The work contained in this paper has been supported by the Dr. Douglas H. Johnson Award for Glaucoma Research from the American Health Assistance Foundation (XL), the William &amp; Mary Greve Special Scholar Award from the Research to Prevent Blindness (XL), the Illinois Society for the Prevention of Blindness (HC) and NIH grant R01EY019034 (XL).

Procedures
C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) are raised at Northwestern University's Animal Care Facility. All animals are used in accordance with protocols approved by Northwestern University Institutional Animal Care and Use Committee and conformed to the guidelines on the Use of Animals in Neuroscience Research from the NIH.
1. Laser Photocoagulation
The procedure of laser photocoagulation is modified from previously published protocols 5-7.
<ol>
	<li>Anesthetize a 40-60 day old mouse by an intraperitoneal injection of ketamine (100 mg/kg, Butler Schein Animal Health, OH) and xylazine (10 mg/kg, Lloyd Inc. of Iowa, Shenandoah, IA).</li>
	<li>Dilate the pupil of the right eye of the experimental animal by topical treatment with one or two drops of 1% atropine sulfate solution (Alcon Labs, Inc., Fort Worth, TX).</li>
	<li>After mydriasis, flatten the anterior chamber to enhance laser induction 6. Insert a glass micropipette with sharp tip (World Precision Instruments Inc, Sarasota, FL) into the anterior space under the slit lamp (SL-3E, Topcon, Oakland, NJ) to drain out the fluid in the anterior chamber.</li>
	<li>Restrain the mouse in a plastic cone holder (Braintree Sci Inc., MA) and tied up on a homemade platform (See Figure 1A). Hold the mouse with restrainer and exposes the right eye of the mouse to the light source behind the slit lamp. Align the right eye of the anesthetized mouse under the slit lamp.</li>
	<li>While holding the mouse restrainer with both hands, apply the laser illumination to the corneal limbus using an Argon laser (Ultima 2000SE, Coherent, Santa Clara, CA). Deliver about 80-100 laser spots (514 nm, 100 mW, 50 msec pulse, and 200 &mu;m spot) perpendicularly around the circumference of the trabecular meshwork. The C57BL/6 mice have pigmented iris which serves as a barrier for any potential stray energy 7.</li>
	<li>Instill topical 0.5% moxifloxacin (Alcon Labs, Inc., Fort Worth, TX) on the ocular surface to disinfect the laser-treated area and 0.5% Proparacaine (Bausch &amp; Lomb, Rochester, NY) to relieve pain.</li>
	<li>Keep the animal on a heating pad (Sunbeam Products Inc, Boca Raton, FL) for recovery for about an hour until it is fully awake.</li>
	<li>The left eye is untreated to serve as a control.</li>
</ol>
2. IOP Measurements
<ol>
	<li>Place the awake mouse into a tube to load into the plastic cone holder and then restrain it on the platform (See Figure 2A).</li>
	<li>Allow five to ten minutes to let the mouse get adapted to the holder position. Approach the rebound tonometer (TonoLab, Colonial Medical Supply, Franconia, NH) to the mouse eye until the probe tip is 2-3 mm away from the surface of cornea 14.</li>
	<li>Press the measurement button to let the probe tip hit the center surface of cornea gently. Three consecutive sets of six-measurements of IOP of the same eye are acquired and averaged as the IOP of the eye. The untreated control eye is always measured first to get a baseline reading for the laser-treated eye that is measured next.</li>
</ol>
3. Optomotor Test
Visual acuity and contrast sensitivity are tested 14,15 . The two eyes of individual mice are examined separately by reversing the drifting grating direction; i.e. a clockwise drifting grating is used to identify the visual function of the left eye and a counter-clockwise drifting grating for the right eye 16. Each test takes about 15 min and is repeated by two observers independently.
<ol>
	<li>Place the mouse and allow the mouse to move freely on an elevated platform surrounded by four computer monitors (Figure 3A-B).</li>
	<li>Set up the monitors so that they display horizontally drifting sinusoidal gratings as visual stimuli with mean luminance of 39 cd/m2. The moving direction of the grating should alternate consecutively between clockwise and counterclockwise.</li>
	<li>Analyze the animal's movements. The animal's movements in-concert with the drifting gratings are considered "positive" within 15 sec after the visual stimulus is on and then gradually increased. The highest response-eliciting visual stimulus is defined as the animal's visual acuity 17.</li>
	<li>Examine the contrast sensitivity at three pre-selected spatial frequencies: 0.075, 0.16, and 0.3 cycles per degree (cpd). The contrast threshold for each eye is defined as the lowest contrast that elicits visual responses at the pre-fixed frequency. The contrast sensitivity is the reciprocal of the threshold 17.</li>
</ol>

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The authors declare that they have no competing financial interests.
	The authors are full-time employees of Northwestern University.
	The authors received NO funding that was provided by companies which produce reagents and instruments used in this article.

