This work was supported partly through grants of the Regione Veneto (Ricerca Sanitaria Finalizzata n.292/2008 and Ricerca Sanitaria Finalizzata 2009). The authors thank Dr C. Griffoni for the 2009/2010 summary data on the use of human ocular globes.

1. In situ Excision of the Cornea from Ocular Globes
<ol>
 <li> Switch on the laminar air flow cabinet approximately 15 minutes before use. Clean the laminar flow hood using 70% isopropyl alcohol. Put on personal protective clothing such as surgical cap and mask. Scrub hands and forearms and dry using a sterile towel. Wear sterile gloves and gown or sleeves using aseptic technique. </li>
 <li> Set up the sterile field by placing a sterile instrument tray aseptically. Verify that the instruments are sterile. Open the sterile instruments pack and flip onto the sterile field avoiding any contamination. </li>
 <li> Position all sterile materials and instruments (Table 2) along with bottles (eye jars) containing eye globes so that they are adjacent to the edge of the sterile field for ease of use. </li>
 <li> Label the bottles with previously prepared preservation/storage medium as indicated in Table 3 and place them on the surface of the laminar air flow hood along with I-PVP (iodine-polyvinylpyrrolidone), sodium thiosulphate and a sterile saline solution PBS (phosphate buffer saline). </li>
 <li> Allow the ocular tissues and solutions to reach normal room temperature as they are preserved in refrigerators at 4 &deg;C. Avoid repeated warming/cooling cycles. Keep the lids of the solution and the eye jars with inner side facing upwards next to their respective jars. </li>
 <li> Remove gauzes and sponges from the eye jar using sterile forceps. Immerse the globe in sterile I-PVP 0.5% for 2 minutes to decontaminate the ocular globes. Place the two forceps on the lid of the sterile tray, outside the sterile field. Transfer the globe in sodium thiosulphate 0.1% for 1 minute using another pair of sterile forceps. Using the same forceps, transfer the globe to the bottle containing sterile saline solution (PBS) and leave it till it is operated. Repeat the same procedure for the other ocular globe from the same donor. </li>
 <li> Working underneath the laminar air flow hood, wrap the globe using sterile gauze (bandages) leaving the cornea and approximately 5 mm sclera from the cornea uncovered. The eye bulb could be simply held in the hand maintaining the adequate pressure. The tissue should be kept moist during the entire surgery. </li>
 <li> Forceps are used along with scissors to remove all eventual remains of the conjunctiva. Keep the instruments used for the above operation separate from other instruments. Use a scalpel blade to perform a scleral incision of 3-4 mm from the limbus region, then extend the incision by 360&deg;, avoiding to perforate the underlying uveal tissue or cause any deformation of the cornea's normal curvature. Cut four big incisions leaving four small gaps avoiding the secretion of the vitreous body. All four gaps are cut ensuring no removal of any other tissue. </li>
 <li> Presence of small scleral plaques may hamper the cutting, so make sure to complete the scleral excision with microsurgery scissors. This operation should be performed without damaging the choroid, retina and vitreous body. </li>
 <li> Inspect the incision to ensure it is complete. If the incision has been performed correctly the corneo-scleral rim adheres to the ciliary bodies only at the point in correspondence with the cornea. </li>
 <li> Set the wrapped ocular globe down near the center of the sterile field. Complete the corneal removal using forceps to hold the scleral rim stationary and the hand used for excision to pull the ciliary body-choroid downward and away from the corneo-scleral button. </li>
 <li> Gently separate any remaining adhesions from the corneo-scleral button. Neither pull the corneo-scleral rim in a way that could cause cross-corneal tension nor allow it to drop back down onto the anterior chamber. </li>
 <li> The endothelial cell density of the cornea is checked using a hypotonic solution and the cell mortality is checked using a trypan blue staining for around 1 min and the cells are then counted under an optical microscope and measured as cells/mm2. The anterior chamber should face the lid while the posterior should face the bottom of the Petri plate. The endothelial cell density could be counted manually using a grid in the objective under 100X magnification. The minimum accepted cell count for transplantation in Italy is 2200 endothelial cells/mm2. </li>
 <li> Transfer the corneo-scleral rim using a corneal claw to previously prepared corneal storage medium preserved at RT (room temperature) as shown in Table 3. </li>
 <li> Examine the posterior chamber for a natural crystalline lens. If the sclera is prepared, remove all the residues of uvea and vitreous humor from the globe. Carefully unwrap and return the remaining posterior segment to its respective eye jar, using aseptic technique. Repeat the procedure for the other globe using new instruments. Fix the cornea with corneal claw and preserve under the storage media (organ culture) at 31 &deg;C. The excised cornea, as shown in Figure 1a, can then be used for keratoplasty or research.</li>
