Pharmacological Inhibition of Transient Receptor Potential Vanilloid 4 Reduces Vasogenic Edema after Traumatic Brain Injury in Mice

Abstract

Vasogenic edema results from blood–brain barrier (BBB) disruption after traumatic brain injury (TBI), and although it can be fatal, no promising therapeutic drugs have been developed as yet. Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable channel that is sensitive to temperature and osmotic pressure. As TRPV4 is known to be responsible for various pathological conditions following brain injury, we investigated the effects of pharmacological TRPV4 antagonists on TBI-induced vasogenic edema in this study. A TBI model was established by inflicting fluid percussion injury (FPI) in the mouse cerebrum and cultured astrocytes. Vasogenic brain edema and BBB disruption were assessed based on brain water content and Evans blue (EB) extravasation into brain tissue, respectively. After FPI, brain water content and EB extravasation increased. Repeated intracerebroventricular administration of the specific TRPV4 antagonists HC-067047 and RN-1734 dose-dependently reduced brain water content and alleviated EB extravasation in FPI mice. Additionally, real-time PCR analysis indicated that administration of HC-067047 and RN-1734 reversed the FPI-induced increase in mRNA levels of endogenous causal factors for BBB disruption, including matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor-A (VEGF-A), and endothelin-1 (ET-1). In astrocytes, TRPV4 level was observed to be higher than that in brain microvascular endothelial cells. Treatment with HC-067047 and RN-1734 inhibited the increase in mRNA levels of MMP-9, VEGF-A, and ET-1 in cultured astrocytes subjected to in vitro FPI. These results suggest that pharmacological inhibition of TRPV4 is expected to be a promising therapeutic strategy for treating TBI-induced vasogenic edema.

INTRODUCTION

Brain edema is characterized by excessive brain water accumulation in the cerebral parenchyma, and can be classified as cytotoxic, osmotic, or vasogenic edema.¹⁾ Vasogenic edema results from excessive outflow of intravascular fluid into the cerebral parenchyma due to blood–brain barrier (BBB) disruption after brain injury, including cerebral ischemia and traumatic brain injury (TBI).^2,3) It is a potentially fatal condition that can induce irreversible neuronal dysfunction even if death is avoided; however, it has not been possible to develop any promising therapeutic drugs for alleviating vasogenic edema.

Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable non-selective cation channel that is sensitive to temperature and osmotic pressure. In the central nervous system, TRPV4 is broadly distributed in various brain cells, including neuronal, endothelial, and glial cells.^4–6) TRPV4 plays a key role in the maintenance of normal physiological conditions and processes, including the modulation of neuronal excitability and proliferation of brain endothelial cells.^4,7) On the other hand, TRPV4 is also known to be involved in various pathological processes, including neuronal death, inflammation, and brain edema after cerebral ischemia and TBI.^8–11) TRPV4 knockout reduced brain edema^12,13) and alleviated BBB disruption^13,14) in animal models of cerebral ischemia. In a mouse model of stab wound TBI, TRPV4 knockout reduced brain edema.¹⁰⁾ HC-067047, a specific TRPV4 antagonist, reduced brain edema and ameliorated BBB disruption after ischemic damage in experimental animals.¹⁵⁾ Administration of RN-1734, another specific TRPV4 antagonist, also attenuated TBI-induced increase in brain water content.⁹⁾ These results indicate that inhibition of TRPV4 may be beneficial in various pathological conditions after brain injury.

TBI-induced BBB disruption due to a direct physical impact is mediated by various vascular permeability accelerating factors. In the damaged brain, reactive astrocytes produce various vascular permeability accelerating factors, and astrocyte-derived these factors accelerated the BBB disruption.^16–18) We also confirmed that the expression of several vascular permeability accelerating factors, including matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor-A (VEGF-A), and endothelin-1 (ET-1), was increased in the mouse cerebrum after TBI due to fluid percussion injury (FPI), and these expressions were also observed in astrocytes.¹⁹⁾ Moreover, an MMP-9 inhibitor, a VEGF-neutralizing antibody, and ET receptor antagonists reduced brain edema and ameliorated BBB disruption in TBI mice.^19,20) These results imply that astrocyte is a producing cell for vascular permeability accelerating factors and inhibition of MMP-9, VEGF-A, and ET-1 can alleviate vasogenic edema.

In the present study, we investigated the effects of pharmacological TRPV4 antagonists on vasogenic edema and vascular permeability accelerating factors after TBI in the mouse cerebrum and cultured cells.

