We are indebted to Eleut Hernandez for animal husbandry, Gabe Gaidosh for microscopy expertise, and Khue Tran and Zhenyang Zhao for video editing. This study was funded by National Eye Institute grants R01-EY022129 to JLG and P30 grants EY022589 to UCSD and EY014801 to UM; the American Heart Association, the James and Esther King Foundation, the doctoral student exchange program fund of Fudan University Graduate School (No. 2010033), and an unrestricted grant from Research to Prevent Blindness, Inc.

All animal procedures were approved by the University of California San Diego and University of Miami institutional animal care and use committees (IACUC) and performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. All reagents and instruments used in surgical procedures are sterile.
1. Anesthetize and Prepare the Rat for Surgery
<ol>
	<li>Prior to procedure, rats are anesthetized with an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (8 mg/kg) acc...

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Here we describe in detail a method for inducing PION in a rat model. The most critical part of the protocol is the exposure and irradiation of the optic nerve &#8211; to expose the nerve as long as possible while avoiding damage caused by the sharp fine tip forceps or from stretching. In rats, the ophthalmic artery enters the optic nerve &#8804;1 mm from the optic nerve head. Therefore, irradiation of the optic nerve 3-4 mm away from the optic nerve head should only result in ischemia of the capillaries feeding the nerv...

In patients over 50 years old, ischemic optic neuropathy (ION) is the most prevalent type of acute optic neuropathy2. The condition can present as one of two subtypes according to the source of specific affected blood supply&#160;and clinical presentation: anterior (AION) or posterior (PION)3. While the pathogenesis and course of AION have&#160;been studied extensively4-7, PION has remained poorly understood due to its low prevalence, variable presentation, ill-defined diagnostic criteria and lack of an animal model. Furthermore, no treatments have been proven to effectively prevent or reverse vision loss from AION or PION. Therefore, a reproducible and reliable animal model of PION is of great value to study the disease process in vivo and test new therapeutic regimens for neuroprotection and axon regeneration.
Photochemically induced ischemic injury to the microvasculature resulting in vasogenic edema and thrombosis effectively creates regional tissue ischemia8-12. After injection into the vascular circulation, the photosensitive dye erythrosin B produces reactive singlet molecular oxygen upon activation by laser irradiation of&#160;target vessels. The singlet oxygen directly peroxidizes the vascular endothelium, stimulating platelet adherence/aggregation and leading to occlusive thrombus formation. Ischemic damage is spread&#160;to neighboring areas and further exacerbated&#160;by microvascular compression due to vasogenic edema. The overall goal of this protocol is to photochemically induce ischemia in&#160;the retrobulbar optic nerve to mirror the damage caused by PION.
To our knowledge, this is the first model of ischemic injury in the posterior optic nerve1. As this model produces ischemia while avoiding physical trauma, the physiological processes of posterior ischemic optic neuropathy are better mimicked and studied. Also, this model offers a novel platform for screening of candidate therapeutics for optic neuropathies and other CNS ischemic disease. Here, a detailed protocol for femoral vein catheterization, optic nerve exposure, intravenous injection of Erythrosin B and laser irradiation in a rat PION model are described.

<table>
	<tbody>
		<tr>
			<td>532-nm Nd:YAG laser&#160;</td>
			<td>Laser glow</td>
			<td>LRS-532-KM-200-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Beam chopper</td>
			<td>custom-made</td>
			<td>custom-made</td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical shutter and corresponding shutter drive timer</td>
			<td>AAM Vincent Associates</td>
			<td>SD-10</td>
			<td></td>
		</tr>
		<tr>
			<td>25-cm focal length spherical lens</td>
			<td>CVI/Mellles-Griot</td>
			<td>01 LPX 293</td>
			<td>plano-convexBK7 glass lens with HEBBARTM antireflection coating</td>
		</tr>
		<tr>
			<td>Erythrosin B&#160;</td>
			<td>MP Biomedicals</td>
			<td>190449</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorogold</td>
			<td>Fluorochrome,LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Cardinal Health</td>
			<td>CAH1203421</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing (PE10)</td>
			<td>BD Intramedic</td>
			<td>427400</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 10 Blade</td>
			<td>Miltex</td>
			<td>4-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>F.S.T.</td>
			<td>91150-20</td>
			<td>DUMONT #5 RUSTLESS NON-MAGNETIC</td>
		</tr>
		<tr>
			<td>Forceps with Teeth</td>
			<td>F.S.T.</td>
			<td>11153-10</td>
			<td>Germany stainless</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>F.S.T.</td>
			<td>18025-10&#160;</td>
			<td>Germany stainless</td>
		</tr>
		<tr>
			<td>Vannas spring scissors</td>
			<td>F.S.T.</td>
			<td>2-220&#160;</td>
			<td>JJECK Stainless</td>
		</tr>
		<tr>
			<td>Polyglactin suture</td>
			<td>Ethicon</td>
			<td>J488G</td>
			<td>7-0 suture</td>
		</tr>
		<tr>
			<td>hemostat</td>
			<td>F.S.T.</td>
			<td>12075-12&#160;</td>
			<td>Germany stainless</td>
		</tr>
	</tbody>
</table>

rat model of photochemically-induced posterior ischemic optic neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The resulting ischemic injury induced by this technique yields a gradual but pronounced death of retinal ganglion cells after ischemic axon injury. This is a clinically relevant outcome similar to that observed in the human disease. FG retrograde labeling is used to quantify RGC survival after PION. The same method is employed to validate a successful model creation as well as to assess the effects of&#160;different therapeutic regimens.&#160;Figure 1 shows representative confocal images of FG-positive c...

Watch this Scientific Journal Video about Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy at JoVE.com

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

rat-model-photochemically-induced-posterior-ischemic-optic

Research

JoVE Journal

Medicine

8.5K visualizações.  University of Miami Miller School of Medicine.  O objetivo deste protocolo é para induzir fotoquimicamente lesão isquêmica ao nervo óptico posterior em ratos. Este modelo é fundamental para estudos de fisiopatologia da neuropatia óptica isquêmica posterior e abordagens terapêuticas para essa e outras neuropatias ópticas, bem como de outras doenças isquêmicas do sistema nervoso central. 