Name: enrichment of bruch's membrane from human donor eyes
Uploaded: 2015-11-15T14:00:00
Duration: 10 min 22 s
Description: Watch this Scientific Journal Video about Enrichment of Bruch's Membrane from Human Donor Eyes at JoVE.com

The Authors would like to acknowledge Dr. Isaac Zambrano and the staff at the Manchester Eye Hospital Eye Bank for the supply of human donor eye tissue, and Mr. Pete Walker of the Faculty of Life Sciences Histology facility for the H&#38;E stained images. Special thanks goes to Mr. Roger Meadows for his help with the microscopy. SJC is a recipient of a Medical Research Council (MRC) Career Development Fellowship (MR/K024418/1) and the authors also acknowledge other recent research funding from MRC (G0900538 and&#160;K004441), Fight for Sight (1866) and The Macular Society.

Human eye tissue consented for research purposes was obtained from the Manchester Royal Eye Hospital Eye Bank following the removal of the cornea for transplantation. The use of human tissue for research was in accordance with the Human Tissue Act (2004) guidelines, and with University Research Ethics Committee approval (reference 11305).
1. Enrichment of Whole Bruch&#8217;s Membrane
<ol>
	<li>Clean the workspace around and underneath the dissecting microscope with 1% disinfectant (v/v) and then wipe down with 70% (v/v) ethanol.</li>
	<li>Ensure the following are to hand: one new disposable scalpel, two sterile cell scrapers (commonly used in tissue culture) and three pairs of tweezers (listed here as A, B, and C, see materials list).</li>
	<li>Place the eye globe into a disposable petri-dish, retaining the lid for later use. Using a new disposable scalpel, remove any residual optic nerve, which may interfere with creating a flat mount of the tissue. Next, make four incisions equally spaced apart to create four quadrants, if proceeding to part 3 (enrichment of macular Bruch&#8217;s membrane) do not cut through the macula region.
	<ol>
		<li>Ensure that the incisions extend through the entire depth of the eye tissue &#8211; from the innermost neurosensory retina (NSR), through the choroid and sclera (white of the eye). Continue the incision all the way round the eye from the margin of the eyecup to the optic nerve. This will allow the eyecup to be flattened out (see Figure 1B).</li>
	</ol>
	</li>
	<li>Holding one quadrant of flattened eyecup using tweezer A (to prevent choroid detachment), use the larger tweezer B to grip the vitreous and underlying NSR and pull this tissue away from the retinal pigment epithelium (RPE) starting at the periphery, moving towards the center of the opened eyecup and then to the opposing side. Usually, but not always both vitreous and NSR detach as one. Use the scalpel to excise any residual NSR anchored at the optic disc.</li>
	<li>Again, whilst holding one quadrant of tissue in place using tweezer A, use the sterile cell scraper to gently dislodge the RPE monolayer from the entire inside surface of the eyecup. These cells readily detach and can be seen as dark brown-pigmented clumps. Rinse with PBS to gently wash away dislodged RPE cells from the eyecup.</li>
	<li>Next, use tweezer A to gently peel off the overlying dark red tissue (comprising both Bruch&#8217;s membrane and the choroid) from all 4 eye quadrants. Place the excised tissue into a clean dish.</li>
	<li>Using the flattened edge of one cell scraper to hold the excised tissue in place, use the second scraper to gently scrape the tissue. Turn the tissue over and repeat, removing as much excess material as possible. Eventually, a translucent membrane will remain: this is the crudely enriched Bruch&#8217;s membrane. The use of the dissecting microscope at a minimum of 20X magnification is essential in aiding the removal of choroid and enrichment of Bruch&#8217;s membrane.</li>
	<li>Using a Pasteur pipette, briefly rinse the Bruch&#8217;s membrane with ultrapure water to remove residual choroid and blood before placing the membrane into a 1.5 ml microcentrifuge tube.</li>
	<li>Suspend the enriched Bruch&#8217;s membrane in approximately 1 ml ultrapure water and vortex briefly (approximately 15 sec) to help lyse any contaminating cellular matter (see Figure 2). Centrifuge the sample at 500 x g for 30 sec&#160;to pellet the membrane and aspirate off the supernatant. 
