We thank the many students and fellows who have worked on this model to improve its effectiveness, and to understand its mechanisms. Special thanks are due to Dr&#39;s. Mary Johnson (University of Maryland-Baltimore), Nitza Goldenberg-Cohen (Schneiderman Childrens Hospital, Petah-Tikva, Israel), Charles Zhang (Einstein Medical College, Bronx, NY), and Valerie Touitou (Hopital Salpetrie, Paris, France). This study was funded in part by RO1 EY015304 to SLB.

This protocol was approved by the University of Maryland Institutional Animal Care and Utilization Committee (IACUC; Baltimore, MD, USA)
1. Experimental Set-up
<ol>
	<li>Make a custom designed contact lens from a clear optical grade circular 7 mm in diameter Plexiglas, of 3 mm thickness. Cut the circular lenses with a drill press. Use a standard drill bit to make the inner curve, and finally polish the outer and inner curves using a contact lens polisher of ultrafine grit (1000/3000).</li>
	<li>Prepare 2.5 mM Rose Bengal (RB) in pH 7.4 phosphate buffer saline (PBS) in advance, filter sterilize with a 0.45 micron syringe filter and store 1 ml aliquots in -20 &#176;C in a light-tight container for up to 6 months. 
	NOTE: The use of albino outbred male animals, such as Sprague Dawley minimizes the strain dependent response differences, enables increased ease of induction and reduces the variability that can occur with estrus cycling female.</li>
	<li>Set up the frequency doubled Neodymium-yttrium aluminum garnet (Fd-YAG) ophthalmic medical laser, which generates a 532 nm laser light. Mount the laser on a Haag-Streit ophthalmic slit lamp using a standard ophthalmic laser adapter. This commercially available device enables simultaneous visualization of the animal&#39;s eye and laser spot application. The medical laser also has an aiming beam for proper focusing and centration using same spot size as laser induction. Laser power parameters are as follows:
	<ol>
		<li>For rat rNAION induction: use 500 &#181;m spot size/50 mW laser power/1,000 msec duration/1,000 msec interval.</li>
		<li>For mouse rNAION induction: change the spot size to 300 &#181;m for smaller optic disc and leave other parameters the same as the rat setting.</li>
		<li>Routinely use a laser power meter to assure the laser power output.</li>
	</ol>
	</li>
</ol>
2. Experimental Procedure
<ol>
	<li>Turn on the laser power and set up the appropriate laser parameter. Warm the laser for at least five minutes before use.</li>
	<li>Weigh the animal to determine the appropriate dose for ketamine/xylazine and RB dye. Anesthetize the animal by intraperitoneal injection of a 1 ml/kg mixture of 80 mg/ml ketamine and 4 mg/ml xylazine.</li>
	<li>Leave the animal in a heated cage until fully anesthetized. Check for no response to aversive stimuli (tail or toe pinch). Check animals for depth of anesthesia every 10 min.</li>
	<li>Dilate the animal&#39;s pupils with 1% tropicamide and anesthetize the surface of the eye with 0.5% proparacaine. If using pigmented animals, such as Long Evans, a 2.5% neosynephrine ocular drops will increase pupil dilation.</li>
	<li>Use scissors to cut the whiskers close to the muzzle on the side to be induced to avoid blocking the view.</li>
	<li>Put a drop of 1% methylcellulose or other ophthalmic coupling drop to the inside of the custom-made contact lens, and then apply the lens onto the rat eye.</li>
	<li>Place the animal on a platform adjusted to the height of the slit lamp. Set the animal&#39;s head at a 45&#176; angle so that the eye is perpendicular to the slit lamp and laser beam.</li>
	<li>Visualize the eye through the ophthalmic slit lamp. Make sure the aiming beam is the right size as well as focused and centered directly on the visualized optic nerve. Photograph the retinal fundus using a digital camera with high ASA (1,200 - 2,000) speed mounted on one of the eye pieces of the slit lamp with a custom made adapter. 
