Today We are demonstrating a simplified technique to exercise corneal and to ate retinal tissue from a human ocular globe of cadaveric donors. The technique described the year will help to exercise good quality tissue to be used for transplantation, surgical, oral research purposes without damaging other tissue of the ocular globe. With 18 years of expertise and Andres of cornea transplanted each year, we have maintained a good standard of our work by developing new techniques and improving our skill every day.
To achieve this, we exercise the cornea first, followed by retinal evis. 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. The cornea comprises five layers, the epithelium, bowmans, layer, stroma, desus membrane, and endothelium.
If the cornea is damaged, it can be replaced with the help of a suitable donor cornea. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the bruss membrane and is responsible for blood flow in the eye. Appropriate evisceration of the choroid is as important as removal of retina.
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 as it is transparent and delicate, it should be eviscerated systematically for a good quality of tissue. Switch on the lanar airflow cabinet.
Clean the lanar flow hood using 70%isop alcohol. Put on personal protective clothing such as surgical cap mosque, sterile gloves, and goner sleeves. Using aseptic technique, set up the sterile field by placing a sterile tole and a pre sterilized instrument tray.
Aseptically open the sterile instruments back and flip onto the sterile field, avoiding any contamination. Position all sterile materials and instruments in the ster field and place eye jars containing eye globes so that they are sistant to the edge of the sterile field. For ease of use, label the bottles with pre-prepared storage medium and place iodine polyol pyro lid on sodium th sulfate, and a sterile phosphate buffer saline solution.
Outside the sterile feed, allow the ocular tissues and solutions to reach normal room temperature as they are preserved in refrigerators at four degrees Celsius. Avoid repeated warming or cooling cycles. Remove gazes and sponges from the eye jar using sterile forceps.
Place the ocular globe on the lid and remove the additional bandages. Carefully pick the ocular globe with the optic nerve and immerse the globe in sterile. IPVP decontaminate the ocular globes in sterile IPVP for two minutes.
Transfer the globe to sterile sodium thous sulfate for one minute. Transfer the globes to sterile phosphate buffer cell line solution and leave till it is operated. Working underneath the lanar airflow hood, wash and remove the ocular globe from PBS and place it under the sterile field.
Wrap the globe using steroid, leaving the cornea and approximately five millimeters sclera from the cornea uncovered the eye bulb could be simply held in the hand. Maintaining the adequate pressure using sterile scissors. Remove all eventual remains of the conjunctiva without damaging the cornea.
Use a disposable scalpel blade to perform a scleral incision of three to four millimeters from the limbus region. Then extend the incision by 360 degrees, 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. Presence of small scleral plaques may hamper the cutting, so ensure the completion of scleral excision with microsurgery scissors. This operation should be performed without damaging the choroid retina and vitreous body.
Inspect the incision to ensure it is complete. If the incision has been performed correctly, the cornal scleral rim adheres to the ciliary body only at the point in correspondence with the cornea, set the rib ocular globe down near the center of the sterile feed. Complete the cornea removal using sterile forceps.
Hold the scleral rim stationary and excise the cornea scleral rim away from the ocular glow. Gently separate any remaining adjacents from the corno scleral button. Never pull the corneal scleral rim in away that could cause cross corneal tension, nor allow it to drop back down onto the anterior chamber.
The endothelial cell density of the cornea is checked using hypotonic solution and cell mortality using trip and blue staining for around one minute. Remove the trip and blue stain on a sterile wire gauze and wash the conia in sterile PBS. Transfer it in a sterile Petri plate filled with sucrose solution.
This solution helps to create a hypotonic environment and helps to identify the cells visually. The cells are then counted under an optical microscope under a hundred x magnification and measured as cells per millimeter square. The cells are usually looked up for cell density morphologic like intercellular borders, polymorphism degeneration, dystrophy, and cell mortality.
The corneal thickness is also an important feature for transplantation, along with a good transparency rate of the excised cornea. Sterile corneal CLO is used to fix the cornea at the scleral rim, bend the corneal CLO to ensure an opening of the hole sufficient to lock the scleral rim, transfer the corneal scleral rim using a corneal clove to previously prepared corneal storage, medium prepared at room temperature. The cornea should then be preserved at 31 degree SIUs during the four week preservation of the cornea.