	Note: The remaining enriched Bruch&#8217;s membrane is now ready for solubilization (as described in section 2 below) or storage at -80 &#176;C for use later.</li>
</ol>
2. Solubilization of Whole Enriched Bruch&#8217;s Membrane and Western Blot Analysis
<ol>
	<li>Extract protein from enriched Bruch&#8217;s membrane by immersing the tissue in 500 &#181;l 8 M urea for 6 hr&#160;at RT&#160;in 1.5 ml microcentrifuge tubes ( Figure 3A).</li>
	<li>Centrifuge solubilized samples at 10,000 x g for 10 min to pellet any remaining extracellular matter. Remove the supernatant and transfer it to 3,500 Da MWCO dialysis membrane, then place the sample in 1 L of PBS to dialyse for 16 hr&#160;at 4 &#176;C.</li>
	<li>For each sample, add 40 &#181;l of protein isolation beads, and rotate sample/bead mix (20 rpm on a rotating mixer) at RT for 30 min.</li>
	<li>Centrifuge samples at 10,000 x g for 5 min to pellet the beads. Remove supernatant and solubilize protein bound to beads in 30 &#181;l 2x sample loading buffer (65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, 0.01% bromophenol blue, 3% &#946;-mercaptoethanol).</li>
	<li>Incubate the samples at 100 &#176;C for 10 min, and then centrifuge at 10,000 x g for 5 min to pellet the remaining beads.</li>
	<li>Load the supernatant that contains the released proteins on to a pre-cast SDS-page gradient gel (see materials list) for protein separation.</li>
	<li>Visualize separated protein bands using a common staining protocol, e.g., Coomassie blue.
	<ol>
		<li>In brief, add 20 ml of the brilliant blue stain (0.1% brilliant blue (w/v), 50% methanol, 10% glacial acetic acid, 40% water) to the gel, and incubate for 30 min at RT&#160;with gentle agitation.</li>
		<li>Discard the staining solution and wash the stained gel with deionized water until background staining is sufficiently reduced to allow stained protein band visualisation.</li>
		<li>Alternatively, transfer separated protein bands to nitrocellulose for western analysis, as described below.</li>
	</ol>
	</li>
	<li>Transfer separated protein bands onto nitrocellulose membrane at 80 mA for 2.5 hr using semidry transfer apparatus in transfer buffer (25 mM Tris, 192 mM glycine, 10% [v/v] methanol).</li>
	<li>Block the membrane in 10 ml PBS, 10% (w/v) milk, and 0.02% (w/v) BSA for 16 hr at 4 &#176;C before the addition of the desired primary antibodies for protein detection. 
	Note: As an example here, Figure 3C shows a representative immunoblot of solubilised Bruch&#8217;s membrane from three separate donors probed for a regulator of the complement cascade, factor H-like protein 1 (FHL-1).</li>
</ol>
3. Enrichment of Macular Bruch&#8217;s Membrane
<ol>
	<li>Follow steps 1.1 - 1.3 as previously described.</li>
	<li>Whilst the NSR is still in place, locate the yellow foveal colouration that represents the central macula region (see Figure 4). Using a sterile 6 mm biopsy punch, place it above the foveal region and firmly cut in to the macula.
	<ol>
		<li>Keeping the biopsy punch in place, make a separate incision with a scalpel from the edge of the eyecup to the edge of biopsy punch blade, and then using tweezer B, peel away the surrounding eye material to achieve a clean biopsy.</li>
	</ol>
	</li>
	<li>Using tweezer C, remove the 6 mm disc of tissue from the punch and place on the clean petri dish lid. Place the petri dish lid and macular punch under the dissecting microscope.</li>
	<li>Remove the thin NSR disc (check for yellow foveal staining to infer accuracy of biopsy). Use a sterile cell scraper to gently remove the RPE cells.</li>
	<li>Secure the macular disc via the sclera using the tips of tweezer A, and slowly peel off the choroid and Bruch&#8217;s membrane complex using tweezer C. Again, use the cell scraper to remove unwanted choroid and blood matter from Bruch&#8217;s membrane.</li>
	<li>Place the macular Bruch&#8217;s membrane into a 1.5 ml microcentrifuge tube and suspend in 1 ml ultrapure water. Vortex briefly (approximately 15 sec) to help lyse any contaminating cellular matter. Centrifuge the sample at 500 x g for 30 sec to pellet the membrane and aspirate off the supernatant. 
	Note: The enriched Bruch&#8217;s membrane isolated from the macula can now be used for proteomic analysis using the user&#8217;s preferred digestion protocols.</li>
</ol>

