העיתון מתאר שיטה פשוטה כדי הקרנית בלו וכדי להוציא את העקר רקמות רשתית העין מן העולם של תורמים גווייה אדם. הטכניקה המתוארת כאן יעזור ובלו רקמות באיכות טובה כדי לשמש להשתלה, למטרות מחקר או ניתוח מבלי לפגוע ברקמות אחרות של הגלובוס העין.
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.
שניהם, כריתה נכונה ושמירה נכונה של רקמות הקרנית הרשתית הם קריטיים כמו פגמים קלים כגון נזק האנדותל או מספר גבוה של קפלים של descemet יכול להוביל לכישלון השתל של הקרנית ואילו שינויים בטמפרטורה או טיפולו הכושל יכול להתפשר על שלמות רקמות הרשתית. מטרת מאמר זה היא להראות כיצד ?…
The authors have nothing to disclose.
עבודה זו נתמכה בחלקה באמצעות מענקים של ונטו Regione (Ricerca Sanitaria Finalizzata n.292/2008 ו Ricerca Sanitaria Finalizzata 2009). המחברים מודים לד"ר ג Griffoni עבור הנתונים 2009/2010 סיכום על השימוש גלובס עיניים אנושיות.
Materials for excision of cornea and retina.
Product description | Dimensions | Company / Institute |
Guarded disposable scalpel | Blade size 15 | Swann-Morton |
Sterile bandages | 5 cm X 5 cm, 8 layered, 5 pcs | Artsana |
Sterile disposable medical towel | 35 X 50 cm | U.Jet |
Sterile scissors | Blades 24 mm / overall length. 95 mm curved, blunt | e.janach |
Sterile forceps | Stainless steel -100 mm 11 X 2 ruled by 0.70 mm teeth | e.janach |
Corneal claw – Disposable medical device | NIIOS (Hippocratech) | |
Preparations | ||
PBS | 100 ml PBS [10x] in 900 ml d/w (distilled water) | Sigma-Aldrich |
Na-thiosulphate | 1 gm Na-thiosulphate in 1 litre of PBS [1x] | Sigma-Aldrich |
I-PVP | 5 gm I-PVP in 1 litre d/w | Sigma-Aldrich |
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).
Components | Supplier | Catalogue number | Concentration | Quantity |
MEM (1X) liquid | Invitrogen | 32360-034 | 1900 ml | |
Sodium pyruvate | Invitrogen | 11360-039 | 1mM (10ml/l) | 20 ml |
L-glutamine | Invitrogen | 25030-032 | 2mM (10ml/l) | 20 ml |
Antibiotic/antimycotic | Sigma-aldrich | A5955-20ML | 10ml/l | 20 ml |
Newborn calf serum | Invitrogen | 26010-74 | 2% (20 ml/l) | 40 ml |
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).
Components | Supplier | Catalogue number | Concentration | Quantity |
MEM (1X) liquid | Invitrogen | 32360-034 | 1800 ml | |
Sodium pyruvate | Invitrogen | 11360-039 | 1mM (10 ml/l) | 20ml |
L-glutamine | Invitrogen | 25030-032 | 2mM (10 ml/l) | 20ml |
Newborn calf serum | Invitrogen | 26010-74 | 2% (20 ml/l) | 40 ml |
Dextran t500 | Pharmacosmos | 551005004007 | 6% (60 gm/litre) | 120 g/l |
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.