This work was funded by the following funding: NEI R01EY033103 (MSS), Foundation Fighting Blindness (MSS),the Shulsky Foundation (MSS), the Joseph Albert Hekimian Fund (MSS), the Juliette RP Vision Foundation (YVL), Research to Prevent Blindness (unrestricted grant to the Wilmer Eye Institute at Johns Hopkins University and the Cullen Eye Institute at Baylor College of Medicine). We thank Dr. Malia Edwards (Johns Hopkins University School of Medicine) for kindly providing training on the microscope.

All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures were approved by the Johns Hopkins University Animal Care and Use Committee (approval M021M459).
1. Animals
<ol>
	<li>Use Rho::EGFP mice (aged P3-P6) as donors of retinal cell suspensions. 
	NOTE: This strain was a kind gift from Dr. T. Wensel, Baylor College of Medicine.</li>
	<li>Use OPN1LW-EGFP/NRL-/- mice14 (aged P3) as donors of retinal sheets.</li>
	<li>Use adult retinal degeneration Rd1/NS mice with immune deficiency (either sex) as recipients.</li>
	<li>House all mice in cages under a 12:12 h light-dark cycle with access to water and food as needed.</li>
</ol>
2. Collect neural retina (Figure 1)
NOTE: All the following steps were performed under sterile conditions. Supplier information of the research tools and products is provided in the Table of Materials.
<ol>
	<li>Donor mice preparation: Euthanize donor mice with an overdose of carbon dioxide and confirm euthanasia with cervical dislocation.</li>
	<li>Isolate mice eyeballs. To do so follow the steps below.
	<ol>
		<li>Carefully open pups&#39; eyelids using micro scissors to expose the eyeball.</li>
		<li>Use smooth forceps to grab the optic nerve and pull out the eyeball with a pair of forceps.</li>
	</ol>
	</li>
	<li>Isolate neural retina 
	NOTE: This step is performed under a dissection microscope.
	<ol>
		<li>Incise a hole in the center of the cornea using a 25 G sterile needle.</li>
		<li>Cut the cornea, through the hole, in half and enlarge the incision to the sclera and RPE, then remove the sclera and RPE.</li>
		<li>Use micro-toothed forceps to gently remove the lens and the vitreous to isolate the neural retina.</li>
		<li>Proceed with section 3 to prepare donor retinal suspension or section 4 to prepare donor retinal sheet, as required.</li>
	</ol>
	</li>
</ol>
3. Prepare donor retinal suspension (Figure 1)
<ol>
	<li>Incubate the neural retina in Papain solution at 37 &#176;C for 20-30 min until no cell clumps are detected. 
	NOTE: Gently pipette the cell mixture 15x every 10 min.</li>
	<li>Follow manufacturer&#39;s instructions of the Papain Dissociation Kit to collect single cells.</li>
</ol>
4. Prepare donor retinal sheets (Figure 1)
NOTE: This step follows section 2 and is performed under a dissection microscope.
<ol>
	<li>Put the isolated neural retina cup into a 35 mm Petri dish with prechilled sterile PBS.</li>
	<li>Use micro scissors to cut the neural retina cup into multiple retinal sheets (around 1 mm x 2 mm).</li>
</ol>
5. Prepare recipient mice
<ol>
	<li>Anesthetize recipient mice with intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine hydrochloride (20 mg/kg body weight).</li>
	<li>Confirm the surgical plane of anesthesia by ensuring the loss of blink and pain reflexes, regular breathing, and respiration.</li>
	<li>Assess the anesthetic depth by the lack of response to the tail pinch or pedal reflexes withdrawal.</li>
	<li>Always recheck the anesthetic depth during the operative procedure and adjust the anesthetic depth if the animal is responsive to pain. Keep the mice on the pre-warmed surgery table to prevent hypothermia.</li>
	<li>Dilate recipient pupils with 1% (wt/vol) tropicamide eye drops 5 min before the surgery. A well-dilated pupil can facilitate transpupillary visualization under the operating microscope.</li>
	<li>Disinfect the operating mouse eye and surrounding ocular tissues with povidone-iodine, wash with sterilized PBS.</li>
	<li>Apply 0.5% Proparacaine hydrochloride on the mouse eye for analgesia.</li>
</ol>
6. Subretinal transplantation of retinal grafts (Figure 2)
NOTE: All the following steps were performed under aseptic conditions. Surgical tools were autoclaved (use instrument trays to protect the tool tips and take the plunger out of the micro syringes to prevent them getting stuck in the lumen). Supplier information of the research tools and products is provided in the Table of Materials.
