Laser doppler probe

Surgical endovascular insertion of a silicon-coated monofilament (602012PK10; Doccol Corporation, Sharon, MA, USA) was performed to induce transient middle cerebral artery occlusion (MCAO) for 30 minutes of ischemia, followed by filament removal to allow reperfusion^{31 (link),33} . Briefly, 10-weeks-old C57BL/6J mice were anesthetized with sevoflurane (4% for induction, 3% for maintenance) in a mixture of O₂/N₂O (30/70%). After surgical exposure of the right carotid artery tree, the filament was inserted through the external carotid artery and advanced through the internal carotid artery until it reached the middle cerebral artery. The regional cerebral blood flow was monitored during surgery with a laser Doppler probe (Moor Instruments, Devon, UK). After 30 minutes of ischemia, the filament was removed to allow reperfusion. AZ67 (60 mg/kg of body weight) or vehicle were administered in a bolus (200 µl) via the jugular vein immediately after reperfusion. Body temperature was maintained at 37 ± 0.5 °C using a heating pad connected to a rectal probe (BAT-12 thermometer; Physitemp Instruments Inc., Clifton, NJ, USA). Mice were then sutured and returned to the cages. Sham-operated mice underwent the same surgical procedure without middle cerebral artery occlusion.

Transient MCAO model was induced by 60 min focal cerebral ischemia and reperfusion using a filament method, as we previously described [16 (link)–18 ]. Briefly, mice were anesthetized by inhalation of 3.5% isoflurane and kept by inhalation of 1.0–2.0% isoflurane in 70% N₂O and 30% O² using a face mask. A 6–0 nylon filament with rounded tip was insert into the right MCA to occlusion for 60 min. Reperfusion was established when the filament was withdrawn back to the common carotid artery. Laser Doppler probe (model P10, Moor Instruments, Wilmington, DE) was used to monitor the cerebral blood flow (CBF) for 5 min both before and after MCAO, as well as during reperfusion for 5 min. Relative CBF post reperfusion had to rise to at least 50% of preischemic levels in order for mice to be included for further analyses. During surgery procedures, body temperature was maintained by an electric warming blanket. The sham-operated group mice underwent identical surgical procedures except that the filament was not advanced to the MCA. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was used to measure the infarct volume at day 1 and day 3 after MCAO and reperfusion.

Relative CBF was continuously monitored at the site of the cranial window using a laser-Doppler probe (Moor Instruments; Axminster, UK) positioned stereotaxically 0.5 to 1 mm from the cortical surface. CBF values were expressed as percent increase relative to the resting level [(CBF_stimulus–CBF_resting)/CBF_resting]. Zero values for CBF were obtained after the heart was stopped by an overdose of isoflurane at the end of the experiment (Capone et al., 2012 (link)).
CBF recordings were started after arterial pressure and blood gases had reached a steady state, as previously described (Capone et al., 2012 (link)). All pharmacological agents and drugs studied were dissolved in a modified Ringer’s solution (Girouard et al., 2006 (link)). The increase in CBF produced by somatosensory activation was assessed by stimulating the whiskers contralateral to the cranial window by side-to-side deflection for 60 s. The endothelium-dependent vasodilator acetylcholine (10 µmol/L; Sigma-Aldrich) was topically superfused for 5 min, and the resulting changes in CBF were monitored. CBF responses to the smooth muscle-dependent relaxant adenosine (400 µM; Sigma-Aldrich) were also examined.