強膜網膜移植ひと網膜色素上皮細胞のラットの目の洗練されたプロトコルの開発

The overall goal of this video is to introduce a reproducible trans-scleral subretinal injection technique to deliver human retinal pigment epithelial, or RPE cells, into the subretinal space of rodent's eyes with minimal risk of retina damage. This is a accomplished by first making a small incision in the temporal conjunctiva posterior to the limbus to expose the underlying sclera. Then, a beveled 31-gauge insulin needle is used to penetrate the sclera core complex by inserting the needle 500 microns or approximately half of the tip into the tissue with the beveled side facing up to create a pilot hole.

Next, a beveled 33-gauge injection needle attached to a syringe is guided into the pilot hole and inserted at a specific angle and depth, the injection hole self seals after the needle is retracted and no stitch or tissue glue is needed. Degenerative retinopathies consist of a wide spectrum of diseases involving different retinal cell types. Worldwide the most common disease, age-related macular degeneration, or AMD, occurs among the elderly and leads to permanent blindness.

The pathological changes occurring in AMD involve an initial degeneration of retinal pigmented epithelial cells, or RPE. RPE loss leads to retinal fluororeceptor dysfunction or death. Current pharmacological treatments for early stage AMD are limited to slowing progression or relieving symptoms.

This coupled with a limited regenerative potential of the retina means there is no effective treatment to restore vision. The Neural Stem Cell Institute recently identified a population of adult stem cells in the RPE cell population. These cells can be used as a source for stem cell replacement therapy.

Currently our institute is running pre-clinical studies to investigate the efficacy and safety of RPE stem cell transplantation therapy in an RPE degenerative rat animal model. In this study, we utilize a trans-scleral, subretinal injection technique to deliver RPE stem cells into the rat eyes, between the retinal and RPE layers. Our method is an easy and reliable means to successful delivery of cells into the subretinal space.

You will need the following reagents and equipment. 1%tropicamide and 2.5%phenylephrine, freshly made from 10%phenylephrine, by diluting it in sterile 0.9%saline on the day of surgery, are used to dilate the pupil. Eye wash is used to rinse the eye and keep the unoperated eye moist.

An eye lubricant is used to keep the unoperated right eye moist during the surgery. 0.5%proparacaine is used as a local anesthetic. An eye lubricant is used on the operated left eye during the OCT scanning.

A minimal amount of material and equipment is needed for this technique. Detailed information of equipment and reagents used in this technique is outlined in the protocol. A 10 microliter syringe is used to inject cells.

Before injection, we first assemble the injector and sterilize it with ethanol. Insert a sterile, 33-gauge beveled needle into the syringe and screw tightly to assemble the injector. Flush the injector with 100%ethanol, then 70%ethanol, and then deionized distilled water, sequentially for five to six times each.

Mark the injector needle with a sterile black marker pen at a position 600 microns away from the tip of the needle. Put the injector on a micro manipulator for injection use. Human RPE cells are prepared on the day of surgery and kept in ice water mixture.

Gently triturate the cell suspension and load the injector with 1.2 microliters, the extra 0.2 microliters is used to compensate for injection backflow, to guarantee a volume of one microliter injection. Replace the injector filled with the cell suspension on the micromanipulator, or have an assistant hold it vertically, as RPE cells tend to sink easily in solutions. Prepare the sterile surgical area by placing a sterile surgery pad with a heating pad underneath on the stage of the dissecting microscope.

All procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee at the State University of New York at Albany. Weight the animal and anesthetize it using the isoflurane vapor delivery system as shown here. Transfer the rat to the surgical area once it's anesthetized, and place it in a nose cone connected to the isoflurane system to maintain anesthesia.

Cover the rat's body with gauze, and pinch the rat's toe to confirm full anesthesia. Apply a drop of eye lubricant on the rat's right eye position the rat onto its right side, with its left eye facing the ceiling for injection, its head towards the surgeon's right hand, and it's back towards the surgeon. Trim away any whiskers that cover the eye.

