Attenuation of Blood–Brain Barrier Disruption in Traumatic Brain Injury via Inhibition of NKCC1 Cotransporter: Insights into the NF-κB/NLRP3 Signaling Pathway

Abstract

Following traumatic brain injury (TBI), inhibition of the Na⁺-K⁺-Cl⁻ cotransporter1 (NKCC1) has been observed to alleviate damage to the blood–brain barrier (BBB). However, the underlying mechanism for this effect remains unclear. This study aimed to investigate the mechanisms by which inhibiting the NKCC1 attenuates disruption of BBB integrity in TBI. The TBI model was induced in C57BL/6 mice through a controlled cortical impact device, and an in vitro BBB model was established using Transwell chambers. Western blot (WB) analysis was used to evaluate NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and nuclear factor-kappaB (NF-κB) pathway proteins. Flow cytometry and transendothelial electrical resistance (TEER) were employed to assess endothelial cell apoptosis levels and BBB integrity. ELISA was utilized to measure cytokines interleukin-1β (IL-1β) and matrix metalloproteinase-9 (MMP-9). Immunofluorescence techniques were used to evaluate protein levels and the nuclear translocation of the rela (p65) subunit. The Evans blue dye leakage assay and the brain wet–dry weight method were utilized to assess BBB integrity and brain swelling. Inhibition of NKCC1 reduced the level of NLRP3 inflammasome and the secretion of IL-1β and MMP-9 in microglia. Additionally, NKCC1 inhibition suppressed the activation of the NF-κB signaling pathway, which in turn decreased the level of NLRP3 inflammasome. The presence of NLRP3 inflammasome in BV2 cells led to compromised BBB integrity within an inflammatory milieu. Following TBI, an upregulation of NLRP3 inflammasome was observed in microglia, astrocytes, vascular endothelial cells, and neurons. Furthermore, inhibiting NKCC1 resulted in a decrease in the positive rate of NLRP3 inflammasome in microglia and the levels of inflammatory cytokines IL-1β and MMP-9 after TBI, which correlated with BBB damage and the development of cerebral edema. These findings demonstrate that the suppression of the NKCC1 cotransporter protein alleviates BBB disruption through the NF-κB/NLRP3 signaling pathway following TBI.

Introduction

Traumatic brain injury (TBI) refers to an acquired injury to the brain caused by external mechanical force and has the potential to cause both temporary and permanent impairment.¹ This condition is recognized as a significant global public health concern and a prominent contributor to mortality and reduced functioning. TBI is estimated to affect 640,000–740,000 individuals annually.² TBI presents in a bimodal distribution pattern, with higher frequency among adolescent and elderly populations and often resulting from falls and vehicular accidents.³ Approximately 43% of those impacted by TBI are expected to experience long-term disability.²

The blood–brain barrier (BBB) is a semipermeable structure consisting of vascular endothelial cells, astrocytes, neurons, microglia, and other components.⁴ It effectively regulates brain tissue metabolism through its selective permeability.⁵ The impairment of the BBB is a common pathological event in secondary craniocerebral injury cases.⁶ Several studies have shown that microglia play a significant role in BBB damage after TBI.^7
–9 As resident macrophages of the central nervous system (CNS), microglia are responsible for maintaining tissue homeostasis.¹⁰ The neuroinflammatory response caused by microglia following TBI is a significant secondary injury factor, and this response is regarded as driving trauma-induced neurodegeneration and BBB leakage.^11,12 Following TBI, the activation of microglia cells triggers an inflammatory response that releases a variety of inflammatory factors, such as cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and matrix metalloproteinases (MMPs), resulting in the breakdown of tight junction proteins within the BBB, further compromising its integrity.^12
–14 Therefore, managing the inflammatory response after TBI is critical for maintaining BBB integrity and improving TBI outcomes.

The Na⁺-K⁺-Cl⁻ cotransporter (NKCC) is a prominent class of ion transporters that belongs to the SLC12 cation–chloride cotransporter protein family. The NKCC1 and NKCC2 subtypes function in facilitating the same-directional cotransmembrane transport of Na⁺, K⁺, and Cl⁻, in addition to regulating cellular volume and ion balance.¹⁶ The hyperactivation of NKCC1 has been associated with a multitude of health concerns, such as epileptic seizures, cerebral swelling, neuronal harm, an overactive inflammatory response, and various psychological disorders.^17

–20 Transcriptomic analyses demonstrated that this protein had observable expression levels in microglia.²¹ Moreover, NKCC1 is a contributor to the amplification of lipopolysaccharide (LPS) stimulated macrophage functions, and the inhibition of NKCC1 has been found to mitigate inflammation and suppress the release of pro-inflammatory cytokines triggered by LPS.²²

In cases of surgical brain injury, inhibition of NKCC1 has been demonstrated to modulate the release of inflammatory cytokines in microglia, ultimately resulting in improved neurological function and enhanced prognoses.¹⁵ In a previous study conducted by our team, we observed that the inhibition of NKCC1 led to a significant improvement in the neurological dysfunction resulting from TBI. This inhibition also resulted in a notable reduction in MMP-9 levels within the brain tissue and a substantial alleviation of BBB damage.²³ Nevertheless, the precise regulatory mechanism by which NKCC1 inhibition impacts BBB integrity in the context of TBI remains elusive. Given the role of NKCC1 in modulating immune cell function, the objective of this study was to elucidate the mechanism by which NKCC1 regulates BBB integrity in TBI, particularly its involvement in the regulation of inflammation. Investigating the role of NKCC1 in modulating BBB integrity following TBI may contribute to the identification of potential intervention targets and the enhancement of patient prognosis.

Methods

Animals and substances

Male C57BL/6 mice, aged 10 weeks, were sourced from Vitonlihua Company. The mice were maintained under controlled environmental conditions, including a stable temperature and relative humidity, and were subjected to a standard light-dark cycle. They had free access to food and water throughout the study. All experimental procedures were approved by the Institute of Animal Care Committee and conducted according to the guidelines for the care and use of laboratory animals established by the National Institutes of Health.

Bumetanide (BMT), a specific inhibitor of NKCC1, was procured from MCE Technologies (USA) and utilized at a concentration of 1 μM in vitro and 20 mg/kg in vivo, dissolved in dimethyl sulfoxide (DMSO).^23,24 SN50, an NF-κB inhibitor that selectively impedes the nuclear translocation of the p65 subunit was obtained from MCE Technologies (USA) and prepared with DMSO at a concentration of 20 μM.²⁵ MCC950, a highly selective NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome inhibitor, was dissolved in 0.9% NaCl solution prior to use. The concentration of MCC950 used in the mice was 10 mg/kg, which was in accordance with a reported study.²⁶ The internal inflammatory cascade within microglia was activated by inducing an inflammatory environment using LPS (Sigma, USA) at a concentration of 1 μg/mL.²⁷ The BV2 cells (mouse microglial cell line) and bEnd.3 cells (mouse brain microvascular endothelial cell line) were purchased from the Beijing Union Cell Bank. The cells were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum and 1% antibiotics. The cell cultures were maintained in a cell culture incubator at 37°C with 5% CO2 concentration.

Immunofluorescence staining

The culture plate containing BV2 cells was subjected to a series of steps for sample preparation. First, the slides were soaked with phosphate-buffered saline (PBS) for three rounds, lasting 3 min each. Following this, the sample was fixed using 4% paraformaldehyde for 15 min and then soaked with PBS again for three rounds, 3 min each. The next step involved permeating the cells using 0.5% TritonX-100 for 20 min at room temperature. The cells were then blocked with goat serum at room temperature for 30 min after soaking in PBS again. The primary antibodies, anti-NF-κB p65 from CST (#8242S), and anti-NLRP3 from Abcam (ab270449) at 1:100 dilution were then applied and incubated overnight at 4°C in a wet box. Subsequently, the tablet was soaked in PBS again for three rounds, each lasting 3 min, before the addition of a dilution of fluorescence secondary antibody (Alexa Fluor® 488 from Abcam) at room temperature for an hour in a wet box. Finally, 4,6-diamidino-2-phenylindole (DAPI) was added to the slipper and incubated for 5 min, allowing for restaining of the nucleus. The slices were then observed and imaged by a fluorescence microscope (Nikon, Tokyo, Japan).