 <li> Dispose the unnecessary materials and bio-hazardous waste into appropriate waste bins. Clean the working area once the work is completed using 70% isopropyl alcohol spray. </li>
 <li> If, after 28 days the cornea maintains all the evaluation parameters [such as morphology, cell count, transparency, thickness and microbiology tests - bacterial and fungal using Bactec 9240 instrument (Becton Dickinson, Milan, Italy)], it can then be transported to the hospitals using the transport medium shown in Table 4. The cornea should be removed and placed from the storage to transport medium under laminar air flow cabinet. </li>
 <li> As, during storage, the cornea gets thicker than its usual size required for transplantation, eye banks use de-swelling agents to reduce thickness. We use 6% dextran T500 to get the cornea back to its normal size in transport medium. Though 4-8% dextran T500 could be used, 6% has served best for us. This makes it easier for the surgeons to transplant the cornea as soon as they receive it. </li>
</ol>
2. Evisceration of Retina from the Ocular Globe after Excising the Cornea
<ol>
 <li> Following excision, the cornea is selected for transplantation or research based on its morphology and endothelial cell density and is placed in the storage or preservation medium at 31 &deg;C. The remaining of the ocular globe is then processed for further use such as sclera isolation for surgical purposes, retina for research etc.</li>
 <li> Prior to retinal excision, make sure that the ocular globe is intact and only the cornea with a part of sclera is removed. If the gelatinous vitreous body is secreting out it becomes difficult to remove a retinal tissue. </li>
 <li> If the donor is below 65 years of age, the sclera is preserved under sterile PBS for surgical purposes after serological testing. In this case, the sclera is not cut, only the vitreous body is removed and the entire sclera is preserved. </li>
 <li> For retinal excision, start with a small cut or incision from the corneal side of the sclera moving towards the optic nerve using sterile forceps and scissors. Hold the part of the sclera using forceps and cut with the scissors straight towards the optic nerve cutting part by part avoiding damage to the vitreous body. </li>
 <li> Choroidal plaques should be separated from the sclera as they are stuck together. Cut the entire sclera to expose the choroid tissue. Once the sclera is completely cut, separate the sclera with the remaining of the vitreous body by cutting it near the optic nerve. An entire ball of vitreous body covered with choroid will be visible as shown in Figure 2a.</li>
 <li> Using two sterile forceps remove the choroid layer gently by picking it up with one pair of forceps and removing it with the other. Alternatively, the choroid layer can be removed using scissors but this should ideally be avoided as it may cause damages to the retina. Remove the choroid layer partially and cut it to facilitate the removal.</li>
 <li> Once the choroid layer is removed, a transparent layer of retina will be seen clearly as shown in Figure 2b. The choroid layer has an interior Bruch's membrane which could also be removed but it is difficult to identify it with a naked eye.</li>
 <li> Similarly, use two forceps to remove the retinal layer as shown in Figure 2c. It is easy to isolate high amount of retina if excised near the optic nerve. All the layers of the eye could be now seen as shown in Figure 2d.</li>
 <li> Once the retinal tissue has been removed, it could be used for different purposes and therefore the subsequent steps and conditions used for preservation may differ accordingly. For instance, the retinal tissue can be placed in tubes containing RNA stabilization reagents (such as RNAlater) or lysis buffer (for RNA or DNA extraction), dispase/trypsin (for isolation of cells), fixing solutions (prior to embed tissues in paraffin or OCT for immunohistochemistry).</li>
 <li> Clear all the working area and clean the area using 70% isopropyl alcohol spray.</li>
</ol>
3. Representative Results
The corneo-scleral rim as seen in Figure 1a is a properly excised cornea with required amount of scleral rim and endothelial morphology, this kind of cornea is generally used for penetrating keratoplasty. If the anterior stroma of the excised cornea is damaged with presence of opacity, the cornea could be used for endothelial keratoplasty / posterior lamellar keratoplasty (endothelium with a part of stroma). Whereas, if the endothelial layer is damaged or if the endothelial cell density is &lt; 2200 cells /mm2 (Italian standard), the cornea could be used for anterior lamellar keratoplasty (epithelium with a part of anterior stroma). If the endothelial cell count is less than the required, the cornea could be used for anterior lamellar keratoplasty or research whereas, if the cornea is damaged completely, it could be used for research as shown in Figure 1b, 3b and 3c or discarded. The damage could be a cause of mishandling the tissue, prior cataract surgeries leading to stromal or descemet endothelial scars, development of physical injury during the patient's life etc. Therefore, it becomes necessary to check every tissue with slit lamp and then under optical microscope for fine scars / folds / detachments. The cornea is usually preserved under two different conditions such as organ culture (approximately 31 &deg;C) usually used in European countries or hypothermic conditions (approximately 4 &deg;C) usually used in the USA. We use organ culture as it helps to regenerate the damaged epithelial cells, to re-vitalize endothelial cell check with a high post mortem time, a storage period of 4 weeks which gives sufficient window for planning a surgery, gives sufficient time for microbiological (bacterial and fungal) and serological testing etc. The cornea can be well preserved in the transport medium for 7 days.
