We thank Wendy Chao for her support in critical review. This work was supported by Bausch &amp; Lomb Vitreoretinal Fellowship (HM), National Eye Institute grant EY014104 (JWM), Research to Prevent Blindness Foundation (DGV), Lions Eye Research Fund (DGV), and a generous donation of the Yeatts family (JWM and DGV).

All experiments on animals were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and the guidelines and regulations set forth by the Massachusetts Eye and Ear Infirmary Animal Care Committee.
1. Pretreatment
<ol>
 <li>Anesthetize an 8-week-old mouse with an intraperitoneal injection of a mixture of 60 mg/kg ketamine and 6 mg/kg xylazine. </li>
 <li>Cut the whiskers. This is done because they interfere with visualization and instrument placement.</li>
 <li>Dilate the pupil with 5% phenylephrine and 0.5% tropicamide. </li>
 <li>Cut the cilia. This is also done because they interfere with visualization and instrument placement. </li>
 <li>Apply topical anesthesia (0.5% proparacaine hydrochloride ophthalmic solution). </li>
</ol>
2. Surgery Under the Microscope
<ol>
 <li>Set the mouse in a lateral position with the nose toward the surgeon. A toe pinch is performed to confirm surgical anesthesia, and sterile gloves are donned prior to starting surgery.</li>
 <li>Incise the temporal conjunctiva at the posterior limbus and separate the conjunctiva from the sclera. Avoid penetrating the sclera. After this step, grasp the conjunctiva at the limbus with forceps to control the eye. </li>
 <li>Make a self-sealing scleral incision using the tip of a 30 G needle with the bevel pointed up. Make a scleral tunnel through the sclera and penetrate the sclera into the choroid (Figure 1). Avoid penetrating the retina. If choroidal bleeding occurs and comes out through the scleral wound, perform an astriction with a surgical spear until the bleeding stops. This scleral wound will be self-sealed with intraocular pressure.</li>
 <li>Puncture the cornea with a 30 G needle to reduce intraocular pressure. Use the needle parallel to the iris to avoid injuring the iris and lens during entry into the anterior chamber. The intraocular pressure will cause the wound to self-seal. </li>
 <li>Insert a 33 G needle connected to a Hamilton 10 &mu;l syringe into the subretinal space with the bevel pointed down (towards the inner eye), and inject 3.5 &mu;l sodium hyaluronate gently to detach the neurosensory retina from the underlying RPE. Avoid penetrating the retina with the 33 G needle because sodium hyaluronate will go into the vitreous space but not the subretinal space if the retina is penetrated. Also, avoid rapid injection because it will create an oral tear, and sodium hyaluronate will leak into the anterior chamber.</li>
 <li>Let the aqueous humor &#xFB02;ow out from the corneal puncture by pushing the cornea around the corneal puncture with forceps to adjust the intraocular pressure. </li>
 <li>Confirm absence of leakage from the scleral wound using a surgical spear. </li>
 <li>To reduce the risk of sodium hyaluronate leakage, place cyanoacrylate surgical glue on the scleral wound. </li>
 <li>Reattach the conjunctiva to its original position using cyanoacrylate surgical glue. This will further reduce the risk of sodium hyaluronate leakage. </li>
 <li>Check the fundus using a cover glass and confirm the creation of a bullous RD without subretinal hemorrhage. </li>
</ol>
3. After Treatment
<ol>
 <li>Apply bacitracin antibiotic ointment to the eye to reduce the risk of infection.</li>
 <li>Keep the mice on a heating pad to prevent the anesthesia (mixture of ketamine and xylazine) from causing low body temperature subsequent to low blood pressure. </li>
 <li>Put the mice back to the animal cage after they awaken from anesthesia. Monitor the mice daily for complications. If any complications arise, euthanize the mice as described below. </li>
</ol>
4. Sacrifice
<ol>
 <li>Euthanize the mice by cervical dislocation after intraperitoneal injection of 100 mg/kg sodium pentobarbital, and enucleate the eyes at appropriate time points for each experiment (not shown). Avoid applying pressure on the eye during enucleation because sodium hyaluronate may leak out. Bullous RDs should be persistent for at least 14 days.
 </li>
</ol>

