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The isolated perfused bovine retina—A sensitive tool for pharmacological research on retinal function

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Abstract

The electroretinogram (ERG) of the isolated bovine retina serves as a proven criterion of retinal activity. It is used as a sensitive pharmacological tool for testing effects of applied drugs and toxins on photoreceptors, and higher order neurons that contribute to the generation of the b-wave. Following isolation and detachment from the underlying pigment epithelium, part of the retina was mounted into a closed chamber and perfused by a nutrient solution. Flow rate of the nutrient solution and its ingredients, incubation temperature and light intensity were optimised empirically to achieve a maximum b-wave amplitude. Under these conditions, a reproducible, high-resolution ERG can be stably recorded for more than 10 h with sufficient oxygenation found to be a prerequisite for the long-lasting stability. Addition of L(+)glutamate to the nutrient solutions was not anymore beneficial for the b-wave amplitude. A well-known inhibitor of oxidative phosphorylation (KCN) and antagonists of voltage-gated Ca2+ channels (isradipine, ω-conotoxin-GVIA and NiCl2) were used to prove the validity of the test system. The recording of the ERG from the isolated and perfused bovine retina serves as a valuable physiological model for a neuronal network in which important questions related to the retinal signalling and metabolism can be investigated.

Section snippets

Type of research

The vertebrate retina represents a highly organised neuronal network containing most neurotransmitter systems of the central nervous system (CNS). Processing of visual information is initiated in rods and cones of the outer retinal layer, further propagated via horizontal and bipolar cells to ganglion cells of the inner retinal layer that finally projects to various visual areas of the thalamus, midbrain and area 17 of the cerebral cortex. On the level of the retina, both photoreceptor subtypes

Time required

After removal from the animal, total time for the isolation of the bovine retina is ∼15 min. When circular pieces from the sclera–choroid–retina segments were obtained and stored in the nutrient solution at 4 °C, the retina detaches from the underlying pigment epithelium within the next 15 min. After transfer of plain retina segments into the recording chamber, stable ERGs could be recorded after 90–120 min, lasting for more than 10 h.

Animals

Bovine eyes from animals younger than 12 month of age were taken from the local abattoirs. All experiments were performed according to the local institutional guidelines of the committee on research animal care from the University of Cologne.

Water and chemicals

Deionised water (<0.1 μS/cm) was additionally glass distilled and autoclaved in glass bottles. All solutions were prepared in autoclaved glass bottles, using sterile distilled water. Glucose, glutamate, NiCl2, KCN and the constituents of the nutrient

Preparation of the retina and incubation conditions

Eyes were removed from the calf in a local slaughterhouse and immediately stored in a nutrient solution consisting of the following (in mM): NaCl (120), KCl (2.0), CaCl2 (0.15), MgCl2 (0.1), NaH2PO4 (1.5), Na2HPO4 (13.5) and glucose (5) with a final pH of 7.8. First, eyes were hemisected at the junction of sclera and cornea followed by removal of lens and vitreous body from the posterior eyecup. Using a 7-mm trephine, circular pieces were obtained from the posterior segment, which had been

Development of maximum b-wave amplitude, and subsequent glucose and oxygen effects

After the retina segment was mounted in a light-tight incubator constructed as a Faraday cage, it was continuously superfused by the optimised nutrient medium on either side allowing the measurement of the slow transretinal electrical potential changes upon a 0.5-s lasting light stimulus. The maximal b-wave developed during an equilibration period of 90–120 min. Optimised conditions for the nutrient solution were a prerequisite for a stable recording of the b-wave amplitude which can be

Discussion

The ERG obtained from vertebrates serves as a proven criterion of retinal activity, and the response of the dark adapted retina to a flash of light provokes the negative-going a-wave generated by the photoreceptor currents [19] and the positive-going b-wave by activating bipolar-cell currents in combination with bipolar cell-dependent K+ currents affecting Müller cells (for a review, see [20], [22]).

The frog retina has been isolated successfully and investigated in great detail during the last

Quick procedure

  • (i)

    Collecting fresh bovine eyes from a local abattoir immediately after killing.

  • (ii)

    Storage of eyes in a pre-cooled nutrient solution, in an amber bottle.

  • (iii)

    Transport to the laboratory, and hemisection of the eye under red dim light.

  • (iv)

    Removal of the posterior eyecup, and cutting circular sclera–choroid–retina pieces.

  • (v)

    Separation of the retina by gentle shaking and transfer to the recording chamber.

  • (vi)

    Superfusion of the oxygen-saturated nutrient solution at 30 °C, and light stimulation for 500 ms at intervals of

Essential literature references

Refs. [1], [5], [23], [24], [26].

Acknowledgments

The work was financially supported by the Köln Fortune Program/Faculty of Medicine, University of Köln and the Center of Molecular Medicine Cologne (CMMC).

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  • Cited by (0)

    1

    Present address: Department of Ophthalmology I, University of Tuebingen, Schleichstr. 12-16, D-72076 Tuebingen, Germany.

    2

    Dedicated to the late Prof. Dr. Werner Sickel.

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