Increased brain edema in aqp4-null mice in an experimental model of subarachnoid hemorrhage

Abstract

We investigated the role of the glial water channel protein aquaporin-4 in brain edema in a mouse model of subarachnoid hemorrhage in which 30 microl of blood was injected into the basal cisterns. Brain water content, intracranial pressure and neurological score were compared in wildtype and aquaporin-4 null mice. We also measured blood-brain barrier permeability, and the osmotic permeability of the glia limitans, one of the routes of edema elimination. Wildtype and aquaporin-4 null mice had comparable baseline brain water content, intracranial pressure and neurological score. At 6 h after blood injection, aquaporin-4 null mice developed more brain swelling than wildtype mice. Brain water content increased by 1.5+/-0.1% vs. 0.5+/-0.2% (Mean+/-Standard Error, P<0.0005) and intracranial pressure by 36+/-5 vs. 21+/-3 mm Hg (P<0.05) above pre-injection baseline, and neurological score was worse at 18.0 vs. 24.5 (median, P<0.05), respectively. Although subarachnoid hemorrhage produced comparable increases in blood-brain barrier permeability in wildtype and aquaporin-4 null mice, aquaporin-4 null mice had a twofold reduction in glia limitans osmotic permeability. We conclude that aquaporin-4 null mice manifest increased brain edema following subarachnoid hemorrhage as a consequence of reduced elimination of excess brain water.

Figure 1. Mouse SAH model

A. (Top) Mouse calvarium. Burr hole 1 is for injection and burr hole 2 for ICP monitoring. (Bottom) The injecting needle is advanced 30° to the perpendicular into the basal cisterns. B. Exposed dura overlying cisterna magna and CSF at T6 before and after injecting blood into the basal cisterns. C. ICP traces. Arrowheads show onset of blood injection. Inset 1: prominent respiratory waves. Inset 2: cardiac ICP pulsations. D. Coronal brain section at 2.8 mm from the frontal poles stained with hematoxylin-eosin showing normal-sized 3^rd ventricle 24 h after sham injection or SAH. Bar = 0.5 mm. E. Brain sections 24 h after needle insertion (Sham) or SAH. Mice had i.v. injection of 10 kDa rhodamine-dextran (red) and 2 MDa FITC-dextran (green). Bar = 50 µm.

Figure 2. Brain edema after SAH

A. Whole brain water content measured at 6 and 24 h after needle insertion or SAH. B. (Top) Typical ICP recordings from 2 WT and 2 KO mice at 6 h after SAH and (Bottom) summary of ICP data measured at 6 and 24 h after needle insertion or SAH. ● = WT (+/+), ○ = KO (−/−), X = dead mice. ■ = Mean of WT, □ = Mean of KO, Error lines = ±SEM. *P < 0.05, **P < 0.01, #P < 0.0005 for WT vs. KO.

Figure 3. Neurological score after SAH

A. Neurological scale. B. Neurological score at 6 and 24 h after needle insertion or SAH in WT and KO mice. ● = WT (+/+), ○ = KO (−/−), X = dead mice. — = median. *P < 0.05 for WT vs. KO.

Figure 4. Blood-brain barrier and glia limitans

A. (Left) Mouse brains in formamide. WT (+/+) and KO (−/−) mice had needle insertion (Sham) or SAH. (Right) Summary of Evans blue data. N = 4 (Sham, 2 +/+ and 2 −/−), 3 (SAH, +/+), 3 (SAH, −/− ). Mean ± SEM. *P < 0.05 compared with sham. B. Setup for measuring glia limitans osmotic permeability. C. Difference in % water content between right and left hemispheres (ΔH₂O) in WT (●, +/+) and KO (○, −/−) mice exposed to 4 % agarose gel in water (hypotonic) or 0.9 % saline (isotonic). ■ = Mean of WT, □ = Mean of KO, Error lines = ±SEM. *P < 0.02.

Figure 5. Aqp4 immunoreactivity after SAH

A. (top) Glia limitants and (bottom) basal cerebral cortex. In the sham WT mouse (+, left), red arrows indicate the pia, asterisks the glia limitants, and arrowheads show microvessels. In the WT mouse 24 h after SAH (++, middle), ‘R’ indicates red blood cells overlying the pia, white asterisks the subpial region, arrowheads show microvessels, and black asterisks show aqp4 immunoreactivity in the brain parenchyma. In the KO mouse 24 h after SAH (0, right), ‘R’ indicates red blood cells covering the pia. Bar = 50 µm. B. Summary of immunohistochemical staining, graded as 0, +, ++ as described in A.