Distal middle cerebral artery occlusion model for real time optical monitoring of cerebral blood flow and metabolism

Background and aims: Optical imaging of cerebral cortex has recently gained momentum with the advent of novel technologies such as laser speckle flowmetry (LSF), multispectral reflectance imaging (MSRI), intrinsic optical signal imaging, and multiphoton confocal microscopy. Here we describe the technique and characterize the hemodynamic, electrophysiological, histological and behavioral outcome features of a distal middle cerebral artery occlusion (dMCAO) model where ischemia and reperfusion can be reproducibly achieved during real-time optical imaging. Methods: Isoflurane-anesthetized, intubated and ventilated mice (C57BL/6) with arterial blood gas and pressure monitoring are placed in a stereotaxic frame, and dorsal skull surface is exposed. After stripping of the temporalis muscle, distal MCA is exposed via a small burr hole in the temporal bone just above the zygomatic arch, and occluded using a microvascular clip. LSF and MSRI are simultaneously and continuously performed before and during dMCAO. Reperfusion is achieved 60 min later by careful removal of the clip, and confirmed by imaging. Infarct volume and tissue swelling are assessed at 24 and 48 hr (TTC), and 1 month (H&E). Neurological deficits are longitudinally assessed using 5-point, corner and cylinder tests (n=5). Results: Infarcts were limited to the dorsolateral cortex sparing the striatum. Infarct volumes were significantly larger at 48h compared to 24 hr after reperfusion (21+/-2 vs. 10+/-2 mm3, n=5 and 8, respectively, p<0.05; +/-SEM). By overlying the dorsal view of the cortical infarct at 48h (TTC) and the dorsal CBF map 60 min after dMCAO (LSF), we determined that residual CBF less than 28+/-2% of preischemic baseline resulted in cerebral infarction (Fig). Ischemic tissue swelling (ipsilateral-contralateral hemisphere) was detectable at 24h (6.2+/-1.0 mm3) and tended to increase at 48h (8.1+/-2.4 mm3). There were 2.8+/-0.4 peri-infarct depolarizations/h, as determined by spreading hypoperfusion waves on LSF and confirmed by electrophysiological recordings using glass micropipettes. Neurologically, cylinder test showed reduced use of contralateral paw during a full rear; this motor deficit fully recovered between days 7 and 14. Corner test was more sensitive at later time points, where an ipsilateral preference was detected at 28d. The traditional 5-point grading scale did not demonstrate deficits at any time point after occlusion. Conclusions: Real time optical and electrophysiological monitoring of focal cerebral ischemia is feasible in a dMCAO mouse model and may be useful for examining the impact of specific genes on cerebrovascular function.