MATERIALS AND METHODS

Design of the Experimental Animal Study

Animal experiments were executed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023). All protocols were accepted by the Animal Experiment Committee of Osaka Ohtani University (Approval No. 1704).^19–21) Male ddY mice (7 weeks old, 32–37 g) (Japan SLC, Inc.; Shizuoka, Japan) were kept in groups of four or five per a cage (size: 225 × 338 × 140 mm) with a 12-hour light and dark cycle in the pathogen-free room. Temperature and humidity were controlled at 23 ± 2 °C and 55 ± 5%, respectively. Mice were divided into different experimental groups based on the pseudo randomization by shuffled cards described as either sham (vehicle or TRPV4 antagonists) or FPI (vehicle or TRPV4 antagonists) as previously described.^20,21) These cards were randomly applied for each experimental mouse. Experimental analysis was executed by individuals who did not know each group. The independent experiments were repeated twice or thrice.

Lateral FPI

FPI was inflicted on the dura of the mouse cerebrum by a device of fluid percussion (AmScien Instruments, model no. FP302) as previously described.¹⁹⁾ For maintaining anesthetic condition during the procedure, intraperitoneal administrations of medetomidine (0.3 mg/kg) (Wako, Cat. No. 135-17473), midazolam (4.0 mg/kg) (Wako, Cat. No. 135-13791), and butorphanol (5.0 mg/kg) (Wako, Cat. No. 021-19001) were executed. Mice were opened a 3-mm-diameter hole in the skull of the left hemisphere. A lure fitting (ISIS Co., Ltd.; Cat. No. VRS306) was put on the hole and fixed with glue gel (Henkel Japan Ltd., Cat. No. 43594). The fixed lure fitting was connected to a tube of FPI device after filled with saline. A hydraulic impact was delivered at 1.2–1.4 atmospheres. To minimize pain after FPI, mice were kept under anesthetic condition for 3 to 6 h. The mortality rate was approximately 8%.

TRPV4 Antagonist Administration

TRPV4 antagonists were administered into lateral ventricle.^19,21) A guide cannula (Eicom, Cat. No. 807100) was set to 0.1 mm posterior and 1.0 mm lateral to the bregma in the right hemisphere in three days before FPI. Intracerebroventricular (i.c.v.) administration of HC-067047 (Santa Cruz Biotechnology, Inc.; Cat. No. sc-361204), RN-1734 (Abcam, Cat. No. ab254522), or dimethyl sulfoxide (DMSO) (Nacalai Tesque, Cat. No. 13445-45) was performed through the cannula and executed twice a day (8 : 00 a.m. and 8 : 00 p.m.) from 3 h to 3 d after FPI. HC-067047 and RN-1734 were dissolved in DMSO.

Cultured Astrocyte

Mouse cerebrum astrocyte cultures were made from ddY mice (2-d old) as previously studies.^20,21) Cerebrums were collected, and then the meninges were stripped. Collected cerebrums were suspended with trypsin (Gibco, Cat. No. 15090-046) and was centrifuged at 181 × g for 5 min. The collected pellet was suspended in Eagle’s minimum essential medium (MEM) (Nissui Pharmaceutical Co., Ltd.; Cat. No. 05900). Prepared cells were seeded at 1 × 10⁴ cells/cm² in 75 cm² flasks (Falcon, Cat. No. 353135) with MEM supplemented 10% fetal bovine serum (Biowest, Cat. No. S1780-500) and maintained at 37 °C in 5% CO₂. After 10 to 12 d, the culture flasks were shaken at 250 rpm for several hours to remove protoplasmic cell layer small process-bearing cells. The monolayer cells were treated with trypsin and reseeded in 35-mm culture dishes (Thermo Scientific, Cat. No. 150318) and then incubated at 37 °C in 5% CO₂ for 5 to 7 d. Before treatment with TRPV4 antagonists, cultured astrocytes were maintained with non-serum MEM for 2 d. HC-067047 and RN-1734 were applied in non-serum MEM immediately before in vitro FPI, after which the cultured astrocytes were further incubated for 3 h.

bEnd.3 Cell

The bEnd.3 cell is a mouse brain vascular endothelial cell. The bEnd.3 cell line was purchased from the American Type Culture Collection (Manassas, VA, U.S.A.). Cells were seeded at 1 × 10⁴ cells/cm² in 75 cm² flasks with Dulbecco's modified Eagle's MEM (Nissui Pharmaceutical Co., Ltd.; Cat. No. 05919) supplemented 10% fetal bovine serum. The confluent cells were trypsinized and reseeded in 35-mm culture dishes, and then incubated at 37 °C in 5% CO₂ for 3 to 5 d.

In Vitro TBI Model

The in vitro TBI was established as previously described.^21,22) Dishes of cultured cells were set in a chamber of the cell trauma device (AmScien Instruments, model No. FP302). An upper half of the chamber was filled with saline and a tube of the chamber was connected to the FPI device. A hydraulic impact was delivered to culture dishes at 5 to 5.5 atmospheres.

