Name: glutamate and hypoxia as a stress model for the isolated perfused vertebrate retina
Uploaded: 2015-03-22T15:00:00
Description: Watch this Scientific Journal Video about Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina at JoVE.com

This paper is dedicated to my beloved wife Maren and our little Karl.

1. Preparation of Bovine Eyes
<ol>
	<li>Obtain bovine eyes directly after the animal is slaughtered.</li>
	<li>Transport the protected eyes in &#8220;Sickel-solution&#8221; 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.</li>
	<li>Perform the preparation of the retina under dark adapted conditions with a dim red light.</li>
	<li>Remove the anterior part of the eye. Pe...

<ol>
	<li>Varma, D. D., Cugati, S., Lee, A. W., Chen, C. 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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 &#181;M glutamate, we did find a statistically significant impact o...

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 concentration4-6. Glutamate under physiologic conditions is encountered as an excitatory transmitter throughout the whole central nervous system and the inner retina7,8. Elevated glutamate levels have been found not only in glaucoma&#160;and retinal detachment5,6 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 retina11,12.
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&#8217;s general reactions13,14.
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.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">120 mM NaCl&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,064,041,000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2 mM KCl,&#160;&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,050,010,250</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">0.1 mM MgCl2,&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">58,330,250</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">0.15 mM CaCl2</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td style="width:119px;">111 TA106282</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1.5 mM NaH2PO4/13.5 mM Na2HPO4&#160;&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,065,860,500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">5 mM glucose</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">40,741,000</td>
			<td></td>
		</tr>
	</tbody>
</table>

glutamate and hypoxia as a stress model for the isolated perfused vertebrate retina

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 &#181;m 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 &#60;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 &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;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.

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...

Watch this Scientific Journal Video about Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina at JoVE.com

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

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 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

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