<ol>
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N., 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=Caspase+Activation+in+an+Experimental+Model+of+Retinal+Detachment.">Caspase Activation in an Experimental Model of Retinal Detachment.</a> Invest. Ophthalmol. Vis. Sci. 44, 1262-1267 (2003).</li><li>Zacks, D. N., Han, Y., Zeng, Y., Swaroop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Signaling+Pathways+and+Stress-Response+Genes+in+an+Experimental+Model+of+Retinal+Detachment.">Activation of Signaling Pathways and Stress-Response Genes in an Experimental Model of Retinal Detachment.</a> Invest. Ophthalmol. Vis. 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Eye Res. 24, 161-172 (2002).</li><li>Nakazawa, 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=Monocyte+chemoattractant+protein+1+mediates+retinal+detachment-induced+photoreceptor+apoptosis.">Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.</a> Proc. Natl. Acad. Sci. 104, 2425-2430 (2007).</li><li>Trichonas, G., 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=Receptor+interacting+protein+kinases+mediate+retinal+detachment-induced+photoreceptor+necrosis+and+compensate+for+inhibition+of+apoptosis.">Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis.</a> Proc. Natl. Acad. Sci. 107, 21695-21700 (2010).</li><li>Roh, M. I., Murakami, Y., Thanos, A., Vavvas, D. G., Miller, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Edaravone,+an+ROS+Scavenger,+Ameliorates+Photoreceptor+Cell+Death+after+Experimental+Retinal+Detachment.">Edaravone, an ROS Scavenger, Ameliorates Photoreceptor Cell Death after Experimental Retinal Detachment.</a> Invest. Ophthalmol. Vis. Sci. 52, 3825-3831 (2011).</li><li>Mantopoulos, D., 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=Tauroursodeoxycholic+acid+(TUDCA)+protects+photoreceptors+from+cell+death+after+experimental+retinal+detachment.">Tauroursodeoxycholic acid (TUDCA) protects photoreceptors from cell death after experimental retinal detachment.</a> PLoS One. 6, e24245 (2011).</li><li>Yang, L., Bula, D., Arroyo, J. G., Chen, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+retinal+detachment-associated+photoreceptor+cell+loss+in+Bax-deficient+mice.">Preventing retinal detachment-associated photoreceptor cell loss in Bax-deficient mice.</a> Invest. Ophthalmol. Vis. Sci. 45, 648-654 (2004).</li><li>Cebulla, C. M., Ruggeri, M., Murray, T. G., Feuer, W. J., Hernandez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+domain+optical+coherence+tomography+in+a+murine+retinal+detachment+model.">Spectral domain optical coherence tomography in a murine retinal detachment model.</a> Exp. Eye Res. 90, 521-527 (2010).</li><li>Secondi, R., Kong, J., Blonska, A. M., Staurenghi, G., Sparrow, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundus+Autofluorescence+Findings+in+a+Mouse+Model+of+Retinal+Detachment.">Fundus Autofluorescence Findings in a Mouse Model of Retinal Detachment.