A Novel Model of Transient Occlusion of the Middle Cerebral Artery in Awake Mice

Abstract

Background: Stroke is one of the leading causes of death and disability worldwide. As a consequence, several excellent rodent models have been developed to gain insight into the pathophysiology of stroke and testing the efficacy of neuroprotective interventions. However, one potential problem is that albeit roughly 80% of strokes occur in awake patients, all existing murine stroke models employ anesthesia. Moreover, epidemiological studies have shown that stroke injury is more severe in the minority of patients that suffer stroke while asleep. To better mimic the clinical situation, it is therefore preferable to analyze the pathobiology of experimental stroke in awake animals.

New method: This model of transient middle cerebral artery occlusion (MCAO) in awake mice is based on insertion of an intraluminal suture via the external carotid artery during isoflurane anesthesia. Isoflurane is discontinued during the 60 min MCAO while cerebral blood flow is continuously monitored using laser doppler in the awake unrestrained mouse.

Results: Sixty minutes of awake MCAO model reliably induced infarction in striatum and cortex (relative infarct volume is 24.34% of contralateral hemisphere volume; absolute infarct volume is 47.94 mm³).

Comparison with existing methods: The animals in this method are awake during the one hour occlusion period, which may provide a more translational research approach than existing methods that apply anesthesia during the occlusion.

Conclusions: Since the state of brain activity likely affects stroke injury and most anesthetics have neuroprotective effects, this model more accurately mimics the condition during which the majority of human ischemic stroke occurs.

Keywords: Awake Mouse; MCAO; Stroke; Transient MCAO.

Figure 1. Schematic diagram of surgical procedure and transient MCAO time line in subjected mice

(A) Summary diagram of awake MCAO surgical procedures. The monofilament suture was inserted from external carotid artery (ECA) into the internal carotid artery (ICA) and pushed forward for a distance of 9–10 mm beyond the bifurcation of common carotid artery (CCA) until a minor resistance is felt. Blood flow in the MCA territory was continuously recorded by laser doppler. One femoral artery was cannulated for recording of mean arterial blood pressure (MABP). (B) Timeline of awake MCAO. The duration of surgery took 30–45 mins. Once the filament was correctly inserted in the trunk of the MCA, cerebral blood flow dropped by >90%. The incision on the neck was closed and the isoflurane anesthesia was discontinued. The animals were returned to a heated cage (Deluxe reusable animal Cages, Tecniplast Inc., West Chester, PA) while blood flow in the MCA territory and MABP were continuously monitored. After 60 minutes, the mice were re-anesthetized with 2% isoflurane for another 20 mins for removing MCA filament, withdrawing femoral artery catheter, closing the skin and cutting the Doppler probe. Anesthesia was then stopped and the mouse was placed into the transparent box again for recovering. 2 hours later, the mice were returned to their home cage. 24 hrs after MCAO, the mice are re-anaesthetized with ketamine/xylazine. When the mice were unresponsive to stimulation they were quickly decapitated. Their brains were removed and coronally sectioned for TTC staining and imaging.

Figure 2. Representative traces of cerebral blood flow (laser doppler) and mean arterial blood pressure as well as images of TTC and quantification of the ischemic lesions

(A) The cerebral blood flow dropped by 90.0 % ± 3.0 % when the MCA was occlusion and remained low during the 60 min artery occlusion. Removal of the filament occluding the MCA resulted in hyperemia (242 % ± 28.9 %), n=6. Mean blood pressure (MBP) remained stable ~ 100mmHg. In coronal sections prepared 24 hours after awake MCAO, clear ischemic lesions were evident in striatum and dorsolateral cortex. Infarct volume corrected for edema in the ischemic hemisphere and normalized to whole brain was 24.34 ± 3.9 %, and absolute infarct volume is 47.94 ± 7.75 mm³. The infarct volume of cortex and striatum is 27.24 ± 6.21 and 15.34 ± 2.02 mm³, n=6.