Neuroprotective effect of aquaporin-4 deficiency in a mouse model of severe global cerebral ischemia produced by transient 4-vessel occlusion

Abstract

Astrocyte water channel aquaporin-4 (AQP4) facilitates water movement across the blood-brain barrier and into injured astrocytes. We previously showed reduced cytotoxic brain edema with improved neurological outcome in AQP4 knockout mice in water intoxication, infection and cerebral ischemia. Here, we established a 4-vessel transient occlusion model to test the hypothesis that AQP4 deficiency in mice could improve neurological outcome following severe global cerebral ischemia as occurs in cardiac arrest/resuscitation. Mice were subjected to 10-min transient bilateral carotid artery occlusion at 24h after bilateral vertebral artery cauterization. Cerebral blood flow was reduced during occlusion by >94% in both AQP4(+/+) and AQP4(-/-) mice. The primary outcome, neurological score, was remarkably better at 3 and 5 days after occlusion in AQP4(-/-) than in AQP4(+/+) mice, and survival was significantly improved as well. Brain water content was increased by 2.8±0.4% in occluded AQP4(+/+) mice, significantly greater than that of 0.3±0.6% in AQP4(-/-) mice. Histological examination and immunofluorescence of hippocampal sections at 5 days showed significantly greater neuronal loss in the CA1 region of hippocampus in AQP4(+/+) than AQP4(-/-) mice. The neuroprotection in mice conferred by AQP4 deletion following severe global cerebral ischemia provides proof-of-concept for therapeutic AQP4 inhibition to improve neurological outcome in cardiac arrest.

Keywords: AQP4; Astrocyte; Brain edema; Cerebral ischemia; Neuroprotection; Water transport.

Fig. 1

4-Vessel occlusion model of severe global cerebral ischemia in mice. (A) Diagram of approach showing permanent vertebral artery (VA) occlusion produced by cautery at 24 h before transient bilateral common carotid artery (CCA) occlusion. ICA: internal carotid artery. (B) Cerebral blood flow measured by Doppler at indicated times in 4-vessel (left) and 2-vessel (right) models (mean ± S.E., 4 mice per group). (C) Cerebrovascular anatomy visualized following intravenous ink injection.

Fig. 2

Improved survival and neurological outcome of AQP4-deficient mice following 4-vessel occlusion. (A) Survival plot from 12 AQP4^+/+ and 12 AQP4^−/− mice undergoing 10-min bilateral carotid artery occlusion 24 h after permanent vertebral artery occlusion. Survival was significantly different with P < 0.05 (log-rank test). (B) Neurological score (see Section 2, score 12 without neurological impairment) shown for individual mice from study in A (S.E., *P < 0.05). (C) Brain water content determined from wet-to-dry weight ratios for control mice and mice at 1 day after 4-vessel occlusion (S.E., 6 mice per group, *P < 0.05).

Fig. 3

Reduced neuronal injury in AQP4-deficient mice following 4-vessel occlusion. (A) Hematoxylin and eosin-stained sections through hippocampus in control mice and at 5 days after 4-vesssel occlusion (top). Higher magnification of boxed areas shown. NeuN (neuronal marker) immunofluorescence (bottom). Micrographs representative of 5 mice studied per condition. (B) Pathological score (see Methods, score 0 without pathology) deduced from analysis of hippocampal sections (S.E., 5 mice per group, **P < 0.05). (C) TTC staining of freshly cut brain of control mice and at 5 days after 4-vessel occlusion. Hippocampus region indicated by box. Representative of TTC staining from 5 mice.

Fig. 4

Assessment of astrocytes, myelin, inflammation and blood–brain barrier integrity following 4-vessel occlusion. Immunostaining of brain sections in control mice and at 5 days after 4-vessel occlusion with stains for astrocytes (AQP4 and GFAP), myelin (MBP), microglia (Iba1), infiltrating leukocytes (CD45) and blood–brain barrier integrity (albumin). Micrographs are representative of 5 mice per group.