<ol>
	<li>Anterior chamber paracentesis: Penetrate peripheral cornea into anterior chamber with an insulin needle to allow aqueous humor to passively egress, to prevent the intraocular pressure (IOP) spikes during transplantation. 
	NOTE: A successful paracentesis is indicated by the efflux of aqueous humor through the cornea hole.</li>
	<li>Put a drop of sodium hyaluronate and a glass coverslip (5 mm diameter) on top of the cornea to enable transpupillary visualization of the fundus under the surgical scope.</li>
	<li>Use toothed forceps to expose the injection locus (1 mm post-corneal limbus) by pressing either the superior or inferior eye wall, as required by the experiment, toward the center of transpupillary vision.</li>
	<li>Use a microinjection needle to perpendicularly penetrate the sclera.</li>
	<li>Carefully rotate the needle direction tangentially to allow complete penetration of the sclera-choroid-RPE complex into the subretinal space.</li>
	<li>Insert the needle tangentially to access the terminal injection locus. 
	NOTE: Verify the complete positioning of the needle&#39;s bevel by transpupillary vision guidance. Use the superficial retinal vessels as an anatomical reference for accurate needle positioning within the subretinal space.</li>
	<li>Inject retinal grafts 
	NOTE: The presence of small bubbles preloaded in the syringe can facilitate validation of the accurate subretinal positioning of donor cells. Elevated IOP resulting from the subretinal injection increases the risk of donor cell reflux.
	<ol>
		<li>If the cornea becomes cloudy24,25, an indicator of high IOP, keep the needle in the subretinal space for at least 3 min until the cornea clears, an indicator that the IOP has reduced sufficiently. 
		NOTE: In rare cases, the IOP may take longer to normalize, and it is recommended keeping the needle in place until the clarity of the cornea is restored even if this would take 8-10 min. Observe under a surgical microscope to prevent damage to retinal structures.</li>
	</ol>
	</li>
	<li>Grasp the injection hole edge with micro-toothed forceps and quickly withdraw the needle. 
	NOTE: The criteria for successful subretinal delivery of a retinal cell suspension and sheet are a constant bleb and a visible white sheet in the subretinal space, respectively.</li>
	<li>Clean the surgical area of the eye with artificial tears and apply ointment if necessary.</li>
	<li>Post-surgery administration
	<ol>
		<li>Place transplanted mice in a pre-warmed (37 &#176;C) clean recovery cage and carefully monitor for distress.</li>
		<li>Apply a drop of topical 0.5% Proparacaine Hydrochloride on the surgical eye 2-3 times if distress occurs.</li>
		<li>Return mice to the house cage after the mice are completely alert and mobile. Place a small number of food pellets and a gel cup in the cage if the mice have trouble reaching the food hopper. 
