Name: a reversible silicon oil-induced ocular hypertension model in mice
Uploaded: 2019-11-15T10:00:00
Description: Watch this Scientific Journal Video about A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice at JoVE.com

This work is supported by NIH grants EY024932, EY023295, and EY028106 to YH.

All procedures have been approved by the Institutional Animal Care and Use Committee (IACUC) of Stanford University.
1. Ocular hypertension induction by intracameral injection of SO
<ol>
	<li>Prepare a glass micropipette for intracameral SO injection by pulling a glass capillary with a pipette puller to generate a micropipette. Cut an opening at the tip of the micropipette and further sharpen the tip with a microgrinder-beveling machine to make a 35&#176;&#8722;40&#176; bevel.</li>
	<li>Poli...

<ol>
	<li>Chang, E. E., 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=Glaucoma+2.0:+neuroprotection,+neuroregeneration,+neuroenhancement.">Glaucoma 2.0: neuroprotection, neuroregeneration, neuroenhancement.</a> Ophthalmology. 119 (5), 979-986 (2012).</li><li>Tham, Y. C., 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=Global+prevalence+of+glaucoma+and+projections+of+glaucoma+burden+through+2040:+a+systematic+review+and+meta-analysis.">Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta-analysis.</a> Ophthalmology. 121 (11), 2081-2090 (2014).</li><li>Pang, I. H., Clark, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+glaucoma+retinopathy+and+optic+neuropathy.">Rodent models for glaucoma retinopathy and optic neuropathy.</a> Journal of Glaucoma. 16 (5), 483-505 (2007).</li><li>Morrison, J. C., Johnson, E., Cepurna, W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+models+for+glaucoma+research.">Rat models for glaucoma research.</a> Progress in Brain Research. 173, 285-301 (2008).</li><li>McKinnon, S. J., Schlamp, C. L., Nickells, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+retinal+ganglion+cell+death+and+glaucoma.">Mouse models of retinal ganglion cell death and glaucoma.</a> Experimental Eye Research. 88 (4), 816-824 (2009).</li><li>Chen, S., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rodent+Model+of+Glaucoma+and+Its+Implications.">The Rodent Model of Glaucoma and Its Implications.</a> Asia Pacific Journal of Ophthalmology (Philadelphia). 4 (4), 236-241 (2015).</li><li>Sappington, R. M., Carlson, B. J., Crish, S. D., Calkins, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbead+occlusion+model:+a+paradigm+for+induced+ocular+hypertension+in+rats+and+mice.">The microbead occlusion model: a paradigm for induced ocular hypertension in rats and mice.</a> Investigative Ophthalmology and Visual Science. 51 (1), 207-216 (2010).</li><li>Chen, H., 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=Optic+neuropathy+due+to+microbead-induced+elevated+intraocular+pressure+in+the+mouse.">Optic neuropathy due to microbead-induced elevated intraocular pressure in the mouse.</a> Investigative Ophthalmology and Visual Science. 52 (1), 36-44 (2011).</li><li>Cone, F. E., Gelman, S. E., Son, J. L., Pease, M. E., Quigley, H. 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Here we demonstrate a simple but effective procedure for inducing sustained IOP elevation in the mouse eye by intracameral injection of SO. This procedure can be learned quickly by anyone with experience in microdissection under a microscope. The primary potential risk of failure is the leakage of SO from the corneal incision. However, one of the advantages of using SO is that because the oil droplet is visible and measurable, we can easily identify mice that received droplets too small to induce stable ocular hypertensi...

The progressive loss of retinal ganglion cells (RGCs) and their axons is the hallmark of glaucoma, a common neurodegenerative disease in the retina1. It will affect more than 100 million individuals 40&#8722;80 years old by 20402. IOP remains the only modifiable risk factor in the development and progression of glaucoma. In order to explore the pathogenesis, progression, and potential treatments of glaucoma, a reliable, reproducible, and inducible experimental ocular hypertension/glaucoma model that replicates key features of human patients is imperative.
IOP depends on aqueous humor inflow to the anterior chamber from the ciliary body in the posterior chamber and outflow through the trabecular meshwork (TM) at the angle of the anterior chamber. Upon reaching a steady state, IOP is maintained. When the inflow exceeds or is less than the outflow, IOP rises or falls respectively. By decreasing the aqueous outflow either by occluding the angle of the anterior chamber or by damaging the TM, several glaucoma models have been established3,4,5,6,7,8,9,10. These models are normally associated with irreversible ocular tissue damage, and the high IOP in the anterior chamber also causes unwanted complications such as corneal edema and intraocular inflammation, which make retinal imaging and visual function assays difficult to perform and interpret.
To develop a model that overcomes these shortcomings, we focused on the well-sudocumented secondary glaucoma caused by silicone oil (SO) that occurs as a postoperative complication of human vitreoretinal surgery11,12. SO is used as a tamponade in retinal surgeries because of its high surface tension. However, SO can physically occlude the pupil because it is lighter than the aqueous and vitreous fluids, which prevents aqueous flow into the anterior chamber. The obstruction causes IOP elevation in the posterior chamber due to the aqueous humor accumulation. This motivated us to develop and characterize a novel ocular hypertension mouse model based on intracameral SO injection and pupillary block13, with key features of the secondary glaucoma: effective pupillary block, significant IOP elevation that can return to normal after SO removal, and glaucomatous neurodegeneration.
Here we present a detailed protocol for SO-induced ocular hypertension in the mouse eye, including SO injection and removal and IOP measurement.

