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) according to body weight. Adequate depth of anesthesia should be determined by a negative response to toe pinch stimulus.</li>
	<li>Once anesthetized, pull the tongue forward to prevent asphyxia and apply lubricating ointment to both eyes to prevent drying of the corneas during surgery.</li>
	<li>Shave the surgical sites using a hair clipper and wipe the area three times with 10% providone-iodine detergent solution and 70% ethanol.</li>
	<li>Drape the animal within a sterile field. Sterile gloves and surgical instruments are used during survival surgery. Re-sterilize the tips of instruments using a hot bead sterilizer between animals.</li>
</ol>
2. Surgical Approach
<ol>
	<li>PION induction
	<ol>
		<li>Femoral vein catheterization
		<ol>
			<li>Prepare and clean the surgical site. Shave the right inner thigh using a hair clipper and wipe the area three times each with 10% providone-iodine detergent solution and 70% ethanol.</li>
			<li>Prepare the tubing. Cut a 40cm length of polyethylene tubing (PE 10) sterilized in 70% ethanol. Flush the tubing with saline and connect it to a 1 ml syringe containing a pre-measured solution of 2% erythrosin&#160;B dye (1 &#956;l/mg, yielding a dose of 20 mg/kg body weight). Mount the syringe into a foot-switch controlled infusion pump set to a rate of 600 &#956;l/min.</li>
			<li>Using a No. 15 blade, make a small horizontal incision at the base of the right thigh. Cut and spread the membrane inside and clean the area with sterile cotton swabs.</li>
			<li>Separate the muscle with forceps until the branch of the femoral vein is visible. A sheath surrounds the artery, vein and the nerve. Pinch and pull this sheath upward&#160;with forceps (fine-tip Dumont forceps), and cut a small incision (2-4 mm is usually adequate) near the base of the triangular-shaped wedge with Vannas spring scissors. Expand the cut as necessary.</li>
			<li>Separate the vein and artery with a blunt micro-surgical hook parallel to the direction of the vein. Be careful not to damage the delicate membrane and vein branches. Then, gently lift the vein and separate it from the underlying connective tissue.</li>
			<li>Obtain a needleless nylon suture and place it next to the femoral vein. Using the micro-surgical hook, elevate the vein and pass the&#160;fine-tip forceps beneath the distal region. Grab one end of the suture and pull it underneath the vein. Ligate the distal vein tightly. Pass a second suture in a similar manner under the proximal vein and make a loose knot.</li>
			<li>Make a small cut in the vein near the distal ligation with Vannas spring scissors. Expand the hole as necessary with the fine-tip forceps. Some blood may leak through the cut. Clean the surgical area with cold, sterile BSS and sterile cotton swabs.</li>
			<li>Holding the vein wall at the edge of the cut, catheterize the vessel with the prepared saline-flushed tubing using a needle holder. Tighten the proximal knot around the vein and tubing. Then, anchor the tubing by tying it to the distal suture.</li>
			<li>Check the quality of the catheterization by pressing the foot-switch to inject saline, &#60;1 ml is adequate. Make sure the tubing is unobstructed and has no leaks. Temporarily close the incision with sutures to protect the catheterization and tissue.</li>
		</ol>
		</li>
		<li>Exposure of the optic nerve
		<ol>
			<li>Prepare the surgical site by wiping the pre-shaved area above the left eye three times each with 10% providone-iodine detergent solution and 70% ethanol.</li>
			<li>Make an incision along the skin 2-3 mm behind the eye with a No. 15 blade. Pinch and lift the connective tissue with serrated forceps, and make a small incision with Vannas spring scissors. This small incision is generally about 5 mm in length, but can be longer to provide greater exposure for a&#160;surgeon in training. Continue to bluntly dissect through the connective tissue along the superior rim of the orbital bone, taking care to avoid disrupting blood vessels. Clean the surgical area with cotton swabs.</li>
			<li>Dissect downward through the conjunctiva until the superior rectus muscle is visible. Pinch and dissect through the muscle; the muscle will be liberated from deep within the orbit. Now, surrounding tissues can be utilized to aid in the retraction and elevation of the eye for ease of visualization.</li>
			<li>Retract the flap of skin and connective tissue laterally and downward, and hold in place with a suture and hemostat. This will rotate the eye forward and outward in order to reveal the fat-containing sheath that surrounds the optic nerve.</li>
			<li>Carefully insert a pair of sharp forceps and expand parallel to the optic nerve to separate the connective tissue surrounding the sheath. Do not touch the optic nerve with the sharp tips of the forceps.</li>
			<li>A 5 mm length of the optic nerve and surrounding sheath should now be visible. A network of microvessels on the sheath surface encircles the optic nerve. These will be targeted during laser irradiation.</li>
		</ol>
		</li>
		<li>Intravenous injection of Erythrosin B and laser irradiation
		<ol>
			<li>Wear orange-colored safety glasses at all times while operating the laser irradiation apparatus to shield yourself from the green laser light. Turn on the laser, open the shutter and adjust the peak and average powers of the laser as necessary. Close the shutter.</li>
			<li>Position the rat in the laser irradiation apparatus. To ensure proper beam placement, a weak aiming beam is produced by spatially filtering the laser through a 100 &#181;m diameter hole drilled in the closed shutter blade. Re-expose the optic nerve with a pair of fine tip forceps, and position the aiming beam onto the intraorbital optic nerve between 3 mm and 4 mm behind the optic nerve head.</li>
			<li>Inject the solution of 2% erythrosin&#160;B via activation of the infusion pump. It may circulate for a few seconds while the surgeon adds a small drop of BSS to moisten the surface of the optic nerve.</li>
			<li>Verify the position of the aiming beam and then click the foot-switch to initiate irradiation. An orange-colored safety filter, which is added into the optical path of the microscope, will be immediately triggered followed by the opening of the shutter after a 1 sec delay.</li>
			<li>Irradiate the optic nerve for 90 sec&#160;with a peak power of 135mW and average power of 18mW produced by a beam chopper rotating at 250 Hz with a duty cycle of 15% (this minimizes thermal effects). Yellow fluorescence, visualized as bright orange through the safety filter, will be emitted from the superior surface of the optic nerve and is sufficient to ensure that the beam irradiates&#160;the nerve symmetrically.</li>
			<li>After the irradiation, the orange-colored safety filter will open automatically. Microhemorrhage can be observed in some cases.</li>
			<li>Relieve the traction on the extra ocular muscles and return the eye to a neutral position. Close the incision with interrupted sutures. Then withdraw the catheter&#160;and tie off the femoral vein tightly to prevent leakage; close with interrupted sutures. Apply antibiotic ointment to both incisions. Check the fundus to verify the vascular integrity of the central retinal vein and artery.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Retrograde Labeling of Retinal Ganglion Cells (RGCs). NOTE: In order to evaluate RGC&#160;survival, retrograde labeling with fluorogold (FG) should be completed one week before PION. The method is described in detail in JoVE protocol 81913.
	<ol>
		<li>In brief: anesthetize the animal with ketamine (60 mg/kg) and xylazine (8 mg/kg) and shave the head.</li>
		<li>Surgically scrub the incision site and make a midline incision across the head to expose the skull.</li>
		<li>Drill bilateral holes through the skull (&#248; 2x2 mm) 0.5 mm from both the sagittal and transverse sutures.</li>
		<li>Carefully aspirate the cerebral content that lies over the dorsal surface of the superior colliculus (SC) using a vacuum pump. Then, place a small piece of gelfoam soaked with 4% FG onto the surface of the SC.</li>
		<li>Close the incision with sutures and care for the animal using standard postoperative care.</li>
	</ol>
	</li>
	<li>Post-operative care and Euthanasia
	<ol>
		<li>After surgery, place animal in a separate clean cage on top of a recirculating heated water pad until the animal is recovered.</li>
		<li>Post-surgical analgesics (buprenorphine HCl, 0.01 mg/kg) should be administered twice per day for three consecutive days to minimize discomfort.</li>
		<li>Rat should be kept separately and observed until they are able to maintain sternal recumbency and regain sufficient consciousness.</li>
		<li>Signs of recovery and good health are monitored daily for at least 5 days after the surgery, or until suture removal and adequate healing of the surgical site, whichever occurs latest.</li>
		<li>Euthanize the animals by perfusion with 4% PFA at scientifically appropriate time points after the surgery according to investigative interest.</li>
	</ol>
	</li>
</ol>