<ol>
	<li>Coleman, H. R., Chan, C. -. C., Ferris, F. L., Chew, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration.">Age-related macular degeneration.</a> Lancet. 372, 1835-1845 (2008).</li><li>Wong, W. 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=Global+prevalence+of+age-related+macular+degeneration+and+disease+burden+projection+for+2020+and+2040:+A+systematic+review+and+meta-analysis.">Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: A systematic review and meta-analysis.</a> Lancet Glob Health. 2 (2), e106-e116 (2014).</li><li>Coleman, 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=Impact+of+age-related+macular+degeneration+on+vision-specific+quality+of+life:+Follow-up+from+the+10-year+and+15-year+visits+of+the+study+of+osteoporotic+fractures.">Impact of age-related macular degeneration on vision-specific quality of life: Follow-up from the 10-year and 15-year visits of the study of osteoporotic fractures.</a> Am J Ophthalmol. 150 (5), 683-691 (2010).</li><li>Hageman, G. 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=A+common+haplotype+in+the+complement+regulatory+gene+factor+H+(HF1/CFH)+predisposes+individuals+to+age-related+macular+degeneration.">A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration.</a> Proc Natl Acad Sci U S A. 102 (20), 7227-7232 (2005).</li><li>Klein, 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=Complement+factor+H+polymorphism+in+age-related+macular+degeneration.">Complement factor H polymorphism in age-related macular degeneration.</a> Science. 308 (5720), 385-389 (2005).</li><li>Haines, J. 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=Complement+factor+H+variant+increases+the+risk+of+age-related+macular+degeneration.">Complement factor H variant increases the risk of age-related macular degeneration.</a> Science. 308 (5720), 419-421 (2005).</li><li>Edwards, A. O., Ritter, R., Abel, K. J., Manning, A., Panhuysen, C., Farrer, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+factor+H+polymorphism+and+age-related+macular+degeneration.">Complement factor H polymorphism and age-related macular degeneration.</a> Science. 308 (5720), 421-424 (2005).</li><li>Fritsche, L. 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=Seven+new+loci+associated+with+age-related+macular+degeneration.">Seven new loci associated with age-related macular degeneration.</a> Nat Genet. 45 (4), 433-439 (2013).</li><li>Clark, S., Bishop, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Factor+H+and+Related+Proteins+in+Regulating+Complement+Activation+in+the+Macula,+and+Relevance+to+Age-Related+Macular+Degeneration.">Role of Factor H and Related Proteins in Regulating Complement Activation in the Macula, and Relevance to Age-Related Macular Degeneration.</a> J Clin Med. 4 (1), 18-31 (2014).</li><li>Booij, J. C., Baas, D. C., Beisekeeva, J., Gorgels, T. G. M. F., Bergen, A. A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+nature+of+Bruch&#8217;s+membrane.">The dynamic nature of Bruch&#8217;s membrane.</a> Prog Retin Eye Res. 29 (1), 1-18 (2010).</li><li>Clark, S. 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=Identification+of+factor+H-like+protein+1+as+the+predominant+complement+regulator+in+Bruch&#8217;s+membrane:+implications+for+age-related+macular+degeneration.">Identification of factor H-like protein 1 as the predominant complement regulator in Bruch&#8217;s membrane: implications for age-related macular degeneration.</a> J Immunol. 193 (10), 4962-4970 (2014).</li><li>Sim, E., Palmer, M. S., Puklavec, M., Sim, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+against+the+complement+control+protein+factor+H+(beta+1+H).">Monoclonal antibodies against the complement control protein factor H (beta 1 H).</a> Biosci Rep. 3 (12), 1119-1131 (1983).</li><li>Ripoche, J., Day, A. J., Harris, T. J., Sim, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complete+amino+acid+sequence+of+human+complement+factor+H.">The complete amino acid sequence of human complement factor H.</a> Biochem J. 249 (2), 593-602 (1988).</li><li>Moore, D. J., Clover, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+age+on+the+macromolecular+permeability+of+human+Bruch&#8217;s+membrane.">The effect of age on the macromolecular permeability of human Bruch&#8217;s membrane.</a> Invest Ophthalmol Vis Sci. I. 42 (12), 2970-2975 (2001).</li><li>McHarg, S., Clark, S. J., Day, A. J., Bishop, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration+and+the+role+of+the+complement+system.">Age-related macular degeneration and the role of the complement system.</a> Mol Immunol. 67 (1), 43-50 (2015).</li></ol>