	NOTE: The ability to see the choroidal vessels reveals the transparency of the retina. This is an important sign in order to be able to detect later retinal ischemia, which can confound the interpretation if it occurs. rNAION is optic nerve ischemia, which results in isolated RGC loss, while retinal ischemia results in retinal damage affecting all of the cells of the inner retinal layers.</li>
	<li>Optionally, further image the retina and optic nerve using a spectral domain-optical coherence tomograph (SD-OCT8) to evaluate the un-induced eye. Scan en face (Figure 3B) as well as 7 cross-sectional scans through the retina (Figure 3C). The SD-OCT imaging uses the same contact lens used for laser induction.</li>
	<li>Inject 1 ml/kg RB intravenously through tail vein, and wait for 30 sec, then activate the laser power. This time delay enables the RB to distribute evenly throughout the circulation. We use a 50 mW laser pulse at one sec/pulse.&#160; Greater energies (&#8805; 60 mW) can damage the retina or cause retinal vascular ischemia. 
	Caution: Make sure everyone has a pair of laser safety filtering glasses of the appropriate blocking wavelength to prevent stray laser light entering investigator&#39;s eye. 
	NOTE: The laser administration must be given rapidly after the IV RB injection since the dye is eliminated quickly from the circulation. The longer the laser induction, the more severe the optic nerve ischemia. Generally, animals are given 7 - 12 sec pulses in a rapid succession. The contact of the laser light with the circulating dye gives the optic nerve vessels a beautiful golden glow that can be seen through the slit lamp (Figure 4B). This proves the dye was injected systemically and distributed into the blood stream. If the glow is faint, or none at all (Figure 4A), the dye was not injected intravenously. In this case, do not give a second injection immediately, since the animal will need to recover for at least two days before reinjection.</li>
	<li>Immediately after induction, remove the contact lens. Cover both eyes with ophthalmic triple antibiotic (Neosporin/polymyxin/bacitracin) ointment with dexamethasone, and place the animal on a 37 &#176;C warming pad in a single housed cage under close observation until full recovery.
	<ol>
		<li>Clean the contact lens with distilled water and wipe dry with a non-abrasive cleaning cloth for future use.</li>
		<li>Two days after the induction evaluate the optic nerve edema by both fundus color photography and SD-OCT analysis8. 
		NOTE: Comparing the degree of optic nerve edema gives an estimate of the severity of the optic nerve ischemia. At two days, the optic nerve disk margin is blurred, and the retinal veins are slightly dilated, compared with the contralateral (un-induced) eye. Perform electroretinography (ERG) and flash visual evoked potential (VEP11) at two and four weeks post induction for electrophysiology analysis.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Banik, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonarteritic+Anterior+Ischemic+Optic+Neuropathy:+An+Update+on+Demographics,+Clinical+Presentation,+Pathophysiology,+Animal+Models,+Prognosis,+and+Treatment.">Nonarteritic Anterior Ischemic Optic Neuropathy: An Update on Demographics, Clinical Presentation, Pathophysiology, Animal Models, Prognosis, and Treatment.</a> J. Clin. Experimental Ophthalmol. 10, 1-5 (2013).</li><li>IONDT study group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+patients+with+nonarteritic+anterior+ischemic+optic+neuropathy+eligible+for+the+Ischemic+Optic+Neuropathy+Decompression+Trial.">Characteristics of patients with nonarteritic anterior ischemic optic neuropathy eligible for the Ischemic Optic Neuropathy Decompression Trial.</a> Arch Ophthalmol. 114, 1366-1374 (1996).