Nicole Vella (Macquarie University) for providing reagents; Alexia Saunder (Bionics Institute; BI), Michelle McPhedran (BI), Chris Williams (BI) for experimental support; the Royal Victorian Eye and Ear Hospital (RVEEH) Biological Research Centre staff for animal care; Sue Pierce (RVEEH) for veterinary advice; Anthony Burkitt (Bionic Vision Australia; BVA), Tamara Brawn (BVA) and the BVA staff for administrative support. 
This research was supported by the Australian Research Council (ARC) through its Special Research Initiative (SRI) in Bionic Vision Science and Technology grant to Bionic Vision Australia (BVA). The Bionics Institute receives Operational Infrastructure Support from the Victorian Government and also acknowledges support from the Bertalli Family Trust and the J T Reid Charitable Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The Bionic Vision Australia Consortia authors for this manuscript are (a-z): 
Penelope J. Allen, Owen Burns, Kate E. Fox, Kumaravelu Ganesan, David J. Garret, Hamish Meffin, Joel Villalobos, and Jonathan Yeoh.

NOTE: All procedures were approved by The Royal Victorian Eye and Ear Hospital&#8217;s Animal Research &#38; Ethics Committee (RVEEH AEC; #10-199AB). The subjects were treated according to the National Health and Medical Research Council&#8217;s &#8220;Australian Code of Practice for the Care and Use of Animals for Scientific Purposes&#8221; (2013) and the &#8220;Prevention of Cruelty to Animals Act&#8221; (1986; and amendments). All surgical, clinical assessment and electrophysiological procedures were carried out under anaesthesia and all efforts were made to minimize suffering.
1. Enucleation and Fixation
NOTE: Follow the enucleation and fixation procedure described in detail in the companion manuscript16, taking extra care around device cables or vitrectomy ports, if present. Briefly, this involves:
<ol>
	<li>Transcardially perfuse the subject with warm saline followed by cold neutral buffered formalin.</li>
	<li>Tie sutures to the eyeball to serve as landmarks.</li>
	<li>Enucleate the eye16, maintaining the device cables and any attachment patches/tabs.</li>
	<li>Post-fix the eye in Davidson&#8217;s solution for 18 - 36 hr.</li>
	<li>Transfer to 50% ethanol for 6 - 8 hr.</li>
	<li>Transfer to 70% ethanol and refrigerate (4 &#176;C) until dissection.</li>
</ol>
2. Electrode Removal and Dissection.
NOTE: Not all epiretinal implants will have the same form factor, but in general there will be an electrode array and some form of flexible and conformable carrier material. Devices that are tacked to the retina feature a tack hole where the metal tack penetrates the array and the back of the eye, keeping these together.
<ol>
	<li>Visualise the implant and the point where it is secured to the retina.
	<ol>
		<li>Using a 15 degree blade make a circumferential trans-corneal incision and remove the corneal cap to expose the underlying iris and lens.</li>
		<li>Using a 15 degree blade, disinsert the zonular fibres of the lens and remove the lens in toto, exposing the posterior chamber with the epiretinal implant and the metal tack in situ.</li>
	</ol>
	</li>
	<li>Depending on the implant and the study being performed, remove extraneous components before further dissection. 
	NOTE: In the present example, the epiretinal array being evaluated consisted of a prototype hermetic diamond electrode and electronics package housed within a conformal silicone carrier (produced in house; refer to 20).
	<ol>
		<li>Here, carefully extract the diamond electrode package by fine dissection with a scalpel. Leave the silicone body of the implant along with the retinal tack and the remnants of the platinum wiring (Figure 2). If an embedded array/housing is not part of the device design under investigation, then omit this step (2.2).</li>
	</ol>
	</li>
	<li>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. Take a sample, which gives a longitudinal cross section of the tack, showing all the retinal layers adjacent to the tack (Figure 2) 
	NOTE: The desired orientation for the sample may differ depending on the particular result desired. For example, if one wishes to examine the proximity of the tack damage to the optic disk then the optic disk should be included within the sample.</li>
</ol>
3. Dehydration, Embedding, Mounting, Grinding, Staining, and Imaging
<ol>
	<li>Dehydrate the sample over three days in progressive ethanol stages:
	<ol>
		<li>Dehydrate the sample in 70% ethanol for 2 hr twice.</li>
		<li>Dehydrate the sample in 80% ethanol for 2 hr twice and then O/N.</li>
		<li>Dehydrate the sample in 90% ethanol for 2 hr twice.</li>
		<li>Dehydrate the sample in 100% ethanol for 2 hr twice and then O/N.</li>
	</ol>
	</li>
	<li>Dehydrate the sample in 100% acetone for 2 hr twice.
	<ol>
		<li>Remove the sample from the acetone and observe it under a light microscope as it air-dries at RT. Transfer the sample to epoxy (refer to step 3.4) just before it starts to curl/collapse. 
		NOTE: Removing liquid from the soft tissues results in collapsed cells and shrinkage. An appreciation of when the tissue curling/collapse occurs is developed with experience.</li>
