[Effects of Governor Vessel electroacupuncture on chloridion homeostasis in the cortex of rats with limb spasm after cerebral ischemia-reperfusion].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of Governor Vessel on chloridion (Cl-) homeostasis and the expression of γ-aminobutyric acid (GABA) and Na+-K+-Cl- cotransporter 1 (NKCC1) in the cerebral cortex of cerebral ischemia-reperfusion injury (CIRI) model rats, so as to explore its mechanism underl-ying alleviating limb spasm after stroke. METHODS: Forty-five male SD rats were randomly divided into normal, sham-operation, model, EA and baclofen groups, with 9 rats in each group. The CIRI model was established by occlusion of the middle cerebral artery and reperfusion. EA(100 Hz) was applied to "Dazhui" (GV14), "Jizhong"(GV6) and "Houhui" for 30 min. Rats of the baclofen group received gavage of baclofen solution (0.4 mg/kg, 1 mL/100 g), once daily for 7 consecutive days. Neurological deficit score was assessed according to Zea Longa's method. The muscular tone of quadriceps femoris of the limb was evaluated by modified Ashworth scale and electrophysiological recor-ding methods, separately. TTC staining was used to detect cerebral infarction volume, and the brain water content of rats in each group was determined by wet and dry weight method. The contents of Cl- and GABA in the cerebral cortex were detected by colorimetric method, and the expression levels of NKCC1 mRNA and protein in the cerebral cortex were detected by quantitative real-time PCR and Western blot, separately. RESULTS: No significant differences were found between the normal and sham-operation groups in all the indexes. Compared with the normal and sham-operation groups, the neurological deficit score, modified Ashworth muscle tone score, brain water content, cerebral infarct volu-me percent, Cl- content and expression levels of NKCC1 mRNA and protein were all evidently increased (P<0.01), and muscle tone of electrophyiological electromyogram (EMG) signal and GABA content were strikingly decreased (P<0.01) in the model group. Compared with the model group, both EA and baclofen groups had an obvious increase in EMG signal displayed muscle tone, and GABA content (P<0.05, P<0.01), and a marked decrease in the neurological deficit score, modified Ashworth score, brain water content, cerebral infarct percent, Cl- content and expression levels of NKCC1 mRNA and protein (P<0.05, P<0.01). CONCLUSION: EA stimulation of acupoints of the Governor Vessel can improve the degree of limb spasm and reduce the degree of cerebral edema and infarction in rats with stroke, which may be related to its functions in protecting Cl- homeostasis, up-regulating GABA concentration, and down-regulating the expression of NKCC1 protein and mRNA in the cerebral cortex.

Inhibition of HMGB1 Attenuates Spinal Cord Edema by Reducing the Expression of Na+-K+-Cl- Cotransporter-1 and Na+/H+ Exchanger-1 in Both Astrocytes and Endothelial Cells After Spinal Cord Injury in Rats.

Sodium/water transport through Na+-K+-Cl- cotransporter-1 (NKCC1) and sodium/hydrogen exchanger-1 (NHE1) in both astrocytes and endothelial cells is critical to cytotoxic and ionic edema following spinal cord injury (SCI). High-mobility group box-1 (HMGB1) promotes spinal cord edema after SCI. Accordingly, we sought to identify both the role of HMGB1 and the mechanism of its effect on NKCC1 and NHE1 expression in astrocytes and endothelial cells as well as the role of the regulation of spinal cord edema after SCI. An SCI model was generated in adult female rats using a heavy falling object, and an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model was generated in rat spinal cord astrocytes and microvascular endothelial cells. The inhibition of HMGB1 reduced NKCC1 and NHE1 expression in the spinal cord of SCI rats, in cultured spinal cord astrocytes, and in cultured microvascular endothelial cells. The effects of HMGB1 on NKCC1 and NHE1 expression were mediated-at least in part-by activation of the Toll-like receptor 4 (TLR4)-Toll/interleukin-1 receptor domain-containing adapter inducing interferon-ß (TRIF)-nuclear factor-kappa B (NF-κB) signaling pathway. The inhibition of NKCC1 or NHE1 decreased the spinal cord water content in rats following SCI, increased the Na+ concentration in the medium of cultured astrocytes after OGD/R, and reduced the astrocytic cell volume and AQP4 expression. These results imply that HMGB1 inhibition results in a reduction in NKCC1 and NHE1 expression in both astrocytes and microvascular endothelial cells and thus decreases spinal cord edema after SCI in rats and that these effects occur through the HMGB1-TLR4-TRIF-NF-κB signaling pathway.

