Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses with hard metal components are not compatible with traditional histological processes. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinal with a metal T.This is accomplished by first nucleating and fixing the eye according to the protocol described in a companion video, and then dissecting a tissue sample, which includes the implant and the retinal T.The second step is to progressively dehydrate the tissue sample and then embed it in an epoxy resin in the desired orientation using a two step embedding process. Next, the final resin block is mounted in a special holder and ground down incrementally until a cross section through the implant and surrounding ocular tissue is exposed at the desired level.

The final step is to surface stain the exposed tissue, and then image and photograph with a high power dissection microscope to visualize the cellular architecture adjacent to the implanted prosthesis. These steps are then repeated several times as required. Ultimately, a series of cross-sectional images through the tissue sample, including the implant in situ, are collected and may be used to refine the design of the device or surgery, as well as assist in assessing the safety of the particular prosthesis.

The main advantage of this technique over existing methods like traditional blade based cutting histology, is that heart implanted objects, including metallic components, can remain in situ during the procedure, allowing visualization of the implant tissue interface. This method can answer key questions in the retinal prosthesis field, such as assessing the structural integrity of the delicate ocular tissue adjacent to an implanted retinal prosthesis. Following enucleation of the eye and fixation as described in a companion JoVE video, visualize the implant and the point where it is secured to the outside of the eye.

Using a 15 degree blade. Make a circumferential trans corneal incision and remove the corneal cap to reveal the inside of the eyeball. Note that the lens has been previously removed during the lensectomy vitrectomy, which is part of the surgical procedure for this epiretinal implant.

Then disin, insert the iris and any residual Z fibers of the lens to reveal the posterior chamber with the epiretinal implant and the metal T in situ. Depending on the implant and the study being performed, remove extraneous components before further dissection. In the present example, the epiretinal array being evaluated consists of a prototype hermetic, diamond electrode and electronics package housed within a conformal silicone carrier.

Carefully dissect a sample that includes the tack and surrounding tissue in the desired orientation. Use fine dissection scissors to cut full thickness strips from the back of the eye, including sclera, choroid, and retina. Here carefully extract the diamond electrode package by fine dissection with the scalpel.

Leave the silicone body of the implant along with the retinal tack and the remnants of the platinum wiring. Take a sample that gives a longitudinal cross-section of the tack showing all the retinal layers adjacent to the tack. Then return the sample to 70%Ethanol.

Dehydrate the sample over three days in progressive ethanol stages. First, dehydrate the sample in 70%ethanol for two hours, two times. Next, dehydrate the sample in 80%ethanol for two hours twice, and then overnight the next day dehydrate the sample in 90%Ethanol for two hours twice proceed to dehydrate the sample in 100%ethanol for two hours twice, and then overnight.

Finally, dehydrate the sample in 100%acetone for two hours twice. Remove the sample from the acetone and observe it under a light microscope as it air dries at room temperature. Removing liquid from the soft tissues results in collapse cells and shrinkage.

The sample will be transferred to epoxy just before its starts to curl and collapse. An appreciation of when the tissue curling and collapse occurs is developed with experience. To embed the sample in clear epoxy resin, immerse the eye tissue in liquified epoxy in an appropriate sealable container.

Gently degas the epoxy in a vacuum chamber and leave it overnight at room temperature to cure. Take care to embed the sample in the desired orientation by resizing the cured epoxy and sample with a bandsaw and reed The resized block containing the eye tissue in fresh liquified epoxy such that the long axis of the tack is oriented parallel to the bottom of the mold. Use a spot of super glue to secure the resize sample to the bottom of the sample holder.

Fill the sample holder with liquified epoxy and degas as before leave to cure overnight. Then mount the resin in a grinding specimen holder and manually grind the sample at 230 to 250 RPM with the water on using silicon carbide paper. For staining, dip the ground surface in toine blue stain for three to five minutes or until stain develops.

After rinsing the sample with tap water, image the ground surface of the specimen with a higher power dissection.Scope. To visualize the cellular layers of the retina, apply a drop of distilled water on the top surface of the epoxy just above the specimen. To smooth the diffraction at the air epoxy interface.

Use an optical fiber gooseneck light source to illuminate the sample. Repeat the grinding and imaging steps each time grinding away a preset thickness of sample. The minimum precise and reproducible grinding increment of specimen holder is 20 microns.

This process is repeated incrementally until a cross section through the implant and surrounding ocular tissue is exposed at the desired level.Shown. Here are example images of retinal tissue visualized immediately adjacent to a titanium retinal T at various points during the grinding process. The images show longitudinal sections of the tack penetrating through the eye and exiting the sclera adjoining silicone and toluidine blue stained and unstained retina.

Non artifactual retinal detachment and folding can be seen on either side of the silicone at both low and high power. The tack shaft is visible embedded in silicone, and the head of the tack has penetrated the retina and sclera. Here it is apparent that there is non artifactual retinal detachment in the unstained retina on either side of the silicone visualized at both low and high power.

The example has shown that there is retinal disorganization adjacent to the tack and compression of the retina at one side due to an oblique insertion angle. While attempting this procedure, it is important to remember to embed, orient, and reed the sample carefully, such that the resulting ground surfaces aligned in the desired plan. Furthermore, multiple photographs should be collected at each grinding stage as the ground samples cannot be recovered Today.

This method can provide insight into retinal prosthesis histology. It can also be applied to visualize the device tissue interface in other systems that utilize hard implanted components such as deep brain implants, vascular stents, or orthopedic prosthesis.