Isolating Short Posterior Ciliary Arteries: A Protocol to Excise Intact Short Posterior Ciliary Microvessels from Porcine Eye

Overview

This video describes the protocol to isolate the short posterior ciliary arteries from the porcine eye. The isolated short posterior arteries are a suitable source for elucidating the molecular insights and protein profiles of the ocular blood vessels.

Protocol

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).

1. 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.
2. 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.
3. 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.
4. Gently cut away the connective tissues surrounding the optic nerve to expose the underlying retrobulbar vasculature with a pair of Student Vannas spring scissors.
5. 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

1. 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.

Representative Results

Figure 1: Representative photographs of the porcine eyes.

(A) Lateral view of the eye globe shows the cornea, which is located in the anterior part of the eye, the sclera, surrounding muscle and optic nerve. (B) Posterior view of the eye shows the optic nerve. (C) Branches of sPCA seen at the back of the eye globe composed of the paraoptic and distal branches. (D) Isolated sPCA with surrounding fat and connective tissues.

Materials

Name	Company	Catalog Number	Comments
Calcium chloride dihydrate (CaCl2)	Carl Roth	5239.1	2.5 mM
Dulbecco's phosphate-buffered saline (PBS)	Thermo Fisher Scientific	14190169
Sodium chloride (NaCl)	Carl Roth	9265.2	118.3 mM
Sodium hydrogen carbonate (NaHCO3)	Carl Roth	965.3	25 mM
α-(D)-(+)- Glucose monohydrate	Carl Roth	6780.1	11 mM
Disposable scalpels #21	pfm Medical	200130021
Dissection pins	Carl Roth	PK47.1
Extra Fine Bonn Scissors	Fine Science Tools	14084-08
Falcon conical centrifuge tubes (50 mL)	Fisher Scientific	14-432-22
Mayo scissors, Tough cut	Fine Science Tools	14130-17
Precision tweezers	Fine Science Tools	11251-10	Type 5
Precision tweezers, straight with extra fine tips	Carl Roth	LH53.1	Type 5
Standard pattern forceps	Fine Science Tools	11000-12
Student Vannas spring scissors	Fine Science Tools	91501-09
Vannas capsulotomy scissors	Geuder	19760	Straight, 77 mm