	</ol>
	</li>
	<li>Prepare medical grade epoxy resin according to manufacturer&#8217;s directions. To cure the resin, use either 55 &#176;C for 1 hr or 24 hr at RT. Place in a vacuum chamber for ~ 5 min at ~ 50 mbar for removal of air bubbles. Regulate the vacuum manually as an excessive vacuum will cause the epoxy mixture to boil and degassing will not occur effectively 
	NOTE: Perform step 3.3 concurrently with step 3.2.</li>
	<li>Embed the sample in clear epoxy resin. Immerse the eye tissue in degassed epoxy in an appropriate sealable container and leave O/N at RT to cure.</li>
	<li>Take care to embed the sample in the desired orientation (Figure 2) by resizing and re-embedding the cured epoxy block. Re-embed the resized block containing the eye tissue such that the long axis of the tack is oriented parallel to the bottom of the mould (yellow cup - Figure 3A).</li>
	<li>Mount the resin in a grinding specimen holder and grind the sample (230 - 250 rpm with water on, manual grinding) using silicon carbide paper (starting with 800 grid; Figures 3C and 3E).</li>
	<li>For staining, dip the ground surface in toluidine blue stain for 3 - 5 min, or until stain develops (Figure 3F).</li>
	<li>Rinse with tap water (Figure 3G).</li>
	<li>Image the ground surface of the specimen with a high power dissection scope to visualize the cellular layers of the retina (Figure 3H). Apply a drop of distilled water on the top surface of the epoxy above the specimen to smooth the diffraction at the air-epoxy interface. Use an optical fibre &#8216;gooseneck&#8217; light source to illuminate the sample.</li>
	<li>Repeat steps 3.6 &#8211; 3.9, each time grinding away a pre-set thickness of sample (minimum precise and reproducible grinding increment of specimen holder is 20 &#956;m).</li>
</ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+perception+in+a+blind+subject+with+a+chronic+microelectronic+retinal+prosthesis.">Visual perception in a blind subject with a chronic microelectronic retinal prosthesis.</a> Vision Res. 43, 2573-2581 (2003).</li><li>Roessler, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiographic+findings+folowing+tack+fixation+of+a+wireless+epiretinal+retina+implant+device+in+blind+RP+patients.">Angiographic findings folowing tack fixation of a wireless epiretinal retina implant device in blind RP patients.</a> Graefes Arch Clin Exp Ophthalmol. 249, 1281-1286 (2011).</li><li>Chow, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+artificial+silicon+retina+microchip+for+the+treatment+of+vision+loss+from+retinitis+pigmentosa.">The artificial silicon retina microchip for the treatment of vision loss from retinitis pigmentosa.</a> Arch Ophthalmol. 122, 460-469 (2004).</li><li>Zrenner, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+electronic+chips+allow+blind+patients+to+read+letters+and+combine+them+to+words.">Subretinal electronic chips allow blind patients to read letters and combine them to words.</a> Proc Biol Sci. 278, 1489-1497 (2011).</li><li>Fujikado, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+of+semichronically+implanted+retinal+prosthesis+by+suprachoroidal-transretinal+stimulation+in+patients+with+retinitis+pigmentosa.">Testing of semichronically implanted retinal prosthesis by suprachoroidal-transretinal stimulation in patients with retinitis pigmentosa.</a> Invest Ophthalmol Vis Sci. 52, 4726-4733 (2011).</li><li>Saunders, A. 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Werkstofftech. 32, 903-912 (2001).</li><li>Seo, J. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicon+retinal+tack+for+the+epiretinal+&#64257;xation+of+the+polyimide+electrode+array.">Silicon retinal tack for the epiretinal &#64257;xation of the polyimide electrode array.</a> Current Applied Physics. 6, 649-653 (2006).</li><li>Hadjinicolaou, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+of+retinal+ganglion+cells+with+diamond+and+the+development+of+an+all+diamond+retinal+prosthesis.">Electrical stimulation of retinal ganglion cells with diamond and the development of an all diamond retinal prosthesis.</a> Biomaterials. 33, 5812-5820 (2012).</li><li>Briggs, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+round+window+and+cochleostomy+approaches+with+a+prototype+hearing+preservation+electrode.">Comparison of round window and cochleostomy approaches with a prototype hearing preservation electrode.</a> Audiol Neurootol. 11, 42-48 (2006).</li><li>Shepherd, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+cochlear+implant+electrode+array+for+use+in+experimental+studies.">An improved cochlear implant electrode array for use in experimental studies.</a> Hear Res. 277, 20-27 (2011).</li><li>Tykocinski, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+electrode+position+in+the+human+cochlea+using+various+perimodiolar+electrode+arrays.">Comparison of electrode position in the human cochlea using various perimodiolar electrode arrays.</a> Am J Otol. 21, 205-211 (2000).</li><li>Hardie, N. 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Authors have nothing to disclose. Irfan Durmo is an employee of Cochlear Ltd.

Standard histological techniques are unable to process hard metal implants in situ due to limitations in cutting these objects with metal, glass or even diamond blades. In our companion paper16, we showed that the use of a modified whole-eye fixation technique could reduce artifactual retinal delamination. In the current manuscript, an established grinding and polishing technique for visualising cochlear implants21-23in situ was modified for retinal prostheses. A titanium tack, used to secure an electrode array to the retina, epiretinally, was embedded in epoxy along with the surrounding eye tissue. This resin block was then oriented appropriately and progressively ground/polished in order to reveal the tissue morphology immediately adjacent to the metal tack. Images of the polished surface of the block at various depths were taken with a powerful dissection microscope. This technique is useful for: visualising and evaluating the tissue response adjacent to the epiretinal implant; to assess the surgical trauma associated with implantation of the implant; to determine the biological reaction to the hard metal components; and to measure the distance between the implant and the surface of the retina.
This technique will be useful in future safety studies for in situ visualisation of the region adjacent to a retinal tack or other hard (e.g., metallic) objects in the eye. This has direct application in assessing the preclinical safety of prostheses tacked to the retina epiretinally. It may also be useful for evaluating tissue damage in retinal regions in contact with implants located in the sub-retinal location.
There are several ways to verify that the technique has been performed correctly. At each stage, the retina should remain attached to the outer layers of the eye. If there is gross artifactual retinal detachment, this may indicate a problem with the fixation. When the sample is embedded and re-orientated in the final resin block the retina should be close to orthogonal with the grinding-face of the block; this will minimize oblique cutting. It is useful to check that the number of incremental grinding steps (of known step size) required to traverse an object (such as a retinal tack) correlate accordingly with the dimensions of the object.
The technique can be optimised in several ways. Scratches on the surface of the epoxy block associated with the grinding process can be reduced with progressively finer grade polishing. For the present study, we used 800, 1000, 1200, 2400, and 4000 grade silicon carbide paper. Diamond paste could also be used to improve the surface finish. A finer surface finish gives a higher quality image but at the cost of additional polishing time. Another critical consideration for improving the outcome of this technique is the choice and quality of the optics and lighting used for the image capture. Other basic histological stains &#8211; particularly Nissl stains, can be used in place of toluidine blue, but may require further optimisation. Some stains will stain the resin as well as the tissue (e.g., Eosin), therefore a shallow polish may be required after the staining to remove background discolouration. Specialised stains, fluorescent dyes and immunohistochemical staining was not attempted, but unless a very specific result is desired, the time required to perform these stains at each grinding level is likely to be prohibitive. However, it may be possible to stain the tissue as a whole before the embedding step (step 3.4)24.
The main limitation of this technique is that once the region of interest has been ground away, it cannot be retrieved, therefore, it is prudent to capture many (possibly redundant) images at a variety of magnifications at each stage of grinding and polishing. It is also important to use small increments for each grinding depth adjustment. Another limitation of this technique is that of the optical magnification and resolution compared with tissue mounted on a glass slide and viewed with a standard (transmission) light microscope. For the purposes of prototyping and assessing the safety of a novel implant device, the gross pathological evaluation is of primary interest. This technique provides an efficient method for observing clinically relevant damage associated with a retinal tack. With practice, the overall time required to gather grind, polish and photograph a given specimen (once embedded) is comparable to the time it would take to section a paraffin block or frozen section.
There is also the potential for the present techniques to be extended to applications outside the scope of retinal implants. This technique is suited for evaluating the tissue adjacent to a hard implant, where implant extraction is not feasible or would damage the interface. For example, this technique could be expanded to evaluate implants made from metal (e.g., platinum, nitinol, etc.) that cannot be cut with conventional histological techniques, such as some deep brain or peripheral nerve electrodes, cannulae for drug delivery, vascular stents or orthopaedic prostheses.

Retinitis pigmentosa (RP) is a hereditary disorder that causes widespread loss of photoreceptors, which are the cells in the outermost layer of the retina responsible for transducing light, in the form of photons, into neural activity. Importantly, patients with RP typically have residual neurons in the other layers of their retina which are still functional. Retinal prostheses are capable of restoring some limited vision to these patients by targeting these surviving neurons with electrical stimulation to activate their visual pathway1,2. Perceptual outcomes from clinical trials have shown promising early results and recently some devices have been approved for commercial use. Currently, there are three main anatomical locations in which clinical retinal prostheses have been positioned: epiretinally3,4, subretinally5,6 and suprachoroidally7,8. Different devices utilise different materials and their form is customised to the location in which they are implanted. However, they all create visual percepts by activating the residual neurons of the retina with electrical pulses.
There is the potential for any medical prosthesis to damage surrounding tissue due to mechanical effects of the initial placement or subsequent ongoing forces. In the case of implantable stimulators, such as retinal prostheses, there is the additional consideration that the electrical parameters should be within safe limits. Patient safety is paramount, so devices must be rigorously tested in preclinical studies before advancing to a clinical setting9-15. In our companion article, we described a method for assessing the localized histopathology of the eye surrounding an implant positioned in the suprachoroidal space16. In the present manuscript, we describe a technique for visualising eye tissue surrounding an electrode array tacked to the retina epiretinally, in a preclinical (feline) model (Figure 1).
The epiretinal location is the most commonly utilised position for locating a visual prosthesis. Electrode arrays situated here are typically affixed to the retina with a metal tack that penetrates all the layers of the eye17-20. Prior to the techniques described in the present manuscript, it was difficult to accurately assess the retinal and other tissues immediately surrounding a tack. Standard eye fixation using neutral buffered formalin resulted in artifactual retinal damage due to the differential movement of the retina and sclera against the fixed point of the tack. Therefore any actual damage caused by the tack and epiretinal array could not be accurately observed. In addition, sectioning the eye tissue could not be performed with the retinal tack in situ as metal objects cannot be readily cut with traditional histological apparatus; removing the tack before histological processing was also undesirable as this also led to artifactual retinal damage.
The aim of the present study was twofold: 1) to reduce retinal detachment artefact so that any damage caused by the tack and the epiretinal implant array can be reliably assessed; and 2) to visualise the retinal architecture adjacent to the tack without removing it. In order to achieve aim 1, a new fixation technique was utilised (as described in the companion article16), which reduces artifactual retinal delamination. In order to achieve aim 2, we modified an embedding, grinding, and polishing technique, originally developed for in situ observation of cochlear implant electrodes21-23. The methods described in this manuscript allow the visualisation of the retina surrounding and adjacent to a tack in situ while minimizing artifactual retinal damage and therefore allowing accurate assessment of any potential damage caused by the tack and epiretinal array.

<table><tbody><tr><td>Acetone</td><td>Chem-Supply</td><td>AA008</td><td>Propanone BHD Medical grade</td></tr><tr><td>Epo-Tek 301 Epoxy</td><td>Epoxy Technology</td><td>Part A 1675-54-3 Part B 9046-10-0</td><td></td></tr><tr><td>Ethanol 70-75% v/v</td><td>Merck PTY LTD</td><td>4.10261</td><td>Alcohol</td></tr><tr><td>Ethanol</td><td>Merck PTY LTD</td><td>90143</td><td>Alcohol</td></tr><tr><td>Toluidine blue O</td><td>Sigma-Aldrich</td><td>T3260</td><td></td></tr><tr><td>Ethylenediamine Tetraacetic Acid</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>TegraPol grinding/polishing machine</td><td>Struers</td><td>TegraPol-25</td><td></td></tr><tr><td>AccuStop specimen holder</td><td>Struers</td><td>Accustop</td><td></td></tr><tr><td>Light microscope</td><td>Leica</td><td>MZ16</td><td></td></tr><tr><td>Objective lens</td><td>Leica</td><td>2.0x Planapo Objective</td><td></td></tr><tr><td>Digital Microscope Camera</td><td>Leica</td><td>DFC-420C</td><td></td></tr><tr><td>Microscope Software</td><td>Leica&amp;nbsp;</td><td>Application Suite v4.1.0</td><td></td></tr></tbody></table>

techniques for processing eyes implanted with a retinal prosthesis for localized histopathological analysis: part 2 epiretinal implants with retinal tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

The fixation protocol substantially reduced artifactual detachment and delamination of the retina16. Orientation of the specimen within the epoxy block was achieved consistently using the described two-step embedding process. The incremental grinding procedure required a moderate level of manual dexterity to achieve optimum results, but was aided by the adjustable specimen holder which provided fine control over the increment resolution. In all cases (n = 5) the tack was located and ground/polished with desirable and consistent results. The retinae adjacent to the tacks were resolvable and stained appropriately. Polishing the surface of the epoxy resin block with a grade P#800 silicon carbide paper was sufficient to image the cellular macrostructure of the embedded tissue. Higher grade paper, or diamond slurry may be used to further polish the surface at any given depth if desired. A dissection microscope and optical fiber &#8216;gooseneck&#8217; light source was found to be suitable for imaging the surface of the ground block and the embedded tissue sample. The position of the light source was varied by trial and error to find a location and angle that gave the best illumination and contrast through the microscope. Adding a drop of distilled water to the surface of the block, above the sample, was useful to reduce light path diffraction and/or smooth distortions at the epoxy air interface. Figure 4 shows example images of retinal tissue visualised immediately adjacent to a titanium retinal tack using this technique. Non-artifactual retinal detachment and folding can be seen on either side of the silicone (Figure 4A). The tack shaft is visible embedded in silicone; the head of the tack has penetrated the retina and sclera. There is non-artifactual retinal detachment in the unstained retina either side of the silicone (Figure 4C). The technique has shown that, in this instance, there is retinal disorganisation adjacent to the tack and compression of the retina at one side due to an oblique insertion angle. Note that the images presented are merely illustrations of the success of the technique, not representative of tack-damage histopathology in general.
<img alt="Figure 1" src="/files/ftp_upload/52348/52348fig1highres.jpg" /> 
Figure 1.&#160;Placement of an epiretinal electrode array.&#160;(A) Schematic diagram of the eye showing a magnified cross-section of the posterior sclera, choroid and degenerate retina (lacking photoreceptors). An electrode array is depicted in blue, affixed epiretinally. (B) Computer-aided-drawing of an epiretinal electrode array. a, integrated-circuit (&#8216;chip&#8217;) and electrode package; b, titanium retinal tack; c, medical grade silicone housing; d, lead exit point. Panel A modified from an original illustration provided courtesy of Bionic Vision Australia, copyright Beth Croce. <a href="https://www.jove.com/files/ftp_upload/52348/52348fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52348/52348fig2highres.jpg" /> 
Figure 2. Electrode removal and dissection of the eye.&#160;High dynamic range macro photographs of an enucleated feline eye with epiretinal tack in situ. (A) Post fixation with Davidson&#8217;s fixative to preserve retinal architecture16, the enucleated eye was dissected. The electrode array package was removed from the silicone carrier (dashed square outline indicates original location of electrode array) with the tack (arrow) and silicone body of the implant remaining. (B) The eye was dissected with the longitudinal cross section adjacent to the tack (dashed line). The tack remains embedded in the posterior wall of the eyecup (arrow), stabilised predominantly by the sclera. The tissue section containing the tack was prepared for resin embedding and grinding (right segment), while the section containing the retina beneath the removed electrode array was prepared for standard histological processing16 (left segment). A ruler with 0.5 mm increments is shown at the bottom edge of each panel. <a href="https://www.jove.com/files/ftp_upload/52348/52348fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52348/52348fig3highres.jpg" /> 
Figure 3.&#160;Epoxy embedment and grinding of the tack and retinal tissue. Photographs of the epoxy embedded and aligned tissue sample, grinding, staining and imaging of the tack and retinal tissue. (A) The tack sample was embedded in an epoxy resin block. Using the mould, the sample was oriented so the longitudinal plane is parallel to the bottom of the mould. (B) The cured epoxy block containing the tack sample. (C) The epoxy block was mounted in a grinding specimen holder, ready for grinding. (D) The sample was orientated so imaged sections of the ground tissue contain the retinal cellular layers and longitudinal axis of the tack. (E) The sample was ground using silicon carbide paper on a rotary grinder. (F) The ground surface of the block was stained with toluidine blue to identify the retinal layers. (G) The block was rinsed in tap water to remove excess stain. (H) The toluidine blue stained retinal layers and tack were imaged using a high powered dissection scope.
<img alt="Figure 4" src="/files/ftp_upload/52348/52348fig4highres.jpg" /> 
Figure 4.&#160;High and low power images of the tack and retina. At various points during the grinding process images were taken with a dissecting scope, showing longitudinal sections of the tack (asterisk) penetrating through the eye and exiting the sclera (&#8216;S&#8217;), adjoining silicone (hash) and toluidine blue stained and unstained retina (&#8216;R&#8217;). Note these are example images only to demonstrate the present visualisation technique, and are not representative of all epiretinal implant or tack insertion histology results. Ground remnants of the platinum wiring are visible in panels A-D (&#8216;W&#8217;). (A) Low power, unstained image of the tack penetrating the retina and sclera. (B) High powered image of the retinal detachment and folding in unstained retina adjacent to the silicone carrier in image A. (C) Low power image at the centre of the tack shaft. (D) High power image of the centre of the tack in image C. (E) In a separate sample, with no silicone carrier, a high powered image of toluidine blue stained retina beneath the tack hilt is shown, immediately prior to grinding the tack shaft. (F) Normal toluidine blue stained retinal architecture (GCL: retinal ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; PR: photoreceptors; T: feline tapetum lucidum) visualised using the same grinding technique. Scale bars in each panel are: A and C = 2 mm; B and D = 500 &#181;m; E = 200 &#181;m; F = 100 &#181;m.

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

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

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Research

JoVE Journal

Medicine

11.5K Views.  Bionics Institute. Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis. 

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

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

With  the recent development of retinal prostheses, it is important to develop  reliable techniques for assessing the safety of these devices in ...

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

Genetics

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Biology

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

Neuroscience

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Development of a Surgical Technique for Subretinal Implants in Rats

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been undertaken to develop regenerative medicine-based therapies for AMD, including stem cell-based therapies. Rodents as animal models for retinal degeneration are a foundation for translational research, due to the broad spectrum of strains that develop retinal degeneration diseases at different stages. However, mimicking human therapeutic delivery of subretinal implants in rodents is challenging, due to anatomical differences such as lens size and vitreous volume. This surgical protocol aims to provide a guided method for transplanting implants into the subretinal space in rats. A user-friendly comprehensive description of the critical steps has been included. This protocol has been developed as a cost-efficient surgical procedure for reproducibility across different preclinical studies in rats. Proper miniaturization of a human-sized implant is required prior to conducting the surgical experiment, which includes adjustments to the dimensions of the implant. An external approach is used instead of an intravitreal procedure to deliver the implant to the subretinal space. Using a small sharp needle, a scleral incision is performed in the temporal superior quadrant, followed by paracentesis to reduce intraocular pressure, thereby minimizing resistance during the surgical implantation. Next, a balanced salt solution (BSS) injection through the incision is carried out to achieve focal retinal detachment (RD). Lastly, insertion and visualization of the implant into the subretinal space are conducted. Post-operative assessment of the subretinal placement of the implant includes imaging by spectral domain optical coherence tomography (SD-OCT). Imaging follow-ups ascertain the subretinal stability of the implant, before the eyes are harvested and fixated for histological analysis.

The present protocol describes the scleral approach for subretinal device implantation, a feasible surgical technique for implementation in animal models of retinal diseases in research.

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

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

Summary

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Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

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Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

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