Name: the rodent model of nonarteritic anterior ischemic optic neuropathy (rnaion)
Uploaded: 2016-11-20T13:00:00
Description: Watch this Scientific Journal Video about The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION at JoVE.com

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>
	...

<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>

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 ap...

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 border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50mW 532nm laser</td>
			<td style="width:99px;">Iridex</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Standard Ophthalmic Laser</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0-100mW 532nm laser</td>
			<td style="width:99px;">Laserglow technologies</td>
			<td style="width:119px;"></td>
			<td>Substitute for iridex</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laser slit lamp adapter</td>
			<td style="width:99px;">Iridex</td>
			<td style="width:119px;"></td>
			<td>SMA coupled adapter for laser output</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cpherent Fieldmate laser meter with thermopile sensor</td>
			<td style="width:99px;">Coherent</td>
			<td style="width:119px;"></td>
			<td>others also appropriate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ophthalmic Examing Slit lamp biomicroscope</td>
			<td style="width:99px;">VArious</td>
			<td style="width:119px;"></td>
			<td>Haag-Streit is the best; cheaper versions available on ebay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rose Bengal</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">330000-1G</td>
			<td>Photoinducing agent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fundus Contact lens or glass cover slip</td>
			<td style="width:99px;">custom/Cantor and Nissel (UK)</td>
			<td style="width:119px;"></td>
			<td>Custom designed planoconvex plastic lens for eye exam and induction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tropicamide 1%</td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tamiya polishing compound</td>
			<td style="width:99px;">Tamiya, INC</td>
			<td align="right" style="width:119px;">87068</td>
			<td>polishing contact lens</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2.5% Hypromellose (Goniovisc)/1% Methycellulose</td>
			<td style="width:99px;">HUB Pharmaceuticals</td>
			<td style="width:119px;"></td>
			<td>contact lens coupling agent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2.5% Neosynephrine Ophthalmic drops</td>
			<td style="width:99px;">Alcon labs</td>
			<td style="width:119px;"></td>
			<td>pupil dilating agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tropicamide 1%</td>
			<td style="width:99px;">Alcon labs</td>
			<td style="width:119px;"></td>
			<td>pupil dilating agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5% Proparacaine</td>
			<td style="width:99px;">Alcon labs</td>
			<td style="width:119px;"></td>
			<td>topical Anesthetic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30ga fused needle insulin syringe</td>
			<td style="width:99px;">Various</td>
			<td style="width:119px;">Various</td>
			<td>for intravenous injection of rose bengal</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ophthamic Antibiotic ointment with dexamethasone added (Triple antibiotic ointment)</td>
			<td style="width:99px;">Various</td>
			<td style="width:119px;">Various</td>
			<td>Apply after induciton to minimize corneal scarring</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Heidelberg Corporation Spectral domain-Optical Coherence Tomograph</td>
			<td style="width:99px;">Heidelberg Corportion</td>
			<td style="width:119px;"></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...

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 ...

The overall goal of this procedure, is to produce a rodent model of anterior ischemic optic neuropathy, for use in characterizing stroke response mechanisms. And for analyzing the ethicacy of neuroprotective agents in the treatment of optic nerve stroke. This measure can help answer key questions in the field of optic nerve stroke.Including, mechanisms of edema, post-stroke inflammation, and pass-ways of protection and repair. The main advantage of this technique is that the ischemic damage is isolated to the anterior portion of optic nerve. Without surgery, the technique is painless.And the severity of the damage can be reproducibly varied at will. This method avoids the pain associated with many nerve damage models. Including, crushing, or cutting the optic nerve.It also enables detailed analysis of the effects of isolated exo-ischemia, on a single neuron class, in vivo. Start by obtaining a custom made contact lens designed according to the specifications shown. Also, prepare 2.5 milimaler rose bengal, in pH 7.4 phosphate buffered saline.Fills are sterilized with a 0.5 micron syringe filter. And prepare one millimeter aliquots, place the aliquots in a light, tight, container and store at negative 20 degrees Celsius for up to six months. On the day of the procedure, set up the frequency doubled neodymium yttrium aluminum garnet ophthalmic medical laser.Which generates a 532 nanometer laser light. Mount the laser on a Haag-Streit ophthalmic slit lamp, using a standard ophthalmic laser adapter, to enable simultaneous visualization of the animals eye, and laser spot application. Turn on the laser, and allow it to warm for at least five minutes, before use.Set the appropriate laser power parameters for the experimental animal used. Mount a high ASA speed digital camera on one of the eye pieces of the slit lamp, with a custom made adapter. After appropriately, anesthetizing the experimental animal, and ensuring the appropriate level of anesthesia, by the absence of the response to a toe pinch.Dilate the animals pupils with one percent tropicamide. And anesthetize the surface of the eye, with 0.5 percent proparacaine. Then, use the scissors to cut the whiskers close to the muzzle on the side of the induction, to allow a clear view of the eye.Next, put a drop of one percent methylcellulose, or other ophthalmic coupling drop, onto the inside of the custom made contact lens. And then apply the lens onto the rats eye. Place the animal on a platform, adjusted to the height of the slit lamp.Set the animals head at a 45 degree angle, so that the eye is perpendicular to the slit lamp and laser beam. Then, visualize the eye through the ophthalmic slit lamp. In a healthy eye, the optic nerve head has a clear border and a reddish surrounding Q.And the retinal vessels are thin and evenly distributed.Make sure the aiming beam is the correct size. As well as focused and centered directly on the visualized optic nerve. Then photograph the retinal fundus, using the digital camera.Next, inject one milligram per kilogram, of rose bengal intravenously through the tail vein. And wait for 30 seconds. A smooth, continuous, intervaneous injection of rose bengal, must be followed after 30 seconds, by laser exposure, of the optic nerve head.Variation in time, between these two steps, result in increase variability of degree of optic nerve stroke damage. Which can bias your results. After 30 seconds, activate the laser power.The optic nerve, shows a golden glow within the vessels, emerging from the disc at the laser spot sight. Indicating rose bengal in the blood stream, illuminated by the green laser light. Immediately after induction, remove the contact lens, cover both eyes, with ophthalmic triple antibiotic ointment, dexamethasone.Single house the animal, in a cage on a 37 degree Celsius warming pad, under close observation until full recovery. This colored fundus picture, shows optic nerve swelling and optic disc pallor. With loss of the surrounding choroidal flush.The veins are enlarged, and curve. This n-face SDOCT picture, post-induction, shows disc edema, and venous dilation. The scan direction is indicated with a green arrow, the cross section in the next image, is generated from within the green box.Here, a cross section, shows optic nerve disc edema, as evidenced by reduced gray intensity, and increased thickness of the retinal nerve fiber layer. Consistent with higher water content. The diameter of the intraretinal optic nerve, indicated between the black arrows, is increased.While attempting this procedure, it is important to remember to have a clean, polished, contact lens. Or cover slip, that enables good visualization of the nerve, and precise focusing, of the laser light over the nerve. Following this procedure, other methods like, immunohistochemistry, cytokine analysis, or gene expression analysis, can be performed in order to answer additional questions.Like inflammatory responses after stroke, or changes in the target tissues.

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Research

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Medicine

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

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 Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...