Analgesic Effect of Bumetanide on Neuropathic Pain in Patients With Spinal Cord Injury.

INTRODUCTION: The current study evaluated the analgesic effects of bumetanide as an adjunctive in the management of neuropathic pain following Spinal Cord Injury (SCI). The peripheral expression of Na-K-Cl Cotransporter-1 (NKCC1) and K-Cl Cotransporter-2 (KCC2) genes in polymorphonuclear lymphocytes (PMLs) was assessed as a possible biomarker indicating central mechanisms underlying the observed response. METHODS: Through an open-label, single-arm, pilot trial of bumetanide (2 mg/d), an add-on treatment was conducted on 14 SCI patients for 19 weeks. This study consisted of 3 phases: pre-treatment (1 month), titration (3 weeks), and active treatment (4 months). Ultimately, 9 patients completed the study. The primary outcome variables were the endpoint pain score using the Numeric Rating Scale (NRS), and also the short-form of the McGill pain questionnaire. Secondary endpoints included the short-form of the health survey that assesses the quality of life. Blood samples were collected and used for determining the expression of NKCC1 and KCC2 genes in transcription and translation levels. RESULTS: Bumetanide treatment significantly decreased average pain intensity according to the NRS and the short-form of the McGill pain questionnaire scores. Baseline expression of KCC2 protein was low between groups and increased significantly following treatment (P<0.05). In the current study, pain improvement was accompanied by the greater mean change from the baseline (improvement) for the overall quality of life. CONCLUSION: These data highlighted the analgesic effect of bumetanide on neuropathic pain and indicated the potential role of the upregulation of KCC2 protein and involvement of GABAergic disinhibition in producing neuropathic pain.

Inhibition of the p­SPAK/p­NKCC1 signaling pathway protects the blood­brain barrier and reduces neuronal apoptosis in a rat model of surgical brain injury.

Surgical brain injury (SBI) can disrupt the function of the blood­brain barrier (BBB), leading to brain edema and neurological dysfunction. Thus, protecting the BBB and mitigating cerebral edema are key factors in improving the neurological function and prognosis of patients with SBI. The inhibition of WNK lysine deficient protein kinase/STE20/SPS1­related proline/alanine­rich kinase (SPAK) signaling ameliorates cerebral edema, and this signaling pathway regulates the phosphorylation of the downstream Na+­K+­Cl­ cotransporter 1 (NKCC1). Therefore, the purpose of the present study was to investigate the role of SPAK in SBI­induced cerebral edema and to determine whether the SPAK/NKCC1 signaling pathway was involved in SBI via regulating phosphorylation. An SBI model was established in male Sprague­Dawley rats, and the effects of SPAK on the regulation of the NKCC1 signaling pathway on BBB permeability and nerve cell apoptosis by western blotting analysis, immunofluorescence staining, TUNEL staining, Fluoro­Jade C staining, and brain edema and nervous system scores. The results demonstrated that, compared with those in the sham group, phosphorylated (p)­SPAK and p­NKCC1 protein expression levels were significantly increased in the SBI model group. After inhibiting p­SPAK, the expression level of p­NKCC1, neuronal apoptosis and BBB permeability were significantly reduced in SBI model rats. Taken together, these findings suggested that SBI­induced increases in p­SPAK and p­NKCC1 expression exacerbated post­traumatic neural and BBB damage, which may be mediated via the ion­transport­induced regulation of cell edema.