</li><li>Tesser, R. A., Niendorf, E. R., Levin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphology+of+an+infarct+in+nonarteritic+anterior+ischemic+optic+neuropathy.">The morphology of an infarct in nonarteritic anterior ischemic optic neuropathy.</a> Ophthalmology. 110, 2031-2035 (2003).</li><li>Bernstein, S. L., Johnson, M. A., Miller, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonarteritic+anterior+ischemic+optic+neuropathy+(NAION)+and+its+experimental+models.">Nonarteritic anterior ischemic optic neuropathy (NAION) and its experimental models.</a> Prog Retin Eye Res. 30, 167-187 (2011).</li><li>Nicholson, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PGJ2+Provides+Prolonged+CNS+Stroke+Protection+by+Reducing+White+Matter+Edema.">PGJ2 Provides Prolonged CNS Stroke Protection by Reducing White Matter Edema.</a> PLoS One. 7 (12), (2012).</li><li>Goldenberg-Cohen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte+Dysfunction+Following+Induction+of+Experimental+Anterior+Optic+Nerve+Ischemia.">Oligodendrocyte Dysfunction Following Induction of Experimental Anterior Optic Nerve Ischemia.</a> Invest Ophthalmol Vis Sci. 46, 2716-2725 (2005).</li><li>Bernstein, S. L., Guo, Y., Kelman, S. E., Flower, R. W., Johnson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+cellular+responses+in+a+novel+rodent+model+of+anterior+ischemic+optic+neuropathy.">Functional and cellular responses in a novel rodent model of anterior ischemic optic neuropathy.</a> Invest Ophthalmol Vis Sci. 44, 4153-4162 (2003).</li><li>Berger, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral-Domain+Optical+Coherence+Tomography+of+the+Rodent+Eye:+Highlighting+Layers+of+the+Outer+Retina+Using+Signal+Averaging+and+Comparison+with+Histology.">Spectral-Domain Optical Coherence Tomography of the Rodent Eye: Highlighting Layers of the Outer Retina Using Signal Averaging and Comparison with Histology.</a> PLoS One. 9 (5), (2014).</li><li>Huang, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+systemic+treatment+with+methylprednisolone+in+a+rodent+model+of+non-arteritic+anterior+ischemic+optic+neuropathy+(rNAION).">Protective effects of systemic treatment with methylprednisolone in a rodent model of non-arteritic anterior ischemic optic neuropathy (rNAION).</a> Exp Eye Res. 131, 69-76 (2015).</li><li>Mantopoulos, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Experimental+Model+of+Optic+Nerve+Head+Injury.">An Experimental Model of Optic Nerve Head Injury.</a> Invest Ophthalmol Vis Sci. 57, 6222 (2014).</li><li>You, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Evoked+Potential+Recording+in+a+Rat+Model+of+Experimental+Optic+Nerve+Demyelination.">Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination.</a> J. Vis. Exp. (101), e52934 (2015).</li><li>Templeton, J. P., Geisert, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+optic+nerve+crush+in+the+mouse.">A practical approach to optic nerve crush in the mouse.</a> Mol Vis. 18, 2147-2152 (2012).</li><li>Magharious, M. M., D'Onofrio, P. M., Koeberle, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+Nerve+Transection:+A+Model+of+Adult+Neuron+Apoptosis+in+the+Central+Nervous+System.">Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System.</a> J Vis Exp. (51), e2241 (2011).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Rodent+Model+of+Posterior+Ischemic+Optic+Neuropathy.">A Novel Rodent Model of Posterior Ischemic Optic Neuropathy.</a> JAMA Ophthalmol. 131 (2), 194-204 (2013).</li><li>Huang, T. L., Chang, C. H., Chang, S. W., Lin, K. H., Tsai, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+Intravitreal+Injections+of+Antivascular+Endothelial+Growth+Factor+Agents+in+a+Rat+Model+of+Anterior+Ischemic+Optic+Neuropathy.">Efficacy of Intravitreal Injections of Antivascular Endothelial Growth Factor Agents in a Rat Model of Anterior Ischemic Optic Neuropathy.</a> IOVS. 56, 2290-2296 (2015).</li></ol>

