Aquaporin (AQP) water channels, essential for fluid homeostasis, are expressed in perivascular brain end-feet regions of astroglia (AQP4) and in choroid plexus (AQP1). At a high concentration, the loop diuretic bumetanide has been shown to reduce rat brain edema after ischemic stroke by blocking Na ϩ -K ϩ -2Cl Ϫ cotransport. We hypothesized that an additional inhibition of AQP contributes to the protection. We show that osmotic water flux in AQP4-expressing Xenopus laevis oocytes is reduced by extracellular bumetanide (Ն100 M). The efficacy of block by bumetanide is increased by injection intracellularly. Forty-five synthesized bumetanide derivatives were tested on oocytes expressing human AQP1 and rat AQP4. Of these, one of the most effective was the 4-aminopyridine carboxamide analog, AqB013, which inhibits AQP1 and AQP4 (IC 50 ϳ20 M, applied extracellularly). The efficacy of block was enhanced by mutagenesis of intracellular AQP4 valine-189 to alanine (V189A, IC 50 ϳ8 M), confirming the aquaporin as the molecular target of block. In silico docking of AqB013 supported an intracellular candidate binding site in rat AQP4 and suggested that the block involves occlusion of the AQP water pore at the cytoplasmic side. AqB013 at 2 M had no effect, and 20 M caused 20% block of human Na ϩ -K ϩ -2Cl Ϫ cotransporter activity, in contrast to Ͼ90% block of the transporter by bumetanide. AqB013 did not affect X. laevis oocyte Cl Ϫ currents and did not alter rhythmic electrical conduction in an ex vivo gastric muscle preparation. The identification of AQP-selective pharmacological agents opens opportunities for breakthrough strategies in the treatment of edema and other fluid imbalance disorders.

Reduced risk and severity of stroke in adult females is thought to depend on normal endogenous levels of estrogen, a well-known neuroprotectant and immunomodulator. In male mice, experimental stroke induces immunosuppression of the peripheral immune system, characterized by a reduction in spleen size and cell numbers and decreased cytokine and chemokine expression. However, stroke-induced immunosuppression has not been evaluated in female mice. To test the hypothesis that estradiol (E2) deficiency exacerbates immunosuppression after focal stroke in females, we evaluated the effect of middle cerebral artery occlusion on infarct size and peripheral and CNS immune responses in ovariectomized mice with or without sustained, controlled levels of 17-β–E2 administered by s.c. implant or the putative membrane estrogen receptor agonist, G1. Both E2- and G1-replacement decreased infarct volume and partially restored splenocyte numbers. Moreover, E2-replacement increased splenocyte proliferation in response to stimulation with anti-CD3/CD28 Abs and normalized aberrant mRNA expression for cytokines, chemokines, and chemokine receptors and percentage of CD4+CD25+FoxP3+ T regulatory cells observed in E2-deficient animals. These beneficial changes in peripheral immunity after E2 replacement were accompanied by a profound reduction in expression of the chemokine, MIP-2, and a 40-fold increased expression of CCR7 in the lesioned brain hemisphere. These results demonstrate for the first time that E2 replacement in ovariectomized female mice improves stroke-induced peripheral immunosuppression.

Background and Purpose Bone marrow derived mononuclear cells (MNCs) are an investigational autologous cell-based therapy for acute ischemic stroke. Both intravenous (IV) and intra-arterial (IA) administration routes have been used in clinical trials. However, the route of administration to optimize the effect of MNCs is unknown. In this study, we compared the effect of IV versus IA route of MNCs in the rat stroke model. Methods Long Evans rats were subjected to transient middle cerebral artery occlusion (MCAo). At 24 hrs after stroke, animals were randomly assigned to either receive autologous bone marrow derived MNCs using IV or IA delivery routes. IV saline served as control. 1 million cells/kg (low dose) and 30 million cells/kg (high dose) were assessed. Neurological testing, cavity size, serum cytokines, neuroregenerative endpoints, and MNC biodistribution were evaluated. Results High dose MNCs improved functional recovery, reduced lesion size and pro-inflammatory cytokines, and increased vessel density and neurogenesis markers compared with saline treatment (p<0.05). However, there were no significant differences between IV and IA MNC treated groups; though, IV MNCs reduced serum IL-1β levels compared with IA MNCs (p<0.05). IA MNCs at high dose led to a greater number of cells in the brain at 1 and 6 hrs after injection but not in the lungs and spleen. Low dose MNCs (by IV or IA) did not improve any functional or structural endpoint compared with saline. Conclusion At low and high doses of MNCs, we found that IV or IA achieves similar structural and functional outcomes after stroke.

Objective We tested the hypothesis that osmotherapy with hypertonic saline (HS) attenuates cerebral edema following experimental cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) by exerting its effect via the perivascular pool of aquaporin-4 (AQP4). We used mice with targeted disruption of the gene encoding α-syntrophin (α-Syn-/-) that demonstrate diminished perivascular AQP4 pool but retain the minor endothelial pool. Design Laboratory animal study. Setting University animal research laboratory. Interventions Isoflurane-anesthetized adult male wild type (WT) C57B/6 or α-Syn-/- mice were subjected to CA/CPR and treated with either a continuous intravenous (IV) infusion of 0.9% saline (NS) or various concentrations of hypertonic saline (HS). Serum osmolality, regional brain water content, blood-brain barrier (BBB) disruption, and AQP4 protein expression were determined at 24 hr after CA/CPR. Measurements and Main Results 7.5% HS treatment significantly attenuated water content in the caudoputamen (CP) complex and cortex as compared with NS-treatment in WT mice subjected to CA/CPR. In contrast in α-syn-/- mice subjected to CA/CPR, 7.5% HS treatment did not attenuate water content. Treatment with 7.5% HS attenuated BBB disruption at 24 hr following CA/CPR in WT mice but not in α-Syn-/- mice. Total AQP4 protein expression was not different between NS and HS-treated WT mice. Conclusions Following experimental CA/CPR: 1) continuous HS therapy maintained to achieve serum osmolality of ∼350 mOsm/L is beneficial for the treatment of cerebral edema; 2) perivascular pool of AQP4 plays a critical role in water egress from brain; 3) HS attenuates BBB disruption via perivascular AQP4 pool.