Transient MCAO and treatments

Transient focal cerebral ischemia was induced in mice by MCAO as we have previously described (tMCAO) (47). Thirty-three mice (male, 24 to 28g; C57BL/6) were used in this study. The criterion for exclusion was the development of subarachnoid hemorrhage. No animals were excluded from this study. Surgeries and all behavioral and histological assessments were performed by an investigator blinded to the drug treatment. Mice were anesthetized with 3% isoflurane vaporized in O₂ for induction and 1.5% isoflurane for maintenance. Temperature was maintained at 36.5° ± 0.5°C, controlled by a thermostatic blanket (CMA 450 Temperature Controller for mice, Harvard Apparatus) throughout the procedure. The left common carotid artery was exposed, and the occipital artery branches of the external carotid artery (ECA) were isolated and coagulated. The ECA was dissected distally and coagulated along with the terminal lingual and maxillary artery branches. The internal carotid artery (ICA) was isolated, and the extracranial branch of the ICA was then dissected. A rubber silicone-coated monofilament suture (filament size, 6-0; diameter, 0.09 to 0.11 mm; length, 20 mm; diameter with coating, 0.23 ± 0.02 mm; coating length, 5 mm; Doccol Corp.) was introduced into the ECA lumen through an incision and then gently advanced about 9 to 9.5 mm in the ICA lumen to block MCA blood flow. For reperfusion, the suture was withdrawn 60 min after MCAO. 2D laser speckle flowmetry (PeriCam PSI HR, Perimed) was used to confirm MCAO and reperfusion. Right after removal of the suture, animals randomly received an intraperitoneal injection of PBS, ApoM-Fc, or ApoM-Fc–TM.

Physiological parameters (arterial O₂ saturation, heart rate, pulse distention, and respiratory rate) were recorded before, during, and after tMCAO using the MouseOx Plus (Starr Life Sciences Corp.). After the surgery, all animals were maintained in a small-animal heated recovery chamber (IMS Vetcare Chamber Recovery Unit, Harvard Apparatus). After recovery, animals were returned to their cages with free access to food and water. The mortality rate was 1/11 in PBS-treated mice, 0/11 in ApoM wild-type mice, and 1/11 in ApoM-TM–treated mice.