		NOTE: Keep the eye surface humid until anesthesia wears off to prevent cataract formation.</li>
	</ol>
	</li>
</ol>
7. Multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging
<ol>
	<li>Two months post-transplantation, anesthetize recipient Rd1/NS mice (n=5) by intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine hydrochloride (20 mg/kg body weight) for imaging.</li>
	<li>Dilate pupils with 1% (wt/vol) tropicamide eye drops.</li>
	<li>Perform standard 55&#176; multimodal imaging using the confocal scanning laser ophthalmoscope cSLO system22.</li>
	<li>Obtain MR and SD-OCT images using 30 frames of ART averaging.</li>
</ol>
8. Histological staining
<ol>
	<li>Prepare frozen slides of transplanted Rd1/NS mice eyes as previously reported 14.</li>
	<li>Block and penetrate the frozen slides of transplanted retinae using a mixture of 5% goat serum and 1% Triton X100 in PBS.</li>
	<li>Incubate samples with primary antibodies over night at 4 &#176;C and rinse in PBS (5 min, 3x). The primary antibodies include goat anti-GFP-FITC (1:200), rabbit anti-recoverin (1:1000), and rabbit anti-S-opsin (1:500).</li>
	<li>Incubate the samples with secondary antibody (goat anti-rabbit Cy3, 1:500) and counterstained with DAPI. 
	NOTE: The suppliers of primary and secondary antibodies are listed in Table of Materials.</li>
</ol>

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MSS is/was a paid advisor to Revision Therapeutics, Johnson &#38; Johnson, Third Rock Ventures, Bayer Healthcare, Novartis Pharmaceuticals, W. L. Gore &#38; Associates, Deerfield, Trinity Partners, Kala Pharmaceuticals, and Acucela. MSS receives sponsored research support from Bayer. These arrangements have been reviewed and approved by the Johns Hopkins University in accordance with its conflict-of-interest policies. KVL is named as inventors on patent applications at University of Colorado. MSS and YVL are named as inventors on patent applications at Johns Hopkins University.

Subretinal transplantation in mice is technically challenging due to the small size of mouse eyes. In this study, we developed a simple and reproducible platform for subretinal transplantation in mouse recipients. The platform allows consistency in donor viability protection, successful subretinal delivery, and ensures a low complication rate.
The subretinal transplantation technique depicted here was developed based on the trans-scleral route, where the injection needle penetrates the outer layers (sclera-choroid-RPE complex) of the eyewall. Compared to the trans-corneal19,20 and trans-vitreous21,26,27&#160;approach, the trans-scleral approach directly enters the subretinal space without penetrating the anterior segment structures and neural retina, thus enabling a harmless transplantation and reducing the risk of donor cell reflux through the penetrating hole. A challenge of the trans-scleral approach is to ensure the needle tip propagates along the subretinal space before arriving at the terminal delivery site, while avoiding potential disturbance of the adjacent RPE and choroid layers. It is challenging to achieve these goals through a traditional trans-scleral approach28,29 without direct visual guidance. In this study, we developed a transpupillary vision-guided platform using a surgical microscope and external illumination of the retina to facilitate subretinal transplantation in mouse models. The platform allows the operator to track the surgical process and the recipients&#39; retinal condition by providing real-time visualization of the recipient fundus under the surgical microscope. For example, successful penetration of the sclera-choroid-RPE complex can be simply validated by a relatively bright reflectance of the needle tip via transpupillary visualization. Importantly, when guided by transpupillary visualization, the operator can accurately modulate the angle and the depth of injection according to the relative position between the needle and anatomic structures in the recipient retina (e.g., retinal vessels and optic nerve head). Moreover, accurate administration of materials using this technique can minimize RPE, choroid trauma, and other surgical complications. Indeed, we found hemorrhages can be minimized by avoiding maneuvers in close proximity to retinal blood vessels with the help of transpupillary visualization.