The sclera could be cut part by part ensuring gentle removal of all choroidal plaques. The vitreous liquid can excrete when the optic nerve, choroid or retina is cut sharply as shown in Figure 4. Retinas if excised for RNA analysis should be preserved in RNAlater as soon as the tissues are excised. Usually 1X1 cm of the choroid and retinal layer is excised and after keeping it in RNAlater for 24 hours at 4 &deg;C is preserved under -80 &deg;C in fresh Eppendorf tubes (safe-lock tubes, 2 ml) until used or shipped with dry ice. The retinal tissue should be excised using two forceps near optic nerve away from iris or ciliary margin to avoid contamination with other pigmented structures and should be transferred to the preserving container immediately as RNA gets unstable while retrieving the retina.
<img src="/files/ftp_upload/3765/3765fig1.jpg" alt="Figure 1" /> 
Figure 1. Comparison between a good and a poor quality cornea: a) A properly excised cornea. Such a cornea is usually preserved under organ culture for 4 weeks. Microbiological (using Bactec 9240 instrument) and morphological (staining the cells with trypan blue for cell mortality determination and observing cells under an optical microscope with 100X magnification) examination is performed before transporting it to the hospital (Figure 3a); b) a bent cornea, which eventually leads to graft rejection due to high descemet's folds and high endothelial cell damage examined using a slit lamp microscope. Such a cornea is usually used for research or is discarded.
<img src="/files/ftp_upload/3765/3765fig2.jpg" alt="Figure 2" /> 
Figure 2. Method used to ensure an accurate excision of the retinal tissue. a) Dissecting the sclera from the ocular globe using 24mm blade with 95 mm curved or blunt end sterile scissors and 100 mm 11X2 ruled by 0.70 mm teeth sterile forceps. The sclera should be cut by holding one side with the forceps cutting it straight towards the optic nerve using the scissors. The choroid layer will now be easily seen; b) Using two 100 mm 11X2 ruled by 0.70 mm teeth sterile forceps, choroid is removed by tearing it apart, which reveals the underlying transparent retinal layer; c) Transparent retinal layer is excised very carefully near the optic nerve without excretion of vitreous fluid using the same forceps. Both the forceps should hold each side of the retina and the tissue should gently be removed starting near the optic nerve moving towards the cornea, to obtain a good amount of retinal tissue. The tissue could be cut into different sizes and used for analysis experiments; d) Different layers of the eye following corneal removal can be viewed in this figure. The choroid body is removed first followed by retina without damaging the other parts of the eye. 
<img src="/files/ftp_upload/3765/3765fig3.jpg" alt="Figure 3" /> 
Figure 3. Comparison of the different types of cell density and morphology viewed under 100X magnification of an optical microscope. a) Good endothelial cell density (&gt;2200 cells / mm2) and morphology (less polymorphism, low degeneration, no dystrophy and 0% mortality) in a cornea which is suitable for transplantation; b) Poor endothelial cell density (approximately 1200 &#x2013; 1400 cells / mm2) and morphology (very high polymorphism and degeneration of cells) along with approximately 30-40% overall mortality are observed after trypan blue staining; c) A part of endothelial cells are damaged due to detachment of an endothelial sheet while retrieving the cornea from ocular globe. This is confirmed by observing the mortality of the cells in the region stained by trypan blue; d) The descemet's folds are formed due to mishandling of cornea by bending, creating cross corneal tension, using the instruments intensely etc at the time of excision. The figure shows the thick developed iatrogenic folds found between the periphery and optic zone of the cornea.
<img src="/files/ftp_upload/3765/3765fig4.jpg" alt="Figure 4" /> 
Figure 4. Damages to the choroid and retinal tissues while excising the cornea eventually leads to excretion of vitreous fluid. The retina gets implicated in the vitreous body making it difficult to excise a good quality tissue without contamination.
<img src="/files/ftp_upload/3765/3765table1.jpg" alt="Table 1" /> 
Table 1. Summary of the range of activities performed using human ocular globes during 2009 and 2010 at Fondazione Banca Degli Occhi (Venezia, Italy). Approximately 60% of tissues were used for transplantation, thus leaving a high number of ocular globes (around 40%) for scientific and medical research. 
<img src="/files/ftp_upload/3765/3765fig1sup.jpg" alt="Supplementary Figure 1" /> 
Supplementary Figure 1. Anatomy of the human eye5. Different parts of human eye are indicated in this figure, used to follow the exact position of the tissues.

