organ culture

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The Novel Induction of Retinal Ganglion Cell Apoptosis in Porcine Organ Culture by NMDA — An Opportunity for the Replacement of Animals in Experiments

Sandra Kuehn, Jose Hurst, Adelina Jashari, Kathrin Ahrens, Teresa Tsai, Ilan M. Wunderlich, H. Burkhard Dick, Stephanie C. Joachim and Sven Schnichels

Some of the advantages of retina organ culture models include their efficient and easy handling and the ability to standardise relevant parameters. Additionally, when porcine eyes are obtained from the food industry, no animals are killed solely for research purposes. To induce retinal degeneration, a commonly used toxic substance, N-methyl-D-aspartate (NMDA), was applied to the cultures. To this end, organotypic cultures of porcine retinas were cultured and treated with different doses of NMDA (0 [control], 50, 100 and 200μM) on day 2 for 48 hours. On day 7, the retinas were cryo-conserved for histological, Western blot and quantitative rt-PCR (qrt-PCR) analyses. NMDA treatment was found to significantly increase retinal ganglion cell (RGC) apoptosis in all the treated groups, without a profound RGC loss. In addition, the intrinsic apoptotic pathway was activated in the 50μM and 100μM NMDA groups, whereas induced nitric oxide synthase (iNOS) expression was increased in the 200μM group. A slight microglial response was detectable, especially in the 100μM group. NMDA treatment induced apoptosis, oxidative stress and a slight microglia activation. All these effects mimic a chronic slow progressive disease that especially affects RGCs, such as glaucoma. A particular advantage of this model is that mediators that can interact in the very early stages of the onset of RGC death, can be easily detected and potential therapies can be tested.

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Richard Clothier: An Appreciation

Michael Balls

The career of Richard Clothier is reviewed in the light of his long-standing collaboration with Michael Balls and Laurens Ruben at the University of East Anglia (UEA), the University of Nottingham, and Reed College, Portland, Oregon, USA. It began with work at UEA on the aetiology of the lymphosarcoma of Xenopus laevis, followed by studies on the effects of exposure to N-nitroso-N-methylurea on T-cell functions, which led to many contributions to comparative immunology. This was followed by the establishment of the FRAME Research Programme, which led to participation in extensive studies on the development of in vitro cytotoxicity tests and their application in acute and topical toxicity testing. A FRAME Trustee since 1983, Richard Clothier was a co-founder, and subsequently Director, of the FRAME Alternatives Laboratory in the University of Nottingham Medical School, where he led successful collaborations with a number of industrial partners and, in particular, with the European Centre for the Validation of Alternative Methods (ECVAM).
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Fish as Research Tools: Alternatives to In Vivo Experiments

Marlien Schaeck, Wim Van den Broeck, Katleen Hermans and Annemie Decostere

The use of fish in scientific research is increasing worldwide, due to both the rapid expansion of the fish farming industry and growing awareness of questions concerning the humane use of mammalian models in basic research and chemical testing. As fish are lower on the evolutionary scale than mammals, they are considered to be less sentient. Fish models are providing researchers, and those concerned with animal welfare, with opportunities for adhering to the Three Rs principles of refinement, reduction and replacement. However, it should be kept in mind that fish should also be covered by the principles of the Three Rs. Indeed, various studies have shown that fish are capable of nociception, and of experiencing pain in a manner analogous to that in mammals. Thus, emphasis needs to be placed on the development of alternatives that replace, as much as possible, the use of all living vertebrate animals, including fish. This review gives the first comprehensive and critical overview of the existing alternatives for live fish experimental studies. The alternative methods described range from cell and tissue cultures, organ and perfusion models, and embryonic models, to in silico computer and mathematical models. This article aspires to guide scientists in the adoption of the correct alternative methods in their research, and, whenever possible, to reduce the use of live fish.

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