Furthermore, frozen brain tissue slices were subjected to immunofluorescence labeling in accordance with the established protocol as reported previously.²⁸ Brain sections were fixed in 4% paraformaldehyde for 15 min, followed by three washes with a PBS solution containing 0.025% TritonX-100 for 5 min each time. After the sections of the brain were incubated in a PBS solution containing 0.25% TritonX-100 at room temperature for 10 min, they were exposed to a mixture of PBS solution containing 10% goat serum, 1% bovine serum albumin, 0.3M glycine, and 0.1% Tween20 for 2 h. The sections were then incubated overnight at 4°C with primary antibodies targeting NLRP3 (Novus, 768319, dilution 1:100), transmembrane protein 119 (TMEM-119) (Proteintech, 27585–1-AP, dilution 1:100), Glial fibrillary acidic protein (GFAP) (Zenbioscience, 381012, dilution 1:50), neuronal nuclear protein (NeuN) (Abcam, ab177487, dilution 1:100), platelet endothelial cell adhesion molecule-1 (CD31) (Abcam, Ab222783, dilution 1:100), and Zonula Occluden-1 (ZO-1) (Santa Cru, sc-33725, dilution 1:100). The following day, secondary antibodies, including Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488 or 594) and Goat Anti-Rat IgG H&L (Alexa Fluor® 488 or 594), were added to the sections (dilution 1:500) and incubated for 2 h at room temperature. The codistribution of the antibodies was detected using a fluorescence microscope (Nikon, Tokyo, Japan).

Western blot analysis

Cells were lysed using a lysis buffer containing 1% protease and phosphatase inhibitors. Electrophoresis was conducted on an SDS–polyacrylamide gel, and the protein was transferred to a PVDF membrane (Millipore, United States). The membranes were then blocked with 5% skimmed milk and subsequently incubated with the appropriate primary antibodies, respectively, including anti-NLRP3 (1:1000; Abcam, ab270449), anti-ASC (1:1000; Cell Signaling Technology, #67824), anti-caspase-1 (1:1000; Cell Signaling Technology, #83383), anti-NF-κBp65 (Cell Signaling Technology, #8242), and anti-pNF-κBp65 (1:100; Cell Signaling Technology, #3033). Following three washes, the membranes were incubated with the suitable Horseradish peroxidase-conjugated secondary antibody (anti-GAPDH, 1:10000; Abcam, ab8245) for 60 min at room temperature. The immunoblots were detected using a gel imaging system (CLiNX, Shanghai, China), and the band intensity was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

ELISA

The concentrations of both cytokines IL-1β and MMP-9 were measured using ELISA kits (Elabscience Biotechnology), in accordance with the manufacturer’s guidelines. The assay was executed, adhering to the stated instructions, while the collected data were expressed relative to the generated standard curve of IL-1β and MMP-9.

The Transwell coculture system

The Transwell coculture system was set up as reported previously.²⁹ In brief, the Transwell coculture system consisted of BV2 cells cultured on the lower compartment of a two-chamber Transwell system (NEST, China, 724121) with a 0.4-mm pore size polyester membrane. The bEnd.3 cells were cultured in DMEM/F12 medium on the upper chamber. Medium was changed daily, and the transepithelial resistance was monitored using a transmembrane resistance meter. Subsequent experiments were conducted upon reaching the maximum resistance level.

Small interfering RNA (siRNA) transfection

BV2 cells cultured in the lower compartment of a two-chamber Transwell system were transfected with either NLRP3-siRNA or NC-siRNA (Hanheng Biological, Beijing, China), using Lipofectamine RNA I MAX (Thermofisher, USA) in accordance with the manufacturer’s instructions. The NLRP3-siRNA was prepared using the following sequences: forward, 5′-GCGAGACCUCUGGGAAAATT-3′; reverse, 5′-UUUUCCCAGAGGUCUCGCCTT-3′. After 24 h of transfection, the treated lower chambers were moved to a new set of wells containing bEnd.3 cells cultured in the upper chambers. Following this coculture setup, both siRNA-transfected BV2 cells and Transwell systems were treated with LPS.

TEER measurement

TEER serves as a crucial indicator of BBB integrity, and measuring TEER is a commonly employed noninvasive and label-free method for monitoring endothelial barrier function in vitro.^30,31 TEER measurements were conducted following established protocols,³² at a temperature of 37°C using Millicell-ERS equipment with a heating plate to minimize temperature fluctuations once the cells had formed a confluent monolayer. The electrical resistance was recorded, taking into account the filter and medium, and was corrected by subtracting background readings. TEER values were then calculated as Ω·cm² by multiplying the surface area of the Transwell insert.

Flow cytometry

The apoptosis rate of bEnd.3 cells was assessed utilizing the Annexin V-FITC/PI Fluorescent Double Stain Apoptosis assay kit (Elabscience Biotechnology, China). The cells were collected in a 15 mL centrifuge tube, washed twice with precooled PBS, and resuspended with Annexin V Binding Buffer solution that was diluted in ultra-pure water; 5 μL of Annexin V-FITC and 5 μL of PI staining solution were added to the cell suspension, the cells were gently vortexed, followed by an incubation at room temperature and away from light for 15 min. Then, the number of cells was determined via flow cytometry.

Animal model of TBI

C57/BL mice weighing between 28 and 30 g, which were purchased from Vital River Laboratory Animal Technology Co., were utilized for this study. General anesthesia was administered using 1% sodium pentobarbital at a dose of 50 mg/kg. Controlled cortical impact (CCI) model of TBI was performed as described previously,^23,33 the animals were positioned in a stereotaxic frame, and a parietal craniotomy was performed under aseptic conditions, specifically located 1.0 mm anterior and 2.0 mm lateral to the bregma, with a diameter of 4.0 mm. CCI was induced utilizing a pneumatically driven cortical impact device (TBI 0310, Precision Systems and Instrumentation, Fairfax Station, VA). A flat-tipped impactor with a diameter of 3 mm was employed to compress the exposed dura mater and the underlying cerebral tissue to a depth of 1.0 mm for a duration of 150 ms, achieving an impact velocity of 3.5 m/s. The Sham group of mice underwent the same procedure, however, without the impact. The animals were placed in incubators at 30°C for 1 h and then raised individually in cages with ample access to both food and water.

Measurement of Evans blue extravasation

Assessment of Evans blue (EB) dye extravasation in our experiment was performed with reference to the procedure in a previous study³⁴; 72 h after CCI, the mice received peritoneal anesthesia through a 1% pentobarbital sodium injection at a dose of 50 mg/kg. To assess BBB integrity, the mice were injected with EB dye solution via the inferior vena cava at a dose of 4 mL/kg. The presence of a blue color in the limbs indicated that EB dye solution had entered the mice’s circulatory system and remained present for 30 min. To eliminate EB dye from the circulatory system, normal saline was perfused through the mice for approximately 15 min. The brain was then separated from the mice, and the damaged and surrounding brain tissues were removed and weighed. The brain tissue was subsequently mixed with 2-mL PBS, homogenized, and then mixed with 2-mL trichloroacetic acid solution with 60% concentration. The protein was deposited at 4°C for 30 min and centrifuged at 18,000 revolutions per minute for 10 min. The absorption value of EB dye in the supernatant was measured using a spectrophotometer at 610 nm. The EB dye content in the tissue was calculated by comparing it to the standard curve.

Cerebral edema and brain water contents

Seventy-two hours following CCI, brain tissue water content was assessed utilizing the wet–dry method. At a predetermined point during the experiment, the mice were anesthetized and then euthanized, and their brains were completely extracted and weighed utilizing an electronic scale. Subsequently, the brains were desiccated in a 99°C oven for 48 h to eliminate moisture from the brain tissue. After 48 h, the mice were reweighed. Brain water content was then calculated as follows: (wet weight of brain tissue − dry weight of brain tissue)/wet weight of brain tissue ×100%.

Statistical analyses

The mean ± standard deviation (SD) was used to represent all data, and statistical analysis was conducted through GraphPad Prism 8.0 software. To assess significant differences among the study groups, K–W one-way ANOVA for multiple comparisons, and Student–Newman–Keuls post hoc tests were utilized. Statistical significance was defined as a p < 0.05.

