Name: an alternative and validated injection method for accessing the subretinal space via a transcleral posterior approach
Uploaded: 2016-12-07T14:00:00
Description: Watch this Scientific Journal Video about An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach at JoVE.com

We gratefully acknowledge support by the Harold and Pauline Price Chair in Ophthalmology and the Jules Stein Eye Institute to MBG, the NEI Core grant (EY00331-43) to SN. Research was supported in part by a generous gift from the Sakaria family to SN and MGB, and from an unrestricted grant from the Research to Prevent Blindness to the Department of Ophthalmology. We thank Charlotte Yiyi Wang at Berkeley School of Optometry for obtaining initial OCT images of subretinal injections.

Animals: Wild type C57Bl/6J mice bred at the University of California at Los Angeles (UCLA). All animals were between 11 - 17 weeks old, and included male and female mice. All mice were group-housed, maintained in a 12:12 light/dark cycle with food and water ad libitum. All experiments were performed in accordance with the institutional guidelines of UCLA and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
NOTE: All drugs and injectable agents are Un...

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Subretinal injections are the method of choice for the delivery of viral vectors and stem cell-derived therapy for manipulating photoreceptors and the RPE in both basic research and clinical treatment. In patients, subretinal injections are typically done with an anterior sclerotomy at the pars plana, a posterior core vitrectomy and penetration of the retina by the needle with direct visualization. As with most vitrectomy procedures, it is common for cataract formation to occur prematurely unless the eye is already pseud...

Subretinal injections are the primary means of delivering cellular and viral agents to the retinas of mice to study their effects on photoreceptors and the underlying RPE1,2. Most subretinal injection protocols in mice use a transcorneal or a transcleral injection site anterior to the equator (Figure 1). This approach can result in inherent collateral damage that includes nicking and resultant clouding of the lens, disruption of the integrity of the vitreous, penetration of the neurosensory retina and iris, retinal hemorrhage, substantial retinal detachments and lasting subretinal edema 3-9. Experimental manipulations must overcome these effects in order to evaluate the effects of therapeutic interventions3,7,10,11. This study provides a detailed description and validation of a posterior transcleral injection method that avoids these complications, minimizes trauma and has a high success rate of targeting the subretinal space.
Injections targeting the subretinal space in mice are often very difficult to perform and most investigators encounter a high frequency of failed attempts in which the vector is delivered to an incorrect location or there is significant retinal damage, for example in a complete retinal detachment6. The number of eyes excluded from analysis because of injection complications is typically not reported in mouse studies, but in our own experience and in discussion with other investigators, the number of failed injections can be as high as 50% and vary dependent on the experience and capabilities of the investigator who is performing the injections. The success of the injection is typically assessed by direct fundus imaging and/or optical coherence tomography (OCT)7,9. An easily mastered method with high success rates for subretinal injections in mice can hasten experimentation and reduce the cost of preclinical studies of treatments for retinal diseases that are major causes of blindness in the United States.
The posterior, transcleral subretinal injection technique described here is an adaptation from clinical and preclinical protocols9,12. The noninvasive diagnostic assessments performed in injected mice demonstrate mild and highly localized damage and lack additional collateral lens, retinal and RPE injury. Furthermore, with relatively little practice, an experimenter can achieve these results with a high success rate (80 - 90% or better), thereby reducing the costs associated with such studies. This procedure can be used to deliver cellular, viral, or pharmacological therapeutic interventions to photoreceptors and/or RPE in preclinical studies and to easily evaluate experimental interventions.

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			<td>Hamilton Model 62 RN SYR</td>
			<td>Hamilton</td>
			<td>87942</td>
			<td>Syringe x 1</td>
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			<td>Hamilton Needle 33G, 1.0&#34;, 20 DEG, point 3 (304 stainless steel)</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td>Needles x 6</td>
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		<tr>
			<td>Vannas Curved Scissors</td>
			<td>Ted Pella, INC.</td>
			<td>1347</td>
			<td>5mm Blade</td>
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		<tr>
			<td>22.5 Degree Microsurgery Knife</td>
			<td>Wilson Ophthalmic Corp.</td>
			<td>91204</td>
			<td></td>
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			<td>Ketaject&#160;</td>
			<td>Phoenix</td>
			<td>NDC 57319-609-02</td>
			<td>Ketamine</td>
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			<td>Anased</td>
			<td>Lloyd Laboratories</td>
			<td>NDC 61311-482-10</td>
			<td>Xylazine</td>
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		<tr>
			<td>Fluorescein 10% AK-Fluor</td>
			<td>Akorn</td>
			<td>NDC 17478-253-10</td>
			<td>100mg/ml</td>
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		<tr>
			<td>0.9% Saline USP</td>
			<td>Hospira</td>
			<td>NDC 0409-4888-50</td>
			<td>0.9% NaCl</td>
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			<td>Antibiotic Ointment</td>
			<td>Akorn</td>
			<td>NDC 17478-235-35</td>
			<td>Ophthalmic</td>
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		<tr>
			<td>Water Circulating Pump</td>
			<td>Gaymar</td>
			<td>TP-500 T/Pump&#160;</td>
			<td>P/N 07999-000</td>
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		<tr>
			<td>sd-OCT</td>
			<td>Bioptigen</td>
			<td>R-Series</td>
			<td>Commercial</td>
		</tr>
		<tr>
			<td>Fundus Camera</td>
			<td>Phoenix Research Laboratories</td>
			<td>MICRON III</td>
			<td></td>
		</tr>
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			<td>Tweezers Type 3</td>
			<td>Ted Pella, INC.</td>
			<td>5385-3SU</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% Phenylephrine</td>
			<td>Paragon BioTeck</td>
			<td>NDC 42702-102-15</td>
			<td>Ophthalmic</td>
		</tr>
		<tr>
			<td>IMARIS8</td>
			<td>Bitplane</td>
			<td></td>
			<td>Version 8.1.2</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>V1.8.0_77</td>
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		<tr>
			<td>Hypromellose&#160;2.5%</td>
			<td>Goniovisc</td>
			<td>AX0401</td>
			<td>Methylcellulose</td>
		</tr>
		<tr>
			<td>Eye Drops (Rinse)</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>Saline Solution</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Fostec</td>
			<td>P/N 20520</td>
			<td>Light source</td>
		</tr>
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an alternative and validated injection method for accessing the subretinal space via a transcleral posterior approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

