We wish to thank Peter Munro PhD for his assistance with optic nerve sectioning. This study was supported by the Medical Research Council (G0901303), and in part by the Dorothy Hodgkin Postgraduate Award/Medical Research Council, the Helen Hamlyn Trust, Fight for Sight, and Moorfields special trustess,.

Ethics statement: All animal experiments have been conducted in accordance with the Institutional Animal Care and Use Committee (IACUC), and were approved in agreement with United Kingdom Home Office guidelines (<a href="http://goo.gl/FLkirW" target="_blank">http://goo.gl/FLkirW</a>, last accessed 10th June, 2014) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research (<a href="http://goo.gl/4LFOjD" target="_blank">http://goo.gl/4LFOjD</a>, last accessed 10th June, 2014).
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, based on the method of Samsel et al.3&#160;Other pigmented rats may be suitable, although these would need to be validated first by the user.</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 IOP7, with access to food and water ad libitum.</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 depth of anesthesia by testing animal&#8217;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 to settle better into the iridocorneal angle, and prevent binding to the lens. Apply ocular ointment to prevent corneal drying on un-operated contralateral eye.</li>
	<li>Wash the operative eye with 5% povidone iodine in&#160;water10 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&#8217;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.&#160;Alternatively, one could use disposable needles if the correct gauge and syringe combination is available. Optional:&#160;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 to a warm box and supply 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 eye9.</li>
	<li>The criteria for including eyes in studies can be: if 1) the IOP is elevated above the contralateral control pressure by 5 mmHg, and 2) does not exceed 60 mmHg.</li>
	<li>Eyes where the pressure returns to baseline (usually by 1 week post-injection) should not be included in the studies, however it is possible to re-inject beads in eyes that fail to develop high IOP if desired.</li>
	<li>Euthanize animals by CO2 asphyxiation at the end of the experiment.</li>
	<li>Dissect eyes and optic nerves, and fix in 4% paraformaldehyde (PFA) overnight for further histological analysis.</li>
</ol>
2. Assessing Retinal Neuron Damage Using TUNEL Staining
<ol>
	<li>To quantify apoptotic cells in whole mount retinasuse the terminal deoxynucleotidyl transferase-mediated dUTP nick-labelling (TUNEL) assay, according to the manufacturer&#8217;s instructions.</li>
	<li>Dissect the retina from the eye cup, wash for 3 x 5 min in 0.3% Triton X-100 in phosphate buffered saline (T-PBS).</li>
	<li>Permeabilize the tissue in 3% T-PBS for 2 hr.</li>
	<li>Pre-equilibrate retinas in Equilibration Buffer for 10 min, before incubation in TUNEL reaction solution for 1 hr at 37 &#176;C.</li>
	<li>Wash the tissue for 3 x 5 min in 0.3% T-PBS, rinse in 0.3% T-PBS containing 5 &#181;M DAPI, and flat-mount in mounting media.</li>
	<li>To quantify TUNEL positive nuclei use a confocal microscope to take 10 &#181;m z-stacks through the ganglion cell layer at 20X magnification. Take 3 images on each of the 4 petals, at sites close to the optic nerve, in the mid-periphery, and at the far periphery of the retina, giving a total of 12 images per whole mount, sampling approximately 7,000 cells. Morphological criteria discriminated non-neuronal (endothelial and glial) cells from neuronal cells.</li>
	<li>Select areas for imaging using only the DAPI channel, and mask investigators to treatment groups.</li>
</ol>
3. Assessing Optic Nerve Damage Using Toluidine Blue Staining
<ol>
	<li>Fix optic nerves overnight in Karnovsky&#8217;s solution at 4 &#176;C.</li>
	<li>Treat specimens for 2 hr in 1% (w/v) osmium tetroxide and then dehydrate in 100% ethanol.</li>
	<li>Incubate optic nerves in propylene oxide for 30 min, and place in a 50:50 mixture of propylene oxide:araldite overnight.</li>
	<li>Change this solution to 100% araldite, followed by incubation overnight at 60 &#176;C.</li>
	<li>Cut semithin sections (0.75 mm thick) and stain with 1% toluidine blue/ borax (TB) in 50% ethanol before examination by light microscopy.</li>
</ol>
4. Statistical Analysis
<ol>
	<li>Carry out statistical analyses using an appropriate statistics software package. A two-way ANOVA with Newman-Keul&#8217;s post-hoc test may be used to calculate statistical significance for IOP changes over time.</li>
	<li>A p value of less than 0.05 may be considered significant.</li>
</ol>