Drip a small amount of eye wash for the temporal side of the left eye, and collect the excess at the nasal side with a cotton applicator to rinse the eye. Dilate the pupil with 1%tropicamide, 2.5%phenylephrine, for a post-injection optical coherence tomography, or OCT exam, by applying one drop of each. Apply a drop of eye wash to keep the eye moist.

Apply a drop of Proparacain on the eye and remove the excess with a cotton applicator. Gently massage the skin surrounding the eye four to six times to open the eyelid, so that the eye temporarily and slightly pops out of its socket to allow for an easy access of the injector needle. Use forces to grip the conjunctiva posterior to the limbus, and rotate the eye around to find a better spot for surgery without damaging the ocular muscles.

Gently lift the conjunctiva to make a tent, and rotate the eye nasally. Then use scissors to cut the top of the tent off, to make a small opening in the conjunctiva and expose the sclera. Next, use forceps to grip the edge of the conjunctiva opening next to the limbus, and rotate the eye nasally, so that the pupillary axis is at an angle of about 30 degrees relative to the ground.

A sterile beveled 31-gauge insulin needle is used to penetrate the sclera core complex with a bevel pointing up and half of the tip, or about 500 microns inserting into the tissue, to create a pilot hole for injection. Carefully withdraw the insulin needle, and a small effusion of blood may be seen. An eye spear can be applied to clear the hole and stop bleeding if necessary.

Guide the RPE cell loaded injector needle into the pilot hole at an angle of about 10 to 15 degrees relative to the local surface of the sclera to reach the subretinal space by inserting the needle with a length of 500 microns completely into the tissue and leaving a 100 micro distance between the point on the needle that is covered by the tissue and the point of the black marker on the needle. Tell the assistant to depress the plunger of the syringe, to inject the appropriate volume of cells. Hold the injector in place for 25 to 30 seconds after injection, then slowly retract the injector.

The injection hole seals automatically, and a small amount of about 0.2 microliters of backflow is commonly observed. Rinse the injection site with sterile eyewash three times, and collect the excess with a cotton applicator. Apply a drop of lubricant on the operated eye, and transfer the rat to the OCT station, to examine the location of transplanted cells, and the size of the subretinal bleb.

Return the rat to the recovery cage under a heat lamp to keep its body temperature once OCT is done. Observe the rat to ensure it comes out of anesthesia, and monitor daily for any signs of distress. Notify the veterinarian immediately if there are concerns.

Using our technique, we consistently and successfully delivered human RPE cells into the subretinal space. Immediately following transplantation, we performed OCT examination to observe the injection site and the subretinal bleb created by the transplanted cells. This serves as a good screening tool for evaluating the quality of injections and the degree of retinal damage, or hemorrhage.

Bright light reflection around the injection site and a subretinal bleb filled with the cell suspension could be seen under the OCT scanning. There is no bleeding, interocular fluid leakage, or retinal detachment, seen around the injection site or the intravitreal space in a successful injection, demonstrating the minimal trauma caused by injection. The operated rat eyes were nucleated, fixed, and sectioned for immunohistological analysis at seven days after transplantation.

The human nuclear marker, Hunu, and the RPE cell marker, OTX2, were used to detect the transplanted cells. A positive stating of both markers showed that the transplanted human RPE cells were located in the subretinal space of the rat eyes. This trans-scleral subretinal injection technique is simple and easy to learn.

It causes less surgery trauma and leaves the retina intact by passing the injection needle through the outer layers of the eye wall, without breaking the neural retina. The quantified drops for the pilot needle and injection needle to go in the tissue and the angle used during injection allow for delivery of cells reliably into the subretinal space with much higher accuracy and success rate. With this injection method, we consistently and successfully delivered human RPE stem cell derived RPE cells into the subretinal space of RCS rats.

This procedure can be easily repeated by anyone who watches the video.