Results

Inhibition of NKCC1 decreased the level of NLRP3 inflammasome and the secretion of inflammatory cytokines in microglia

To investigate the impact of NKCC1 on NLRP3 inflammasome and downstream signaling molecules in vitro-cultured microglial cells, we utilized the specific inhibitor BMT to modulate NKCC1 activity and subsequently assessed the levels of NLRP3 inflammasome and downstream inflammatory factors IL-1β and MMP-9. The LPS group exhibited significantly increased levels of NLRP3 inflammasome, ASC, and Caspase-1 compared to the Control group. In contrast, the LPS + BMT group showed significantly reduced levels of NLRP3 inflammasome, ASC, and Caspase-1 compared to the LPS group, as confirmed by immunofluorescence results (Fig. 1E and F). These findings suggest that BMT could mitigate the level of LPS-induced NLRP3 inflammasome-related proteins in BV2 cells. To assess the effect of NKCC1 on cytokine secretion in microglial cells, we measured the levels of IL-1β and MMP-9 in the cell supernatant using ELISA (Fig. 1G and H). The LPS group demonstrated significantly higher levels of IL-1β and MMP-9 in the cell supernatant compared to the Control group. Conversely, the LPS + BMT group exhibited significantly lower levels of IL-1β and MMP-9 in the cell supernatant compared to the LPS group. These findings indicate a close association between NKCC1 and the secretion of inflammatory factors IL-1β and MMP-9 in microglial cells.

FIG. 1.

Effects of NKCC1 on NLRP3 inflammasome expression and secretion of IL-1β and MMP-9 in microglia. (A) WB analysis of NLRP3 inflammasome from different treated cells. (B) Quantitative analysis results of NLRP3 in Panel A (F _{(3, 8)} = 34.04, p < 0.0001). (C) Quantitative analysis results of Caspase-1 in panel A (F _{(3, 8)} = 57.49, p < 0.0001). (D) Quantitative analysis results of ASC in Panel A (F _{(3, 8)} = 17.44, p = 0.0007). (E) Representative photographs of immunofluorescence staining of BV2 cells in each group. (F) Quantification of result in panel E (F _{(3, 8)} = 91.13, p < 0.0001). (G) Detection of IL-1β level in BV2 cells of each group by ELISA (F _{(3, 8)} = 34.85, p < 0.0001). (H) Detection of MMP-9 level in BV2 cells of each group by ELISA (F _{(3, 8)} = 112.5, p < 0.0001). The data were analyzed using one-way analysis of variance, and all data are expressed as the mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 represent a statistically significant difference between the two groups. IL-1β, interleukin-1β; NKCC1, Na⁺-K⁺-Cl⁻ cotransporter; MMP-9, matrix metalloproteinase-9; NLRP, NOD-like receptor family pyrin domain containing 3; WB, Western blot.

The inhibition of NKCC1 disrupted the phosphorylation process and nuclear translocation of the p65 subunit within the NF-κB pathway

The NF-κB signaling pathway is a key factor in regulating inflammation and immune function in vivo.³⁵ In the CNS, injury signals can stimulate cells by activating the NF-κB signaling pathway, which in turn promotes the inflammatory response.³⁶ The phosphorylated p65 (p-p65) and p65 subunit ratio is a key marker of NF-κB signaling pathway activation.³⁷ In this study, we utilized BMT to interfere with NKCC1 and subsequently measured changes of p-p65 and p65 subunit in different groups by WB. Our results demonstrated a significant increase in p-p65/p65 subunit ratio in the LPS group compared with the Control group, indicating activation of the NF-κB pathway (Fig. 2A and B). Additionally, the p-p65/p65 ratio was significantly reduced in the LPS + BMT group compared with the LPS group, indicating that the inhibition of NKCC1 resulted in decreased activation of the NF-κB pathway. The activation of the NF-κB pathway involves the translocation of the p65 subunit into the nucleus. To investigate the intracellular distribution of the p65 subunit in microglia cells, an immunofluorescence study was conducted as presented in Figure 2C. In the Control group, the p65 subunit was predominantly observed in the cytoplasm of microglia cells. Conversely, in the LPS group, activation of microglia cells resulted in the increase of p65 subunit in nucleus with increased overlapping blue DAPI signal, which indicated that the p65 subunit had entered the nucleus. In the LPS + BMT group, the p65 subunit was mostly located in the cytoplasm. Notably, compared to the LPS group, there was a considerable increase in the p65 subunit signal in the cytoplasm of the LPS + BMT groups, which suggested that inhibiting NKCC1 can prevent the nuclear translocation of the p65 subunit. These findings indicate that the inhibition of NKCC1 might offer a strategy to hinder the activation of the NF-κB signaling pathway.

FIG. 2.

NKCC1 regulated the expression of NLRP3 inflammasome through NF-κB signaling in microglia. (A) WB analysis of p65 and p-p65 from different treated cells. (B) Quantitative analysis results of p-p65/p65 in panel A (F _{(3, 8)} = 49.65, p < 0.0001). (C) Representative photographs of p65 immunofluorescence staining of BV2 cells in different group. (D) WB analysis of NLRP3 inflammasome from different treated cells. (E) Quantitative analysis results of NLRP3 in panel D (F _{(3, 8)} = 39.54, p < 0.0001). (F) Quantitative analysis results of Caspase-1 in Panel D (F _{(3, 8)} = 27.85, p = 0.0001). (G) Quantitative analysis results of ASC in Panel D (F _{(3, 8)} = 10.59, p = 0.0037). The data were analyzed using one-way analysis of variance, and all data are expressed as the mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 represent a statistically significant difference between the two groups. NKCC1, Na⁺-K⁺-Cl⁻ cotransporter; NF-κB, nuclear factor-kappaB; NLRP, NOD-like receptor family pyrin domain containing 3; WB, Western blot.

The level of NLRP3 inflammasome could be reduced by blocking the NF-κB signaling pathway

The NF-κB signaling pathway is crucial in the process of inflammation activation. To investigate the regulatory effects of the NF-κB signaling pathway on NLRP3 inflammasome in microglia, we utilized SN50, a specific inhibitor of the p65 subunit, to inhibit the nuclear translocation of p65. The LPS group exhibited significantly increased levels of NLRP3 inflammasome, ASC, and Caspase-1 compared to the Control group (Fig. 2D–G). However, in the LPS + SN50 group, the levels of NLRP3 inflammasome, ASC, and Caspase-1 were significantly decreased compared to the LPS group. These findings suggested that the inhibition of the NF-κB signaling pathway may effectively reduce the level of NLRP3 inflammasome in microglia. Overall, these results indicate that NKCC1 could regulate the NLRP3 inflammasome of microglia through the NF-κB signaling pathway.

The NLRP3 inflammasome in BV2 cells could aggravate the damage of bEnd.3 cells and compromise BBB integrity in the inflammatory environment

To investigate the impact of activated BV2 cells on the BBB, we established an in vitro BBB model using Transwell chambers and coculturing bEnd.3 cells with BV2 cells. By analyzing the changes in the apoptosis rate of the bEnd.3 cells on the upper layer of the Transwell chambers under different treatment conditions, we evaluated the damage to the endothelial cells by BV2 cells. The results of flow cytometry are shown in Figure 3A and C. Compared to the bEnd.3 group, the apoptosis rate of endothelial cells in the bEnd.3 + LPS group was significantly higher. Furthermore, the apoptosis rate of bEnd.3 cells in the bEnd.3 + BV2 + LPS group was significantly elevated in comparison to the bEnd.3 + LPS group. These findings suggested that, under an inflammatory environment, activated BV2 cells could prompt apoptosis of bEnd.3 cells. To further explore the potential impact of the NLRP3 inflammasome in BV2 cells on the BBB, siRNA was utilized to interfere with the expression of NLRP3 inflammasome in BV2 cells, and flow cytometry was employed to determine the apoptosis rate of bEnd.3 cells. The Transwell coculture system was conducted as reported previously.²⁹ The LPS concentration was 1 μg/mL, and the duration of exposure was 24 h. The results, illustrated in Figure 3B and D, revealed that compared to the Control group, the apoptosis rate of endothelial cells was substantially increased in the bEnd.3 + BV2 + LPS group. Furthermore, in the bEnd.3 cells + BV2 cells + LPS + NLRP3 siRNA group, the apoptosis rate of bEnd.3 cells was significantly reduced when compared to the bEnd.3 + BV2 + LPS group. These findings suggest that the activation of NLRP3 inflammasome in BV2 cells may play a crucial role in mediating the detrimental effects of BV2 cells on bEnd.3 cells within an inflammatory microenvironment.

