Упрощенная Техника<em> В месте</em> Удаление роговицы и сетчатки Evisceration ткани человека глобус Глазные

Summary

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 исследовательских целей, не повреждая другие ткани глазного шара.

Abstract

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 vision^4,5,6. 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 retina^5,6. 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 nerve^5,6.

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.

Protocol

1. В местах иссечения роговицы от глазных глобус Включите ламинарного потока воздуха шкаф примерно 15 минут перед использованием. Очистите капот ламинарного потока с использованием 70% изопропилового спирта. Положите на индивидуальную защитную одежду, таких как хирургически?…

Discussion

И, правильное удаление и надлежащую сохранность роговицы и сетчатки тканей являются критическими как незначительные дефекты, такие как повреждение эндотелия или большим количеством складок Десцеметова может привести к недостаточности трансплантата роговицы в то время как температ?…

Materials

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.