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			<td height="21" style="height:21px;width:257px;">0.5% proparacaine hydrochloride</td>
			<td style="width:232px;">Akorn, Somerset</td>
			<td style="width:147px;"></td>
			<td style="width:279px;"></td>
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			<td height="21" style="height:21px;width:257px;">10mL syinge</td>
			<td style="width:232px;">BD</td>
			<td style="width:147px;"></td>
			<td>Luer-Lok Tip</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:26px;">18G needle</td>
			<td style="width:232px;">BD</td>
			<td style="width:147px;"></td>
			<td>with Regular Bevel, Needle Length:25.4 mm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:257px;">2,2,2-Tribromoethanol (Avertin)</td>
			<td style="width:232px;">Fisher Scientific</td>
			<td style="width:147px;">CAS# 75-80-9</td>
			<td>50g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">32G nano</td>
			<td style="width:232px;">BD</td>
			<td style="width:147px;">320122</td>
			<td>BD Nano Ultra Fine Pen Needle-32G 4mm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:257px;">33G ophalmology needle</td>
			<td style="width:232px;">TSK/ VWR</td>
			<td style="width:147px;">TSK3313/ 10147-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">5mL syinge</td>
			<td style="width:232px;">BD</td>
			<td style="width:147px;"></td>
			<td>Luer-Lok Tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">AnaSed Injection (xylazine)</td>
			<td style="width:232px;">Butler Schein</td>
			<td style="width:147px;"></td>
			<td>100 mg/ml, 50 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">artificial tears</td>
			<td style="width:232px;">Alcon Laboratories</td>
			<td style="width:147px;">300651431414</td>
			<td>Systane Ultra Lubricant Eye Drops</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">BSS PLUS Irrigating solution</td>
			<td style="width:232px;">Alcon Laboratories</td>
			<td style="width:147px;">65080050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-Stage Glass Micropipette Puller</td>
			<td style="width:232px;">NARISHIGE</td>
			<td style="width:147px;">PC-10</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:257px;">EZ-7000 Classic System</td>
			<td style="width:232px;">EZ system</td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Isoflurane</td>
			<td style="width:232px;">VetOne</td>
			<td style="width:147px;">502017</td>
			<td>isoflurane, USP, 250ml/bottle</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:257px;">IV Administration sets</td>
			<td style="width:232px;">EXELint/ Fisher</td>
			<td style="width:147px;">29081</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:257px;">KETAMINE HYDROCHLORIDE INJECTION</td>
			<td style="width:232px;">VEDCO</td>
			<td style="width:147px;">50989-996-06</td>
			<td>KETAVED 100mg/ml * 10ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microgrind bevelling machine</td>
			<td style="width:232px;">NARISHIGE</td>
			<td style="width:147px;">EG-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Miniature EVA Tubing</td>
			<td style="width:232px;">McMaster-Carr</td>
			<td style="width:147px;">1883T4</td>
			<td>0.05&#34; ID, 0.09&#34; OD, 10 ft. Length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">silicon oil (SILIKON)</td>
			<td style="width:232px;">Alcon Laboratories</td>
			<td style="width:147px;">8065601185</td>
			<td>1,000 mPa.s</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Standard Glass Capillaries</td>
			<td style="width:232px;">WPI/ Fisher</td>
			<td style="width:147px;">1B150-4</td>
			<td>4 in. (100mm) OD 1.5mm ID 0.84mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TonoLab tonometer</td>
			<td style="width:232px;">Colonial Medical Supply, Finland</td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">veterinary antibiotic ointment</td>
			<td style="width:232px;">Dechra Veterinary</td>
			<td style="width:147px;">1223RX</td>
			<td>BNP ophthalmic ointment, Vetropolycin</td>
		</tr>
	</tbody>
</table>

a reversible silicon oil-induced ocular hypertension model in mice

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.

Soon after the injection we can easily identify mice that do not produce stable ocular hypertension because of the SO droplets being too small (&#8804;1.5 mm)13. These animals are excluded from subsequent experiments. Following the injection procedures, more than 80% of SO injected mice end up with droplets larger than 1.6 mm. We measured the IOP of these mouse eyes once a week for 8 weeks after a single SO injection. The IOP of the eye receiving SO remained high, generally double the IOP of the c...

Watch this Scientific Journal Video about A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice at JoVE.com

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing ...

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Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Creation of a reversible cholestatic rat model can be challenging in view of the smaller size and unique hepatopancreatobiliary anatomy in rats. This video article demonstrates the creation of a reversible cholestatic model.

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Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats ...