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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160;&#160;&#160;&#160;
1. Ocular Hypertension Induction
<ol>
	<li>Induce experimental glaucoma by elevating the intraocular pressure (IOP) via unilateral injection of paramagnetic microspheres into the anterior chamber of Brown Norway rats. Other pigmented rats may be suitable, although the user would need to validate them first.</li>
	<li>House 250-300 g female ex-breeder Brown Norway rats in a constant low-light environment (40-60 lux) to minimize diurnal fluctuations in IOP, with access to food and water.</li>
	<li>Take baseline IOP measurements in awake animals8 prior to anesthesia and bead injection, using a rebound tonometer calibrated for use in the rat eye9. IOP is taken as the mean of five readings.</li>
	<li>Anaesthetize rats with 37.5 mg/kg ketamine and 0.25 mg/kg medetomidine hydrochloride delivered intraperitoneally. Confirm the depth of anesthesia by testing the animal&#39;s rear foot reflexes prior to povidone-iodine application (see step 1.5) and bead injection (see step 1.8). Administer 0.5% proparacaine hydrochloride for analgesia. 
	NOTE: Do not dilate the pupil at any stage. This will help the beads settle better into the iridocorneal angle and prevent binding to the lens. Apply ocular ointment to prevent corneal drying on the unoperated contralateral eye.</li>
	<li>Wash the operative eye with 5% povidone-iodine in water 5 min prior to injection.</li>
	<li>After 5 min, wick the povidone iodine off using sterile gauze and wash the eye with 0.9% sterile saline solution. Keep the eye moist during anesthesia with regular application of sterile saline.</li>
	<li>Place a toroidal magnet around the eye.</li>
	<li>Inject 25 &#956;l of a solution containing 30 mg/ml of gamma-irradiation sterilized 8 &#956;m magnetic microspheres in Hank&#39;s Balanced Salt Solution (HBSS) into the anterior chamber using a 33 G beveled needle.
	<ol>
		<li>To prepare beads, wash by re-suspending, then centrifuging 3 times at 10,000 x g for 5 min with 1 ml HBSS before making the final 30 mg/ml solution. Maintain sterile conditions throughout.</li>
		<li>For injection, be careful to avoid inserting the needle to the iris, to minimize the risk of iris trauma. This can be prevented by orientating the needle tangential to the corneal surface, as parallel to the iris as possible. This will also help to minimize bead loss from the injection site. 
		NOTE: Inject beads at a rapid rate to ensure an even distribution around the iridocorneal angle, which is critical to raising IOP. Additionally, store beads, needles, and magnetic rings separately so that the beads do not form clusters, making them difficult to load into the syringe and inject, and the needle does not become magnetized.</li>
	</ol>
	</li>
	<li>Leave the needle in place for 1 min post-injection to ensure that beads settle into the iridocorneal angle to impede aqueous drainage from the trabecular meshwork. Slightly angle the needle after the beads have initially settled to allow some leakage of aqueous, to minimize transient increases in IOP. At the end of the surgery, flush the needle through with first phosphate buffered saline (PBS), then 70% ethanol, followed by distilled water, to ensure continued use of the needle in separate procedures. Alternatively, one could use disposable needles if the correct gauge and syringe combination is available. Optional: needles may be sharpened using a beveller to prolong their use.</li>
	<li>At this stage if necessary, remove the magnet, and use it to draw beads into areas of incomplete coverage.</li>
	<li>Leave the magnet in place around the eye for a further 10 min post-injection to ensure beads settle well into the iridocorneal angle.</li>
	<li>Reverse anesthesia using 0.25 mg/kg atipemezole hydrochloride.</li>
	<li>Administer chloramphenicol or other antibiotic ointment, for example, gentamycin or terramycin topically to prevent infection, and 0.5% proparacaine hydrochloride for analgesia. Leave animals to recover on a heat mat until they regain movement, then transfer them to a warm box and supply them with additional nutrition, such as a food supplement or moistened regular diet, until recovery is complete. Systemic or local analgesia should be given to animals displaying signs of pain 24 hr after surgery. If these symptoms persist despite treatment, animals should be humanely culled.</li>
	<li>Use the contralateral eye as an unoperated control.</li>
	<li>Take IOP measurements every 2-3 days following bead administration and every 2-3 days thereafter using a rebound tonometer calibrated for use in the rat eye.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>250-300g female Brown Norway ex-breeder rats</td>
			<td>Harlan UK</td>
			<td>203</td>
			<td></td>
		</tr>
		<tr>
			<td>Tonolab Rebound Tonometer</td>
			<td>Tiolat</td>
			<td>TV02</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketaset (Ketamine)</td>
			<td>Fort Dodge Animal health</td>
			<td>BN1000118</td>
			<td>37.5 mg/kg</td>
		</tr>
		<tr>
			<td>Domitor (medetomidine hydrochloride)</td>
			<td>Orion Pharma</td>
			<td>140-999</td>
			<td>0.25 mg/kg</td>
		</tr>
		<tr>
			<td>Povidone iodine</td>
			<td>Ecolab</td>
			<td>BN4369LE10</td>
			<td>5% in H2O</td>
		</tr>
		<tr>
			<td>Minim&#39;s Saline Solution</td>
			<td>Bausch and Lomb</td>
			<td>PL00033/5017</td>
			<td></td>
		</tr>
		<tr>
			<td>Toroidal magnet</td>
			<td>Supermagnete</td>
			<td>R-10-07-03-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Microspheres</td>
			<td>Bangs Laboratories</td>
			<td>UMC4N/9692</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14025</td>
			<td></td>
		</tr>
		<tr>
			<td>33-guage bevelled needle</td>
			<td>Hamilton</td>
			<td>7747-01</td>
			<td>Custom needle</td>
		</tr>
		<tr>
			<td>Luer tip syringe</td>
			<td>Hamilton</td>
			<td>80601</td>
			<td></td>
		</tr>
		<tr>
			<td>Antisedan (atipemezole hydrochloride )</td>
			<td>Orion Pharma</td>
			<td>141-003</td>
			<td>0.25 mg/kg</td>
		</tr>
		<tr>
			<td>Chloramphenicol ointment</td>
			<td>Medicom</td>
			<td>18956-0005</td>
			<td></td>
		</tr>
	</tbody>
</table>

experimental glaucoma induction via iridocorneal angle obstruction in a mouse model

Source: Bunker, S., et al. <a href="https://app.jove.com/v/52400">Experimental glaucoma induced by ocular injection of magnetic microspheres.</a> J. Vis. Exp. (2015)
In this video, an anesthetized rat undergoes an ocular procedure where magnetic beads are injected into the anterior chamber under a magnetic field to block the iridocorneal angle. This blockage obstructs the trabecular meshwork, preventing aqueous humor drainage and leading to increased intraocular pressure, damaging the retinal ganglion cells and optic nerve. This develops a glaucoma-like condition.

Experimental Glaucoma Induction via Iridocorneal Angle Obstruction In a Mouse Model

Source: Bunker, S., et al. Experimental glaucoma induced by ocular injection of magnetic microspheres. J. Vis. Exp. (2015)
In this video, an anesthetized ...

Start with an anesthetized rat with a sterilized eye, and place a magnetic ring covering the eyeball.
Pierce the eye tangentially with a syringe containing magnetic beads.
Inject the beads into the anterior chamber of the eye, holding the syringe in position.
The magnet pulls the beads to the iridocorneal angle and begins to block the outflow of aqueous humor fluid from the eye through the trabecular meshwork.
Angle the needle slightly when removing the syringe to avoid a sudden increase in eye pressure.
Remove the magnet and reposition it to direct beads into areas with incomplete coverage.
Then, place the magnet back around the eyeball.
As the beads settle, they completely block aqueous humor outflow, causing fluid buildup in the anterior chamber and increased eye pressure.
The elevated pressure induces mechanical stress on the retina, promoting optic nerve degeneration and developing a glaucoma-like condition.

experimental-glaucoma-induction-via-iridocorneal-angle-obstruction

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

42 Views. Source: Bunker, S., et al. Experimental glaucoma induced by ocular injection of magnetic microspheres. J. Vis. Exp. (2015) In this video, an anesthetized rat undergoes an ocular procedure where magnetic beads are injected into the anterior chamber under a magnetic field to block the iridocorneal angle. This blockage obstructs the trabecular meshwork, preventing aqueous humor drainage and leading to increased intraocular pressure, damaging the retinal ganglion cells and optic nerve. This develop...

Video: Experimental Glaucoma Induction via Iridocorneal Angle Obstruction In a Mouse Model - Concept

Ultrasound Cyclo Plasty in Eyes with Glaucoma

Glaucoma is a chronic disease caused by the progressive degeneration of the optical nerve fibers, resulting in decreased visual field that can lead to severe visual impairment, and eventually blindness. This manuscript describes a simple, surgeon-friendly, non-incisional technique, named Ultrasound Cyclo Plasty (UCP), for reducing intraocular pressure (IOP) in glaucoma patients. The technique determines a selective coagulation necrosis of the ciliary body; in addition, the stimulation of supra-choroidal and trans-scleral portions of the uveo-scleral outflow pathway has been recently proposed. UCP shows several technical improvements in ultrasound technology compared to previous techniques, providing more precise focusing on the target zone. The procedure is performed in the operating room under peribulbar anesthesia. Briefly, the coupling cone is put in contact with the eye and the ring probe, that contains six piezoelectric transducers which produce the ultrasound beams, is inserted inside it. Their proper centering over the ocular surface represents a crucial step for the correct targeting of the ciliary body. Sterile balanced salt solution is used to fill the empty spaces to ensure ultrasound acoustic propagation. Surgical treatment consists in the sequential automatic activation of each of the six transducers, for a total duration of less than 3 min. The patient leaves the hospital 1 h after the procedure with the treated eye patched. In the present study, 10 patients with open-angle glaucoma were followed-up during at least 12 months after the procedure. IOP was reduced at each interval compared to pre-operative, as well as the number of hypotensive medications. Twenty percent of patients did not respond to the treatment, and needed subsequent surgery to better control IOP. Treatment tolerability was good, with no cases of hypotony or phthisis. The UCP procedure is simpler, faster, safer, and less invasive than traditional cyclodestructive procedures with similar results in reducing IOP.

Glaucoma is a chronic disease with progressive degeneration of optic nerve fibers resulting in decreased visual field. Elevated intraocular pressure is considered the most important and the only treatable risk factor. This manuscript describes a simple, surgeon-friendly, non-incisional technique, named Ultrasound Cyclo Plasty, for reducing intraocular pressure in glaucoma patients.

Glaucoma is a chronic disease caused by the progressive degeneration of the optical nerve fibers, resulting in decreased visual field that can lead to ...

JoVE Journal

Behavior

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Medicine

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

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

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

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

Experimental Glaucoma Induction via Iridocorneal Angle Obstruction In a Mouse Model

Transkrypcja

Inducing Experimental Glaucoma Induction via Iridocorneal Angle Obstruction with Magnetic Beads

Ultrasound Cyclo Plasty in Eyes with Glaucoma

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

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