The overall goal of this procedure is to improve the evaluation of retinal prosthesis safety. This is accomplished by first labeling the surface of an optimally fixed enucleated eye with tissue dyes to indicate the position of an implanted electrode. In the second step, the electrode array is removed and the eye tissue is dissected into strips.
Next, the strips are stabilized in agar and then embedded in paraffin. In the final step, the marked areas of interest are sectioned and collected on slides for staining. Ultimately, the health of the implanted areas adjacent to each electrode can be evaluated by brightfield and immunofluorescence microscopy.
The main advantage of this technique is that fixation related artifact and delamination can be minimized while electrode adjacent tissue regions can be accurately tracked in large samples. It can also be used in evaluating the safety of new implants for other eye diseases. Generally, individuals new to this method will find it challenging because a retina is prone to delamination and a high level of dexterity is required for handling the samples.
Visual demonstration of this method is important because marking and handling the fragile tissue is difficult to learn. Prior to this study, after implantation with a suprachoroidal electrode array, the eyes were previously processed using a non-optimal technique as indicated by the star. This first image shows a representative orthogonal section through the electrode array cavity.
Note that the retina is detached from the outer eye tissue. This is particularly evident beneath the implanted region as indicated by the arrowhead. Note also that it is not possible to determine which portion of the retina was adjacent to each individual electrode of the array.
In this higher magnification, there are several regular release based artifacts in the retinal layers as indicated by the arrowheads, as well as major detachment from the torpedo layer, the reflective layer in the feline eye. Upon further magnification, it can be observed that the outer segments of the photoreceptors, as well as the pigmented epithelium, however, remain intact, suggesting that previously observed detachments or artifactual side effects of the processing as opposed to resulting from in vivo trauma or pathology. Thus, the following technique was implemented to minimize these fixation related artifacts and delamination.
After nucleating and fixing the eyes begin by removing an implanted eye globe from ethanol and carefully trimming away the excess tissue, including the conjunctiva and tendon's capsule. Trim the optic nerve to a two to three millimeter length. The electrode array is visible through the semi translucent sclera.
Then use a fine tipped paintbrush to carefully apply histological dyes to the tissue to label the electrodes with a predefined color code. Taking care not to smudge the dye. Extra dye marking can be made to define points of interest or help to align sections.
Here, the maximally stimulated electrodes have been labeled with green. The other active electrodes have been labeled with red. The larger diameter return electrodes have been colored yellow, and any additional guides or anatomical markings have been labeled with blue dye.
After five minutes of air drying, return the globe to the 70%ethanol to dehydrate the dye. Then after trimming the excess tissue from the unplanted control eye globe as just demonstrated, use the eight nylon sutures attached during the nucleation and fixation step to align the control and implanted eyes as a mirrored pair. Then place a silicone template with the same dimensions as the electrode array onto a mirrored location on the control I corresponding to the location of the array in the implanted eye.
Then label the control globe as just demonstrated so that each implanted electrode site has a control pair in an anatomically comparable location. Next, remove the implant array from the eye and then remove the front of the eye, including the cornea, iris, and lens. Next, remove the vitreous fluid from the remaining eye cup.
Now dissect the implanted eye into multiple two millimeter thick strips. Each containing a subset of the die marked regions. The orientation of the strips should be selected to assist in the assessment of various aspects of the tissue response to the implant, carefully document the position of the die markings on the samples for future reference.
Next place the strip with the side to be cut first facing downwards into a shallow pool of liquified agar. Once the agar has set, cut around the sample and place it into a tissue cassette supported by foam inserts. After dissecting and embedding the control eye, place the cassettes in neutral buffered formin and process them overnight via a standard automated 12 hour paraffin processing technique.
The next day, embed the processed tissue in paraffin with the side to be cut first facing downwards. Now, cut the paraffin blocks into five micron sections. Then mount the sections from each of the dyed regions onto slides.
In order to locate the DY region regularly check the slides under a microscope. The regions immediately adjacent to each dyed spot of the sclera will be those that were in closest proximity to the corresponding electrode from the array. Finally, stain the sections as desired.
High dynamic range. Macro photographs of an enucleated feline eye with a suprachoroidal electrode array in situ are shown after fixation with davidson's fixative and prior to array removal. The translucent sclera enables visualization of the individual electrodes in the array.
As indicated with the arrowhead, the electrodes are marked with a predefined dye color scheme. These dye markings placed with regular spacing from anatomical landmarks are used to maintain consistency across experiments. Upon removal of the electrode array, the eye is dissected by hand into sample strips.
The arrowheads indicate two of the electrode adjacent sites on the sclera. The dashed line indicates the plane of sectioning in this representative section obtained from cutting the sample from the previous figure, the gross shape of the sample strip was preserved with minimal differential tissue artifacts. Both dye markings are still visible on the sclera as indicated by the arrowheads.
Although the green dye was more resilient than the red, the location of the dye indicates the position of an electrode. Therefore, the adjacent retinal tissue can be assessed for the damage, if any, caused by electrical stimulation as seen at this magnification, the retinal layers adjacent to the green dye seen in the previous image were not factually detached, and the retinal morphology was preserved here. Representative special staining and immunohistochemistry of retinal tissue sections processed according to the current protocol are shown in this first image.
Hematin stained the cell nuclei blue while the eoin stained the cell cytoplasm, collagen and supportive tissue, various shades of pink In this image, the luxal fast blue stained the myelin blue. The crestal violet counter stain was used to identify the ganglion cells by staining the missile bodies within the peric blue and by highlighting the surrounding satellite cells. In this mason's trione blue treated section, the BRIC Scarlet acid fusion solution stained the muscle fibers red while the lin blue stained the collagen.
In this case, the sclera blue for this section stained by periodic acid shiff the glycoprotein components of the basement membrane and the connective tissue components appear purple while the hemat toin counterstain cell nuclei appear blue. The sub scleral section shown in this image was treated with pearl's Prussian blue at the site of hemorrhage. The formation of hemosiderin from degraded red blood cells and the release of iron complexes produced a purple color.
The addition of neutral red stained the lysosomes red in this image, anti glutamine Synthes was stained. This neurotransmitter degrading enzyme found in Mueller cells in green extends in both directions with the Mueller cell bodies within the inner nuclear layer and the Mueller cell end feed forming the inner limiting membrane for staining of the neurofilament protein. This heavy chain cytoskeletal protein was labeled green found in the cell soma and processes the neurofilament protein cross-links with other neurofilaments to maintain the structure of neurons.
Ganglion cells and their axons can be observed in the ganglion cell and nerve fiber layers while the axons of the horizontal cells located at the outer border of the inner nuclear layer in the feline retina appear green. In this final image, glial fibrillary acidic protein is shown this cytoskeletal protein in red, proliferates in Mueller cells and astrocytes during gliosis and can be seen lining the nerve fiber layer with Mueller cells forming thin extensions through the inner to the outer retinal layers. Counter staining with a DPI in blue allows for visualization of the retinal layers.
While attempting this procedure, it's important to remember to visualize a sample workflow in three dimensions and to regularly consider the orientation of the samples throughout the procedure. Following this procedure, all the histological methods can be used in order to answer additional safety related questions After its development. This technique paved the way for researchers in bionic eye development to assess the safe stimulation levels long-term for blind patients.
After watching this video, you should have a good understanding of how to evaluate the safety of a bionic eye implant.