The authors declare that they have no competing financial interests.

Here we describe the enrichment of Bruch&#8217;s membrane for further analysis allowing subsequent analyses including western blotting11, mass spectrometry and the diffusion properties of Bruch&#8217;s membrane utilising modified Ussing chambers11,14. Critical steps include a) the thorough scraping and washing of the inner surface of the eye to remove all RPE cells b) the repeated and careful scraping of the underlying choroid without causing the Bruch&#8217;s to disintegrate and c) washing of the excised Bruch&#8217;s in ultrapure water to ensure lysis of residual cells. Furthermore, if the enriched Bruch&#8217;s membrane is to be utilized for proteomic analysis, protein extraction by urea treatment is the most effective in breaking down this resilient tissue as compared to other methods such as mechanical homogenisation. Histology of enriched Bruch&#8217;s membrane indicates the ECM of the choriocapillaris is not removed. This is to be expected as the outer layer of the pentilaminar Bruch&#8217;s membrane is formed by choriocapillaris endothelial cell basement membranes. Indeed it may be advantageous that the ECM of the choriocapillaris remains as changes occur in this in AMD including the deposition of terminal complement complex/membrane attack complex (MAC)15. It should be noted that for detailed histological analysis of Bruch&#8217;s membrane, its structure would be better preserved if sectioned in the presence of sclera, choroid, and Bruch&#8217;s membrane. Indeed, the histology presented in Figure 2 is solely intended to demonstrate the degree of enrichment resulting from the technique described in this manuscript.
Enrichment of macular Bruch&#8217;s requires careful dissection of the eyecup, ensuring that the NSR and particularly the fovea are not damaged; otherwise identification and thus isolation of the macula are not possible. The integrity of the tissue is also important to this process and it is therefore recommended that samples under 48 hr post-mortem time be used. The degree of enrichment required varies between donors and can be affected by donor age, post-mortem time, and the presence of any eye pathology. The use of the term enriched is deliberate in acknowledging a limitation of the technique, this being that &#8216;purification&#8217; or &#8216;isolation&#8217; of Bruch&#8217;s membrane using this method simply is not possible. Indeed, given that the ECM of the choriocapillaris merges into Bruch&#8217;s membrane there complete separation is not feasible.
Understanding the biochemical changes of the respective ocular tissues is essential in understanding eye disease progression. This unique enrichment of Bruch&#8217;s membrane facilitates this understanding; ongoing studies are investigating the proteome of enriched macular Bruch&#8217;s membrane using mass spectrometry at different stages of AMD and in samples originating from subjects with different genotypes to improve understanding of the molecular pathology of AMD. Already the technique described here has been used to successfully identify FHL-1 as the predominant complement regulator in Bruch&#8217;s membrane 11 and further studies on enriched Bruch&#8217;s membrane are likely to result in more new insights into the molecular pathology of AMD.

