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 loss¹, with only 8% of patients recovering visual acuity significantly¹. Although arterial fibrinolysis has been suggested as a treatment option, the benefit could not be shown in a randomized clinical trial³.

Glaucoma and retinal detachment both have an increase in glutamate concentration^4-6. Glutamate under physiologic conditions is encountered as an excitatory transmitter throughout the whole central nervous system and the inner retina^7,8. Elevated glutamate levels have been found not only in glaucoma and retinal detachment^5,6 but also in proliferative diabetic retinopathy⁹. An increase in glutamate possibly leads to excitotoxicity and, therefore, nerve cell damage¹⁰. In most cases of retinal detachment and in some cases of proliferative diabetic retinopathy surgery on the retina (pars plana vitrectomy) are necessary. During pars plana vitrectomy mechanical manipulation, bright light of the optic fiber or shear stress exerted by high flow rates of irrigation solutions during long operations exert an additional stress on the retina^11,12.

All the mentioned diseases have in common that the pathology is localized to the retina alone and pose the ophthalmologic community with the challenge to find ways to protect the retina as a neurosensory system.

The electroretinogram (ERG) is the standard method for the evaluation of in vivo photoreceptor function (a-wave) and the function of the inner retina (b-wave). The ERG is measured by silver-electrodes introduced into the cornea and the eyes are being stimulated by an increasing level of light to detect defects in rods or cones or in the inner retina. Different defects in the retina can be detected by changes in the amplitude (the strength of the response) or the latency (the time-to-response-interval) of the ERG. Different ERG protocol and measurement methods (pattern-ERG, multifocal-ERG or bright field ERG) allow further differentiation of defects. The technique of the isolated retina has been introduced recently, making it possible to evaluate effects on the retina without interferences from e.g. a study animal’s general reactions^13,14.

It was the purpose of this study to evaluate and introduce a defined and standardized stress model for hypoxia and glutamate stress on the superfused isolated retina. Thus, we are hoping to lay the foundations for future studies on neuroprotective effects of certain agents or intraocular irrigation solutions.

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 Sickel-solution.
5. Mechanically loosen the vitreous attachments to the retinal surface and remove the vitreous from the open eye cup.
6. Afterwards divide the eye into four quadrants and punch out round areas of ca. 7 mm diameter using a trephine.
7. Gently separate the retina from the pigment epithelium and place it on a recording device inside a box protected from light. The recording device consists of a plastic maintainer with a mesh in the middle; place the retina on the mesh and then fix with a plastic ring directly on the electrodes.
   NOTE: The plastic maintainer has two channels to allow a constant flow of the medium.

2. Recording the Electroretinogram (ERG)

1. In order to record the electroretinogram, use two silver/silver-chloride electrodes on either side of the retina and perfuse the retina at a constant perfusion velocity of ca. 1 ml/min and constant temperature of 37 °C. Use the “Sickel-solution” saturated with oxygen.
2. Before starting the measurement, dark adapt the retina (protect it from light during all measurements) and use stimulus intervals of five min. Use a 1 Hz single white xenon flash for stimulation with an intensity set to 6.3 mlx at the retinal surface.
3. Use calibrated neutral density filters and a light stimulus of 10 µsec controlled by a timer in order to have optimal responses.
4. To measure and process the data, filter the ERG and amplify it (100 Hz high pass filter, 50 Hz notch filter, 100,000 x amplification) using a Grass RPS312RM Amplifier. Try to filter out possible disturbing frequencies that may disturb the signal. In order to process the data, use an analog-to-digital data acquisition board on a desktop computer (PC compatible).
5. After the dark adaptation period under constant perfusion, measure the amplitudes of the electrical signal until stable b-wave amplitudes are recorded.
   NOTE: Amplitudes are considered stable if five single measurements reach a mean value and deviate less than 10%. A good example of single measurements is given in Figure 1.
6. To start the testing, replace pure oxygen by either pure nitrogen (number of single experiments, n = 5) to test for hypoxia or 250 µm glutamate (n = 5).
7. Record the electrical responses every 5 min for 45 min.
8. After the testing period, perfuse the retinas with standard medium saturated with oxygen for 75 min and look at the changes of the b-wave amplitude. This is the washout phase. Measure the b-wave amplitude from the trough of the a-wave to the peak of the b-wave.
9. To investigate the effect of hypoxia or glutamate on the photoreceptor potential under scotopic conditions, suppress the b-wave by adding 1 mM to the nutrient solution.
10. After recording a stable photoreceptor potential for 30 min, carry out the procedure as before, exposing the retinas 45 min to the different irrigation solutions with 1 mM aspartate. Use the same washout period (step 2.8) as mentioned earlier.

3. Data Analysis

1. In order to statistically evaluate the data, ensure a normal distribution for all data, e.g. using the Kolmogorov-Smirnov test¹⁵.
2. Calculate the reduction of the a- and b-wave amplitudes in percentages after the exposure phase in comparison to the last measurement before the exposition. Compare the reduction of the ERG-amplitudes after 45 min – at the end of the exposition period – to ERG measured before application.
3. Compare the a- and b- wave at the end of the washout phase to the corresponding amplitude before exposition to examine a possible recovery.
4. For statistical analysis, use the software JMP statistical software or SPSS software. Calculate data throughout as the mean ± standard deviation. Estimate significance by the appropriate statistical test.
   NOTE: These tests might be different depending on the experimental scope. In this setting, use the Student’s paired t-test.

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