The overall goal of this procedure is to investigate the mechanism of Corneal Endothelial-mesenchymal Transition In Vitro and In Vivo. This method can help answer questions about Corneal Endothelial-mesenchymal Transition in the field of Corneal Endothelial regeneration. The advantage of this technique is that it can be applied to both In Vitro and the In Vivo models.
And is easy to manipulate. The implications of this technique is tend towards therapy of disease related to Corneal Endothelial-mesenchymal Transition. Such as formation, because the models can be used to select to us that inhibits the process of Corneal Endothelial-mesenchymal Transition.
The the procedure will be miss Jung-Shen Chang, a technician from a laboratory. To begin this procedure, disinfect fresh bovine eyes in a 10%weight per volume Povidone-iodine solution for three minutes. Then, wash them with PBS.
Harvest the corneal button with a scalpel and scissors and wash it with PBS Under a dissecting microscope, peel the Descemet's Membrane with forceps. After that, incubate the Descemet's Membrane in one milliliter of Trypsin at 37 degrees Celsius for 30 minutes. Next, collect the bovine CECs by centrifugation at 112 times g for five minutes.
After centrifugation, re-suspend the cells in one milliliter of SHEM. Seed the cells in a six centimeter dish and culture them in SHEM. Subsequently, incubate the cells at 37 degrees Celsius in 5%CO2 and change the culture medium every three days.
When the cells reach confluence, wash them in PBS. Then, incubate them in one millimeter of Trypsin at 37 degrees Celsius for five minutes. Collect them by centrifugation at 112 times g for five minutes.
After five minutes, re-suspend the cell pellet in one millimeter of SHEM. Next, count the cells in a Hemacytometer. Following that, seed the cells on cover slides in a 24-well plate and culture the cells in SHEM.
For investigating the ENMT-suppressing effect of Maramastat, incubate the cells in SHEM with 10 micromolar Maramastat in the culture medium and change the culture medium every three days. Then, fix the cells at an indicated time point with 250 microliters of 4%Paraformaldehyde for 30 minutes at room temperature. Afterward, permeablize them with 250 microliters of 0.5%Triton X-100 for five minutes.
And block them with 10%BSA for 30 minutes. Subsequently, incubate the cells with primary antibodies against ABC, snail, and slug overnight at four degrees Celsius. The next day, wash the cells twice with PBS for 15 minutes each time.
And incubate them with the Alexa Fluor conjugated secondary antibody at room temperature for one hour. After that, counterstain the cell nucleus by covering the cells with DAPI at two micrograms per milliliter. Wash the cells twice with PBS for 15 minutes each time.
And mount them with anti-fading mounting solution before obtaining the immunoflourescent images. After anesthetizing a 12-week old male SD rat, gently pinch the skin of the animal to confirm proper anesthesia in the absence of skin twitching. Then, apply a drop of 0.5%Proparacaine Hydrochloride to the rat's right eye to minimize pain and the blink reflex.
Subsequently, apply Tetracycline ointment to the left eye to prevent corneal dryness. Next, cool a stainless steel probe in liquid Nitrogen. Apply the stainless steel probe to the central cornea of the right eye for 30 seconds.
Frequently instill PBS to the right eye during the procedure to prevent corneal dryness and apply 0.1%Atropine and 0.3%Gentamicin Sulfate immediately after cryoinjury once daily to relieve ocular pain resulting from Ciliary spasm and to prevent infection. After the procedure, keep the rat warm using a heat lamp and observe its recovery until it regains motor control. Apply Tetracycline ointment to the right eye to prevent corneal dryness during the recovery period and repeat the corneal cryoinjury procedure for three consecutive days.
For delivering Maramastat or basic FGF into the interior chamber of the rat eyes, apply a drop of 0.5%Proparacaine Hydrochloride to the right eye of the anesthetized rat to minimize pain and the blink reflex. Next, irrigate the ocular surface with sterile PBS. Under an operating microscope, perform anterior chamber paracentesis by inserting a 30 gauge needle attached to a one millimeter syringe at the paralimbal clear cornea in a plain, above, and parallel to the iris Then, turn the needle bevel up and slightly depress the corneal wound to drain some aqueous humor and reduce the interocular pressure.
Inject 0.02 milliliters of the drug intracamerally and gently compress the needle tract with a cotton tip during the needle withdraw. This figure presents the phase contrast images of the bovine CECs and cultures. The hexagonal shape of the cells at confluence indicates that the cells are not contaminated by corneal stromal fibroblasts during cell isolation.
During the In Vitro culture of bovine CECs, the nuclear translocation of ABC, snail, and slug was detected through day 14. This figure illustrates the effect of Maramastat, a broad spectrum MMP inhibitor on the ENMT process of the In Vitro cultured bovine CECs. Here, are the external eye photographs of rats after cryoinjury, followed by intracameral injection.
The photographs show reduced corneal edema after basic FGF injection. Whereas Maramastat further reduced corneal edema compared with basic FGF alone. And this figure shows immunostaining of the rat corneal button using antibody against ABC after cryoinjury followed by intracameral injection.
The nuclear translocation of ABC is observed in the PBS group and is significantly increased in the basic FGF group, indicating activation of Wnt Beta-catenin signalling and the ENMT process. After intracameral injection, a basic FGF followed by Maramastat, the nuclear staining of ABC is diminished, suggesting the ENMT-inhibiting effect of Maramastat. After its development, this technique pave the way for researchers in the field of Corneal Endothelial-mesenchymal Transition, In Vitro and the In Vivo.
After watching this video, you should have a good understanding of how to isolate bovine corneal endothelial cells and to perform corneal cryoinjury as well as intracameral injection
