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 border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Name of Material/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:324px;">250-300g female Brown Norway ex-breeder rats</td>
			<td style="width:175px;">Harlan UK</td>
			<td style="width:119px;">203</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Tonolab Rebound Tonometer</td>
			<td style="width:175px;">Tiolat</td>
			<td style="width:119px;">TV02</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:324px;">Ketaset (Ketamine)</td>
			<td style="width:175px;">Fort Dodge Animal health</td>
			<td>BN1000118</td>
			<td>37.5 mg/kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Domitor (medetomidine hydrochloride)</td>
			<td style="width:175px;">Orion Pharma</td>
			<td style="width:119px;">140-999</td>
			<td>0.25 mg/kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Povidone iodine</td>
			<td style="width:175px;">Ecolab</td>
			<td>BN4369LE10</td>
			<td>5% in H2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Minim&#39;s Saline Solution</td>
			<td style="width:175px;">Bausch and Lomb</td>
			<td>PL00033/5017</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Toroidal magnet</td>
			<td style="width:175px;">Supermagnete</td>
			<td>R-10-07-03-N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Magnetic Microspheres</td>
			<td style="width:175px;">Bangs Laboratories</td>
			<td>UMC4N/9692</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">HBSS</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">14025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">33-guage bevelled needle</td>
			<td style="width:175px;">Hamilton</td>
			<td>7747-01</td>
			<td>Custom needle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Luer tip syringe</td>
			<td style="width:175px;">Hamilton</td>
			<td>80601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Antisedan (atipemezole hydrochloride )</td>
			<td style="width:175px;">Orion Pharma</td>
			<td style="width:119px;">141-003</td>
			<td>0.25 mg/kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Chloramphenicol ointment</td>
			<td style="width:175px;">Medicom</td>
			<td>18956-0005</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">TUNEL staining kit</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">G3250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Triton X-100</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">DAPI</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Vectashield Mounting Media</td>
			<td style="width:175px;">Vector Labs</td>
			<td style="width:119px;">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

Research

JoVE Journal

Medicine

17.0K visualizações.  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.

Vídeo: Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

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 ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

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 ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

We describe methods to develop an experimental model of diet-induced metabolic syndrome (MetS) in rabbits using a high-fat, high-sucrose diet. Animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia, thus reproducing the main components of human MetS. This chronic model will allow acquisition of knowledge underlying mechanisms of disease progression.

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...

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 Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...