<ol>
	<li>Fay, A., Goldman, L., Ausiello, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diseases+of+the+visual+system.">Diseases of the visual system.</a> Cecil Medicine. , (2007).</li><li>Parekh, M., Megaw, R., Ray-Chaudhuri, A., Ahmad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patents+in+Limbal+Stem+Cell+Biology.">Patents in Limbal Stem Cell Biology.</a> Recent patents on Regenerative Medicine. 1, 207-212 (2011).</li><li>Pharm, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Eye:+The+physiology+of+human+perception.">The Eye: The physiology of human perception.</a> Britannica educational publishing. 36, 19-27 (2011).</li><li>Lang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ophthalmology: A pocket textbook atlas. , 115-117 .</li><li>Liesbeth, P. e. l. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+Culture:+The+method+of+choice+for+preservation+of+human+donor+corneas.">Organ Culture: The method of choice for preservation of human donor corneas.</a> Br. J. Ophthalmol. 81, 523-525 (1997).</li><li>Meyer, J. S., Howden, S. E., Wallace, K. A., Verhoeven, A. D., Wright, L. S., Capowski, E. E., Pinilla, I., Martin, J. M., Tian, S., Stewart, R., Pattnaik, B., Thomson, J., Gamm, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+vesicle-like+structures+derived+from+human+pluripotent+stem+cells+facilitate+a+customized+approach+to+retinal+disease+treatment.">Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment.</a> Stem Cells. , (2011).</li><li>Trifunovic, D., Karali, M., Camposampiero, D., Ponzin, D., Banfi, S., Marigo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-resolution+RNA+expression+atlas+of+retinitis+pigmentosa+genes+in+human+and+mouse+retinas.">A high-resolution RNA expression atlas of retinitis pigmentosa genes in human and mouse retinas.</a> Invest. Ophthalmol. Vis. Sci. 49, 2330-2336 (2008).</li><li>Mora, P., Montanini, L., Ferrari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Retina. 30, 1555 (2010).</li><li>Claybon, A., Bishop, A. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+a+Mouse+Eye+for+a+Whole+Mount+of+the+Retinal+Pigment+Epithelium.">Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium.</a> J. Vis. Exp. (48), e2563 (2011).</li><li>Skeie, J. M., Tsang, S. H., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evisceration+of+Mouse+Vitreous+and+Retina+for+Proteomic+Analyses.">Evisceration of Mouse Vitreous and Retina for Proteomic Analyses.</a> J. Vis. Exp. (50), e2795 (2011).</li></ol>