</a> Invest. Ophthalmol. Vis. Sci. 53, 5190-5197 (2012).</li><li>Machemer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+retinal+detachment+in+the+owl+monkey.+IV.+The+reattached+retina.">Experimental retinal detachment in the owl monkey. IV. The reattached retina.</a> Am. J. Ophthalmol. 66, 1075-1091 (1968).</li><li>Ross, W., Lavina, A., Russell, M., Maberley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+correlation+between+height+of+macular+detachment+and+visual+outcome+in+macula-off+retinal+detachments+of+&lt;+or+=+7+days'+duration.">The correlation between height of macular detachment and visual outcome in macula-off retinal detachments of &lt; or = 7 days' duration.</a> Ophthalmology. 112, 1213-1217 (2005).</li><li>Glatt, H., Machemer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+subretinal+hemorrhage+in+rabbits.">Experimental subretinal hemorrhage in rabbits.</a> Am. J. Ophthalmol. 94, 762-773 (1982).</li><li>Toth, C. A., Morse, L. S., Hjelmeland, L. M., Landers, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+directs+early+retinal+damage+after+experimental+subretinal+hemorrhage.">Fibrin directs early retinal damage after experimental subretinal hemorrhage.</a> Arch. Ophthalmol. 109, 723-729 (1991).</li><li>Benner, J. D., Hay, A., Landers, M. B., Hjelmeland, L. M., Morse, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinolytic-assisted+removal+of+experimental+subretinal+hemorrhage+within+7+days+reduces+outer+retinal+degeneration.">Fibrinolytic-assisted removal of experimental subretinal hemorrhage within 7 days reduces outer retinal degeneration.</a> Ophthalmology. , 101-672 (1994).</li><li>Bhisitkul, R. 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The Massachusetts Eye and Ear Infirmary has an ownership interest in three U.S. patents directed to the use of verteporfin. In addition, the Massachusetts Eye and Ear Infirmary has ownership interest in certain patent applications directed to the selective destruction of subretinal choroidal neovasculature for the treatment of macular degeneration and other disorders. The Massachusetts Eye and Ear Infirmary receives royalties as a result of these patents and patent applications and Dr. Miller receives a share of the same in accordance with the Massachusetts Eye and Ear Infirmary's institutional Patent Policy and Procedures, which includes royalty-sharing provisions. The author, Dr. Joan W. Miller, is a past board member and former shareholder of Alcon, Inc., and currently serves as a consultant for Alcon; Imagen Biotech, Inc.; ISIS Pharmaceuticals; Kalvista Pharmaceuticals; Regeneron Pharmaceuticals, Inc and ONL Therapeutics, LLC. Dr. Demetrios G. Vavvas serves as consultant for Roche, Genentech, Kala Pharmaceuticals.

