This meta-analysis aimed to describe the efficacy of bumetanide in improving infarct volume, brain edema, and behavioral outcomes in animal models of cerebral ischemia. Embase, PubMed and Web of Science databases were searched from their inception to February 2024 (INPLASY:202430023). Data on the animal species, stroke model, drug dose, time of treatment, method of administration, study quality, and outcomes were extracted and pooled in a meta-analysis. The combined standardized mean difference (SMD) or mean difference (MD) estimates and 95% confidence intervals (CIs) were calculated using random- or fixed-effects models. Thirteen eligible studies involving >200 animals fulfilled the inclusion criteria and were included in this meta-analysis. Meta-analyses demonstrated that bumetanide treatment significantly reduced cerebral infarct volume (SMD: -0.42; 95% CI: -0.75, -0.09; p < 0.01; n = 186 animals) and consistently relieved brain edema (SMD: -1.39; 95% CI: -2.06, -0.72; p < 0.01; n = 64 animals). Subgroup analyses demonstrated that bumetanide treatment reduced infarct volume in transient but not permanent cerebral ischemia models. When administered after the stroke, it was more effective than treatment initiation before the stroke. Eight studies assessed the effect of bumetanide on behavioral function and the results showed that bumetanide treatment significantly improved neurobehavioral deficits (SMD: -2.35; 95% CI: -2.72, -1.97; p < 0.01; n = 250 animals). We conclude that bumetanide appears to be effective in reducing infarct volume and brain edema and improving behavioral recovery in animal models of cerebral ischemia. This mechanism needs to be confirmed through further investigation.

BACKGROUND: The inhalational anesthetic isoflurane is commonly utilized in clinical practice, particularly in the field of pediatric anesthesia. Research has demonstrated its capacity to induce neuroinflammation and long-term behavioral disorders; however, the underlying mechanism remains unclear [1]. The cation-chloride cotransporters Na+-K+-2Cl--1 (NKCC1) and K+-2Cl--2 (KCC2) play a pivotal role in regulating neuronal responses to gamma-aminobutyric acid (GABA) [2]. Imbalances in NKCC1/KCC2 can disrupt GABA neurotransmission, potentially leading to neural circuit hyperexcitability and reduced inhibition following neonatal exposure to anesthesia [3]. Therefore, this study postulates that anesthetics have the potential to dysregulate NKCC1 and/or KCC2 during brain development.
METHODS: We administered 1.5% isoflurane anesthesia to neonatal rats for a duration of 4 h at postnatal day 7 (PND7). Anxiety levels were assessed using the open field test at PND28, while cognitive function was evaluated using the Morris water maze test between PND31 and PND34. Protein levels of NKCC1, KCC2, BDNF, and phosphorylated ERK (P-ERK) in the hippocampus were measured through Western blotting analysis. Pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified using ELISA.
RESULTS: We observed a decrease in locomotion trajectories within the central region and a significantly shorter total distance in the ISO group compared to CON pups, indicating that isoflurane induces anxiety-like behavior. In the Morris water maze (MWM) test, rats exposed to isoflurane exhibited prolonged escape latency onto the platform. Additionally, isoflurane administration resulted in reduced time spent crossing in the MWM experiment at PND34, suggesting long-term impairment of memory function. Furthermore, we found that isoflurane triggered activation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α; downregulated KCC2/BDNF/P-ERK expression; and increased the NKCC1/KCC2 ratio in the hippocampus of PND7 rats. Bumetadine (NKCC1 specific inhibitors) reversed cognitive damage and effective disorder induced by isoflurane in neonatal rats by inhibiting TNF-α activation, normalizing IL-6 and IL-1β levels, restoring KCC2 expression levels as well as BDNF and ERK signaling pathways. Based on these findings, it can be speculated that BDNF, P-ERK, IL-1β, IL-6 and TNF - α may act downstream of the NKCC1/KCC2 pathway.
CONCLUSIONS: Our findings provide evidence that isoflurane administration in neonatal rats leads to persistent cognitive deficits through dysregulation of the Cation-Chloride Cotransporters NKCC1 and KCC2, BDNF, p-ERK proteins, as well as neuroinflammatory processes.

A salt of vandetanib, namely, 4-({4-[(4-bromo-2-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}methoxy)-1-methylpiperazin-1-ium 2-(butylamino)-4-phenoxy-6-sulfamoylbenzoate acetonitrile monosolvate, C22H25BrFN4O2+·C17H19N2O5S-·C2H3N, composed of kinase inhibitor vandetanib and sulfamyl diuretic bumetanide in a 1:1 molar ratio, is reported. There is proton transfer between the piperidine ring of vandetanib and the carboxyl group of bumetanide to form the salt. In the vandetanib cation, the arene and pyrimidine rings are not coplanar, their planes subtending a dihedral angle of 60.47 (14)°. The roles of the intermolecular interactions in the crystal packing were clarified using Hirshfeld surface analysis, and two-dimensional fingerprint plots indicate that the most important contributions to the crystal packing are from H...H (40.5%), O...H/H...C (20.7%), C...H/ H...C (18.8%) and N...C/C...N (9.0%) contacts.