Authors have nothing to disclose.

While there are a number of models of optic nerve damage (optic nerve crush12, optic nerve transection13, and PION14), the rNAION model is humane, adaptable to both rats and mice. It more closely resembles the human clinical condition of NAION. This condition includes progressive anterior optic nerve edema, an anterior optic nerve compartment syndrome, focal axonal ischemia, isolated retinal ganglion cell axonal damage and loss over a prolonged time course. The current report gives the appropriate steps for rNAION induction, discusses potential problems during induction, and described early post induction analyses that can be used to evaluate the quality of the induced lesion prior to data inclusion. The advantage of the rNAION model is that with practice, relatively consistent levels of damage can be achieved. Typically 10 - 11 sec exposure results in 40 - 65% RGC loss. The success rate can vary from individual to individual, depending on experience and skill, but an experienced investigator can achieve nearly a 100% induction rate.
In addition to its ease of induction, the severity and time course of the lesion can be easily monitored. An important early part of the quality control in the rNAION model, which can add to model advantages, is early post induction analysis. We normally evaluate the animals two days after the induction, when the optic nerve edema is maximal. The rNAION is characterized by optic nerve edema that resolves over a five day period (roughly 5 times faster than that which occurs in humans), followed by optic nerve pallor and isolated retinal ganglion cell loss. One can identify retinal ischemia (as distinct from optic nerve ischemia) by loss of retinal transparency and whitening of the retina. Retinal ischemia is functionally confirmed by loss of inner retinal signal with electroretinography (ERG). If there is widespread loss of retinal transparency and whitening of the retina, it suggests diffuse retinal ischemia, which is consistent with central retinal vein occlusion, and not rNAION alone. If the induction results in severe retinal ischemia in a number of animals (characterized by sectional or total retinal whitening), the induction parameters can be reduced by one or two seconds exposure time. This way one can optimize both the degree of optic nerve damage, and get consistency of induction in animals. Animals with significant loss of ERG signal should be eliminated from the study. Isolated loss of optic nerve function can be confirmed by flash visual evoked potential measurement (VEP).
There are a number of variables to be kept in mind when using the rNAION model. Individual animals can differ in the overall severity of the lesion, so that multiple animals need to be used in the neuroprotective assays, and a power analysis should be performed to determine the minimum number of animals needed to achieve a statistically valid results, particularly when a more modest protective effect is seen or predicted. We have found that 10 - 15 animals are required for determining a 25% RGC protective effect in rats, and 15 - 20 animals when mice are used. Since the RB dye used in the induction is rapidly eliminated, once the animal is injected, variability can also be dependent on the speed of the time required to perform the induction. Slight differences on the focus on the optic disc, differences in the angle of laser illumination, and a difference in the speed of induction can also affect the outcome. One dedicated individual should be selected to perform the technique in each lab, to further reduce variability. Roughly 10 - 15% of induced animals may need to be eliminated after early post-induction evaluation due to severity of induction (central or branch retinal vein occlusion). This report does not discuss myriad other intrinsic variables that may influence outcome, such as from sex differences, circadian rhythm, and age or strain differences. These questions must be satisfied by the individual investigator. There are other modified parameter settings recently reported, such as 80 mW laser power15. These modifications used different contact lens or laser wavelength but resulted in similar outcomes.
It is important to realize that despite the similarities of rNAION to many aspects of clinical NAION, rNAION is a model, and no model is a perfect duplicate of a human disease, since the actual causative factors in NAION are unknown, and the vascular and inflammatory physiological control of the rodent retina and optic nerve are different in many ways from the human and nonhuman primate. Findings generated by the model need to be interpreted in light of these differences. Regardless of this, the rNAION model is a valuable method of rapidly dissecting many of the potential pathophysiological mechanisms responsible for visual loss and approaches to neuroprotection in a living mammalian system.