WNK4-SPAK modulates lipopolysaccharide-induced macrophage activation.

Dysregulation of alveolar macrophage activation has been recognized as the major mechanism in the pathogenesis of acute lung injury. The aim of the present study was to investigate the role of NKCC1 regulating mechanism in modulating macrophage activation. Knockout (SPAK-/- and WNK4-/-) and knockin (WNK4D561A/+) mice were used in this study. LPS induced expression of p-NKCC1 and activation of NFκB in the primary culture of alveolar macrophages. WNK4 or SPAK knockout suppressed p-NKCC1 expression and inflammation cascade activation, whereas WNK4 knockin enhanced these responses. Intrapulmonary administration of LPS induced in vivo expression and phosphorylation of NKCC1 in alveolar inflammation cells and caused a shift in the cell population from macrophage to neutrophil predominance. WNK4 or SPAK knockout attenuated the LPS-induced alveolar cell-population shifting, macrophage NKCC1 phosphorylation, and acute lung injury, whereas WNK4 knockin augmented the inflammatory response. In summary, our results demonstrated the presence of NKCC1 in alveolar macrophage, which is inducible by lipopolysaccharide. Our results also showed showed that the WNK4-SPAK-NKCC1 cascade plays an important role in modulating macrophage activation to regulate LPS-induced lung inflammation and lung injury.

Off-Label Use of Bumetanide for Brain Disorders: An Overview.

Bumetanide (BTN or BUM) is a FDA-approved potent loop diuretic (LD) that acts by antagonizing sodium-potassium-chloride (Na-K-Cl) cotransporters, NKCC1 (SLc12a2) and NKCC2. While NKCC1 is expressed both in the CNS and in systemic organs, NKCC2 is kidney-specific. The off-label use of BTN to modulate neuronal transmembrane Cl- gradients by blocking NKCC1 in the CNS has now been tested as an anti-seizure agent and as an intervention for neurological disorders in pre-clinical studies with varying results. BTN safety and efficacy for its off-label use has also been tested in several clinical trials for neonates, children, adolescents, and adults. It failed to meet efficacy criteria for hypoxic-ischemic encephalopathy (HIE) neonatal seizures. In contrast, positive outcomes in temporal lobe epilepsy (TLE), autism, and schizophrenia trials have been attributed to BTN in studies evaluating its off-label use. NKCC1 is an electroneutral neuronal Cl- importer and the dominance of NKCC1 function has been proposed as the common pathology for HIE seizures, TLE, autism, and schizophrenia. Therefore, the use of BTN to antagonize neuronal NKCC1 with the goal to lower internal Cl- levels and promote GABAergic mediated hyperpolarization has been proposed. In this review, we summarize the data and results for pre-clinical and clinical studies that have tested off-label BTN interventions and report variable outcomes. We also compare the data underlying the developmental expression profile of NKCC1 and KCC2, highlight the limitations of BTN's brain-availability and consider its actions on non-neuronal cells.

Bumetanide attenuates acute lung injury by suppressing macrophage activation.

Bumetanide is a potent loop diuretic that acts as an inhibitor of sodium-potassium-chloride cotransporter 2 (NKCC2) and its isoform NKCC1. Although the expression of NKCC2 is limited to the kidney, NKCC1 is widely expressed in various cells, where it participates in a variety of physiological functions including ion transport, alveolar fluid secretion, and cell volume regulation. We investigated the role of NKCC1 in modulation of host immunity. Lipopolysaccharide (LPS) stimulated the expression and phosphorylation of NKCC1 in RAW264.7 cells in vitro and activated these cells to produce inflammatory cytokines. Enlarging the cell volume in a low-osmotic microenvironment amplified the LPS-induced inflammatory responses and phagocytosis activity of RAW264.7 cells. Pretreatment with the NKCC1 inhibitor bumetanide attenuated LPS-induced activation of inflammatory cells and cell volume-related function. Mice treated with an intratracheal bumetanide spray showed greater resistance to LPS-induced tissue inflammation and acute lung injury in vivo. Our studies suggest that NKCC1 plays a unique role as an amplifier of LPS-induced macrophage functions and that NKCC1 might be a novel target for treating sepsis-related acute respiratory distress syndrome.