Reflux of donor cells is a major cause of surgical failure post-injection29,30. There are multiple factors involved in promoting donor cell reflux. The high intraocular pressure (IOP) of the recipient eyes caused by the injected donor cell mixtures is an important factor that promotes the reflux of cells out of the eye. Our protocol reduces the recipients&#39; IOP by anterior chamber penetration at the beginning of the surgery and enables the IOP to lower spontaneously by aqueous fluid egress before pulling out the vitreous needle from the vitreous cavity. A second factor is the unsealed injection tunnel in the recipients&#39; eyes. To prevent graft reflux back through the injection tunnel, we use a small size micro-injection needle (34G) to create a self-sealing tunnel when penetrating the eyewall and toothed micro-forceps to hold the edges of the external tunnel opening when pulling out the needle. In conventional transplantation, refluxed grafts are hardly detected because they may occur rapidly or dissipate quickly. The sodium hyaluronate, upon application of the coverslip, almost invariably slides off the cornea to cover most of the limbus and sclera including the sclerotomy site. Following cell injection, the sodium hyaluronate at the sclerotomy site can help to slow down the bulk flow of refluxed contents, if any, and help to make them visually detectable under the surgical microscope. Moreover, the absence of reflux can also be verified by tracing the constancy of the subretinal bleb for cell suspensions and the intraocular location of the retinal sheet using our protocol, which could be useful for laboratories where optical coherence tomography (OCT) is unavailable.
While a substantial number of photoreceptor cells survived in the transplanted retinal sheets, no discernible cellular orientation, or sheet orientation, could be detected. Although delivery of 3D retinal sheets to large animals has been established26,31,32, the subretinal space in mice, coupled with the lack of intraoperative retinal imaging capability, makes it very challenging to ensure the maintenance of sheet orientation intraoperatively as well as in the immediate postoperative period during which the transplanted cells or sheets may become unstable as the mouse regains ambulation.
We describe a trans-scleral surgical platform with direct transpupillary vision guidance for the subretinal transplantation in mouse recipients. This platform enables high accuracy of injection and a low rate of surgical complications. This platform enables precise delivery of known doses of cells, is relatively easy to learn, and facilitates subretinal delivery in addition to intra-retinal or intravitreal injections for different types of therapeutic agents including gene therapy.

Transplantation of photoreceptor and retinal pigmented epithelial (RPE) cells provides potential therapy for retinal degenerative diseases such as age-related macular degeneration (AMD), Stargardt disease, and retinitis pigmentosa (RP)1,2,3,4,5,6,7. To replenish or replace the diseased photoreceptors and RPE cells in degenerated retinae, the subretinal space is particularly well suited as a transplantation target given the laminar anatomy of host photoreceptors and RPE cells. While surgical procedures of sub-retinal transplantation of RPE cells are well-established in large animals8,9,10 and clinical trials11,12,13, challenges facing photoreceptor transplantation research include the scarcity of transgenic large animal models and the limited understanding of in-depth synaptogenesis mechanisms involved in neuronal transplantation, among other concerns. Genetically modified murine models, with various types of retinal degeneration mutants, provide useful tools for studying molecular mechanisms in the context of transplantation and steering the development of effective cell replacement therapies at the preclinical stage14,15,16,17,18.
Unlike the relatively large eye and small crystalline lens in large animals (e.g., pig, monkey), the small size and large crystalline lens of mouse eyes make them difficult surgical targets, especially for subretinal transplantation in which physical space constraints and limited direct visualization are the core challenges.
Current approaches can be classified into three major types based on the injection route. First, in the case of the trans-corneal approach, the needle is passed through the cornea into the vitreous cavity and then into the subretinal space19,20. Successful subretinal delivery can be achieved using this method but the damage to anterior segment structures (i.e., cornea, iris, lens) is a major risk that may severely impede downstream in vivo analysis. Second, in the case of the trans-vitreous approach, the needle enters the vitreous cavity through the pars plana and then into the subretinal space21. This approach is widely used in humans and large animals. However, there is a potential risk of lens damage in rodents as the lens occupies a larger relative volume of the vitreous cavity. Notably, both trans-corneal and trans-vitreous protocols require penetration of the neural retina to arrive at the subretinal space, which causes damage to the host retina and increases the risk of donor cell reflux through the penetration hole. Third, in the case of the trans-scleral approach22,23, the needle penetrates through the scleral-choroid-RPE complex and directly enters the subretinal space. This approach reduces the potential trauma of anterior segment structures and the vitreous cavity. However, without direct visualization of the host fundus, surgical failures caused by trans-retinal punctures, RPE detachment, and choroid hemorrhage are commonly detected.