The use of post mortem human eye tissue in ophthalmic research is an invaluable resource for the understanding of eye disease pathogenesis. Analysis of human donor eyes has made important contributions to discerning the mechanisms underpinning age-related macular degeneration (AMD), which is the leading cause of blindness in the western world1. Globally, AMD accounts for approximately 8.7% of all blindness and with an increasingly aging population, it is predicted to affect 196 million people by 20202. AMD results in the loss of central vision and has a profound impact on patient independence and quality of life3. Early AMD is characterised by the formation of drusen and it can progress to geographic atrophy (sometimes referred to as &#8216;dry&#8217; AMD), or choroidal neovascularization (also referred to as neovascular or &#8216;wet&#8217; AMD). Whilst anti-VEGF injections can stabilise or improve vision in neovascular AMD it is not curative, and at present, no treatment exists for geographic atrophy.
Whilst certain genetic variants have been shown to modify AMD risk 4&#8211;8, less is known about how these variants affect the macula at a biochemical level. An important site in AMD pathogenesis is Bruch&#8217;s membrane. Drusen form within this structure and various studies have provided evidence of complement activation in and around Bruch&#8217;s membrane in AMD9. Bruch&#8217;s membrane is a sheet of extracellular matrix that separates the retinal pigment epithelial (RPE) cells from the choroid, and is approximately 4 &#181;m thick. Bruch&#8217;s membrane merges into the choriocapillaris, a layer of the choroid that contains fenestrated capillaries and external to this are layers containing larger blood vessels10 (Figure 1A).
Bruch&#8217;s membrane is a pentilaminar sheet of extracellular matrix (ECM), and this methodology details how to isolate Bruch&#8217;s membrane, along with the ECM of the underlying choriocapillaris (henceforth referred to enriched Bruch&#8217;s membrane), which is largely decellularized and free from red blood cells. Enriched Bruch&#8217;s membrane was then used for western blotting and histology experiments. Furthermore, methodology for enriching Bruch&#8217;s membrane solely from the macula region of the eye is described, which will be of particular interest to those investigating the biochemical aspects of AMD.

<table border="0">
	<tbody>
		<tr>
			<td>Auxillary objective 0.5x</td>
			<td>GT-Vision Ltd</td>
			<td>3638</td>
			<td>Useful (but not essential) for lower magnification imaging of whole eye flat-mount</td>
		</tr>
		<tr>
			<td>Biopsy Punch 6mm</td>
			<td>Oncall Medical Supplies</td>
			<td>SCH-33-36</td>
			<td>6mm Biopsy Punches</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma - Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Sigma - Aldrich</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scrapers</td>
			<td>Sarstedt</td>
			<td>83.183</td>
			<td>For membrane enrichment</td>
		</tr>
		<tr>
			<td>CyroPure Cyrovials</td>
			<td>Sarstedt</td>
			<td>72.38</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpels</td>
			<td>Fisher Scientific</td>
			<td>12387999</td>
			<td>Sterile, individually wrapped Swann-Morton scalpels.</td>
		</tr>
		<tr>
			<td>Dual Gooseneck LED spot lights on 30cm arms</td>
			<td>GT-Vision Ltd</td>
			<td>0153</td>
			<td>Flexible light source essential for dissection and imaging</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma - Aldrich</td>
			<td>D8537</td>
			<td>Used to prevent macula drying out and during membrane enrichment.</td>
		</tr>
		<tr>
			<td>(A) Forceps</td>
			<td>Scientific Laboratory Supplies</td>
			<td>INS4280</td>
			<td>(A) Fine (100mm) and straight</td>
		</tr>
		<tr>
			<td>(B) Forceps</td>
			<td>Scientific Laboratory Supplies</td>
			<td>INS4272</td>
			<td>(B) Broad and blunt (150mm). Useful for lifting large portions of sample</td>
		</tr>
		<tr>
			<td>(C) Forceps</td>
			<td>Scientific Laboratory Supplies</td>
			<td>INS4323</td>
			<td>(C) Straight fine points</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma - Aldrich</td>
			<td>G1345</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma - Aldrich</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>GXCAM 5 digital colour camera &#38; GT-Vision software</td>
			<td>GT-Vision Ltd</td>
			<td>1122</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma - Aldrich</td>
			<td>M/4000/17</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Life Technologies</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4-12% Bis-Tris Protein Gels</td>
			<td>Life Technologies</td>
			<td>NP0322BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sterilin</td>
			<td>101VR20</td>
			<td>90mm, used as a dish for samples</td>
		</tr>
		<tr>
			<td>Protein isolation beads (Strataclean resin)</td>
			<td>Agilent</td>
			<td>400714-61</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Bio-Rad</td>
			<td>161-0301</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereozoom microscope GXMXTL3T (magnification range 7x-45x)</td>
			<td>GT-Vision Ltd</td>
			<td>5595</td>
			<td>Stereozoom microscope attached to boom stand for ease of use during dissection</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma - Aldrich</td>
			<td>T3253</td>
			<td></td>
		</tr>
	</tbody>
</table>