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	<li>Wang, Y., Brown, D. P., Duan, Y., Kong, W., Watson, B. D., Goldberg, J. L. <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 Ophthalmology. 131, 194-204 (2013).</li><li>Rucker, J. C., Biousse, V., Newman, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+optic+neuropathies.">Ischemic optic neuropathies.</a> Curr Opin Neurol. 17, 27-35 (2004).</li><li>Hayreh, S. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-arteritic+anterior+ischemic+optic+neuropathy:+role+of+systemic+corticosteroid+therapy..">Non-arteritic anterior ischemic optic neuropathy: role of systemic corticosteroid therapy..</a> Graefe's archive for clinical and experimental ophthalmology. 246, 1029-1046 (2008).</li><li>Prado, R., Dietrich, W. D., Watson, B. D., Ginsberg, M. D., Green, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+Induced+Graded+Spinal-Cord+Infarction+-+Behavioral,+Electrophysiological,+and+Morphological+Correlates..">Photochemically Induced Graded Spinal-Cord Infarction - Behavioral, Electrophysiological, and Morphological Correlates..</a> J Neurosurg. 67, 745-753 (1987).</li><li>Dietrich, W. D., Busto, R., Watson, B. D., Scheinberg, P., Ginsberg, M. 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The authors have nothing to disclose.

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&amp;nbsp;</td><td>Laserglow</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>Vincent Associates</td><td>SD-10</td><td></td></tr><tr><td>25-cm focal length spherical lens</td><td>CVI/Melles-Griot</td><td>01 LPX 293</td><td>plano-convexBK7 glass lens with HEBBAR&lt;sup&gt;TM&lt;/sup&gt; antireflection coating</td></tr><tr><td>Erythrosin B&amp;nbsp;</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&amp;nbsp;</td><td>Germany stainless</td></tr><tr><td>Vannas spring scissors</td><td>F.S.T.</td><td>2-220&amp;nbsp;</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&amp;nbsp;</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

8.6K Views.  University of Miami Miller School of Medicine.  Het doel van dit protocol is ischemische letsel fotochemisch ertoe te bewegen de achterste oogzenuw in de rat. Dit model is kritisch voor onderzoek van de pathofysiologie van posterieure ischemische optische neuropathie, en therapeutische toepassingen voor deze en andere optische neuropathieën, evenals andere CNS ischemische ziekten. 

Video: Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

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, ...

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 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.

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

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 ...

Neuroscience

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

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 ...

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. ...

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

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