The storage media undergoes several microbiological tests using a B 9 2 4 0 instrument. As during storage, the cornea gets thicker than its usual size required for transplantation. Eye banks use D swelling agents to reduce thickness 6%Dextron T 500 is used to get the cornea back to its normal size and transport medium.
Clean the sterile instruments with RNA app to create a RA free environment prior to retinal excision. Prepare the sterile field and make sure that the ocular globe is intact and only the cornea and scleral rim is removed. If the gerus vitreous body is secreting out, it becomes difficult to remove the retinal tissue.
If the donor is below 65 years of age, the sclera is preserved under 70%alcohol for surgical purposes after serological testing. In such cases, the sclera is not cut, only the vitreous body is removed and the entire sclera is preserved for retinal excision. Start with a small cut or incision near the corneal side of the sclera, moving towards the optic nerve using sterile forceps and scissors.
Hold the part of the sclera using sterile forceps and cut with the scissors straight towards the optic nerve part by part, avoiding damage to the vitreous body. The choroidal plugs should be separated from the sclera as they're stuck together. Cut the entire sclera to expose the choroid tissue.What?
Once the sclera is completely cut, separate the sclera with the remaining tissues by cutting it near the optic nerve, an entire layer of choroid covering the vitreous body will now be visible. Fill these sterile RNAs free append off tubes with two ml RL later using two sterile forceps. Remove the choroid layer gently by picking up the optic nerve with one pair of forceps and removing the choroid with the other.
Separate the choroid partially and cut it to facilitate the removal. The poid layer could be cut using scissors avoiding damages to the retina. Place the excised choroidal layer inside the RNA later solution and preserve them at four degrees Celsius for 24 hours.
Remove all the additional choroid to expose the retinal tissue. Once the entire choroid layer is removed, a transparent layer of retina will be seen. Clearly, the choroid layer has an interior brus membrane, which could also be removed, but it is difficult to identify it with the naked eye.
Similarly, use two forceps to remove the retinal layer. 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.
Place the excise retina inside the RNA later solution and preserve it at four degrees Celsius for 24 hours. Clean the working area using 70%ISO prop profile alcohol. The endothelial cells stain using trip and blue can be viewed under a hundred x magnification of an inverted microscope.
A suitable cornea with more than 200 cells per millimeter square is usually preserved for further use. Rating them from fair to excellent quality will help for comparative studies. Usually once the cornea is ready for transplantation thickness and the transparency is noted, a suitable cornea is used for penetrating kersty where entire cornea is transplanted.
Cell mortality is checked by observing stained blue cells and rated in percentage of dead cells. Overall cell mortality of more than 2%is considered as unsuitable during preservation. If the cell density is lower than 2200 cells per millimeter square or damage due to mortality, the cornea could be used for anterior laminar kersty.
If the anterior trauma is damaged and the cornea has a good endothelial cell density, the cornea could be used for posterior laminar kersty. Thickness of the cornea is usually increased during organ culture. Transport medium helps to regain its normal shape and structure back.
Also, organ culture helps re epithelialization of the cornea and due to its long storage period, it helps for a proper donor selection. Microbiological and serological testing. It gives enough time to arrange a surgery and helps to check the endothelial cell density and morphology when it is not possible due to high postmortem time.
Thus, if followed the procedure as described, the corneas could be excised without any damage to other tissues. If the cornea is not suitable for transplantation, the cornea could be used for research purposes to develop a new media and preservation conditions to study the ocular surface diseases, to isolate limbal stem cells and to study the effect of drugs on cornea. For retina, we can avoid cross-contamination of RNA samples with cent tissues and preserve RNA integrity, which is of fundamental importance.
These tissues indispensable for research purposes are aimed at characterizing the transcriptome of the ocular tissues, isolating stem cells for regenerative medicine projects, and evaluating histological differences between tissues from normal or affected subjects. 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 r nator or thisis buffer for RNA or DNA extraction, displaced or trypsin for isolation of cells.
Fixing solutions prior to embed tissues in paraffin or OCT for immunohistochemistry. Thanks for watching this video and good luck with your research.