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the anterior portion of the optic nerve (ON)1. NAION is the most common cause of sudden optic nerve related vision loss in individuals over the age of 502. The mechanism is believed to be a compartment syndrome that results in intraneural edema, and causes compression of the capillaries supplying the axons within the optic nerve3.
Since the ON is actually a central nervous system (CNS) tract, the rodent NAION (rNAION) model can be used to study the mechanisms and responses to isolated CNS white matter strokes. The rNAION model may therefore be useful in dissecting many problems associated with stroke related damage to white matter. It can be used to evaluate different neuroprotective strategies and agents in white matter stroke.
One of the most attractive features of the model is that it is a painless, noninvasive procedure. The laser power can be adjusted to produce various degree of ischemic damage. Another feature is that it relies on laser induced superoxide radicals to damage the capillary endothelium, producing a progressive capillary dysfunction. It is this dysfunction and progressive edema that is believed to be remarkably similar to the mechanism that causes NAION. Research has shown that it does not cause direct capillary clotting, but works through at least two mechanisms: superoxide induced death and stripping of some of the capillary endothelial cells4, and NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells) associated inflammatory up regulation in remaining endothelium, with increased fluid transport across the cell membranes into the interstitium5 . The closure of the optic nerve capillaries and compression caused by interstitial fluid accumulation result in optic nerve head ischemia. A schematic picture is shown in Figure 2. The rNAION model can be used in both rat and mouse species6,7, and can be varied in the level of its severity, from a mild lesion to a complete, but painless destruction of the optic nerve and the retina, such as central retinal artery occlusion(CRAO).

<table><tbody><tr><td>50 mW 532 nm laser</td><td>Iridex</td><td></td><td>Standard Ophthalmic Laser</td></tr><tr><td>0 - 100 mW 532 nm laser</td><td>Laserglow technologies</td><td></td><td>Substitute for iridex</td></tr><tr><td>Laser slit lamp adapter</td><td>Iridex</td><td></td><td>SMA coupled adapter for laser output</td></tr><tr><td>Coherent Fieldmate laser meter with thermopile sensor</td><td>Coherent</td><td></td><td>others also appropriate</td></tr><tr><td>Ophthalmic Examing Slit lamp biomicroscope</td><td>Various</td><td></td><td>Haag-Streit is the best; cheaper versions available on ebay</td></tr><tr><td>Rose Bengal</td><td>Sigma</td><td>330000-1G</td><td>Photoinducing agent</td></tr><tr><td>Fundus Contact lens or glass cover slip</td><td>custom/Cantor and Nissel (UK)</td><td></td><td>Custom designed planoconvex plastic lens for eye exam and induction</td></tr><tr><td>Tropicamide 1%</td><td></td><td></td><td></td></tr><tr><td>Tamiya polishing compound</td><td>Tamiya, INC</td><td>87068</td><td>polishing contact lens</td></tr><tr><td>2.5% Hypromellose (Goniovisc)/1% Methycellulose</td><td>HUB Pharmaceuticals</td><td></td><td>contact lens coupling agent</td></tr><tr><td>2.5% Neosynephrine Ophthalmic drops</td><td>Alcon labs</td><td></td><td>pupil dilating agent</td></tr><tr><td>Tropicamide 1%</td><td>Alcon labs</td><td></td><td>pupil dilating agent</td></tr><tr><td>0.5% Proparacaine</td><td>Alcon labs</td><td></td><td>topical Anesthetic</td></tr><tr><td>30 G fused needle insulin syringe</td><td>Various</td><td>Various</td><td>for intravenous injection of rose bengal</td></tr><tr><td>Ophthamic Antibiotic ointment with dexamethasone added (Triple antibiotic ointment)</td><td>Various</td><td>Various</td><td>Apply after induciton to minimize corneal scarring</td></tr><tr><td>Heidelberg Corporation Spectral domain-Optical Coherence Tomograph</td><td>Heidelberg Corporation</td><td></td><td>For Optical coherence measurements baseline and post-induction; not essential for induction</td></tr></tbody></table>

the rodent model of nonarteritic anterior ischemic optic neuropathy (rnaion)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

