The results suggest that isoflurane administrated after OGD or brain ischemia provides neuroprotection. Mitochondrial adenosine 5'-triphosphate-sensitive potassium channels may be involved in this protection.

BACKGROUND AND PURPOSE WNK kinases, including WNK3, and the associated downstream SPAK and OSR1 kinases, comprise an important signaling cascade that regulates the cation-chloride cotransporters. Ischemia-induced stimulation of the bumetanide-sensitive Na+-K+-Cl- cotransporter (NKCC1) plays an important role in the pathophysiology of experimental stroke, but the mechanism of its regulation in this context is unknown. Here, we investigated the WNK3-SPAK/OSR1 pathway as a regulator of NKCC1 stimulation and their collective role in ischemic brain damage. METHOD Wild-type WNK3 (WT) and WNK3 knockout (KO) mice were subjected to ischemic stroke via transient middle cerebral artery (MCA) occlusion. Infarct volume, brain edema, blood brain barrier (BBB) damage, white matter demyelination, and neurological deficits were assessed. Total and phosphorylated forms of WNK3 and SPAK/OSR1 were assayed by immunobloting and immunostaining. In vitro ischemia studies in cultured neurons and immature oligodendrocytes were conducted using the oxygen-glucose deprivation/reoxygenation method. RESULTS WNK3 KO mice exhibited significantly decreased infarct volume and axonal demyelination, less cerebral edema, and accelerated neurobehavioral recovery compared to WNK3 WT mice subjected to MCA occlusion. The neuroprotective phenotypes conferred by WNK3 KO were associated with a decrease in stimulatory hyper-phosphorylations of the SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites Thr203/Thr207/Thr212, as well as with decreased cell surface expression of NKCC1. Genetic inhibition of WNK3 or siRNA knockdown of SPAK/OSR1 increased the tolerance of cultured primary neurons and oligodendrocytes to in vitro ischemia. CONCLUSION These data identify a novel role for the WNK3-SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury, and suggest the WNK3-SPAK/OSR1 kinase pathway as a therapeutic target for neuroprotection following ischemic stroke.

Postoperative cognitive decline is a clinical syndrome. Volatile anesthetics are commonly used during surgery. It is conceivable that volatile anesthetics may contribute to postoperative cognitive decline. Isoflurane can impair cognitive functions of animals under certain conditions. However, the mechanisms for this impairment are not clear. Here, male 18-month old Fisher 344 rats or 10-week old mice were exposed to 1.2 or 1.4% isoflurane for 2 h. Our studies showed that isoflurane impaired the cognitive functions of the rats in Barnes maze. Isoflurane-exposed rats had reduced freezing behavior during the training sessions in the fear conditioning test. This isoflurane effect was attenuated by lidocaine, a local anesthetic with anti-inflammatory property. Rats that had training sessions and were exposed to isoflurane 30 min later had freezing behavior similar to that of control animals. Isoflurane increased the expression of interleukin 1β (IL-1β), interleukin-6 and activated caspase 3 in the hippocampus of the 18-month old rats. IL-1β positive staining was co-localized with that of NeuN, a neuronal marker. The increase of IL-1β and activated caspase 3 but not interleukin-6 was attenuated by lidocaine. Isoflurane also impaired the cognitive functions of 10-week old C57BL/6J mice and increased IL-1β in their hippocampi. However, isoflurane did not affect the cognitive functions of IL-1β deficient mice. Our results suggest that isoflurane impairs the learning but may not affect the recall of the aged rats. IL-1β may play an important role in this isoflurane effect.

Obesity is a major health issue. Obesity started from teenagers has become a major health concern in recent years. Intermittent fasting increases the life span. However, it is not known whether obesity and intermittent fasting affect brain functions and structures before brain aging. Here, we subjected 7-week old CD-1 wild type male mice to intermittent (alternate-day) fasting or high fat diet (45% caloric supplied by fat) for 11 months. Mice on intermittent fasting had better learning and memory assessed by the Barnes maze and fear conditioning, thicker CA1 pyramidal cell layer, higher expression of drebrin, a dendritic protein, and lower oxidative stress than mice that had free access to regular diet (control mice). Mice fed with high fat diet was obese and with hyperlipidemia. They also had poorer exercise tolerance. However, these obese mice did not present significant learning and memory impairment or changes in brain structures or oxidative stress compared with control mice. These results suggest that intermittent fasting improves brain functions and structures and that high fat diet feeding started early in life does not cause significant changes in brain functions and structures in obese middle-aged animals.