Here, we developed a trans-scleral surgical platform with direct transpupillary vision guidance for subretinal transplantation in mouse recipients. Validation of the viability of donor retinal sheets and cell suspensions before transplantation was performed. Successful delivery of the donor cells into the subretinal space of a retina degeneration mouse model was confirmed. Only rare surgical complications were detected. In addition, transplanted photoreceptors in the retinal grafts survived and showed evidence of advanced maturation into &#34;adult&#34; rods and cones two months post-transplantation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Artificial tears</td>
			<td>CareAll</td>
			<td>P31447-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips (5mm in diameter)</td>
			<td>Deckglaser</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-GFP (FITC)</td>
			<td>Abcam</td>
			<td>Ab6662</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti-rabbit Cy3&#160;</td>
			<td>Invitrogen</td>
			<td>A10520</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe (30G)</td>
			<td>Easy Touch</td>
			<td>08496-3015-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>VETone Zetamine</td>
			<td>AH2017J</td>
			<td></td>
		</tr>
		<tr>
			<td>Live Dead Viability Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissor</td>
			<td>Harvard Apparatus</td>
			<td>72-8503</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro smooth forceps</td>
			<td>ASICO</td>
			<td>AE-4360</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro toothed forceps</td>
			<td>World Precision Instruments</td>
			<td>555041FT</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection needle (26G)</td>
			<td>Hamilton</td>
			<td>7804-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection needle (34G)</td>
			<td>Hamilton</td>
			<td>207434</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection syringe</td>
			<td>Hamilton</td>
			<td>7633-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain dissociation kit</td>
			<td>Worthington Biochemical</td>
			<td>LK003150</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri-dish (35 mm)</td>
			<td>Thermo Fisher Scientific</td>
			<td>FB012920</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine (10%)</td>
			<td>Betadine Solution</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Proparacaine Hydrochloride (0.5%)</td>
			<td>Keeler</td>
			<td>AX0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-recoverin</td>
			<td>Millipore</td>
			<td>Ab5585</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-S-opsin</td>
			<td>Millipore</td>
			<td>Ab5407</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hyaluronate</td>
			<td>Johnson &#38; Johnson Vision&#160;</td>
			<td>10-2400-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cotton swabs</td>
			<td>Puritan</td>
			<td>25-806 2PC</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile needle (25G)</td>
			<td>BD PrecisionGlide Needle</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile towel drapes</td>
			<td>Dynarex</td>
			<td>4410</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical materials/reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tropicamide ophthalmic solution</td>
			<td>Henry Schein</td>
			<td>112-7192</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>AnaSed Injection</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

transpupillary-guided trans-scleral transplantation of subretinal grafts in a retinal degeneration mouse model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

Retinal grafts are successfully delivered into the subretinal space and survived in vivo. 
The performance of our subretinal transplantation platform was assessed in Rd1/NS recipient&#160;mice aged 6-8 weeks when their residual retinae were severely thinned due to the almost complete outer nuclear layer (ONL) degeneration. Given the fragility of the retina, the scarcity of cone photoreceptors, and the relatively poor viability of cells in suspension compared to sheet donors, cone-rich mice (OPN1LW-EGFP;NRL-/-)14 were used as the sources of donor retinal sheets and rod-rich mice (Rho::EGFP) as the donor for cell suspensions. Cone-rich retinal sheet and rod-rich cell suspensions were successfully delivered into the subretinal space of all recipient mice using our surgical platform, as confirmed by the subretinal bleb and white sheet seen using transpupillary visualization immediately following injection. Two months post-transplantation, multimodal cSLO imaging was performed to track the states of retinal grafts in vivo. Cross-sectional OCT scanning showed that