<ol>
	<li>Quigley, H. A., Broman, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+number+of+people+with+glaucoma+worldwide+in+2010+and+2020.">The number of people with glaucoma worldwide in 2010 and 2020.</a> Br J Ophthalmol. 90 (3), 262-267 (2006).</li><li>Weinreb, R. N., Aung, T., Medeiros, F. 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+pathophysiology+and+treatment+of+glaucoma:+a+review.">The pathophysiology and treatment of glaucoma: a review.</a> JAMA. 311 (18), 1901-1911 (2014).</li><li>Samsel, P. A., Kisiswa, L., Erichsen, J. T., Cross, S. D., Morgan, J. 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+novel+method+for+the+induction+of+experimental+glaucoma+using+magnetic+microspheres.">A novel method for the induction of experimental glaucoma using magnetic microspheres.</a> Invest Ophthalmol Vis Sci. 52 (3), 1671-1675 (2011).</li><li>Libby, R. T., 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=Inherited+glaucoma+in+DBA/2J+mice:+pertinent+disease+features+for+studying+the+neurodegeneration.">Inherited glaucoma in DBA/2J mice: pertinent disease features for studying the neurodegeneration.</a> Vis Neurosci. 22 (5), 637-648 (1017).</li><li>Morrison, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+intraocular+pressure+and+optic+nerve+injury+models+in+the+rat.">Elevated intraocular pressure and optic nerve injury models in the rat.</a> J Glaucoma. 14 (4), 315-317 (2005).</li><li>Levkovitch-Verbin, 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=Translimbal+laser+photocoagulation+to+the+trabecular+meshwork+as+a+model+of+glaucoma+in+rats.">Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats.</a> Invest Ophthalmol Vis Sci. 43 (2), 402-410 (2002).</li><li>Moore, C. G., Johnson, E. C., Morrison, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+of+intraocular+pressure+in+the+rat.">Circadian rhythm of intraocular pressure in the rat.</a> Curr Eye Res. 15 (2), 185-191 (1996).</li><li>Morrison, J. C., Jia, L., Cepurna, W., Guo, Y., Johnson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+and+sensitivity+of+the+TonoLab+rebound+tonometer+in+awake+Brown+Norway+rats.">Reliability and sensitivity of the TonoLab rebound tonometer in awake Brown Norway rats.</a> Invest Ophthalmol Vis Sci. 50 (6), 2802-2808 (2009).</li><li>Wang, W. H., Millar, J. C., Pang, I. H., Wax, M. B., 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=Noninvasive+measurement+of+rodent+intraocular+pressure+with+a+rebound+tonometer.">Noninvasive measurement of rodent intraocular pressure with a rebound tonometer.</a> Invest Ophthalmol Vis Sci. 46 (12), 4617-4621 (2005).</li><li>Isenberg, S. 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+ocular+application+of+povidone-iodine.">The ocular application of povidone-iodine.</a> Community Eye Health. 16 (46), 30-31 (2003).</li><li>Morrison, J. 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=A+rat+model+of+chronic+pressure-induced+optic+nerve+damage.">A rat model of chronic pressure-induced optic nerve damage.</a> Exp Eye Res. 64 (1), 85-96 (1997).</li><li>Foxton, R. 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=VEGF-A+is+necessary+and+sufficient+for+retinal+neuroprotection+in+models+of+experimental+glaucoma.">VEGF-A is necessary and sufficient for retinal neuroprotection in models of experimental glaucoma.</a> Am J Pathol. 182 (4), 1379-1390 (2013).</li></ol>

David T. Shima is an employee of Hofmann-La Roche, Switzerland. The remaining authors declare they have no competing financial interests.

Here we demonstrate a method for inducing elevated IOP in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. This method is simple to carry out, and requires little surgical expertise, or hours of practice and refinement. Furthermore, the procedure is effective; rarely requiring more than a single injection of beads to induce a strong, robust rise in pressure (approximately 10% reinjection rate). This may provide an advantage over existing inducible methods, such as the technically challenging episceral vein sclerosis11 model, or laser photocoagulation protocol6, which can require multiple procedures to maintain raised IOP.
In order for the method to be successful however, there are some small critical steps that need to be taken. Firstly it is useful to use a toroidal-shaped magnet to draw beads into the iridocorneal angle. This step is a modification of the original protocol, where the beads were injected into the anterior chamber, and then moved freehand around the eye3. Using a toroidal magnet means that microspheres should settle evenly around the angle, requiring minimal manual redistribution. Secondly, the rate of injection should be fast &#8211; too slow and the beads will predominantly accumulate on one side of the angle, leading to incomplete coverage, and potentially no pressure rise. Generally speaking though, the method is straightforward enough that the user could easily make modifications to the protocol, such as varying the size or volume of the microsphere particles, perhaps to attempt to alter the degree of the IOP elevation.
However, one potential drawback of the method is that one has little control over the extent of the hypertension, which in about 5-10% of cases we observed rose above 60 mmHg. Excessive rises in IOP can be very destructive to retinal tissue, and may make studying the mechanisms and biology of cell death challenging. However, the method produces a consistent neuronal pathology, both in the retina and optic nerve, which can be manipulated pharmacologically12. This may make the model attractive for developing novel therapeutics for treating glaucoma. Additionally, because the beads are directed into the iridocorneal angle, this leaves the visual axis free for live imaging of the retina or optic disc. We anticipate that this model will be adapted and used for future applications in other species, including mouse.

Primary glaucoma is a devastating eye disease affecting an estimated 60.5 million people throughout the world1, which can lead to life-altering vision loss and blindness2. Research into the disease mechanisms, and development of novel therapeutics for glaucoma, are dependent on good models of the disease which recapitulate some of the hallmarks of the pathology.
We present here a rat glaucoma model based on the method of Samsel et al.3 The overall goal of this technique is to increase intraocular pressure (IOP) in the eye by injecting magnetic microspheres into the anterior chamber, and using a magnetic ring, direct them into the iridocorneal angle. This impedes aqueous outflow, which increases IOP, leading to neuronal damage and cell loss. The protocol was developed to attempt to provide a simpler, inducible model of glaucoma.
This protocol may have some advantages over existing techniques. Genetic mouse models such as the DBA/2J are available, which do not require procedures to initiate; however these may have an unpredictable onset of disease progression4. In contrast, inducible models, most of which rely on surgically elevating IOP in rodents, have the advantage that initiation can be controlled by the user. Some of these methods may have drawbacks of their own however, including being technically challenging5, and can require multiple procedures to maintain elevated IOP6.
In contrast, the inducible method detailed in this manuscript is a simple, effective, and reproducible technique that produces stable, robust increases in pressure, with minimal need for reinjection. Additionally, it does not involve expensive equipment, and only requires basic surgical skills to perform. This protocol may be appropriate for readers who are looking to set up a less technically demanding inducible glaucoma model in their laboratory.

<table><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 H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Minim&#039;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 G beveled 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><tr><td>TUNEL staining kit</td><td>Promega</td><td>G3250</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Vectashield mounting media</td><td>Vector Labs</td><td>H-1000</td><td></td></tr></tbody></table>

experimental glaucoma induced by ocular injection of magnetic microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

Injection of magnetic beads into the iridocorneal angle consistently induced a prolonged and robust rise in pressure (Figure 1), which was readily observable at the first time point, 3 days post-injection. Furthermore, the increase in pressure was maintained throughout the duration of the experiment, and although our time course finished at 18 days post-injection, others have reported that pressure persists long-term3. The mean IOP averaged over the full length of the experiment for control, non-bead-injected eyes was 19.7 &#177; 0.3 mmHg, compared with 40.5 &#177; 2.8 mmHg for bead-injected eyes (P &#60;0.001). Additionally, peak IOP increased from 22.8 &#177; 0.3 mmHg to 49.9 &#177; 2.3 mmHg.
To determine whether the elevation in IOP leads to death of retinal ganglion cells, we performed TUNEL staining on retinas, and histology on transverse optic nerve sections (Figure 2). In the retina we observed an increase in TUNEL staining (Figure 2A) in bead-injected eyes with elevated IOP. The number of apoptotic nuclei rose approximately 15-fold, from 1.6 &#177; 0.5 cells in contralateral controls, to 24.5 &#177; 0.5 cells in hypertensive retinas (Figure 2B; P &#60;0.05). Furthermore, in eyes in which magnetic beads were injected but pressure did not increase (likely due to incomplete blockage of the iridocorneal angle), the numbers of TUNEL-positive cells were not significantly different from those of uninjected controls (P &#62;0.05). This suggests that cell death was related to pressure increases, not due to direct toxicity of the magnetic microspheres. Finally, we investigated optic nerve pathology in the glaucoma model, and saw accumulation of toluidine blue in many of the axons, indicating degeneration of these cellular processes (Figure 2C).
<img alt="Figure 1" src="/files/ftp_upload/52400/52400fig1highres.jpg" /> 
Figure 1. Elevation of intraocular pressure using magnetic microspheres. Injection of magnetic microspheres into the anterior chamber induced a robust, significant rise in intraocular pressure (IOP) in comparison to contralateral control, non-injected eyes. Y-axis units = millimeters of mercury (mmHg). Data = mean &#177; SEM. * = P &#60;0.001; N = 12. This figure has been modified from Foxton et al., Am. J. Pathol. 182(4): 1379-1390.
<img alt="Figure 2" src="/files/ftp_upload/52400/52400fig2highres.jpg" /> 
Figure 2. Elevation of IOP by injection of magnetic microspheres into the iridocorneal angle, induced neuronal death in the ganglion cell layer (GCL). (A) Representative images of retinas from control (left) and glaucoma (right) eyes stained for apoptotic nuclei by TUNEL (green; white arrows) and DAPI (blue), indicating the number of apoptotic nuclei increased as IOP rose. (B) Quantification of TUNEL positive cells in the GCL, showing that eyes with elevated IOP (middle), had significantly more apoptotic cells in comparison to control (left). In contrast, in bead-injected eyes where pressure did not rise (right), no significant increase in TUNEL staining was observed. Data = mean &#177; SEM. * = P &#60;0.05; N = 7 - 8. (C) Representative images of optic nerve staining, demonstrating an increase in toluidine blue accumulation (black arrows) in damaged axons from glaucoma (right), but not control eyes (left). Scale bars = 50 &#956;m. This figure has been modified from Foxton et al., Am. J. Pathol. 182(4): 1379-1390.

Watch this Scientific Journal Video about Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres at JoVE.com

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

experimental-glaucoma-induced-ocular-injection-magnetic

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17.1K Views.  University College London Institute of Ophthalmology. We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Video: Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

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

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

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

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

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Neuroscience

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 ...

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

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

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

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

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

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

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.

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 ...

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

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

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

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

A Hypertonic Saline Injection Technique for Inducing Glaucoma in Rats

Source: Gossman, C. A. et al. <a href="https://app.jove.com/v/53831">Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation.</a> J. Vis. Exp. (2016)
This video demonstrates glaucoma induction in an anesthetized rat by injecting hypertonic saline into the episcleral vein. The saline enters the trabecular meshwork via Schlemm&#39;s canal, causing tissue scarring, which blocks aqueous humor outflow and raises intraocular pressure. The elevated pressure damages retinal ganglion cells, leading to glaucoma.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Neuropathology

Epilepsy and Seizure Disorders

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.

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

Neurodegenerative Diseases

Inducing Experimental Glaucoma Induction via Iridocorneal Angle Obstruction with Magnetic Beads

After anesthetizing the rats, apply ocular ointment to the unoperated contralateral eye to keep it moist during anesthesia. Apply 0.5% pro procaine hydrochloride local anesthetic to the operative eye. Next, wash this eye with 5% povidone iodine in water. After 5 minutes, wick off the povidone iodine and wash the eye with 0.9% sterile saline solution.
Keep the eye moist during anesthesia with regular application of sterile saline. Now, place a toroidal magnet around the eye. To minimize the risk of iris trauma and minimize bead loss, orient the needle tangential to the corneal surface as parallel to the iris as possible. Proceed to inject 25 microliters of 30 milligrams per millimeter gamma irradiated magnetic microspheres into the anterior chamber.
Leave the needle in place for one minute post-injection, to ensure that beads settle into the iridocorneal angle to impede aqueous drainage from the trabecular meshwork. Slightly angle the needle a few seconds after the beads have initially settled to allow some leakage of aqueous, and to minimize transient increases in intraocular pressure.
To ensure continued use of the needle in separate procedures, flush the needle with PBS, then 70% ethanol, followed by distilled water. At this stage, if necessary, remove the magnet and use it to draw beads into areas of incomplete coverage. Leave the magnet in place around the eye for a further 10 minutes post-injection, to ensure beads settle well into the iridocorneal angle.

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Summary

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

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

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

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

A Hypertonic Saline Injection Technique for Inducing Glaucoma in Rats

Experimental Glaucoma Induction via Iridocorneal Angle Obstruction In a Mouse Model

Inducing Experimental Glaucoma Induction via Iridocorneal Angle Obstruction with Magnetic Beads