Epithelial disruptions, but not immune cell invasion, induced secretory dysfunction following innate immune activation in a novel model of acute salivary gland injury.

BACKGROUND: Salivary gland (SG) injurious agents are all translated into loss of salivation (xerostomia). An association has been established between activation of innate immunity and SG injury and dysfunction. However, it remains unclear how the secretory epithelia respond by halting saliva production. METHODS: C57BL/6 submandibular glands (SMGs) were acutely challenged using a single dose of the innate immune stimulant: polyinosinic-polycytidylic acid (poly (I:C)). Secretory capacity of the infected SMGs was substantiated by assessing the flow rate in response to pilocarpine stimulation. Depletion of the acute inflammatory cells was achieved by pre-treating mice with RB6-8C5 depletion antibody. Flow cytometry, histology and immunohistochemistry were conducted to verify the immune cell depletion. Epithelial expression of saliva-driving molecules: muscarinic 3 receptor (M3R), aquaporin 5 water channel (AQP5), Na-K-CL-Cotransporter 1 (NKCC1) and transmembrane member 16A (TMEM16A), was characterized using RT-qPCR and immunohistochemistry. Tight junction (TJ) protein; zonula occludens (ZO-1) and basement membrane (BM) protein; and laminin were assessed by immunohistochemistry. RESULTS: Innate immune challenge prompted dysfunction in the exocrine SGs. Dysregulated gene and protein expression of molecules that drive saliva secretion was substantiated. Aberrant expression of TJ and BM proteins followed innate immune activation. Hyposalivation in the current model was independent of myeloperoxidase (MPO)-positive, acute inflammatory cells. CONCLUSIONS: In this study, we developed a novel injury model of the SGs, featuring acute secretory dysfunction and immediate structural disruptions. Our results ruled out the injurious role of aggressively infiltrating inflammatory cells.

Expression of aquaporin 4 in 1,2-dichloroethane-induced toxic brain edema in rats / 中国职业医学

OBJECTIVE: To explore the effects of aquaporin 4( APQ4) in rat toxic brain edema induced by subacute 1,2-dichloroethane( 1,2-DCE) exposure. METHODS: Thirty-two specific pathogen free healthy adult female SD rats were randomly divided into control( 8 rats),low-dose( 12 rats) and high-dose( 12 rats) groups. The treatment groups were exposed to 1,2-DCE( low-dose: 600 mg / m3; high-dose: 1 800 mg/m3,nose-only) and the control group was exposed to fresh air by dynamic inhalation for 8 hours per day for consecutive 7 days. After exposure,histopathologic changes were examined in the cerebral cortex. Real-time polymerase chain reaction was used to detect the mRNA relative expression of matrix metalloproteinase 2( MMP2),Na-K-Cl cotransporter-1( NKCC1) and AQP4. The Western blotting was used to detect the expression of AQP4 protein in the cerebral cortex. RESULTS: The pathological results showed that the cerebral cortex tissues were loose around the peripheral vessels and the vessels tissue space appeared widen in low-dose exposure group. The pathological change was more serious in high-dose group than low-dose group,with obvious loosen vessels and vacuole. Compared with those of the control group and the low-dose group,the relative expression level of MMP2 mRNA in the high-dose group increased significantly[( 1. 07 ± 0. 41) vs( 1. 56 ± 0. 55),( 1. 21 ± 0. 59) vs( 1. 56 ± 0. 55),P <0. 05],while the the relative expression level of AQP4 mRNA in the high-dose group significantly decreased [( 1. 03 ±0. 25) vs( 0. 81 ± 0. 12),( 1. 00 ± 0. 20) vs( 0. 81 ± 0. 12),P < 0. 05]. The relative expression levels of NKCC1 mRNA in all groups showed no statistical difference [( 1. 03 ± 0. 31) vs( 1. 14 ± 0. 43) vs( 1. 36 ± 0. 50),P > 0. 05]. The relative expression level of AQP4 protein in the high-dose group was lower than that of the control group [( 0. 80 ± 0. 25) vs( 1. 19 ± 0. 42),P < 0. 05]. CONCLUSION: The brain edema induced by subacute inhalation of 1,2-DCE is of mixed types with vasogenic edema as its main symptom. Its pathogenesis is related to the changes of AQP4 expression.

