Organ culture of the porcine retina
Models with simulated retina degeneration are necessary to understand the pathological alterations of the retina. The most models based on animals experiments [1-3]. However, the aim is to reduce the animal models through organ cultures.
A good alternative to study the retina is the use of porcine organ cultures, which are obtained from a slaughterhouse. This way the number of experimental animals will be reduced. Additionally, the size and form of the porcine eye is quite similar to the human eye in contrast to the commonly used rodent eyes [4]. Furthermore, the complex structure and form of the retina remained intact and organ culture is a reproducible and manageable method. A higher number of samples are producible in contrast to animal models. But, organ cultures also have disadvantages. The disconnection to the optic nerve leads to a decreased supply of the retina with nutrients, neurotransmitter and electrical signals. Therefore, the retinas are only cultivable for a short period of time [5]. Yet, a simulation of a retinal disease over several days is possible, as the retina explants are treated with toxic substances [6, 7]. One of these substances is N-Methyl-D-Aspartate (NMDA) [8], a synthetic glutamate analogue, which was also used in an intraocular injection model.
For the retina organ culture, pieces of the retina were explanted and cultivated. At day two and three, the retinae were treated with the toxic substance (figure 1). In pilot projects, a moderate cell decline was noted at day seven. Interestingly, the microglia reacted at this point in time [9], which is consistent with results of the animal model.
Cultivation of the porcine retina
First, retina pieces were explanted and cultivated on cell culture insert in 6-well plates. The explants remained in the culture for seven days. The toxic substances were added at day two and three. At day seven, the retinas were cryo-conserved for, among other things, histological analysis. Abbreviations: GCL=ganglion cell layer, IPL=inner plexiform layer, INL=inner nuclear layer, OPL=outer plexiform layer, ONL=outer nuclear layer, scale bar=20 µM.
This form of organ culture should be used in the future for a better understanding of the degeneration processes and to test the effect of new therapy agents.
This project is performed in cooperation with the group of Dr. rer. nat. Sven Schnichels, University Eye hospital, Tübingen.
Conference contributions
Kuehn S, Hurst J, Jashari A, Ahrens K, Wunderlich MI, Dick HB, Schnichels S, Joachim SC. NMDA triggered microglia activation in a porcine retina organ culture. Association for Research in Vision and Ophthalmology Meeting, 2015.
Joachim SC, Schnichels S, Januschowski K: Gibt es in der Retinaforschung Alternativen zum Tiermodell?. Symposium auf der Jahrestagung der Deutschen Ophthalmologischen Gesellschaft, 2015.
Literature
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[2] Joachim SC, Reinehr S, Kuehn S, Laspas P, Gramlich OW, Kuehn M et al. Immune response against ocular tissues after immunization with optic nerve antigens in a model of autoimmune glaucoma. Mol Vis, 2013;19:1804-14.
[3] Yang Q, Cho KS, Chen H, Yu D, Wang WH, Luo G et al. Microbead-induced ocular hypertensive mouse model for screening and testing of aqueous production suppressants for glaucoma. Invest Ophthalmol Vis Sci, 2012;53:3733-41.
[4] Hendrickson A, Hicks D. Distribution and density of medium- and short-wavelength selective cones in the domestic pig retina. Exp Eye Res, 2002;74:435-44.
[5] Winkler J, Hagelstein S, Rohde M, Laqua H. Cellular and cytoskeletal dynamics within organ cultures of porcine neuroretina. Exp Eye Res, 2002;74:777-88.
[6] Luo X, Heidinger V, Picaud S, Lambrou G, Dreyfus H, Sahel J et al. Selective excitotoxic degeneration of adult pig retinal ganglion cells in vitro. Invest Ophthalmol Vis Sci, 2001;42:1096-106.
[7] Ulyanova T, Szel A, Kutty RK, Wiggert B, Caffe AR, Chader GJ et al. Oxidative stress induces heme oxygenase-1 immunoreactivity in Muller cells of mouse retina in organ culture. Invest Ophthalmol Vis Sci, 2001;42:1370-4.
[8] Ghosh F, Taylor L, Arner K. Exogenous glutamate modulates porcine retinal development in vitro. Dev Neurosci, 2012;34:428-39.
[9] Kuehn S, Hurst J, Jashar iA, Ahrens K, Wunderlich M, Dick H et al. NMDA triggered microglia activation in a porcine retina organ culture. accepted for ARVO; 2015.