Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Summary

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N₂) or glutamate stress (250 µM glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

Abstract

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.

Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N₂) or glutamate stress (250 µm glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.

For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p <0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p <0.01, decrease of 25.5% at the end of the washout, p = 0.03).

For 250 µm glutamate, an initial 7.8% reduction of a-wave amplitudes (p >0.05) followed by a reduction of 1.9% (p >0.05). A reduction of 83.7% of b-wave amplitudes (p <0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

Introduction

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades. The retina is a highly sensitive neuronal network that depends significantly on oxygenation and is influenced strongly by the metabolism of its surrounding cells. Major ocular pathologies related to nerve cell damage are retinal vascular occlusions, glaucoma, and retinal detachment.

Retinal artery occlusion, as an example for retinal vascular occlusion, leads to a sudden loss of vision due to hypoxia of the inner retina¹. It is often associated with general vascular pathologies² and leads to a persistent visual lo....

Protocol

1. Preparation of Bovine Eyes

1. Obtain bovine eyes directly after the animal is slaughtered.
2. Transport the protected eyes in “Sickel-solution” a special medium containing 120 mM NaCl, 2 mM KCl, 0.1 mM MgCl₂, 0.15 mM CaCl₂, 1.5 mM NaH₂PO₄, 13.5 mM Na₂HPO₄ and 5 mM glucose at RT.
3. Perform the preparation of the retina under dark adapted conditions with a dim red light.
4. Remove the anterior part of the eye. Perform an equatorial incision ca. 4 mm posterior to the limbus. Thereafter remove cornea, iris, ciliary body and lens in one piece. Keep the retinas in....

Results

After 1 hr of perfusion of the retinal preparations with oxygen-saturated standard solution (Figure 1A and B) ERG-amplitudes showed stabilization and less variation of amplitudes between single measurements. pH, osmotic pressure, temperature, and pO₂ (except for hypoxia testing) were kept constant for all tests.

To isolate the photoreceptor signal from the signal of the inner retina, 1 mM aspartate was added to the standard solution to suppress the b-wave (Figure 1A.......

Discussion

In this study, a significant impact on the b-wave amplitude after 45 min of hypoxia was found. This reduction was still significant after the washout phase. A similar effect on the photoreceptor potential could be observed.

The results are supported by other published data¹⁶ and give us the opportunity to study possible neuroprotective effects after hypoxia.

After 45 min exposition of 250 µM glutamate, we did find a statistically significant impact o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
120 mM NaCl	Merck Pharma, Germany	1,064,041,000
2 mM KCl,	Merck Pharma, Germany	1,050,010,250
0.1 mM MgCl2,	Merck Pharma, Germany	58,330,250
0.15 mM CaCl2	Merck Pharma, Germany	111 TA106282
1.5 mM NaH2PO4/13.5 mM Na2HPO4	Merck Pharma, Germany	1,065,860,500
5 mM glucose	Merck Pharma, Germany	40,741,000