Bumetanide protects focal cerebral ischemia-reperfusion injury in rat.

OBJECTIVE: Bumetanide has been reported to attenuate ischemia-evoked cerebral edema. However, whether bumetanide can protect cerebral ischemia-reperfusion injury (IRI) in vivo is unclear. In the present study, we aim to determine whether intravenously injection bumetanide can attenuate cerebral IRI and if its protection effect might be related to the modification of cerebral NKCC1 and KCC2 protein expression. METHODS: Focal cerebral ischemia was induced by occluding the right middle cerebral artery (MCAO) for 2-h, followed by 3-h, 24-h or 48-h of reperfusion respectively. Brain edema, neurological deficits, and infarction volume were determined by (wet weights-dry weights)/dry weights×100, 5-point neurological function score evaluation system, and TTC staining, respectively. The expression levels of NKCC1 and KCC2 were determined by immunohistochemical staining. RESULTS: Reperfusion increased brain edema, neurological deficits, and infarction volume. Bumetanide decreased brain edema, attenuated the neurological defects and reduced post-ischemic cerebral infarction. Cerebral ischemia-reperfusion injury increased NKCC1 expression level and decreased KCC2 expression level. Interestingly, bumetanide down-regulated the NKCC1 protein expression level without changing the KCC2 protein expression level in rat brain cortex. CONCLUSION: These results suggest that bumetanide protects focal cerebral ischemia-reperfusion injury in rat, which might through the inhibition of NKCC1.

Characterization of CFTR High Expresser cells in the intestine.

The CFTR High Expresser (CHE) cells express eightfold higher levels of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel compared with neighboring enterocytes and were first identified by our laboratory (Ameen et al., Gastroenterology 108: 1016, 1995). We used double-label immunofluorescence microscopy to further study these enigmatic epithelial cells in rat intestine in vivo or ex vivo. CHE cells were found in duodenum, most frequent in proximal jejunum, and absent in ileum and colon. CFTR abundance increased in CHE cells along the crypt-villus axis. The basolateral Na(+)K(+)Cl(-) cotransporter NKCC1, a key transporter involved in Cl(-) secretion, was detected at similar levels in CHE cells and neighboring enterocytes at steady state. Microvilli appeared shorter in CHE cells, with low levels of Myosin 1a, a villus enterocyte-specific motor that retains sucrase/isomaltase in the brush-border membrane (BBM). CHE cells lacked alkaline phosphatase and absorptive villus enterocyte BBM proteins, including Na(+)H(+) exchanger NHE3, Cl(-)/HCO3(-) exchanger SLC26A6 (putative anion exchanger 1), and sucrase/isomaltase. High levels of the vacuolar-ATPase proton pump were observed in the apical domain of CHE cells. Levels of the NHE regulatory factor NHERF1, Na-K-ATPase, and Syntaxin 3 were similar to that of neighboring enterocytes. cAMP or acetylcholine stimulation robustly increased apical CFTR and basolateral NKCC1 disproportionately in CHE cells relative to neighboring enterocytes. These data strongly argue for a specialized role of CHE cells in Cl(-)-mediated "high-volume" fluid secretion on the villi of the proximal small intestine.