The authors have no conflict of interest.

Both, correct excision and proper preservation of corneal and retinal tissues are critical as minor defects such as endothelial damage or high number of descemet's folds can lead to graft failure of cornea whereas temperature alterations or mishandling can compromise the integrity of retinal tissues. The aim of this paper is to show how corneal and retinal tissues can be isolated optimally, without inducing damages or alterations that could compromise their use for transplantation or research purposes. The required minim...

Materials for excision of cornea and retina.
<table border="1">
<tbody>
 <tr>
 <td>Product 			description</td>
 <td>Dimensions</td>
 <td>Company 			/ Institute</td>
 </tr>
 <tr>
 <td>Guarded 			disposable scalpel</td>
 <td>Blade 			size 15 </td>
 <td>Swann-Morton</td>
 </tr>
 <tr>
 <td>Sterile 			bandages</td>
 <td>5 			cm X 5 cm, 8 layered, 5 pcs</td>
 <td>Artsana</td>
 </tr>
 <tr>
 <td>Sterile 			disposable medical towel</td>
 <td>35 			X 50 cm</td>
 <td>U.Jet</td>
 </tr>
 <tr>
 <td>Sterile 			scissors</td>
 <td>Blades 			24 mm / overall length. 95 mm curved, blunt</td>
 <td>e.janach</td>
 </tr>
 <tr>
 <td>Sterile 			forceps</td>
 <td>Stainless 			steel -100 mm 11 X 2 ruled by 0.70 mm teeth</td>
 <td>e.janach</td>
 </tr>
 <tr>
 <td>Corneal claw &#x2013; Disposable medical device</td>
 <td></td>
 <td>NIIOS 			(Hippocratech)</td>
 </tr>
 <tr>
 <td>&nbsp;
 </td>
 <td>Preparations</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>100 			ml PBS [10x] in 900 ml d/w (distilled water)</td>
 <td>Sigma-Aldrich</td>
 </tr>
 <tr>
 <td>Na-thiosulphate</td>
 <td>1 			gm Na-thiosulphate in 1 litre of PBS [1x]</td>
 <td>Sigma-Aldrich</td>
 </tr>
 <tr>
 <td>I-PVP</td>
 <td>5 			gm I-PVP in 1 litre d/w </td>
 <td>Sigma-Aldrich</td>
 </tr>
 </tbody>
</table>
Table 2. The table describes the materials used for excision of cornea and retina and the company they are received from. 
Materials for storage medium (2000 ml).
<table border="1">
<tbody>
 <tr>
 <td>Components </td>
 <td>Supplier</td>
 <td>Catalogue 			number</td>
 <td>Concentration </td>
 <td>Quantity</td>
 </tr>
 <tr>
 <td>MEM 			(1X) liquid</td>
 <td>Invitrogen</td>
 <td>32360-034</td>
 <td>&nbsp;</td>
 <td>1900 			ml</td>
 </tr>
 <tr>
 <td>Sodium 			pyruvate</td>
 <td>Invitrogen</td>
 <td>11360-039</td>
 <td>1mM 			(10ml/l)</td>
 <td>20 			ml</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Invitrogen</td>
 <td>25030-032</td>
 <td>2mM 			(10ml/l)</td>
 <td>20 			ml</td>
 </tr>
 <tr>
 <td>Antibiotic/antimycotic</td>
 <td>Sigma-aldrich</td>
 <td>A5955-20ML</td>
 <td>10ml/l</td>
 <td>20 			ml</td>
 </tr>
 <tr>
 <td>Newborn 			calf serum</td>
 <td>Invitrogen</td>
 <td>26010-74</td>
 <td>2% 			(20 ml/l)</td>
 <td>40 			ml</td>
 </tr>
 </tbody>
</table>
Table 3. Materials for storage medium.
Preparation of storage medium
Add all the ingredients using the specific concentrations as given above in a jar and mix well. Filter them using pore size of 0.2 micron filter (Millipore, Milan, Italy) with help of a peristaltic pump. Preserve the medium in the bottles at RT.
Materials for transport medium (2000 ml).
<table border="1">
<tbody>
 <tr>
 <td>Components </td>
 <td>Supplier </td>
 <td>Catalogue 			number</td>
 <td>Concentration </td>
 <td>Quantity</td>
 </tr>
 <tr>
 <td>MEM 			(1X) liquid </td>
 <td>Invitrogen</td>
 <td>32360-034</td>
 <td>&nbsp;</td>
 <td>1800 			ml</td>
 </tr>
 <tr>
 <td>Sodium 			pyruvate</td>
 <td>Invitrogen</td>
 <td>11360-039</td>
 <td>1mM 			(10 ml/l)</td>
 <td>20ml</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Invitrogen</td>
 <td>25030-032</td>
 <td>2mM 			(10 ml/l)</td>
 <td>20ml</td>
 </tr>
 <tr>
 <td>Newborn 			calf serum</td>
 <td>Invitrogen</td>
 <td>26010-74</td>
 <td>2% 			(20 ml/l)</td>
 <td>40 			ml</td>
 </tr>
 <tr>
 <td>Dextran 			t500</td>
 <td>Pharmacosmos</td>
 <td>551005004007</td>
 <td>6% 			(60 gm/litre)</td>
 <td>120 			g/l</td>
 </tr>
 </tbody>
</table>
Table 4. Material for transport medium.
Preparation of transport medium
Add Dextran 6% in ~ 1.5 litre of MEM and leave it overnight. Add the rest of ingredients in the media and filter using 0.2 micron filter (Millipore, Milan, Italy) using a vacuum pump. Preserve the medium in the bottles at RT.