enrichment of bruch's membrane from human donor eyes

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of the center&#160;of the retina, called the macula, resulting in the loss of central vision. Early AMD is characterised by the presence of small, yellowish lesions called soft drusen that can progress onto late AMD such as geographic atrophy (dry AMD) or neovascularisation (wet AMD). Although the clinical changes are well described, and the understanding of genetic influences on conferring AMD risk are getting ever more detailed, one area lacking major progress is an understanding of the biochemical consequences of genetic risk. This is partly due to difficulties in understanding the biochemistry of Bruch&#8217;s membrane, a very thin extracellular matrix that acts as a biological filter of material from the blood supply and a scaffold on which the retinal pigment epithelial (RPE) cell monolayer resides. Drusen form within Bruch&#8217;s membrane and their presence disrupts nutrient flow to the RPE cells. Only by investigating the protein composition of Bruch&#8217;s membrane, and indeed how other proteins interact with it, can researchers hope to unravel the biochemical mechanisms underpinning drusen formation, development of AMD and subsequent vision loss. This paper details methodologies for enriching either whole Bruch&#8217;s membrane, or just from the macula region, so that it can be used for downstream biochemical analysis, and provide examples of how this is already changing the understanding of Bruch&#8217;s membrane biochemistry.

The protocol described above to produce enriched Bruch&#8217;s membrane results in the isolation of Bruch&#8217;s membrane, the ECM of the choriocapillaris and removes most of the cells/cellular debris including all of the RPE (Figure 2). The washing of the enriched Bruch&#8217;s membrane in water is a key step in lysing any remaining choroidal cells. The pressure exerted during scrapping of the choroid appears to compact some of the few remaining cell nuclei into the region defined as Bruch&#8217;s membrane (Figure 2B).
The solubilisation of enriched Bruch&#8217;s membrane allows the downstream analysis of its protein content. The separation of protein bands from solubilised enriched Bruch&#8217;s membrane using a 4 - 12% gradient SDS-PAGE gel, while not useful in its own right to identify protein content, does demonstrate the ability of this technique to minimise contaminating blood proteins that are non-specifically bound (Figure 3B). In this regard, one notable absentee from the protein gel run is a sizeable band of human serum albumin (~66 kDa) that may otherwise make subsequent western blotting more difficult to interpret: the histological analysis of enriched Bruch&#8217;s membrane shown in Figure 2 also supports this. Indeed, as part of work described previously11, solubilised Bruch&#8217;s membrane from a series of individual donors were probed for a number of proteins using specific antibodies via western blotting (Figure 3C). In the case shown here, an antibody (termed OX23)12 directed against a 155 kDa regulator of innate immunity, called complement factor H (FH), was used and demonstrated the presence of a lower molecular weight band in Bruch&#8217;s membrane samples from three separate donors (Figure 3C). This subsequently turned out to be factor H-like protein 1 (FHL-1), a 49 kDa protein related to FH and arising from a splice variation of the same gene13. FHL-1 appears to be the main complement regulator present in Bruch&#8217;s membrane11.
<img alt="Figure 1" src="/files/ftp_upload/53382/53382fig1.jpg" /> 
Figure 1. Dissection of the Human Eye and Enrichment of Bruch&#8217;s Membrane.&#160;(A) Bruch&#8217;s membrane is an extracellular matrix barrier. The choroid contains a layer of fenestrated capillaries that continuously merge into Bruch&#8217;s membrane (called the choriocapillaris) and these separate the rest of the choroid (containing large blood vessels) from the retinal pigment epithelium (RPE) cell monolayer, which physiologically supports the photoreceptor cells of the neurosensory retina (NSR). (B) Four incisions into the human eyecup are made allowing it to be opened to create a flat mount. The vitreous can be removed with the use of tweezers. The NSR may come off along with the vitreous but can also be removed with tweezers. A cell scraper is used to remove the RPE cell layer from Bruch&#8217;s membrane while it is still anchored to the choroid and sclera. The Bruch&#8217;s membrane/choroid complex can be peeled away from the sclera and using a new cell scraper, the outer choroid can be removed. A final wash in ultrapure water lyses any remaining cellular matter. <a href="https://www.jove.com/files/ftp_upload/53382/53382fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53382/53382fig2.jpg" /> 
Figure 2. Histology of Bruch&#8217;s Membrane and the Choroid. H&#38;E stained sections before (A) and after (B) scraping away the outer choroidal structures from Bruch&#8217;s membrane and washing in ultrapure water to generate enriched Bruch&#8217;s membrane. Scale bars represent 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/53382/53382fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53382/53382fig3.jpg" /> 
Figure 3. Solubilisation of Enriched Bruch&#8217;s Membrane for Protein Content Analysis.&#160;(A) Protein can be extracted from enriched Bruch&#8217;s membrane by incubating in 8 M urea for 6 hr at RT, before being dialysed against PBS. For Western analysis, proteins from the whole Bruch&#8217;s membrane dialysate can be isolated using protein isolation beads and eluted by boiling with laemmli reducing sample loading buffer. (B) A representative Coomassie blue stained SDS-PAGE gel, showing both neat and diluted solubilised Bruch&#8217;s membrane (BM) using the method described in (A). (C) A representative western blot of solubilised Bruch&#8217;s membrane from 3 individual donors (lanes 1 - 3) probed for factor H-like protein 1 (FHL-1). <a href="https://www.jove.com/files/ftp_upload/53382/53382fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53382/53382fig4.jpg" /> 
Figure 4. Isolation of Enriched Bruch&#8217;s Membrane from the Macula. The macula region of the retina is a 5 mm area that can be readily identified by the yellow colouration of the overlying fovea, and its proximity to the adjacent optic disc. Using the fovea as a guide, place a 6 mm biopsy punch on the flat mounted human eye to excise a 6 mm macula tissue block. Remove the overlying NSR and scrape off RPE cells from the excised macula. The remaining Bruch&#8217;s membrane/choroid complex can then be peeled away from the sclera and thoroughly scraped to remove choroid, lyse remaining cellular material by submersion in ultrapure water. <a href="https://www.jove.com/files/ftp_upload/53382/53382fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Enrichment of Bruch's Membrane from Human Donor Eyes at JoVE.com