Several methods for establishing an RD model in rodent eyes have been reported3-15, 22. Most of them utilize subretinal injection of sodium hyaluronate because it is a viscous material commonly used during intraocular surgery in humans, and it is not associated with any known ocular toxicity1-15. Sodium hyaluronate, rather than normal saline or phosphate-buffered saline (PBS), increases the duration of the RD.
The methods for subretinal injection of sodium hyaluronate use one of two approaches: a transvitreal approach3-6 or a transscleral approach7-15. Both methods are performed with observation of the fundus. In the transvitreal approach, a subretinal injector is introduced into the vitreous cavity, a retinotomy is created in the peripheral retina, and the sodium hyaluronate is injected into the subretinal space. In this method, two retinal tears are made, which increases the risk of retinal hemorrhage that might go into the subretinal space. In addition, there is a risk of lens injury when the retinotomy is created. There are several modified methods for the transscleral approach. In most of these methods7-12, after reducing intraocular pressure with an anterior chamber puncture, a 30 G needle connected to a syringe filled with sodium hyaluronate is directly inserted into the subretinal space through the conjunctiva, sclera, choroid, and RPE. The sodium hyaluronate is then injected into the subretinal space. The risk of retinal tear and lens injury using this transscleral method is less than that using the transvitreal approach. However, the hole made in the sclera by a 30 G needle is large, especially for mouse eyes, and the sodium hyaluronate injected into the subretinal space easily leaks out of the eye through the scleral wound. This leads to a lower, less persistent RD and more variable photoreceptor cell death. Moreover, if choroidal bleeding occurs in the scleral perforation step, the hemorrhage will spread into the subretinal space because the intraocular pressure has been reduced prior to the subretinal hyaluronate injection.
Several factors can influence the effect of RD on the detached retina, including both subretinal hemorrhage and the height and persistence of RD16-21. Photoreceptor cell death increases with increasing height of RD16, 17, and photoreceptor cell damage may be more extensive because of reduced diffusion of oxygen and essential nutrients from the choriocapillaris with higher RD compared to shallow RD. Subretinal hemorrhage is also toxic for photoreceptor cells18-21; possible mechanisms of this toxicity in the detached retina include hypoxia and metabolic disruption by subretinal hemorrhage as a diffusion barrier, and direct neurotoxicity induced by blood components (such as iron). Lens injury, which has been reported to have a protective effect on retinal ganglion cells23, may also affect photoreceptor cell death after induction of RD. In addition, if the entry wound site is not sealed, sodium hyaluronate may leak out with eye manipulation during enucleation. This may lead to erroneous classification of an RD as shallow, which may in turn affect the interpretation of the results.
We modified the transscleral method for subretinal injection of sodium hyaluronate to increase the reproducibility of RDs and reduce the rate of subretinal hemorrhage. The critical step of this protocol is creating a self-sealing scleral incision using a 30 G needle, which prevents leakage of sodium hyaluronate after injection. Unlike previous methods, this protocol is performed without observation of the fundus so greater attention is paid to the scleral wound. Application of glue also prevents the sodium hyaluronate from leaking out of the eye. In our experience, the rate of subretinal hemorrhage with this protocol was significantly less than that with other protocols. If choroidal bleeding occurs during the scleral incision step, it will exit the eye through the scleral wound because this step is performed before reducing intraocular pressure. If choroidal bleeding does occur after reduction of the intraocular pressure and detachment of the neurosensory retina, blood will dissect into the subretinal space. We found this to happen in about 5% of the cases in contrast to about 10-20% with the other techniques. These animals should be excluded from analysis.
This technique can also be used for subretinal injection of vector-mediated gene transfer to target photoreceptor or RPE cells24, 25. Because the typical vehicle (PBS, saline) for these injections is significantly less viscous than sodium hyaluronate, standard techniques are plagued by more leaks. The technique described herein, by reducing this risk, makes vector transfer experiments more reproducible and reliable.