retinal grafts survived in the subretinal space and reconstituted the ONL in all recipient mice (Figure 3A). Infrared imaging detected no obvious cataracts in all transplanted eyes (Figure 3B). Other surgical complications including hemorrhage were rarely detected in transplanted retinae (n = 1/10 eyes) by multicolor reflectance imaging at two months post-transplantation (Figure 3C). We performed histological staining to further assess the survival and extent of maturation of photoreceptors in retinal grafts. Abundant cone photoreceptors expressing OPN1LW:EGFP and S-opsin in transplanted retinal sheets were observed. Likewise, transplanted retinal cell suspensions showed a large proportion of Rec+ photoreceptors in vivo, including numerous Rho::EGFP+ rods (Figure 3D). The non-transplanted control mice showed severe ONL degeneration with sparse residual cone photoreceptors (Rec+). No EGFP signal was detected in non-transplanted Rd1/NS retina (Figure 3D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65448/65448fig01.jpg" /> 
Figure 1: Schematic of collecting donor retinal sheet and cell suspensions. Schematics showing the key steps of collecting retinal cell suspensions and sheets from donor Rho::EGFP mice. Bright-field and fluorescent imaging showed representative images of dissociated cells and a dissected sheet isolated from Rho::EGFP mice. Yellow dotted line: incisional margin. <a href="https://www.jove.com/files/ftp_upload/65448/65448fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65448/65448fig02v3.jpg" /> 
Figure 2: Procedures of subretinal transplantation of retinal sheets and cell suspension in Rd1/NS mice. (A)&#160;Schematic images of&#160; recipient mice preparation. (B) Key surgical procedures and corresponding diagrams showing subretinal transplantation of retinal cell suspensions and sheets. Surgical steps include: 1. penetrate the anterior chamber; 2. drop the sodium hyaluronate on the cornea, then mount the coverslip on top of the sodium hyaluronate to facilitate the transpupillary visualization; 3. penetrate outer layers of the eye wall (sclera-choroid-RPE complex). Penetration angles of the needle are on the top right panel of the illustration; 4. insert injection needle; 5. inject grafts. Representative images show the successful delivery of retinal cell suspensions and sheet in two individual recipients, respectively. Asterisk: optic nerve head; Red arrowheads: retinal blood vessel; White arrowhead: penetrated needle tip; White arrows: grafted retinal suspensions or sheet. <a href="https://www.jove.com/files/ftp_upload/65448/65448fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65448/65448fig03.jpg" /> 
Figure 3: Successful delivery of retinal grafts into the subretinal space and survival in vivo. (A)&#160;Representative SD-OCT images showed the subretinal distribution of transplanted retinal sheets and cell suspensions in two individual Rd1/NS mice, respectively. Non-transplanted Rd1/NS mice were collected as a control. Region of Interest (ROI) is indicated by yellow-dotted boxes on infrared (IR) fundus images. Inner retina is designated as laminae of retinal ganglion cell layer (RGC), inner plexiform layer (IPL), and inner nuclear layer (INL). (B) Representative infrared (IR) image of a transplanted Rd1/NS mouse showed no cataract through the dilated pupil. (C) Representative multicolor reflectance (MR) images of transplanted and non-transplanted Rd1/NS eyes. The transplanted eyes presented no obvious surgical complications including hemorrhage. (D) Immunohistochemistry (IHC) staining of transplanted- (sheet and suspension) and non-transplanted (control) Rd1/NS mice. The data showed numerous grafted photoreceptors expressing specific markers of photoreceptors (Rec), rods (Rho::EGFP), L/M cones (OPN1LW:EGFP), and S-cones (S-opsin) at two months post-transplantation. Recipient retinal laminae were identified by DAPI staining (blue). Retinal grafts were identified by EGFP reporter (green). The non-transplanted mice retina showed severe ONL degeneration with sparse residual cone photoreceptors (Rec+). No EGFP signal was detected in non-transplanted Rd1/NS retinae. Magnified images were presented on the right two panels. Abbreviations: RGC: retinal ganglion cell layer; INL: inner nuclear layer. ONL: outer nuclear layer. <a href="https://www.jove.com/files/ftp_upload/65448/65448fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model at JoVE.com

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

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967 Views.  Johns Hopkins University School of Medicine. This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.