The contact lens enabled central retina visualization (Figure 1). The focal laser spot illuminates the optic disc at the back of the retina (Figure 2). The normal un-induced retina is shown imaged by slit lamp bio-microscope (Figure 3A) and by SD-OCT (Figure 3B and 3C). During laser induction, when no dye is present in the circulation, laser light does not result in vessel and disk fluorescence (Figure 4A). Intravenous RB and the laser light illumination on the optic disk results in a golden color fluorescence on the optic nerve (Figure 4B). Two days following rNAION induction, the optic nerve is swollen (Figure 5A). En face and cross-sectional SD-OCT reveal disk swelling (Figure 5B) and expansion of the optic nerve (Figure 5C).
<img alt="Figure 1" src="/files/ftp_upload/54504/54504fig1.jpg" /> 
Figure 1. Schematic of the Rat Planoconcave Contact Lens. The custom made contact lens is made from 3 mm Plexiglas (design shown), with outer diameter 7 mm, inner diameter 5 mm. This lens is designed to fit over the cornea of the rat eye, and enable direct visualization of the retina. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54504/54504fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54504/54504fig2.jpg" /> 
Figure 2. Schematic of rNAION Model. Longitudinal cross section through the back of the eye. The laser spot is centered on the optic disc (green arrows). The green cell and axon represents the retinal ganglion cell neuron. Immediately following intravenous RB administration, the RB circulates through the vasculature in the back of the eye and optic nerve. The laser beam is used to illuminate the disc for 7 - 12 sec depending on the ischemic severity desired. The laser activates the photosensitizing RB to generate superoxide radicals, which causes capillary closure in the anterior ON head (axonal ischemia; seen on the left side of the nerve as loss of small red lines) while sparing the larger intraretinal vessels emerging from the ON head into the eye. The focal ischemia produces localized axonal dysfunction (detached green line). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54504/54504fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54504/54504fig3.jpg" /> 
Figure 3. Baseline Normal Retina (RET) and Optic Nerve (ON) Analysis using Color Photography and SD-OCT. A. Picture of a normal Sprague-Dawley rat retinal fundus by slit lamp fundus photography. The retinal vessels emerge from the optic disc to supply the inner layers of the retina. The disc has a clear border and a reddish surrounding hue (choroidal flush). The retinal vessels are thin and evenly distributed. The optic nerve disk margin is usually well demarcated before rNAION induction. B. Picture of a normal fundus by en face SD-OCT imaging. Individual cross sectional images of the retina shown in panel C are generated from within the green box. Scan direction is shown with a green arrow. C. Single SD-OCT cross section scan of normal retina and optic disc showing the retinal layers. The retinal nerve fiber layer (RNFL, small white arrow) has a greyish look, but is lighter than the underlying outer nuclear layer (ONL, small white arrow). The RNFL is flat against the optic nerve. The optic nerve shadow is narrow (indicated by two arrows). ON: optic nerve. RNFL: retinal ganglion cell/nerve fiber layer. ONL: Outer nuclear layer. Scale bar: 200 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54504/54504fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54504/54504fig4.jpg" /> 
Figure 4. rNAION Induction Appearance. A. 532 nm/500 &#181;m spot laser illumination of the optic nerve disc and retina without systemic RB injection. The optic nerve disc and retina are dark. B. 532 nm laser illumination 30 sec following systemic RB injection. The ON shows a golden glow within the vessels emerging from the disk at the laser spot site, indicating systemic RB in the blood stream illuminated by the green laser light. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54504/54504fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54504/54504fig5.jpg" /> 
Figure 5. Retinal and Optic Nerve Analysis 2 days after rNAION Induction. A. Color fundus picture shows ON swelling and optic disc pallor, with loss of the surrounding choroidal flush. The veins are usually enlarged and curved, sometimes with boxed (interrupted flow) veins. B. En face SD-OCT picture post-induction shows disc edema and venous dilation. Individual cross section is generated from within the green box. Scan direction is shown with a green arrow. C. Cross section shows ON disk edema, as evidenced by increased thickness of the RNFL and reduced grey intensity (more white, consistent with higher water content). The diameter of the intra-retinal ON (indicated between the black arrows) is increased. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54504/54504fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION at JoVE.com

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

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8.9K Views.  University of Maryland-Baltimore School of Medicine. The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

Video: The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Neuroscience

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

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 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 ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

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