A correction was made to <a href="http://www.jove.com/video/50660/">Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate</a>. The footnotes were removed from the abstract due to redundancy in the introduction.

Erratum: Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Photoreceptor cell death and subsequent visual decline occurs when the photoreceptors are separated from the underlying retinal pigment epithelium. Physical separation of photoreceptors is seen in various retinal disorders, including age-related macular degeneration (AMD), central serous chorioretinopathy, diabetic retinopathy, and retinopathy of prematurity, as well as rhegmatogenous (i.e. caused by a break in the retina) retinal detachment (RD). Subretinal injection of sodium hyaluronate is a widely accepted model to create an RD that leads to photoreceptor cell death, providing insight into the pathophysiology of photoreceptor degeneration1-15.
Photoreceptor degeneration induced by subretinal sodium hyaluronate injection, first introduced in 20017, has the advantage of a reasonable time course (days to weeks). However, it can have significant variability of photoreceptor cell loss from animal to animal due to two major factors that affect photoreceptor cell death after RD: 1) height and duration of the RD, and 2) the occurrence of subretinal hemorrhage. There is a steep technical learning curve to the method that contributes to both factors. Photoreceptor cell degeneration increases with the height of the RD, as the distance between the retinal pigment epithelial (RPE) layer and photoreceptors increases16-17. Consistent with these reports, our previous experiments showed more photoreceptor death in bullous RDs than shallow RDs. It has also been reported that subretinal hemorrhage is toxic for photoreceptor cells and affects photoreceptor cell death18-21. Likewise, we have observed more photoreceptor death in RDs with subretinal hemorrhage than RDs without subretinal hemorrhage. Hence, techniques to minimize variability have to focus on achieving consistent heights of RD while avoiding subretinal hemorrhage.
Our modified method of inducing RD can make reproducible bullous and persistent RD at the same position of the eye with rare occurrence of subretinal hemorrhage. We performed surgery using a temporal approach because it is easier to achieve a wider operative field compared with other sites. After the conjunctival incision, a self-sealing scleral incision is made using a 30 G needle. A scleral tunnel is created, followed by scleral penetration into the choroid. If choroidal bleeding occurs in this step, the hemorrhage will exit the eye through the scleral wound, and the bleeding can be stopped by astriction with a surgical spear. An anterior chamber puncture is then performed from the cornea to reduce intraocular pressure. This is an important step because subretinal injection alone will lead to increased intraocular pressure with resultant retinal artery occlusion and ischemia of the inner retina. A 33 G needle connected to a Hamilton 10 &#956;l syringe is then inserted into the subretinal space, and 3.5 &#956;l sodium hyaluronate is gently injected to detach the neurosensory retina from the underlying RPE. In contrast to other methods of inducing RD that are performed under fundus observation, this technique is performed under direct observation. Since the scleral wound is self-sealing, sodium hyaluronate will not leak out after injection. Finally, glue is put on the scleral wound, and the conjunctiva is reattached to its original position. These lasts steps also reduce the risk of sodium hyaluronate leakage. The injection of 3.5 &#956;l sodium hyaluronate creates reproducible RDs (50% of the retina) in the eyes of 8-week-old mice. Creating the self-sealing wound is the most important step in our modified procedure because it prevents the injected sodium hyaluronate from leaking out of the eye, thus allowing reproducible bullous and persistent RDs.

<table border="1">
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Ketaject</td>
 <td>Phoenix</td>
 <td>2010025</td>
 <td></td>
 </tr>
 <tr>
 <td>AnaSed</td>
 <td>LLOYD</td>
 <td>4004821</td>
 <td></td>
 </tr>
 <tr>
 <td>5% Phenylephrine / 0.5% Tropicamide</td>
 <td>Massachusetts Eye and Ear Pharmacy</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>0.5% Proparacaine Hydrochloride Ophthalmic Solution</td>
 <td>AKORN</td>
 <td>17478-263-12</td>
 <td></td>
 </tr>
 <tr>
 <td>Provics</td>
 <td>Alcon</td>
 <td>8065183085</td>
 <td></td>
 </tr>
 <tr>
 <td>Webglue</td>
 <td>Patterson Veterinary</td>
 <td>07-8566128</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td>Leica</td>
 <td>MG90</td>
 <td></td>
 </tr>
 <tr>
 <td>30G1/2 PrecisionGlide Needle</td>
 <td>BD</td>
 <td>305106</td>
 <td></td>
 </tr>
 <tr>
 <td>Weck-Cel Eye Spears</td>
 <td>Beaver-Visitec</td>
 <td>0008685</td>
 <td></td>
 </tr>
 <tr>
 <td>10 Microliter Syringe</td>
 <td>Hamilton</td>
 <td>7635-01</td>
 <td></td>
 </tr>
 <tr>
 <td>33 gauge, 0.5 inch needle</td>
 <td>Hamilton</td>
 <td>7803-05</td>
 <td></td>
 </tr>
 <tr>
 <td>18x18mm Cover Glass</td>
 <td>Fisher Scientific</td>
 <td>18-548A</td>
 <td></td>
 </tr>
 </tbody>
</table>

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).