Cerebral ischemia-hypoxia leads to excitotoxicity-mediated neuronal damage and cognitive dysfunction, especially in the elderly. Excessive intracellular [Cl- ]i accumulation weakens γ-aminobutyric acid (GABA) compensatory effects. Sub-anesthetic dose of propofol protected the brain against ischemia-hypoxia, which was abolished by blocking Cl- efflux transporter K+ /Cl- cotransporter 2 (KCC2). We aimed to determine whether low-dose anesthetic combined with [Cl- ]i regulators could restore the compensatory GABAergic system and improve cognitive function.
Chronic cerebral hypoxia (CCH) model was established by bilateral carotid artery ligation in aged rats. Sub-dose of anesthetics (propofol and sevoflurane) with or without KCC2 agonist N-ethylmaleimide (NEM) or Na+ /K+ /Cl- cotransporter 1 (NKCC1) antagonist bumetanide (BTN) was administered systemically 30 days post-surgery. Primary rat hippocampal neuronal cultures were subjected to hypoxic injury with or without drug treatment. Memory function, hippocampal neuronal survival, GABAergic system functioning, and brain-derived neurotrophic factor (BDNF) expressions were evaluated.
Sub-anesthetic dose of combined propofol (1.2 μg mL-1 ) and sevoflurane [0.7 MAC (minimum alveolar concentration)] did not aggravate the hypoxic brain injury in rats or cell damage in neuronal cultures. Adding either BTN or NEM protected against hypoxic injury, associated with improved cognitive function in vivo, less intracellular accumulation of [Cl- ]i , reduced cell death, restored GABAergic compensation, and increased BDNF expression both in vivo and in vitro.
Sub-anesthetic dose of propofol and sevoflurane is a recommended anesthesia regimen in at-risk patients. Restoration of [Cl- ]i homeostasis and GABAergic could further reduce the brain damage caused by ischemia-hypoxia.

OBJECTIVE: Although absorbed NaCl increases intestinal blood flow to facilitate absorption and transportation, it is unclear if it can directly mediate mesenteric arterial relaxation. We aimed to investigate and test our hypothesis that Cl- induces mesenteric arterial vasorelaxation via endothelium-dependent hyperpolarization (EDH).
METHODS: We used wire myograph to study NaCl-induced vasorelaxation of mesenteric arteries isolated from mice. Cl-, Ca2+ and K+ imaging was performed in human vascular endothelial cells pre-treated with pharmacological agents.
RESULTS: The Cl- concentration-dependently induced vasorelaxation of mesenteric arteries likely through EDH. The Cl--induced vasorelaxation was attenuated in TRPV4 KO mice and inhibited by selective blockers of Na+-K+-2Cl- cotransporter 1 (NKCC1) (bumetanide, 10 μM), transient receptor potential vanilloid 4 (TRPV4) (RN-1734, 40 μM), and small conductance Ca2+-activated K+ channels (SKCa) (apamin, 3 μM)/ intermediate conductance Ca2+-activated K+ channels (IKCa) (TRAM-34, 10 μM) and myoendothelial gap junction (18α-glycyrrhetinic acid, 10 μM), but enhanced by a selective activator of IKCa/SKCa (SKA-31, 0.3 μM). Cl- decreased intracellular K+ concentrations in endothelial cells, which was reversed by apamin (200 nM) plus TRAM-34 (500 nM). Extracellular Cl- raised intracellular Cl- concentrations in endothelial cells, which was attenuated by bumetanide (10 μM). Finally, Cl- induced a transient Ca2+ signaling via TRPV4 in endothelial cells, which became sustained when the Ca2+ exit mode of Na+-Ca2+ exchanger (NCX) was blocked.
CONCLUSIONS: Cl- induces a pure EDH-mediated vasorelaxation of mesenteric arteries through activation of endothelial NKCC1/TRPV4/NCX axis. We have provided a novel insight into the role of Cl--induced vasorelaxation via EDH mechanism.

Sevoflurane (Sevo) is one of the most commonly used general anesthetics for infants and young children. We investigated whether Sevo impairs neurological functions, myelination, and cognition via the γ-aminobutyric acid A receptor (GABAAR) and Na+-K+-2Cl- cotransporter (NKCC1) in neonatal mice. On postnatal days 5-7, mice were exposed to 3% Sevo for 2 h. On postnatal day 14, mouse brains were dissected, and oligodendrocyte precursor cell line level lentivirus knockdown of GABRB3, immunofluorescence, and transwell migration assays were performed. Finally, behavioral tests were conducted. Multiple Sevo exposure groups exhibited increased neuronal apoptosis levels and decreased neurofilament protein levels in the mouse cortex compared with the control group. Sevo exposure inhibited the proliferation, differentiation, and migration of the oligodendrocyte precursor cells, thereby affecting their maturation process. Electron microscopy revealed that Sevo exposure reduced myelin sheath thickness. The behavioral tests showed that multiple Sevo exposures induced cognitive impairment. GABAAR and NKCC1 inhibition provided protection against Sevo-induced neurotoxicity and cognitive dysfunction. Thus, bicuculline and bumetanide can protect against Sevo-induced neuronal injury, myelination impairment, and cognitive dysfunction in neonatal mice. Furthermore, GABAAR and NKCC1 may be mediators of Sevo-induced myelination impairment and cognitive dysfunction.

Previous studies have confirmed the relationship between chloride homeostasis and pain. However, the role of sodium potassium chloride co-transporter isoform 1 (NKCC1) in dorsal horn and dorsal root ganglion neurons (DRGs) in spinal cord injury (SCI)-induced neuropathic pain (NP) remains inconclusive. Therefore, we aimed to explore whether suppression of NKCC1 in the spinal cord and DRGs alleviate the NP of adult rats with thoracic spinal cord contusion. Thirty adult female Sprague-Dawley rats (8 week-old, weighing 250-280 g) were randomly divided into three groups with ten animals in each group (sham, SCI, and bumetanide groups). The paw withdrawal mechanical threshold and paw withdrawal thermal latency were recorded before injury (baseline) and on post-injury days 14, 21, 28, and 35. At the end of experiment, western blotting (WB) analysis, quantitative real-time Polymerase Chain Reaction (PCR) and immunofluorescence were performed to quantify NKCC1 expression. Our results revealed that NKCC1 protein expression in the spinal cord and DRGs was significantly up-regulated in rats with SCI. Intraperitoneal treatment of bumetanide (an NKCC1 inhibitor) reversed the expression of NKCC1 in the dorsal horn and DRGs and ameliorated mechanical ectopic pain and thermal hypersensitivities in the SCI rats. Our study demonstrated the occurrence of NKCC1 overexpression in the spinal cord and DRGs in a rodent model of NP and indicated that changes in the peripheral nervous system also play a major role in promoting pain sensitization after SCI.

BACKGROUND: General anesthetics (eg, propofol and volatile anesthetics) enhance the slow-delta oscillations of the cortical electroencephalogram (EEG), which partly results from the enhancement of (γ-aminobutyric acid [GABA]) γ-aminobutyric acid-ergic (GABAergic) transmission. There is a GABAergic excitatory-inhibitory shift during postnatal development. Whether general anesthetics can enhance slow-delta oscillations in the immature brain has not yet been unequivocally determined.
METHODS: Perforated patch-clamp recording was used to confirm the reversal potential of GABAergic currents throughout GABAergic development in acute brain slices of neonatal rats. The power density of the electrocorticogram and the minimum alveolar concentrations (MAC) of isoflurane and/or sevoflurane were measured in P4-P21 rats. Then, the effects of bumetanide, an inhibitor of the Na + -K + -2Cl - cotransporter (NKCC1) and K + -Cl - cotransporter (KCC2) knockdown on the potency of volatile anesthetics and the power density of the EEG were determined in vivo.
RESULTS: Reversal potential of GABAergic currents were gradually hyperpolarized from P4 to P21 in cortical pyramidal neurons. Bumetanide enhanced the hypnotic effects of volatile anesthetics at P5 (for MAC LORR , isoflurane: 0.63% ± 0.07% vs 0.81% ± 0.05%, 95% confidence interval [CI], -0.257 to -0.103, P < .001; sevoflurane: 1.46% ± 0.12% vs 1.66% ± 0.09%, 95% CI, -0.319 to -0.081, P < .001); while knockdown of KCC2 weakened their hypnotic effects at P21 in rats (for MAC LORR , isoflurane: 0.58% ± 0.05% to 0.77% ± 0.20%, 95% CI, 0.013-0.357, P = .003; sevoflurane: 1.17% ± 0.04% to 1.33% ± 0.04%, 95% CI, 0.078-0.244, P < .001). For cortical EEG, slow-delta oscillations were the predominant components of the EEG spectrum in neonatal rats. Isoflurane and/or sevoflurane suppressed the power density of slow-delta oscillations rather than enhancement of it until GABAergic maturity. Enhancement of slow-delta oscillations under volatile anesthetics was simulated by preinjection of bumetanide at P5 (isoflurane: slow-delta changed ratio from -0.31 ± 0.22 to 1.57 ± 1.15, 95% CI, 0.67-3.08, P = .007; sevoflurane: slow-delta changed ratio from -0.46 ± 0.25 to 0.95 ± 0.97, 95% CI, 0.38-2.45, P = .014); and suppressed by KCC2-siRNA at P21 (isoflurane: slow-delta changed ratio from 16.13 ± 5.69 to 3.98 ± 2.35, 95% CI, -18.50 to -5.80, P = .002; sevoflurane: slow-delta changed ratio from 0.13 ± 2.82 to 3.23 ± 2.49, 95% CI, 3.02-10.79, P = .003).
CONCLUSIONS: Enhancement of cortical EEG slow-delta oscillations by volatile anesthetics may require mature GABAergic inhibitory transmission during neonatal development.

With the current limited drug therapy for the core symptoms of autism spectrum disorder (ASD), we herein report a randomized, double-blind, placebo-controlled trial to investigate the efficacy, safety, and potential neural mechanism of bumetanide in children with ASD aged 3-6 years old. A total of 120 children were enrolled into the study and randomly assigned to either 0.5 mg bumetanide or placebo. In the final sample, 119 children received at least one dose of bumetanide (59 children) or placebo (60 children) were included in the final analysis. The primary outcome was a reduction in the Childhood Autism Rating Scale (CARS) score, and the secondary outcomes were the Clinical Global Impressions Scale (CGI) -Global Improvement (CGI-I) score at 3 months and the change from baseline to 3-month in the Autism Diagnostic Observation Schedule (ADOS). Magnetic resonance spectroscopy (MRS) was used to measure γ-aminobutyric acid (GABA) and glutamate neurotransmitter concentrations in the insular cortex (IC) before and after the treatment. As compared with the placebo, bumetanide treatment was significantly better in reducing the severity. No patient withdrew from the trial due to adverse events. The superiority of bumetanide to placebo in reducing insular GABA, measured using MRS, was demonstrated. The clinical improvement was associated with a decrease in insular GABA in the bumetanide group. In conclusion, this trial in a large group of young children with predominantly moderate and severe ASD demonstrated that bumetanide is safe and effective in improving the core symptoms of ASD. However, the clinical significance remains uncertain, and future multi-center clinical trials are required to replicate these findings and confirm the clinical significance using a variety of outcome measures.

UNASSIGNED: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, severely distressing clinical syndrome characterized by bladder pain and pressure perceptions. The origin and pathophysiology of IC/BPS are currently unclear, making it difficult to diagnose and formulate successful treatments. Our study is aimed at investigating the role of immune-related genes in the diagnosis, progression, and therapy of IC/BPS.
UNASSIGNED: The gene expression datasets GSE11783, GSE11839, GSE28242, and GSE57560 were retrieved from the GEO database for further analysis. Immune-related IC/BPS differentially expressed genes (DEGs) were identified by limma. Three distinct machine learning approaches, least absolute shrinkage and selection operator (LASSO), support vector machine-recursive feature elimination (SVM-RFE), and random forest (RF), were used to find the immune-related IC characteristic genes. Nomogram and receiving operator curves (ROC) were plotted to measure characteristic effectiveness. Using the CMap database and the molecular docking approach, potential small-molecule medicines were found and verified. Consensus cluster analysis was also performed to separate the IC/BPS samples into immunological subtypes.
UNASSIGNED: A total of 24 immune-related IC/BPS-DEGs were identified. When compared to the normal control group, the IC/BPS cohort had significantly more immune cell infiltration. Integrative machine learning methods discovered 5 IC/BPS characteristic genes (RASGRP1, PPBP, RBP4, CR2, and PROS2) that may predict IC/BPS diagnosis and immune cell infiltration. Furthermore, two immunological subgroups with substantial variations in immune cell infiltration across IC/BPS samples were identified, which were named cluster1 and cluster2, with the hallmark genes having greater expression in cluster2. Finally, bumetanide was shown to have the potential to be a medication for the treatment of IC/BPS, and it performed well in terms of its molecular binding with RASGRP1.
UNASSIGNED: We found and validated 5 immune-related IC/BPS genes (RASGRP1, PPBP, RBP4, CR2, and PROS2) and 2 IC/BPS immune subtypes. In addition, bumetanide was discovered to be a potential drug for treating IC/BPS, which may provide new insight into the diagnosis and immune therapy of IC/BPS patients.