the lab in DPBS. Upon arrival, the ex vivo eyes were sterilized by rinsing with PBS and immersing in 1% betadine solution for 5 minutes. In the study group without orbital tissue, orbital fat, connective tissues and majority of the extraocular tissues were carefully removed under a surgical microscope. To maintain pH, DPBS were replaced every 24 hours. Cornea and optic nerve head (ONH) of the whole ex vivo eyes were imaged using an SD-OCT (Spectralis, Heidelberg Engineering; modified arrangement in Balasubramanian, M et al, IOVS June 2017, Vol.58, 4024). The retinal function was assessed using a full-field flash electroretinography (HMsERG, OcuScience). After 4 days in saline suspension, in a dark room, eyes were dissected near ora serrata (e.g. open-sky procedure followed by iris-lens removal) and the retina was carefully removed and floated in a buffer solution. 1 mm2 retinal sections were dissected and perfused with ames' media in a perfusion system. Fig. 1 shows the experimental arrangement for measuring electrophysiological signals from an explant retina.
Results :
Eyes were dark adapted for a minimum of 3 hours before testing. ISCEV standard scotopic threshold responses were measured from porcine retinal explants. Fig. 2. shows representative ISCEV standard scotopic threshold responses of a porcine eye with orbital tissue intact during saline suspension.
Conclusions :
In study eyes with orbital tissues in saline suspension, cornea was transparent, ONH could be imaged and retinal function was intact. In contrast, in eyes without orbital tissues, though the cornea was transparent and ONH could be imaged, the retinal function was not viable. These results confirm that saline-suspension of ex vivo eyes with orbital tissue immediately after harvesting can sustain the structural and functional viability for several days.
This is a 2020 ARVO Annual Meeting abstract.