a simplified technique for in situ excision of cornea and evisceration of retinal tissue from human ocular globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

Watch this Scientific Journal Video about A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe at JoVE.com

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

a-simplified-technique-for-situ-excision-cornea-evisceration-retinal

Research

JoVE Journal

Medicine

20.8K Views.  Fondazione Banca Degli Occhi del Veneto O.N.L.U.S.. В статье описывается упрощенная техника акцизного роговицы и сетчатки потрошить тканей глазной шар трупа человека доноров. Техника, описанная здесь поможет акцизного хорошее качество ткани использовать для трансплантации, хирургическому или исследователь...

Video: A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

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

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

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

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Human Subcutaneous Adipose Tissue Sampling Using a Mini-Liposuction Technique

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate training and strict adherence to aseptic technique, subcutaneous adipose samples can be safely and efficiently obtained in a non-clinical setting by researchers. Following the administration of local anesthetic lateral to the umbilicus, a 14 G needle attached to a 5 or 10 mL syringe is inserted through the skin into the subcutaneous tissue. Under suction, the syringe is moved in a reciprocating, slicing motion to isolate fragments of adipose tissue. Withdrawing the plunger is enough to ensure that adipose tissue fragments are aspirated through the needle into the syringe. A single biopsy can collect about 200 mg of tissue. This biopsy technique is very safe for both participants and research staff. Following the biopsy, participants can resume most everyday activities, although they should avoid swimming and overly strenuous activities for 48 h to avoid excessive bleeding. Participants can safely undergo 2 biopsies within a single day, meaning that the technique can be applied in before-after acute intervention studies.

The manuscript and associated video demonstrate a percutaneous biopsy technique to obtain samples of subcutaneous adipose tissue from areas surrounding the umbilicus. This method is a low-risk and efficient way to investigate a range of parameters (e.g., gene or protein expression, enzyme activity, lipid content) within adipose tissue.

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate ...