Retroorbital injections

Rat serum was administered retro-orbitally in injured fish 1 h post-pHIFU injury. This method has previously been shown as an effective method to introduce drugs and cells into the circulation of adult zebrafish with minimal mortality and complications (Pugach et al., 2009). In brief, fish anesthetized with clove oil (200 ppm) were placed laterally onto the left side onto a damp sponge. A 26 G Hamilton micro-syringe was backfilled with 4 μl of serum. The needle tip was introduced adjacent to the eye at the 9:00 position. The needle was inserted approximately 1−2 mm into the retroorbital cavity followed by injection of the serum. Successful injection was confirmed by the protrusion of the eye during the injection procedure. The eye protrusion returned to normal by 12 h. Fish were recovered in normal tank water and returned to their holding tanks in the animal facility. We observed no mortality as a result of the retro-orbital injection procedure in this study. All fish received a retroorbital injection by a technician who was blinded to the contents of the injectable solutions. Sham fish were injected with filtered water, while pHIFU injured fish received RIC serum or serum from rats exposed only to isoflurane anesthesia (Iso).