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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Isolation of Short Posterior Ciliary Arteries
Porcine eyes together with optic nerve and extraocular tissues were obtained immediately post-mortem. Enucleated eyes were transported to the laboratory in ice-cold phosphate buffered saline (PBS) and used immediately.
NOTE: The porcine eye is basically divided into the anterior (Figure 1A) and posterior sections (Figure 1B).
- Place the eye globe in a dissection chamber containing ice-cold Krebs-Henseleit (K-H) buffer. Carefully cut away surrounding muscle and tissues with a pair of sharp Mayo scissors.
NOTE: To prepare Krebs-Henseleit (K-H) buffer, weigh the following chemicals (27.68 g of NaCl, 8.4 g of NaHCO3,1.4 g of KCl, 1.18 g of MgSO4, and 0.64 g of KH2PO4) into one dry and clean 50 mL conical centrifuge tube and, 1.47 g of CaCl2 and 8.0 g of glucose into another tube. The powder mixture of these chemicals should be stored in a cool and dry place. The CaCl2 and glucose powder mixture is best prepared fresh to prevent absorption of moisture and clumping.
- Make an incision with a scalpel and cut the globe along the equatorial plane with a pair of sharp Mayo scissors until the eye is separated into the anterior and posterior halves. Remove as much vitreous body as possible from the posterior half of the eye using a pair of standard pattern forceps.
NOTE: Separation of the eye globe into two halves facilitates the isolation of short posterior ciliary arteries (sPCA) with ease without having a moving eye in the dissection dish. However, this step is not mandatory. The vessels can also be isolated without opening the eye globe.
- Use dissection pins to carefully pin down the posterior half of the eye with the retina side down and the optic nerve facing up towards the experimenter.
- Gently cut away the connective tissues surrounding the optic nerve to expose the underlying retrobulbar vasculature with a pair of Student Vannas spring scissors.
- The sPCA can be seen as short branches of vessels (between 5-8 branches) that penetrate the sclera and circumferentially surround the optic nerve head (Figure 1C). Isolate the paraoptic and distal sPCA together with the surrounding connective tissues with a pair of type 5 precision tweezers and Vannas capsulotomy scissors (Figure 1D).
NOTE: Ensure that the K-H buffer remains ice-cold throughout the isolation procedure. It is highly recommended to change the buffer every 20-30 min, depending on the ambient temperature.
2. Sample Preparation
- Gently remove connective tissues from the arterial segments using extra fine-tipped type 5 precision tweezers and Vannas capsulotomy scissors under a stereomicroscope and rinse the isolated arteries in ice-cold PBS to remove contaminants and blood residues.
NOTE: If samples are not subjected to the next steps immediately, snap-freeze them in liquid nitrogen and store in -80 °C until further use.