To assess the persistence of the RD made by this protocol, cryosections were made on days 3, 7, and 14 after induction of RD. Six eyes were used for each time point. Hematoxylin and eosin (HE) staining was used to visualize the sections. All sections showed a bullous RD approaching the lens (Figure 2). One eye showed subretinal hemorrhage on day 7. No eyes showed any signs of infection or lens injury.
<img src="/files/ftp_upload/50660/50660fig1.jpg" alt="Figure 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50660/50660fig1highres.jpg" /> 
Figure 1. Creating a self-sealing scleral incision. This schematic shows a cross-sectional image of a normal mouse eye. Red line indicates the self-sealing scleral incision. A scleral tunnel followed by scleral penetration into the choroid with a 30 G needle creates a self-sealing scleral incision. This scleral wound will be self-sealed with intraocular pressure, which can prevent leakage of sodium hyaluronate after injection into the subretinal space and achieve persistent bullous retinal detachments.
<img src="/files/ftp_upload/50660/50660fig2.jpg" alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50660/50660fig2highres.jpg" /> 
Figure 2. Time course of retinal detachment. This fundus photo shows a bullous retinal detachment without subretinal hemorrhage immediately after surgery. Cryosections with hematoxylin and eosin (HE) staining show persistent bullous retinal detachment without subretinal hemorrhage through at least day 14.

Watch this Scientific Journal Video about Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate at JoVE.com

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

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 ...

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Research

JoVE Journal

Medicine

20.7K Views.  Massachusetts Eye and Ear Infirmary, Harvard Medical School.  Creazione di distacchi di retina sperimentali con un'altezza riproducibile e sostenuto di distacco, e senza emorragia subretinica, è importante per lo studio della fisiopatologia della perdita di cellule fotorecettori della retina nella malattia e valutare possibili interventi terapeutici. Qui riportiamo tale metodo in dettaglio. 

Video: Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

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 ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents

To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, mice undergo RR upon removal of the aortic constriction. In this paper, we describe a step-by-step procedure to perform a minimally invasive surgical aortic debanding in mice. Echocardiography was subsequently used to assess the degree of cardiac hypertrophy and dysfunction during LV remodeling and RR and to determine the best timing for aortic debanding. At the end of the protocol, terminal hemodynamic evaluation of the cardiac function was conducted, and samples were collected for histological studies. We showed that debanding is associated with surgical survival rates of 70-80%. Moreover, two weeks after debanding, the significant reduction of ventricular afterload triggers the regression of ventricular hypertrophy (~20%) and fibrosis (~26%), recovery of diastolic dysfunction as assessed by the normalization of left ventricular filling and end-diastolic pressures (E/e&#39;&#160;and LVEDP). Aortic debanding is a useful experimental model to study LV RR in rodents. The extent of myocardial recovery is variable between subjects, therefore, mimicking the diversity of RR that occurs in the clinical context, such as aortic valve replacement. We conclude that the aortic banding/debanding model represents a valuable tool to unravel novel insights into the mechanisms of RR, namely the regression of cardiac hypertrophy and the recovery of diastolic dysfunction.

Here we describe a step-by-step protocol of surgical aorta debanding in the well-established mice model of aortic-constriction. This procedure not only allows studying the mechanisms underlying the left ventricular reverse remodeling and regression of hypertrophy but also to test novel therapeutic options that might accelerate myocardial recovery.

To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, ...

Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the pathophysiological changes of human OA. However, as the main symptom in the late stage of OA, pain affects the patients&#39; daily life, and there are not many available models. The mono-iodoacetate (MIA)-induced model is the most widely used OA pain model, mainly used in rodents. MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which causes chondrocyte death, cartilage degeneration, osteophyte, and measurable changes in animal behavior. Besides, expression changes of matrix metalloproteinase (MMP) and pro-inflammatory cytokines (IL1 &#946; and TNF &#945;) can be detected in the MIA-induced model. Those changes are consistent with OA pathophysiological conditions in humans, indicating that MIA can induce a measurable and successful OA pain model. This study aims to describe the methodology of intra-articular injection of MIA in rats and discuss the resulting pain-related behaviors and histopathological changes.

This study describes the method of intra-articular injection of mono-iodoacetate in rats and discusses the resulting pain-related behaviors and histopathological changes, which provide references for future applications.

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.