We report above that sustained ocular hypertension can be induced by laser illumination in mouse eyes. Compared to the saline injection model 18 and the vein cautery model 11 both of which require extensive microsurgical skills, the laser illumination is relatively simple and easy to perform. Usually we can perform the laser illumination for 4-6 mice in 2-3 hr. The critical steps to achieve sustained IOP elevation are the anterior chamber flattening before laser and the parameters for laser illumination. Draining out the fluid in the anterior chamber facilitated to target the laser to the trabecular meshwork area and minimize injury to nearby ciliary body and blood vessels 6. Different types of laser have also been reported; for example, some studies used a diode laser with wavelength of 532 nm 5,6 and others used 810 nm energy pulses 7 to target the trabecular meshwork and episcleral veins in the limbal region. In order to maximize the angle damage, we have increased the number of laser spots compared to the previously reported laser models 5-7. With our experimental setup, almost every laser-treated mouse had more than 50% increase of IOP by the first week after laser treatment, among which about 60% had elevated IOP for more than 2 months. By contrast, one intraocular injection of microbeads into mouse eyes can elicit an ~30% elevation in IOP for a couple weeks 8 (another study suggested a longer effect of IOP elevation 9,10), and the occlusion of limbal and episcleral veins in albino CD-1 mice only induced an acute IOP elevation for a few days 13.
The accurate measurement of IOP is important in determining the laser effects on mouse eyes. Anesthesia significantly altered the IOP measurement and behavioral training of mice reduced IOP variation in awake animals 9,19. Here, the experimental animals were given a few minutes to rest and adapt to the restrained position before measurement in order to get consistent readings of IOP. To confirm the IOP measurement is reliable and not dependent on the person who performed the test, the same animals were examined by two or three different testers and their differences of IOP readings are generally within 5-15%.
Because of the variability of the duration and degree of IOP elevation, different RGC loss has been reported in different animal models. For example, a 20% loss of axons has been observed in mouse eyes with microbeads injection 8. Around 20% of RGCs died in rat eyes at six weeks after laser illumination at the trabecular meshwork, while around 60% of RGCs died with laser illumination at both the trabecular meshwork and the episcleral veins 5. Our data showed a 20-30% of RGC loss at 2 months after laser treatment in mouse eyes. Nevertheless, all these different animal models of chronic ocular hypertension without significant inflammation or damage to other parts of the eyes provide us the potential to measure the long-term effects of ocular hypertension on retinal structure and visual function over time.
Taking advantage of the non-invasive nature of the visual behavioral assay that allows serial tests as a function of changing conditions, the changes of visual acuity and contrast sensitivity can be monitored for months after the induction of ocular hypertension. The optomotor test provides a rapid assessment of visual function; moreover, the two eyes can be tested separately, which greatly facilitates our experiments because one eye of the target mouse is laser-treated and the other is left intact as the control. At the same time, it is noted that the optomotor reflex sometimes is difficult to use due to the high activity and wandering attention of some mice 12.
Combined with the power of mouse genetics, our model provides a superb readout with which to investigate the pathological mechanisms in high-tension glaucoma. For example, using Thy-1-YFP transgenic mice, which has a small number of RGCs labeled 10,20-22, the dendritic structural changes of individual RGCs can be imaged in eyes with sustained ocular hypertension. We have demonstrated that the dendritic degeneration of RGCs depends on location and subtypes in ocular hypertensive eyes 23. Cell apoptosis or neuroprotective signaling pathways can be further manipulated in vivo to identify the underlying molecular mechanisms of RGC degeneration and survival in glaucoma.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>moxifloxacin</td><td>Alcon Labs, Inc.</td><td>NDC 0065-4013-03</td><td>0.5 %, Rx only</td></tr><tr><td>Proparacaine Hydrochloride</td><td>Bausch Lomb</td><td>NDC 24208-730-06</td><td>0.5 %, Rx only</td></tr><tr><td>Ophthalmic Solution USP</td><td>Bausch Lomb</td><td>NDC 24208-730-06</td><td>.5 %, Rx only</td></tr><tr><td>ketamine</td><td>Butler Schein Animal Health</td><td>NDC 11695-0550-1</td><td>100 mg / kg </td></tr><tr><td>xylazine</td><td>LLOYD Inc. of Iowa</td><td>NADA 139-236</td><td>10 mg / kg </td></tr><tr><td>atropine sulfate solution</td><td>Alcon Labs, Inc.</td><td>NDC 61314-303-02</td><td>1 %, Rx only</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment &lt;/strong&gt;</td></tr><tr><td>Slit Lamp, TOPCON </td><td>Visual Systems Inc</td><td>SL-3E</td><td>powered by PS-30A</td></tr><tr><td>OptoMotry 1.8.0 virtual</td><td>CerebralMechanics Inc.</td><td></td><td></td></tr><tr><td>opto-kinetic testing system</td><td>CerebralMechanics Inc.</td><td></td><td></td></tr><tr><td>Tonometer, TonoLab, for mice</td><td>Colonial Medical Supply</td><td></td><td></td></tr><tr><td>Heating pad</td><td>Sunbeam Products Inc</td><td>722-810</td><td></td></tr><tr><td>Argon laser </td><td>Coherent Inc</td><td>Ultima 2000SE</td><td></td></tr><tr><td>DECAPICONE Plastic cone holder </td><td>Braintree Sci Inc.</td><td>MDC-200</td><td>for mouse</td></tr></tbody></table>

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.

As described in the Procedures, laser illumination is aimed at the trabecular meshwork in the limbal region to photocoagulate the aqueous outflow, inducing angle closure (Figure 1). Most lasered eyes exhibited no significant physical damage, pigment detachment or infection, consistent with previous findings 6. When a small group of mice (less than 5% of all lasered animals) exhibited physical signs of severe damage such as deflated eye balls, severe cataract, significant pigment detachment, or bleeding, we euthanized them immediately. Around 30% of lasered eyes developed minor corneal scars, and most of them recovered within 1-2 weeks after laser treatment.
We found elevated IOP in nearly every laser-treated eyes from more than one hundred mice. The IOP of experimental animals is monitored using a rebound tonometer (Figure 2). Figure 2B shows one example of the changes of IOP before and after the laser treatment. Before laser treatment, the IOP baselines of the two eyes of the mouse showed no difference: 15.7 mmHg (right) vs. 14.7 mmHg (left, Day 0, Figure 2B). Seven days after laser-treatment, the IOP of the treated (right) eye increased almost 2-fold to 30.7 mmHg, compared to the untreated eye (15.7 mmHg). The IOP of laser-treated eye remained elevated at 26-28 mmHg for about 4 months: at 4 months after treatment, the mean IOP of treated eye was 26 mmHg, significantly higher than that of untreated left eye (16.3 mmHg). Subsequently, the IOP of the treated eye slowly decreased and reached 18.7 mmHg at 6 months (24 weeks) after treatment (untreated eye: 15.3 mmHg). Our data demonstrate that sustained IOP elevation is achieved for more than 4 months.
We next confirmed the vision loss using the optomotor test (Figure 3). The optomotor test measures aspects of spatial vision via reflexive head-tracking movements. Figure 3C shows the decrease of visual acuity of the animal examined in Figure 2B. Before the laser-treatment, both eyes exhibited normal acuity (Left: 0.375 cpd; Right: 0.397 cpd; Figure 3C). At two-months after laser treatment, the acuity of the right eye (elevated IOP) decreased significantly compared with the left control eye (Left: 0.45 cpd; Right: 0.228 cpd; Figure 3C). The acuity of the eye with elevated IOP remained low at 5-6 months after laser treatment (Left: 0.378 cpd; Right: 0. 258 cpd; Figure 3C). Similarly, a lower contrast sensitivity of the right eye with elevated IOP was observed (Figure 3D). At two-months after laser treatment, the contrast sensitivity of the control left eye was 6.13, while the right eye was 1.91 at 0.075 cpd. The contrast sensitivity of the control left eye was 5.53 and 2.67 at 0.16 and 0.3 cpd, while the right eye was 4.28 and 1.45, respectively (Figure 3D).
<img alt="Figure 1" src="/files/ftp_upload/50440/50440fig1.jpg" /> 
 Figure 1. Laser photocoagulation of the aqueous humor outflow in mouse eyes. (A) A photo of the slit lamp for laser treatment. The operator holds the mouse with restrainer and then aligns the right eye of the mouse to the light source of the slit lamp. (B-C) Schematic side-view and front-view of the eye. The operator holds the mouse restrainers with both hands while 80-100 laser spots are applied to the area between the episcleral veins and the dilated pupil.
<img alt="Figure 2" src="/files/ftp_upload/50440/50440fig2.jpg" /> 
 Figure 2. IOP increased after laser treatment. (A) The setup to measure IOP using a rebound tonometer. (B) Changes of IOP of one experimental mouse after laser treatment. Each point is the average of three consecutive sets of six-measurements of IOP.
<img alt="Figure 3" src="/files/ftp_upload/50440/50440fig3.jpg" /> 
 Figure 3. Decreases of visual acuity and contrast sensitivity with IOP elevation. (A) Schematic drawing of the optomotor setup. (B) A mouse on the center platform in the optomotor apparatus with gratings displayed on four surrounding monitors. (C) The acuity and contrast sensitivity of the mouse examined in Figure 2B.

Watch this Scientific Journal Video about A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects at JoVE.com

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

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 ...

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12.9K Views.  Northwestern University. 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.

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

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

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A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

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 ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Neuroscience

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Bioengineering

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 ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

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

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

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

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 ...

Mouse Retina OCT Fibergram Alignment with Confocal Images

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

Author Spotlight: Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

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

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Chapters in this video

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

A Mouse Model for Laser-induced Choroidal Neovascularization

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

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

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

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

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

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy