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>

<ol>
	<li>Shepherd, R. K., Shivdasani, M. N., Nayagam, D. A., Williams, C. E., Blamey, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+prostheses+for+the+blind.">Visual prostheses for the blind.</a> Trends Biotechnol. 31, 562-571 (2013).</li><li>Santos, A., 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=Preservation+of+the+inner+retina+in+retinitis+pigmentosa.">Preservation of the inner retina in retinitis pigmentosa.</a> Arch Ophthalmol. 115, 511-515 (1997).</li><li>Humayun, M. 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. L., 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=Development+of+a+surgical+procedure+for+implantation+of+a+prototype+suprachoroidal+retinal+prosthesis.">Development of a surgical procedure for implantation of a prototype suprachoroidal retinal prosthesis.</a> Clin Experiment Ophthalmol. , (2013).</li><li>Lee, S. W., 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=Development+of+microelectrode+arrays+for+artificial+retinal+implants+using+liquid+crystal+polymers.">Development of microelectrode arrays for artificial retinal implants using liquid crystal polymers.</a> Invest Ophthalmol Vis Sci. 50, 5859-5866 (2009).</li><li>Sakaguchi, H., 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=Transretinal+electrical+stimulation+with+a+suprachoroidal+multichannel+electrode+in+rabbit+eyes.">Transretinal electrical stimulation with a suprachoroidal multichannel electrode in rabbit eyes.</a> Jpn J Ophthalmol. 48, 256-261 (2004).</li><li>Majji, A. B., 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=Long-term+histological+and+electrophysiological+results+of+an+inactive+epiretinal+electrode+array+implantation+in+dogs.">Long-term histological and electrophysiological results of an inactive epiretinal electrode array implantation in dogs.</a> Invest Ophthalmol Vis Sci. 40, 2073-2081 (1999).</li><li>Walter, P., 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=Successful+long-term+implantation+of+electrically+inactive+epiretinal+microelectrode+arrays+in+rabbits.">Successful long-term implantation of electrically inactive epiretinal microelectrode arrays in rabbits.</a> Retina. 19, 546-552 (1999).</li><li>Ray, A., Chan, L., Thomas, B., Weiland, J. 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X., 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=Chronic+Electrical+Stimulation+with+a+Suprachoroidal+Retinal+Prosthesis:+A+Preclinical+Safety+and+Efficacy+Study.">Chronic Electrical Stimulation with a Suprachoroidal Retinal Prosthesis: A Preclinical Safety and Efficacy Study.</a> PLoS One. 9, e97182 (2014).</li><li>Nayagam, D. A. X., 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=Techniques+for+Processing+Eyes+Implanted+With+a+Retinal+Prosthesis+for+Localized+Histopathological+Analysis.">Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis.</a> Journal of visualized experiments : JoVE. , e50411 (2013).</li><li>Laube, 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=Development+of+surgical+techniques+for+implantation+of+a+wireless+intraocular+epiretinal+retina+implant+in+Gottingen+minipigs.">Development of surgical techniques for implantation of a wireless intraocular epiretinal retina implant in Gottingen minipigs.</a> Graefe's Archive For Clinical And Experimental Ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie. 250, 51-59 (2012).</li><li>Gerding, H., 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=Successful+long-term+evaluation+of+intraocular+titanium+tacks+for+the+mechanical+stabilization+of+posterior+segment+ocular+implants.">Successful long-term evaluation of intraocular titanium tacks for the mechanical stabilization of posterior segment ocular implants.</a> Mat.-wiss. u. 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 border="0" cellpadding="0" cellspacing="0" width="853">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:337px;">Name of the reagent / equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:176px;">Catalogue number</td>
			<td style="width:151px;">Comments (optional)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">Acetone</td>
			<td style="width:191px;">Chem-Supply</td>
			<td style="width:176px;">AA008</td>
			<td style="width:151px;">Propanone BHD Medical grade</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">Epo-Tek 301 Epoxy</td>
			<td style="width:191px;">Epoxy Technology</td>
			<td style="width:176px;">Part A 1675-54-3 Part B 9046-10-0</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:337px;">Ethanol 70-75% v/v</td>
			<td style="width:191px;">Merck PTY LTD</td>
			<td style="width:176px;">4.10261</td>
			<td style="width:151px;">Alcohol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:337px;">Ethanol</td>
			<td style="width:191px;">Merck PTY LTD</td>
			<td style="width:176px;">90143</td>
			<td style="width:151px;">Alcohol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:337px;">Toluidine blue O</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:176px;">T3260</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:337px;">Ethylenediamine Tetraacetic Acid</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:176px;"></td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">TegraPol grinding/polishing machine</td>
			<td>Struers</td>
			<td>TegraPol-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">AccuStop specimen holder</td>
			<td style="width:191px;">Struers</td>
			<td style="width:176px;">Accustop</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light microscope</td>
			<td>Leica</td>
			<td>MZ16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective lens</td>
			<td>Leica</td>
			<td>2.0x Planapo Objective</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Microscope Camera</td>
			<td>Leica</td>
			<td>DFC-420C</td>
			<td></td>
		</tr>
		<tr height="21">
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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.

Watch this Scientific Journal Video about Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks at 

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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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

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

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 ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

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 ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Biotribological Testing and Analysis of Articular Cartilage Sliding against Metal for Implants

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are now available for the shoulder, hip, ankle and the first metatarsalphalangeal joint. While providing pain reduction and clinical improvement, progressive degenerative changes of the opposing cartilage are observed in many patients. The mechanisms leading to this damage are not fully understood. This protocol describes a tribological experiment to simulate a metal-on-cartilage pairing and comprehensive analysis of the articular cartilage. Metal implant material is tested against bovine osteochondral cylinders as a model for human articular cartilage. By applying different loads and sliding speeds, physiological loading conditions can be imitated. To provide a comprehensive analysis of the effects on the articular cartilage, histology, metabolic activity and gene expression analysis are described in this protocol. The main advantage of tribological testing is that loading parameters can be adjusted freely to simulate in vivo conditions. Furthermore, different testing solutions might be used to investigate the influence of lubrication or pro-inflammatory agents. By using gene expression analysis for cartilage-specific genes and catabolic genes, early changes in the metabolism of articular chondrocytes in response to mechanical loading might be detected.

This protocol describes the preparation, biotribological testing, and analysis of osteochondral cylinders sliding against metal implant material. Outcome measures included in this protocol are metabolic activity, gene expression and histology.

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...