FIG. 3.

Effect of microglia and NLRP3 inflammasome on apoptosis rate of vascular endothelial cells. (A) Representative photographs of flow cytometry of bEnd.3 cells under isolated culture and Transwell coculture condition. (B) Representative photographs of flow cytometry of bEnd.3 cells under Transwell coculture condition. (C) Quantitative analysis concerning the ratio of apoptosis in bEnd.3 cells in Panel A (F _{interaction (1, 8)} = 6.625, p = 0.0329). (D) Quantitative analysis concerning the ratio of apoptosis in bEnd.3 cells in Panel B (F _{(3, 8)} = 51.23, p < 0.0001). (E) Quantification of TEER of bEnd.3 cells under isolated culture and Transwell coculture condition (F _{(1, 8)} = 45.73, p = 0.0001). (F) Quantification of TEER of bEnd.3 cells under Transwell coculture condition (F _{(3, 8)} = 156.9, p < 0.0001). The data were analyzed using one-way (D and F) or two-way (C and E) analysis of variance and all data are expressed as the mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 represent a statistically significant difference between the two groups. NLRP, NOD-like receptor family pyrin domain containing 3; TEER, transendothelial electrical resistance.

Activated microglia could disrupt the integrity of BBB in vitro by regulating the level of NLRP3 inflammasome

To determine the influence of the NLRP3 inflammasome in microglia on the integrity of the BBB, we utilized TEER values to assess BBB integrity. Our data from TEER measurements (Fig. 3E and F) were corroborated with the trends observed through flow cytometry (Fig. 3C and D). In both the isolated culture environment and the Transwell coculture environment, we observed a significant reduction in the TEER values when exposed to LPS compared to the Control group. Moreover, the decrease in TEER values was more pronounced in the Transwell coculture environment with BV2 cells and bEnd.3 cells under LPS stimulation compared to the isolated culture environment of bEnd.3 cells. Additionally, in the Transwell coculture environment, the NLRP3 siRNA group exhibited significantly higher TEER values than the LPS group. These findings suggest that activated microglia might disrupt the integrity of BBB by regulating the level of NLRP3 inflammasome.

NLRP3 inflammasome levels increased in microglia, astrocytes, vascular endothelial cells, and neurons after TBI

The upregulation of the NLRP3 inflammasome is a significant factor in the inflammatory reaction following secondary craniocerebral trauma. In this experiment, the level of NLRP3 inflammasome in various cell types was investigated through immunofluorescence double staining and specific cell markers, including TMEM-119 for microglia, GFAP for astrocytes, CD31 for endothelial cells, and NeuN for neurons. The experiment was divided into two distinct groups: the Sham group and the TBI group. In the TBI group, a standard CCI model was employed to induce TBI. Conversely, the Sham group underwent an identical craniotomy procedure as the TBI group, but did not experience any physical impact. Subsequently, after 72 h, brain tissues were extracted from both groups of mice for the purpose of immunofluorescence staining, allowing for an analysis of the specific cellular localization patterns of the NLRP3 inflammasome. These findings indicate a notable rise in the favorable ratio of NLRP3 inflammasome across microglia, astrocytes, endothelial cells, and neurons in the TBI group compared to the Sham group (Fig. 4A–D). Of the four cellular entities, microglia showed the highest positive rate of NLRP3 inflammasome (Fig. 4E).

FIG. 4.

Effects of TBI on NLRP3 inflammasome expression in microglia, astrocytes, vascular endothelial cells, and neurons in mouse brain tissue. (A) Representative photographs of TMEM119 and NLRP3 costaining of frozen brain sections in different group. (B) Representative photographs of GFAP and NLRP3 costaining of frozen brain sections in different group. (C) Representative photographs of NeuN and NLRP3 costaining of frozen brain sections in different group. (D) Representative photographs of CD31 and NLRP3 costaining of frozen brain sections in different group. (E) Quantification of NLRP3⁺ cells in panels A–D (F _{interaction (3, 40)} = 16.58, p < 0.0001). The data were analyzed using two-way analysis of variance, and all data are expressed as the mean ± standard deviation. *p < 0.05, **p < 0.01, and ****p < 0.0001 represent a statistically significant difference between the two groups. GFAP, glial fibrillary acidic protein; NLRP, NOD-like receptor family pyrin domain containing 3; TBI, traumatic brain injury.

Inhibition of NKCC1 could reduce the positive rate of NLRP3 inflammasome in microglia and the levels of inflammatory cytokines IL-1β and MMP-9 after TBI

This experiment aimed to investigate the potential regulatory effect of NKCC1 on NLRP3 inflammasome in microglia after TBI. TMEM-119 and NLRP3 inflammasome fluorescent antibodies were utilized to identify and visualize cells through immunofluorescence double staining. The findings indicated that, compared to the Sham group, the TBI group exhibited a significantly higher positive rate of NLRP3 (Fig. 5A,B); however, the positive rate of NLRP3 in the TBI + BMT group was significantly decreased compared to the TBI group (Fig. 5A and B). Additionally, we assessed the levels of IL-1β and MMP-9 in and around injured brain tissue using ELISA. The results suggested that the inhibition of NKCC1 significantly decreased the levels of these cytokines associated with BBB injury in the TBI + BMT group compared to the TBI group (Fig. 5C and D). These results indicate that NKCC1 is associated with the activation of NLRP3 inflammasome in microglia and the secretion of IL-1β and MMP-9 in brain tissue after TBI.

FIG. 5.

NKCC1 inhibitor BMT decreased NLRP3 expression and IL-1β and MMP-9 secretion in microglia of TBI mice. (A) Representative photographs of TMEM119 and NLRP3 costaining of frozen brain sections in different group. (B) Quantification of NLRP3⁺ cells in panel A (F _{(3, 20)} = 54.68, p < 0.0001). (C) Detection of IL-1β level in brain tissue of each group by ELISA (F _{(3, 20)} = 30.19, p < 0.0001). (D) Detection of MMP-9 level in brain tissue of each group by ELISA (F _{(3, 20)} = 51.53, p < 0.0001). The data were analyzed using one-way analysis of variance, and all data are expressed as the mean ± standard deviation. ***p < 0.001 and ****p < 0.0001 represent a statistically significant difference between the two groups. BMT, Bumetanide; IL-1β, interleukin-1β; MMP-9, matrix metalloproteinase-9; NKCC1, Na⁺-K⁺-Cl⁻ cotransporter; NLRP, NOD-like receptor family pyrin domain containing 3; TBI, traumatic brain injury.

Elevated NLRP3 inflammasome level after TBI displayed a correlation with BBB damage and the development of cerebral edema

To clarify the influence of NLRP3 inflammasome on BBB integrity after TBI, we utilized a specific inhibitor of NLRP3 inflammasome, MCC950, to disrupt the activity of the inflammasome in mice. Using immunofluorescence, we have measured the fluorescence intensity of ZO-1, a tight junction protein (Fig. 6A). The results showed that the fluorescence intensity of ZO-1 in the TBI group was significantly lower than that in Sham group (Fig. 6B). Additionally, the TBI + MCC950 group showed higher fluorescence intensity when compared to the TBI group (Fig. 6B), suggesting a correlation between NLRP3 inflammasome and ZO-1 tight junction proteins in TBI. BBB integrity was evaluated by measuring the permeability of EB dye, and the morphological characteristics of the mouse brain tissue are illustrated in Figure 6A. In comparison to the Sham group, the TBI group exhibited a noticeable increase in EB leakage, while the TBI + MCC950 group demonstrated less leakage compared to the TBI group (Fig. 6A). The quantitative results corroborated the morphological observations of brain tissue, and the EB dye leakage was notably less in the TBI + MCC950 group compared to the TBI group (Fig. 6C), indicating a positive correlation between NLRP3 inflammasome and increased BBB permeability following TBI. The level of cerebral edema among various groups was assessed through the wet-dry method. According to the outcomes depicted in Figure 6D, it was observed that the water content within the brain tissue of the TBI group exhibited a significant increase in comparison to the Sham group (Fig. 6D). On the contrary, the TBI + MCC950 group manifested a marked decline in brain water content, in contrast to the TBI group (Fig. 6D). We further assessed the impact of NLRP3 inflammasome inhibition on the levels of IL-1β and MMP-9 cytokines in the injured brain tissue using ELISA analysis (Fig. 6E and F). The results revealed a significant upregulation of IL-1β and MMP-9 in the TBI group, indicating an inflammatory response following TBI. Notably, the TBI + MCC950 group exhibited a substantial reduction in IL-1β and MMP-9 levels compared to the TBI group, indicating a potential association between NLRP3 inflammasome activation and the inflammatory response in the injured brain tissue. The results imply a correlation between the NLRP3 inflammasome and the impairment of BBB integrity, as well as the emergence of neuroinflammation and cerebral edema in TBI.