Enrichment of Bruch&#039;s Membrane from Human Donor Eyes

Identifying proteins specifically associated with Bruch&#8217;s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

Enrichment of Bruch's Membrane from Human Donor Eyes

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of ...

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Research

JoVE Journal

Neuroscience

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments

There is a current unmet need for a new preclinical human model that can target disease etiology ex vivo using intracranial pressure (ICP) and intraocular pressure (IOP) which&#160;can identify various pathogenic paradigms related to the glaucoma pathogenesis. Ex vivo human anterior segment perfusion organ culture models have previously been successfully utilized and applied as effective technologies for the discovery of glaucoma pathogenesis and testing of therapeutics. Preclinical drug screening and research performed on ex vivo human organ systems can be more translatable to clinical research. This article describes in detail the generation and operation of a novel ex vivo human translaminar pressure model called the translaminar autonomous system (TAS). The TAS model can independently regulate ICP and IOP using human donor posterior segments. The model allows for studying pathogenesis in a preclinical manner. It can reduce the use of living animals in ophthalmic research. In contrast to in vitro experimental models, optic nerve head (ONH) tissue structure, complexity, and integrity can also be maintained within the ex vivo TAS model.

We describe and detail the use of the translaminar autonomous system. This system utilizes the human posterior segment to independently regulate the pressure inside the segment (intraocular) and surrounding the optic nerve (intracranial) to generate a translaminar pressure gradient that mimics features of glaucomatous optic neuropathy.

There is a current unmet need for a new preclinical human model that can target disease etiology ex vivo using intracranial pressure (ICP) and intraocular ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

OCT-Based Multimodal Imaging of Human Donor Eyes for AMD Research

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Author Spotlight: Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration  

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...