ProtocolThe isolated perfused bovine retina—A sensitive tool for pharmacological research on retinal function
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).
References (35)
- et al.
Electrophysiological properties of a new isolated rat retina preparation
Vision Res.
(1999) - et al.
Molecular basis of glutamate toxicity in retinal ganglion cells
Vision Res.
(1997) - et al.
In vitro retina as an experimental model of the central nervous system
J. Neurochem.
(1981) - et al.
Role of the beta(2) subunit of voltage-dependent calcium channels in the retinal outer plexiform layer
Invest Ophthalmol. Visual Sci.
(2002) - et al.
Functional characterization of the L-type Ca2+ channel Cav1.4alpha1 from mouse retina
Invest. Ophthalmol. Visual Sci.
(2004) - et al.
Loss-of-function mutations in a calcium-channel a1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night blindness
Nat. Genet.
(1998) - et al.
The effect of aspartate on the electroretinogram of the vertebrate retina
Experientia
(1970) - et al.
Effects of daunorubicin and CD95L on retinal function in superfused vertebrate retina
J. Ocular Pharmacol. Ther.
(2005) - et al.
Glutamate transporters and retinal excitotoxicity
GLIA
(2002) - et al.
Involvement of glutamate in ischemic neurodegeneration in isolated retina
Vis. Neurosci.
(2003)
Drug action and cellular calcium regulation
Adv. Drug Res.
Protective effect of arachidonic acid on glutamate neurotoxicity in rat retinal ganglion cells
Invest. Ophthalmol. Visual Sci.
Effects of antiviral agents on retinal function in vertebrate retina
Adv. Ocul. Toxicol.
Effects of etoposide (VP16) on vertebrate retinal function
J. Toxicol., Cutan. Ocul. Toxicol.
Treatment of cytomegalovirus retinitis clinically resistant to ganciclovir and foscarnet with intravitreal etoposide
J. Toxicol., Cutan., Ocul. Toxicol.
Effects of protein tyrosine kinase inhibitor genistein on retinal function in superfused vertebrate retina
J. Ocular Pharmacol. Ther.
A Ni2+-sensitive component of the ERG–b-wave from the isolated bovine retina is related to E-type voltage-gated Ca2+ channels
Graefes Arch. Clin. Exp. Ophthalmol.
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.