Posterior approach transcleral subretinal injections were performed on 31 healthy eyes from 16 wild type mice with injections of 0.3 &#181;l (n = 18), 0.5 &#181;l (n = 8) and 1.0 &#181;l (n = 5) of 0.01% fluorescein. One eye was excluded from injection due to a pre-existing corneal opacity that prevented structural and functional analysis. Every injected eye is included in this report. No unintended retinal detachments, punctures of the neurosensory retina, or leakage into the vitreous we...

Watch this Scientific Journal Video about An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach at JoVE.com

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

The overall goal of this study is to describe a Novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, or induce pluripotent stem cells to the subretinal space in mice. This method can answer key questions in the vision science and ophthalmology fields, such as assessing the efficacy of Novel therapeutic interventions for retinal degenerations. The main advantage of this technique is that it can be used to deliver material to the subretinal space, with high efficacy, minimal damage, and fast recovery of retinal structure and function.Demonstrating the procedure will be Sachin P, a technician from my laboratory. To begin this procedure, trim the whiskers of an an anesthetised mouse to facilitate visualization, and maintain the animal's body temperature at 37 degrees Celsius with a circulating water pad. Next, dilate its pupils with 2.5%phenylephrine eye drops.Then, apply methylcellulose eye drops to prevent dryness, and minimize anesthetic induced transient cataracts. Sterilize the instruments prior to surgery. Afterward, prepare the diluted fluorescein in a biosafety cabinet.Fill the syringe with the appropriate amount of fluorescein. Next, perform a toe pinch to ensure the animal is deeply anesthetised, and position the mouse so that the eye to be injected is facing up and clearly visible in the dissecting microscope. Gently pinch the temporal conjunctiva with a pair of fine-tipped forceps.Then, make a circumferential incision of approximately 90 degrees using the curved Vannas'scissors. Repeat the procedure on the underlying Tenon's capsule. After that, resect the surrounding connective issue with fine-tipped forceps, while rotating the globe nasally.Work towards the injection site at approximately 0.5 millimeters away from the optic nerve and take great care to avoid disrupting the retro-orbital sinus. A critical step is exposing the injection site which requires rotation of the eye, without damaging the eye itself, or associated structures, particularly avoiding damage to the orbital blood sac. In this procedure, make a small incision at the sclera of the injection site, by gently scratching the eye cup with a 22.5 degree ophthalmic blade.This incision should just be large enough to allow the tip of the needle to pass through the sclera. Next, insert the beveled 33 gauge needle into the sclerotomy with the bevel facing and angled parallel to the retina. Inject the desired amount of 0.01%fluorescein by pressing the plunger slowly with even pressure.Note that when the needle is in the subretinal space, a slight resistance will be felt while pressing the plunger. There will be no resistance if the needle punctures the retina, but high resistance if the needle does not penetrate the sclera or RPE. Another critical step is the injection targeting subretinal space.This must be accomplished without penetrating through the neurosensory retina, to the vitreous cavity. Wait several seconds before withdrawing the needle, to minimize backflow. Subsequently, rinse the eye with sterile saline, and ensure the eye has rotated back to its normal position.Then, apply a thick coat of triple antibiotic ophthalmic cream to the corneal surface of the injected eye. Afterward, place the mouse in a clean solitary cage for recovery. Monitor its respiration and temperature during anesthesia recovery, and check that it can maintain sternal recumbency.Perform additional appropriate post-operative monitoring and treatment, including a subcutaneous injection of carprofen, for post-surgical pain management. Here is a 3D reconstruction of the dome-shaped bleb at the injection site. The dome-shaped bleb is then filled in dense white to show the extent and margins of the retinal detachment.Retinal cross-sections from the OCT can be seen in white, while the fluid-filled space at the level of the RPE and photoreceptors appears black. And in this figure, the representative OCTB scans at the site of maximal retinal detachment are shown for pre-injection, 10 minutes post-injection, and four weeks post-injection. This image shows the formation and resolution of a flat bleb from a 0.3 microliter injection.And this image shows the formation and resolution of a domed bleb from a 0.5 microliter injection, whereas this image shows the formation and resolution of a domed bleb from a one microliter injection. Lastly, here is an example of severe choroidal scarring and retinal thinning at the injection site. Retinas retain normal function after bleb resolution.At nine illumination intensities, the waveforms for scotopic rod mediated responses are shown for pre-injection and four weeks post-injection for 0.3 microliter, 0.5 microliter, and 1 microliter injections. Once mastered, this technique can be completed in 10 to 15 minutes per eye. While attempting this procedure, it is important to evaluate the health of the eyes before commencing.Exclude eyes with corneal or lens opacities, or deep-set eyes for which this procedure would be very difficult. Following this procedure, other methods such as OCT and fundus imaging can be performed in order to evaluate the quality of the injection.

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