Name: a simplified technique for in situ excision of cornea and evisceration of retinal tissue from human ocular globe
Uploaded: 2012-06-12T13:00:00
Description: 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

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
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 <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 steril...

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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 ...

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