FIG. 6.

The role of NLRP3 inflammasome in TBI-induced BBB injury. (A) Fluorescence of ZO-1 of frozen brain sections and the leakage of EB staining solution in different group. (B) Quantification of ZO-1 in panel A (F _{(3, 20)} = 195.7, p < 0.0001). (C) Quantitative analysis of EB staining solution leakage in brain tissue across different groups (F _{(3, 8)} = 134.5, p < 0.0001). (D) Quantitative analysis of water content in brain tissue across different groups (F _{(3, 8)} = 29.52, p = 0.0001). (E) Detection of IL-1β level in brain tissue of each group by ELISA (F _{(3, 20)} = 138.8, p < 0.0001). (F) Detection of MMP-9 level in brain tissue of each group by ELISA (F _{(3, 20)} = 93.3, p < 0.0001). The data were analyzed using two-way analysis of variance, and all data are expressed as the mean ± standard deviation. **p < 0.01, ***p < 0.001, and ****p < 0.0001 represent a statistically significant difference between the two groups. BBB, blood–brain barrier; EB, Evans blue; IL-1β, interleukin-1β; MMP-9, matrix metalloproteinase-9; NLRP, NOD-like receptor family pyrin domain containing 3; TBI, traumatic brain injury.

Discussion

The present study investigated the operative mechanism by which NKCC1 regulates BBB integrity after TBI. The study revealed that inhibiting NKCC1 could reduce the level of NLRP3 inflammasome and the release of IL-1β and MMP-9 in microglial cells through the NF-κB signaling pathway, thereby providing a protective effect on the BBB following TBI.

The inflammatory response in secondary TBI is of immense importance to neurodegeneration and neurological dysfunction after TBI, promoting the aggregation of macrophages, neutrophils, and lymphocytes at the site of injury and the release of inflammatory and anti-inflammatory cytokines.^38,39 Excessive neuroinflammation has been shown to be closely associated with disruption of the BBB following TBI. As an important component of CNS immunity, microglia continuously monitor harmful stimuli and infections in the microenvironment, playing a vital surveillance role in CNS diseases such as brain trauma and ischemia.^40,41 In response to TBI, microglia typically react to the site of injury within minutes, inducing postinjury neuroinflammation and secondary cascade reactions.⁴² During this process, the NLRP3 inflammasome serves as an essential component, its activation being the main driving force behind excessive immune and neuroinflammatory responses.^43,44 When exposed to microbial infections, endogenous danger signals, and environmental stimuli, the combination of NLRP3 and ASC initiates a cascade reaction of Caspase-1, which under its action hydrolyzes pro-IL-1β into active mature IL-1β, rapidly releasing it into the extracellular space, exacerbating inflammatory reactions. Additionally, as an upstream regulatory molecule, IL-1β promotes the level of MMP-9, and excessive levels of IL-1β and MMP-9 cytokines in the extracellular space have been shown to hydrolyze a variety of tight junction proteins, leading to increased BBB permeability.^45

–48

The NLRP3 inflammasome has been demonstrated to be expressed in multiple cell types within the CNS.^49,50 Notably, microglia are the primary cell type to express the NLRP3 inflammasome, and the level of this complex in various other cell types is dependent on microglial induction, indicating that the NLRP3 inflammasome in microglia may act as a signal amplifier.⁵¹ In this study, we found that NKCC1 promotes the level of NLRP3 inflammasome-associated proteins in microglia. Specific inhibition of NKCC1 resulted in a significant reduction of NLRP3 inflammasome, as well as downstream cytokine IL-1β and MMP-9 secretion, indicating the importance of NKCC1 in promoting microglia-induced immune-inflammatory responses. Additionally, we found that NKCC1 promotes the activation of the NF-κB signaling pathway, which in turn elevates the level of NLRP3 inflammasome. The NF-κB signaling pathway is considered a central mediator of the inflammatory process and an important participant in both innate and adaptive immune responses.⁵² It has been demonstrated that inhibition of the NKCC1 receptor significantly attenuates macrophage inflammatory response and reduces lung injury in acute lung injury models induced by LPS, and this effect is also mediated through the NF-κB signaling pathway.²² As macrophages share similarities in immune function and origin with microglia, our study further confirms the regulatory role of NKCC1 in immune cell inflammatory response.

The endothelial cells of blood vessels are the primary component of the BBB. The tight junction proteins, such as ZO-1, Occludin, and Claudin-5, found in brain microvascular endothelial cells, form the structural basis for the low permeability of the BBB.^53,54 ZO-1 is one of the key scaffold proteins that is connected to the intracellular actin cytoskeleton, which is indispensable in maintaining stable tight junction structure.⁵⁵ Following TBI, microglia may become activated by damage-associated molecular pattern stimuli, which leads to the release of several signaling molecules, including inflammatory cytokines, neurotoxic compounds, reactive oxygen species, MMPs, and nitric oxide.⁵⁰ Many of these factors have been shown to have detrimental effects on tight junction proteins.^28,45,56 Research has revealed that the activation of microglia is linked to the remodeling of the BBB in rat models of post-traumatic stress disorder. Inhibition of microglial activation resulted in altered permeability of the hippocampus, as evidenced by decreased permeability of luciferin sodium, and a notable increase in tight junction protein level within the hippocampal brain tissue.⁵⁷ The findings demonstrate the significance of microglial activity in BBB integrity and highlight potential therapeutic targets for BBB-related disorders. In this study, we established a classical in vitro BBB model composed of microglia and endothelial cells using a Transwell system. We found that activated microglia induced apoptosis in endothelial cells and disrupted the integrity of the in vitro BBB. However, knockdown of the NLRP3 inflammasome in microglia using siRNA greatly improved BBB function, indicating that the NLRP3 inflammasome in activated microglia plays an important role in BBB disruption in vitro.

NKCC1 is essential in maintaining intracellular ion balance and regulating water movement in and out of cells. Studies have shown that transcription and expression of NKCC1 increase significantly in animal models of TBI. Activation of NKCC1 can enhance the release of various inflammatory factors by immune cells, exacerbating damage to the BBB and brain tissue.^22,23,58,59 Inhibition of NKCC1 can alleviate brain edema, reduce the frequency of post-TBI seizures, and lessen neuronal apoptosis while maintaining neuronal excitability.^60
–62 Recent studies have also suggested that activation of NKCC1 in TBI enhances levels of IL-1β and its downstream cytokine MMP-9 in brain tissue. These cytokines have been demonstrated to be closely associated with BBB damage, but the specific regulatory mechanisms remain unclear.^15,23,63 In the context of the CNS, microglia cells are the initial responders to external signal stimuli and express NLRP3 inflammasome, which facilitates the regulation of downstream inflammatory cytokine IL-1β. Furthermore, microglia serve as the primary source of IL-1β.^51,64 Given these considerations, it is imperative to examine NKCC1’s regulatory mechanism on the disruption of BBB integrity in TBI from the perspective of microglia. In this study, we found that the level of NLRP3 inflammasome in microglia cells increased significantly 3 days after TBI. Intervention of NKCC1 with the specific receptor inhibitor BMT significantly reduced the proportion of NLRP3-positive microglia cells, which is consistent with our in vitro cellular experiments. Additionally, it was observed that following intervention targeting the NLRP3 inflammasome, the mice brain tissue exhibited a significant reduction in the amount of EB dye leakage and brain water content, and the fluorescence intensity of the ZO-1 tight junction protein was improved. Combining our cellular experiments that revealed the damaging effect of NLRP3 inflammasome in microglia cells on an in vitro BBB model, our study results suggest that NKCC1 can regulate BBB integrity by modulating NLRP3 inflammasome after TBI (Fig. 7). Both primary and secondary brain injuries can result in disruption of BBB integrity. In primary brain injury, direct impact from external forces on the skull and brain often renders the loss of BBB integrity difficult to prevent and intervene with drugs. In contrast, during secondary cranial injury, immune cells in the CNS become activated upon sensing external danger signal molecules such as genomic DNA, ATP, and mitochondrial DNA.^65,66 Consequently, a significant release of inflammatory factors takes place, including interleukin-1β (IL-1β), IL-6, TNF-α, and members of the MMPs family. The abundant secretion of these inflammatory factors is significantly linked to the degradation of tight junction proteins between vascular endothelial cells.¹⁵ Therefore, investigating strategies for reducing excessive inflammation activation following TBI may be an effective approach to preserving BBB integrity. Our research indicates that NLRP3 inflammasome regulated by NKCC1 in microglia cells exerts a substantial influence on TBI-induced BBB damage, providing insight into the mechanism by which NKCC1 improves BBB integrity following TBI. These findings have promising implications for the development of therapeutic methods to improve outcomes for TBI patients.

FIG. 7.

A schematic illustration of the mechanism by which NKCC1 causes BBB integrity failure after TBI. After TBI, NKCC1 induces NLRP3 inflammasome activation in microglia through the NF-κB signaling pathway. This leads to an increase in downstream cytokines such as IL-1β and MMP-9 release, causing degradation of tight junction proteins in endothelial cells, ultimately resulting in the disruption of BBB integrity. BBB, blood–brain barrier; IL-1β, interleukin-1β; MMP-9, matrix metalloproteinase-9; NLRP, NOD-like receptor family pyrin domain containing 3; NKCC1, Na⁺-K⁺-Cl⁻ cotransporter; NF-κB, nuclear factor-kappaB; TBI, traumatic brain injury.

The primary limitation of this study lies in the construction of the in vitro BBB model. The Transwell chamber-based BBB model used in this study has deficiencies in simulating complex signaling communication among various cell types, and it is also limited in mimicking the shear forces induced by blood flow in a physiological environment. The advancement of organoid culture techniques may provide a potential avenue to address this deficiency. Furthermore, a limitation of this study is the employment of intraperitoneal sodium pentobarbital for anesthesia, rather than the conventionally utilized isoflurane inhalation anesthesia. Sodium pentobarbital has been shown in several studies to possess potential neuroprotective properties, which could introduce a degree of bias into the experimental outcomes.^67,68 To mitigate this potential bias, we administered an identical dose of pentobarbital across all groups, including the control group, to control the variable and thereby minimize bias, enhancing the validity of our findings. Isoflurane is known for its rapid onset, effective anesthetic properties, ease of adjustment, and high safety profile. However, recent discoveries have highlighted the adverse effects of isoflurane on CNS function. Isoflurane activates the NLRP3-caspase-1 signaling pathway, resulting in elevated NLRP3 levels, which is a central focus of our research. This activation induces an inflammatory response in glial cells, leading to hippocampal inflammation and subsequent cognitive impairment.^69,70 Furthermore, the inhaled dose of anesthetic in gas anesthesia correlates with the duration of surgery, which varies across groups, potentially leading to inconsistent anesthetic exposure. Given this evidence, employing isoflurane for inhalation anesthesia would not have been sufficiently rigorous for our study. Sodium pentobarbital provides effective anesthesia and safety, and its liquid form allows for precise dosage control, facilitating variable consistency and reducing confounding factors. We propose that a systematic experimental design and investigation into the comprehensive effects of various anesthetics on neurological function in TBI mice models would offer valuable insights for future anesthetic selection in related research.

Conclusion

Our study indicates that activated microglia cells have a negative impact on the integrity of the BBB. In TBI, NKCC1 located in microglia cells can elevate the level of NLRP3 inflammasome and the release of IL-1β and MMP-9 via the NF-κB signaling pathway, ultimately damaging the integrity of the BBB.

Transparency, Rigor, and Reproducibility Summary

The study and analysis plan were registered before beginning data collection with the National Natural Science Foundation (https://www.nsfc.gov.cn/, grant No. 81971167). Although the analysis plan was not formally preregistered, the team member primarily responsible for the analysis, Jun Zhang, verifies that the plan was prespecified. We planned for a sample size of 150 subjects based on the availability of mice. Out of these, data from 120 mice were usable. Among the mice that generated usable data, a total of 48 mice were used for immunofluorescence analysis, 48 mice were used to assess the levels of IL-1β and MMP-9, and 24 mice were used to evaluate brain tissue water content and the leakage of EB dye. The remaining 30 mice did not yield usable data; specifically, 14 died during the induction phase of the CCI model, which involved anesthesia and hypothermia. Additionally, 12 mice were allocated for preliminary experiments, and data from 4 mice were deemed unusable due to brain section contamination. Regarding data analysis for the pathological experiments, each group consisted of six mice, and three coronal brain slices from the injured area were taken from each mouse. Under fluorescence microscopy, we selected three fields of view from a 1-mm perimeter surrounding the injured area for quantifying the detection indicators and averaged the data to represent the experimental data for each mouse. The average values for each group (n = 6/group) were then used for statistical comparisons between groups. In regard to the molecular biology experiments, each mouse sample underwent three technical replicates in the in vivo studies, and the average was calculated. The six average values obtained from the six mice in each group (n = 6/group) were used for statistical comparisons between groups. In the in vitro experiments, each experiment was conducted in three wells to obtain an average count, and three independent biological replicate experiments were performed. Investigators blinded to the participants’ relevant characteristics performed biomechanical quality control decisions and analyses. All mice were tested between 0800 and 1200 in a fed state. Histological analyses were conducted in three batches, with samples randomly assigned to batches. We established negative controls for positive results by setting up a solvent group and a sham operation group. All equipment and analytical reagents used for experimental manipulations and measurements may be available upon request from CST and Abcam. To our knowledge, no replication or external validation studies have been performed or are planned at this time. Deidentified data from this study are not publicly archived but will be made available (as allowable according to institutional IRB standards) by emailing the corresponding author as of 30/12/2025. A limited number of histological samples from each experimental group are available for future analyses upon request. The authors have agreed to publish the article using the Mary Ann Liebert Inc. “Open Access” option under the appropriate license.

Footnotes

Authors’ Contributions

Z.Z., H.W., and B.T. contributed substantially to the experimental design, data analysis, and experimental procedures. X.S., G.C., and H.M. provided experimental help. R.P. and J.Z. planned the overall direction of the experiment, supervised the experiment, and reviewed the article.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by Beijing Natural Science Foundation (L244003).

Supplementary Material

Supplementary Data

References

George

, Behera

, Homme

, et al. Rebuilding microbiome for mitigating traumatic brain injury: Importance of restructuring the gut-microbiome-brain axis. Mol Neurobiol, 2021; 58(8):3614–3627; doi: 10.1007/s12035-021-02357-2

Haarbauer-Krupa

, Pugh

, Prager

, et al. Epidemiology of chronic effects of traumatic brain injury. J Neurotrauma, 2021; 38(23):3235–3247; doi: 10.1089/neu.2021.0062

Capizzi

, Woo

, Verduzco-Gutierrez

. Traumatic brain injury: An overview of epidemiology, pathophysiology, and medical management. Med Clin North Am, 2020; 104(2):213–238; doi: 10.1016/j.mcna.2019.11.001

Liebner

, Dijkhuizen

, Reiss

, et al. Functional morphology of the blood-brain barrier in health and disease. Acta Neuropathol, 2018; 135(3):311–336; doi: 10.1007/s00401-018-1815-1

Kadry

, Noorani

, Cucullo

. A blood-brain barrier overview on structure, function, impairment, and biomarkers of integrity. Fluids Barriers CNS, 2020; 17(1):69; doi: 10.1186/s12987-020-00230-3

Sulhan

, Lyon

, Shapiro

, et al. Neuroinflammation and blood-brain barrier disruption following traumatic brain injury: Pathophysiology and potential therapeutic targets. J Neurosci Res, 2020; 98(1):19–28; doi: 10.1002/jnr.24331

Thal

, Neuhaus

. The blood-brain barrier as a target in traumatic brain injury treatment. Arch Med Res, 2014; 45(8):698–710; doi: 10.1016/j.arcmed.2014.11.006

van Vliet

, Ndode-Ekane

, Lehto

, et al. Long-lasting blood-brain barrier dysfunction and neuroinflammation after traumatic brain injury. Neurobiol Dis, 2020; 145:105080; doi: 10.1016/j.nbd.2020.105080

, Wang

, Chen

, et al. Hydroxychloroquine attenuates neuroinflammation following traumatic brain injury by regulating the TLR4/NF-κB signaling pathway. J Neuroinflammation, 2022; 19(1):71; doi: 10.1186/s12974-022-02430-0

10.

Lee

, Eo

, Lee

, et al. Distinct features of brain-resident macrophages: Microglia and non-parenchymal brain macrophages. Mol Cells, 2021; 44(5):281–291; doi: 10.1434/8/molcells.2021.0060

11.

Cai

, Gong

, Qi

, et al. ACT001 attenuates microglia-mediated neuroinflammation after traumatic brain injury via inhibiting AKT/NFκB/NLRP3 pathway. Cell Commun Signal, 2022; 20(1):56; doi: 10.1186/s12964-022-00862-y

12.

, Li

, Peng

, et al. Abrocitinib attenuates microglia-mediated neuroinflammation after traumatic brain injury via inhibiting the JAK1/STAT1/NF-κB pathway. Cells, 2022; 11(22); doi: 10.3390/cells11223588

13.

Devanney

, Stewart

, Gensel

. Microglia and macrophage metabolism in CNS injury and disease: The role of immunometabolism in neurodegeneration and neurotrauma. Exp Neurol, 2020; 329:113310; doi: 10.1016/j.expneurol.2020.113310

14.

Fei

, Wang

, Zhou

, et al. Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury. Biochem Biophys Res Commun, 2020; 523(2):361–367; doi: 10.1016/j.bbrc.2019.12.003

15.

Gong

, Wu

, Shen

, et al. Inhibition of the NKCC1/NF-κB signaling pathway decreases inflammation and improves brain edema and nerve cell apoptosis in an SBI rat model. Front Mol Neurosci, 2021; 14:641993; doi: 10.3389/fnmol.2021.641993

16.

Serranilla

, Woodin

. Striatal chloride dysregulation and impaired GABAergic signaling due to cation-chloride cotransporter dysfunction in Huntington’s disease. Front Cell Neurosci, 2021; 15:817013; doi: 10.3389/fncel.2021.817013

17.

Liu

, Wang

, Liang

, et al. Role of NKCC1 and KCC2 in epilepsy: From expression to function. Front Neurol, 2019; 10:1407; doi: 10.3389/fneur.2019.01407

18.

Lam

, Newland

, Faull

RLM

, et al. Cation-chloride cotransporters KCC2 and NKCC1 as therapeutic targets in neurological and neuropsychiatric disorders. Molecules, 2023; 28(3); doi: 10.3390/molecules28031344

19.

Hung

, Peng

, Yang

, et al. WNK4-SPAK modulates lipopolysaccharide-induced macrophage activation. Biochem Pharmacol, 2020; 171:113738; doi: 10.1016/j.bcp.2019.113738

20.

, Cheng

, Wu

, et al. NKCC1-mediated traumatic brain injury-induced brain edema and neuron death via Raf/MEK/MAPK cascade. Crit Care Med, 2008; 36(3):917–922; doi: 10.1097/ccm.0b013e31816590c4

21.

Bennett

, Bennett

, Liddelow

, et al. New tools for studying microglia in the mouse and human CNS. Proceedings of the National Academy of Sciences of the United States of America, 2016; 113(12):E1738–E46; doi: 10.1073/pnas.1525528113

22.

Hung

, Peng

, Wu

, et al. Bumetanide attenuates acute lung injury by suppressing macrophage activation. Biochem Pharmacol, 2018; 156:60–67; doi: 10.1016/j.bcp.2018.08.013

23.

Zhang

, Pu

, Zhang

, et al. Inhibition of Na(+)-K(+)-2Cl(-) cotransporter attenuates blood-brain-barrier disruption in a mouse model of traumatic brain injury. Neurochem Int, 2017; 111:23–31; doi: 10.1016/j.neuint.2017.05.020

24.

Silvestre de Ferron

, Vilpoux

, Kervern

, et al. Increase of KCC2 in hippocampal synaptic plasticity disturbances after perinatal ethanol exposure. Addict Biol, 2017; 22(6):1870–1882; doi: 10.1111/adb.12465

25.

Cheng

, Yu

, Yan

, et al. Nuclear factor-κB is involved in oxyhemoglobin-induced endothelin-1 expression in cerebrovascular muscle cells of the rabbit basilar artery. Neuroreport, 2016; 27(12):875–882; doi: 10.1097/wnr.0000000000000615

26.

Krishnan

, Ling

, Huuskes

, et al. Pharmacological inhibition of the NLRP3 inflammasome reduces blood pressure, renal damage, and dysfunction in salt-sensitive hypertension. Cardiovasc Res, 2019; 115(4):776–787; doi: 10.1093/cvr/cvy252

27.

, Chen

, Kang

, et al. Neuroprotective effects of natural compounds on LPS-induced inflammatory responses in microglia. Am J Transl Res, 2020; 12(6):2353–2378.

28.

Wang

, Ding

, Chen

, et al. Hypertonic saline mediates the NLRP3/IL-1β signaling axis in microglia to alleviate ischemic blood-brain barrier permeability by downregulating astrocyte-derived VEGF in rats. CNS Neurosci Ther, 2020; 26(10):1045–1057; doi: 10.1111/cns.13427

29.

, Wang

, Li

, et al. Diazoxide attenuates postresuscitation brain injury in a rat model of asphyxial cardiac arrest by opening mitochondrial ATP-Sensitive potassium channels. Biomed Res Int, 2016; 2016:1253842; doi: 10.1155/2016/1253842

30.

, Xu

, Zhu

, et al. Transendothelial electrical resistance measurement by a microfluidic device for functional study of endothelial barriers in inflammatory bowel disease. Front Bioeng Biotechnol, 2023; 11:1236610; doi: 10.3389/fbioe.2023.1236610

31.

Wimmer

, Tietz

, Nishihara

, et al. PECAM-1 stabilizes blood-brain barrier integrity and favors paracellular T-Cell diapedesis across the blood-brain barrier during neuroinflammation. Front Immunol, 2019; 10:711; doi: 10.3389/fimmu.2019.00711

32.

Hao

, Guo

, Zou

, et al. Hyperbaric oxygen preconditioning ameliorates blood-brain barrier damage induced by hypoxia through modulation of tight junction proteins in an in vitro model. Croat Med J, 2016; 57(1):51–57; doi: 10.3325/cmj.2016.57.51

33.

Xie

, Wang

, Lin

, et al. Inhibition of ferroptosis attenuates tissue damage and improves long-term outcomes after traumatic brain injury in mice. CNS Neurosci Ther, 2019; 25(4):465–475; doi: 10.1111/cns.13069

34.

Ahishali

, Kaya

. Evaluation of blood-brain barrier integrity using vascular permeability markers: Evans blue, sodium fluorescein, albumin-alexa fluor conjugates, and horseradish peroxidase. Methods Mol Biol, 2021; 2367:87–103; doi: 10.1007/7651_2020_316

35.

Barnabei

, Laplantine

, Mbongo

, et al. NF-κB: At the borders of autoimmunity and inflammation. Front Immunol, 2021; 12:716469; doi: 10.3389/fimmu.2021.716469

36.

Liu

, Zhang

, Xu

, et al. SARM1 promotes neuroinflammation and inhibits neural regeneration after spinal cord injury through NF-κB signaling. Theranostics, 2021; 11(9):4187–4206; doi: 10.7150/thno.49054

37.

Liu

, Li

, Wang

, et al. Role and mechanisms of the NF-ĸB signaling pathway in various developmental processes. Biomed Pharmacother, 2022; 153:113513; doi: 10.1016/j.biopha.2022.113513

38.

Kalra

, Malik

, Singh

, et al. Pathogenesis and management of traumatic brain injury (TBI): role of neuroinflammation and anti-inflammatory drugs. Inflammopharmacology, 2022; 30(4):1153–1166; doi: 10.1007/s10787-022-01017-8

39.

, Lee

AYW

. Traumatic brain injuries: Pathophysiology and potential therapeutic targets. Front Cell Neurosci, 2019; 13:528; doi: 10.3389/fncel.2019.00528

40.

Bolte

, Lukens

. Neuroimmune cleanup crews in brain injury. Trends Immunol, 2021; 42(6):480–494; doi: 10.1016/j.it.2021.04.003

41.

Gaire

. Microglia as the critical regulators of neuroprotection and functional recovery in cerebral ischemia. Cell Mol Neurobiol, 2022; 42(8):2505–2525; doi: 10.1007/s10571-021-01145-9

42.

Shao

, Wang

, Wu

, et al. Microglia and neuroinflammation: Crucial pathological mechanisms in traumatic brain injury-induced neurodegeneration. Front Aging Neurosci, 2022; 14:825086; doi: 10.3389/fnagi.2022.825086

43.

, Yin

, Ren

, et al. Selective NLRP3 inflammasome inhibitor reduces neuroinflammation and improves long-term neurological outcomes in a murine model of traumatic brain injury. Neurobiol Dis, 2018; 117:15–27; doi: 10.1016/j.nbd.2018.05.016

44.

Freeman

, Swartz

. Targeting the NLRP3 inflammasome in severe COVID-19. Front Immunol, 2020; 11:1518; doi: 10.3389/fimmu.2020.01518

45.

Kangwantas

, Pinteaux

, Penny

. The extracellular matrix protein laminin-10 promotes blood-brain barrier repair after hypoxia and inflammation in vitro . J Neuroinflammation, 2016; 13:25; doi: 10.1186/s12974-016-0495-9

46.

Tasaki

, Shimizu

, Sano

, et al. Autocrine MMP-2/9 secretion increases the BBB permeability in neuromyelitis optica. J Neurol Neurosurg Psychiatry, 2014; 85(4):419–430; doi: 10.1136/jnnp-2013-305907

47.

Qin

, Li

, Zhu

, et al. Melatonin protects blood-brain barrier integrity and permeability by inhibiting matrix metalloproteinase-9 via the NOTCH3/NF-κB pathway. Aging (Albany NY), 2019; 11(23):11391–11415; doi: 10.1863/2/aging.102537

48.

Yang

, Zhao

, Zhang

, et al. ATP induces disruption of tight junction proteins via IL-1 Beta-Dependent MMP-9 activation of human blood-brain barrier In Vitro. Neural Plast, 2016; 2016:8928530; doi: 10.1155/2016/8928530

49.

Liu

, Li

, Chen

, et al. Expression of the NLRP3 inflammasome in cerebral cortex after traumatic brain injury in a rat model. Neurochem Res, 2013; 38(10):2072–2083; doi: 10.1007/s11064-013-1115-z

50.

Irrera

, Russo

, Pallio

, et al. The role of NLRP3 inflammasome in the pathogenesis of traumatic brain injury. Int J Mol Sci, 2020; 21(17); doi: 10.3390/ijms21176204

51.

Gong

, Pan

, Shen

, et al. Mitochondrial dysfunction induces NLRP3 inflammasome activation during cerebral ischemia/reperfusion injury. J Neuroinflammation, 2018; 15(1):242; doi: 10.1186/s12974-018-1282-6

52.

Haftcheshmeh

, Abedi

, Mashayekhi

, et al. Berberine as a natural modulator of inflammatory signaling pathways in the immune system: Focus on NF-κB, JAK/STAT, and MAPK signaling pathways. Phytother Res, 2022; 36(3):1216–1230; doi: 10.1002/ptr.7407

53.

Reinhold

, Rittner

. Barrier function in the peripheral and central nervous system-a review. Pflugers Arch, 2017; 469(1):123–134; doi: 10.1007/s00424-016-1920-8

54.

Hudson

, Campbell

. Tight junctions of the neurovascular unit. Front Mol Neurosci, 2021; 14:752781; doi: 10.3389/fnmol.2021.752781

55.

Chen

, Ju

, Lu

, et al. Propofol improved hypoxia-impaired integrity of blood-brain barrier via modulating the expression and phosphorylation of zonula occludens-1. CNS Neurosci Ther, 2019; 25(6):704–713; doi: 10.1111/cns.13101

56.

Wang

, Jin

, Sonobe

, et al. Interleukin-1β induces blood-brain barrier disruption by downregulating Sonic hedgehog in astrocytes. PLoS One, 2014; 9(10):e110024; doi: 10.1371/journal.pone.0110024

57.

, Zhu

, Xu

, et al. Hippocampal activated microglia may contribute to blood-brain barrier impairment and cognitive dysfunction in post-traumatic stress disorder-like rats. J Mol Neurosci, 2022; 72(5):975–982; doi: 10.1007/s12031-022-01981-4

58.

Weidenfeld

, Kuebler

. Cytokine-regulation of Na(+)-K(+)-Cl(-) cotransporter 1 and cystic fibrosis transmembrane conductance regulator-potential role in pulmonary inflammation and edema formation. Front Immunol, 2017; 8:393; doi: 10.3389/fimmu.2017.00393

59.

Lan

, Wu

, Peng

, et al. Surfactant attenuates air embolism-induced lung injury by suppressing NKCC1 expression and NF-κB activation. Inflammation, 2021; 44(1):57–67; doi: 10.1007/s10753-020-01266-1

60.

Sawant-Pokam

, Vail

, Metcalf

, et al. Preventing neuronal edema increases network excitability after traumatic brain injury. J Clin Invest, 2020; 130(11):6005–6020; doi: 10.1172/jci134793

61.

Liang

, Huang

. Elevated NKCC1 transporter expression facilitates early post-traumatic brain injury seizures. Neural Regen Res, 2017; 12(3):401–402; doi: 10.4103/1673-5374.202939

62.

Zhang

, Cui

, et al. Astaxanthin alleviates cerebral edema by modulating NKCC1 and AQP4 expression after traumatic brain injury in mice. BMC Neurosci, 2016; 17(1):60; doi: 10.1186/s12868-016-0295-2

63.

Hui

, Rao

, Zhang

, et al. Inhibition of Na(+)-K(+)-2Cl(-) Cotransporter-1 attenuates traumatic brain injury-induced neuronal apoptosis via regulation of Erk signaling. Neurochem Int, 2016; 94:23–31; doi: 10.1016/j.neuint.2016.02.002

64.

Garcia-Bonilla

, Sciortino

, Shahanoor

, et al. Role of microglial and endothelial CD36 in post-ischemic inflammasome activation and interleukin-1β-induced endothelial activation. Brain Behav Immun, 2021; 95:489–501; doi: 10.1016/j.bbi.2021.04.010

65.

Klegeris

. Regulation of neuroimmune processes by damage- and resolution-associated molecular patterns. Neural Regen Res, 2021; 16(3):423–429; doi: 10.4103/1673-5374.293134

66.

McCarty

, Lerner

. The second phase of brain trauma can be controlled by nutraceuticals that suppress DAMP-mediated microglial activation. Expert Rev Neurother, 2021; 21(5):559–570; doi: 10.1080/14737175.2021.1907182

67.

Roberts

, Sydenham

. Barbiturates for acute traumatic brain injury. Cochrane Database Syst Rev, 2012; 12(12):Cd000033; doi: 10.1002/14651858.CD000033.pub2

68.

Salort

, Álvaro-Bartolomé

, García-Sevilla

. Pentobarbital and other anesthetic agents induce opposite regulations of MAP kinases p-MEK and p-ERK, and upregulate p-FADD/FADD neuroplastic index in brain during hypnotic states in mice. Neurochem Int, 2019; 122:59–72; doi: 10.1016/j.neuint.2018.11.008

69.

Wang

, Meng

, Cao

, et al. Critical role of NLRP3-caspase-1 pathway in age-dependent isoflurane-induced microglial inflammatory response and cognitive impairment. J Neuroinflammation, 2018; 15(1):109; doi: 10.1186/s12974-018-1137-1

70.

Fan

, Du

, Fu

, et al. Inhibiting the NLRP3 inflammasome with MCC950 ameliorates isoflurane-induced pyroptosis and cognitive impairment in aged mice. Front Cell Neurosci, 2018; 12:426; doi: 10.3389/fncel.2018.00426

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