Isoflurane preconditioning improved short-term neurological outcome after focal brain ischemia in adult rats. It is not known whether desflurane induces a delayed phase of preconditioning in the brain and whether isoflurane preconditioning-induced neuroprotection is long-lasting. Two-month-old Sprague-Dawley male rats were exposed to or were not exposed to isoflurane or desflurane for 30 min and then subjected to a 90-min middle cerebral arterial occlusion (MCAO) at 24 hr after the anesthetic exposure. Neurological outcome was evaluated at 24 hr or 4 weeks after the MCAO. The density of the terminal deoxynucleotidyl transferase biotinylated UTP nick end labeling (TUNEL) positive cells in the penumbral cerebral cortex were assessed 4 weeks after the MCAO. Also, rats were pretreated with isoflurane or desflurane for 30 min. Their cerebral cortices were harvested for quantifying B-cell lymphoma-2 expression 24 hr later. Here, we showed that pretreatment with 1.1% or 2.2% isoflurane, but not with 6% or 12% desflurane, increased B-cell lymphoma-2 expression in the cerebral cortex, improved neurological functions and reduced infarct volumes evaluated at 24 hr after the MCAO. Isoflurane preconditioning also improved neurological functions and reduced brain infarct volumes in rats evaluated 4 weeks after the MCAO. Isoflurane preconditioning also decreased the density of TUNEL-positive cells in the penumbral cerebral cortex. We conclude that isoflurane preconditioning improves short-term and long-term neurological outcome and reduces delayed cell death after transient focal brain ischemia in adult rats. Bcl-2 may be involved in the isoflurane preconditioning effect. Desflurane pretreatment did not induce a delayed phase of neuroprotection.

Isoflurane preconditioning improved the long-term neurologic outcome after brain ischemia. Inducible nitric oxide synthase may be involved in this neuroprotection.

Excitatory amino-acid transporters (EAATs) transport glutamate into cells under physiologic conditions. Excitatory amino-acid transporter type 3 (EAAT3) is the major neuronal EAAT and also uptakes cysteine, the rate-limiting substrate for synthesis of glutathione. Thus, we hypothesize that EAAT3 contributes to providing brain ischemic tolerance. Male 8-week-old EAAT3 knockout mice on CD-1 mouse gene background and wild-type CD-1 mice were subjected to right middle cerebral artery occlusion for 90 minutes. Their brain infarct volumes, neurologic functions, and brain levels of glutathione, nitrotyrosine, and 4-hydroxy-2-nonenal (HNE) were evaluated. The EAAT3 knockout mice had bigger brain infarct volumes and worse neurologic deficit scores and motor coordination functions than did wild-type mice, no matter whether these neurologic outcome parameters were evaluated at 24 hours or at 4 weeks after brain ischemia. The EAAT3 knockout mice contained higher levels of HNE in the ischemic penumbral cortex and in the nonischemic cerebral cortex than did wild-type mice. Glutathione levels in the ischemic and nonischemic cortices of EAAT3 knockout mice tended to be lower than those of wild-type mice. Our results suggest that EAAT3 is important in limiting ischemic brain injury after focal brain ischemia. This effect may involve attenuating brain oxidative stress.

Application of isoflurane, a volatile anesthetic, after brain ischemia can reduce ischemic brain injury in rodents (isoflurane postconditioning). This study is designed to determine whether isoflurane postconditioning improves long-term neurological outcome after focal brain ischemia and whether this protection is mediated by attenuating neuroinflammation. Adult male Sprague–Dawley rats were subjected to a 90-min middle cerebral arterial occlusion (MCAO). Isoflurane postconditioning was performed by exposing rats to 2% isoflurane for 60 min immediately after the MCAO. Isoflurane postconditioning reduced brain infarct volumes, apoptotic cells in the ischemic penumbral brain tissues and neurological deficits of rats at 4 weeks after the MCAO. Isoflurane postconditioning reduced brain ischemia/reperfusion-induced nuclear transcription factor (NF)-κB (NF-κB) activation as well as interleukin 1β (IL-1β) and interleukin-6 production in the ischemic penumbral brain tissues at 24 h after the MCAO. IL-1β deficient mice had smaller brain infarct volumes and better neurological functions than wild-type mice at 24 h after a 90-min focal brain ischemia. Isoflurane posttreatment failed to induce neuroprotection in the IL-1β deficient mice. Our results suggest that isoflurane postconditioning improved long-term neurological outcome after transient focal brain ischemia. This protection may be mediated by inhibiting NF-κB activation and the production of the proinflammatory cytokine IL-1β.