Brain Water Content

Brain water content was evaluated as previously described.^19,20) Mice were euthanized by the carbon dioxide device (Natume, Cat. No. KN-750-2A) and brain coronal sections from 0 to 5 mm posterior to the bregma were made by a brain slicer (Muromachi, Tokyo, Japan). The prepared tissues were weighed as a wet weight and dried at 70 °C overnight; thereafter, dried brain tissues were weighed as a dry weight. Brain water content was evaluated by the determined formula. Brain water content (%) = (wet weight – dry weight) × 100/wet weight.

Determination of BBB Disruption

BBB disruption was determined based on assessing Evans blue (EB) extravasation as previously described.^19,21) Intravenous administration of saline containing 4% EB (Sigma-Aldrich, Cat. No. E2129-10G) was performed at 3 mL/kg. After 60 min of EB administration, 100 µL circulating blood was collected to determine the EB concentration in serum. Mice were perfused from the left ventricle with phosphate-buffered saline under anesthetic condition. After perfusion, coronal brain tissues from 0 to 5 mm posterior to the bregma were made by a brain slicer. The collected tissues were weighed and incubated with formamide (Wako, Cat. No. 065-00436) at 55 °C overnight. The EB content was evaluated by measure of absorbance at 655 nm. Brain EB extravasation was determined by the determined formula. EB extravasation (%) = Brain EB content (µg/g) × 100/serum EB content (µg/mL).

Quantitative RT-PCR

Mice were euthanized by the carbon dioxide device and brain coronal tissues from 0 to 5 mm posterior to the bregma were collected. RNA was extracted from the collected tissues by using the RNAiso Plus (TaKaRa Bio Inc., Cat. No. 9109) and first-strand cDNA was made from the extracted RNA. The mRNA levels were evaluated by quantitative RT-PCR. The collected reverse-transcription products were reacted with the SYBR Green Master Mix (Toyobo Co., Ltd.; Cat. No. QPK-212) in a CFX Connect™ thermal cycler (BioRad; Hercules, CA, U.S.A.). The mRNA levels of prepro-ET-1, MMP-9, and VEGF-A were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The used primer pairs were described below.

• Prepro-ET-1
  • Sense: 5′-TGTGTCTACTTCTGCCACCT-3′
  • Antisense: 5′-CACCAGCTGCTGATAGATAC-3′

• MMP-9
  • Sense: 5′-ACTCACACGACATCTTCCAG-3′
  • Antisense: 5′-AGAAGGAGCCCTAGTTCAAG-3′

• VEGF-A
  • Sense: 5′-CCTGGTGGACATCTTCCAGGAGTACC-3′
  • Antisense: 5′-GAAGCTCATCTCTCCTATGTGCTGGC-3′

• GAPDH:
  • Sense: 5′-ACCGACCCCTTCATT-3′
  • Antisense: 5′-TCCACGACATACTCAGCAC-3′

Immunoblotting of TRPV4

Brain coronal tissues and cultured cells were collected in the radio immunoprecipitation assay buffer (Nacalai Tesque, Cat. No. 16488-34) supplemented with a protease inhibitor cocktail (Nacalai Tesque, Cat. No. 04080-24) and phosphatase cocktail (Nacalai Tesque, Cat. No. 07574-61). The prepared lysate was centrifuged at 21400 × g for 10 min and protein concentration in the supernatant samples was evaluated by a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Cat. No. 23228). The prepared protein samples were applied to the 7.5% polyacrylamide gel for electrophoresis, and transferred to polyvinylidene difluoride membranes (Millipore, Cat. No. IPVH304F0). Transferred membranes were reacted with anti-TRPV4 antibody (1 : 2000; Cell Signaling, Cat. No. 65893S) and then reacted with peroxidase-conjugated antibody (1 : 4000; Invitrogen, Cat. No. 31466). To detect the proteins, a chemiluminescence kit (Chemi-Lumi One^® L, Nacalai Tesque, Cat. No. 07880-70) was used. Membranes were also reacted with antibody for β-actin (1 : 4000; Sigma-Aldrich, Cat. No. A2066). The intensities of the protein bands were determined by the ImageJ (https://imagej.nih.gov/ij/).

Statistical Analysis

Statistical analysis was determined by the Ekuseru-Toukei 2015 (ver. 1.00; Social Survey Research Information Co., Ltd.). Data are represented by the mean ± standard error of the mean and were determined using one-way ANOVA followed by a post-hoc test. The p-values <0.05 were determined as a statistically significant.

RESULTS

Increased TRPV4 in the Mouse Cerebrum and Cultured Astrocytes after TBI

Western blot analysis showed that expression of TRPV4 was observed in the mouse cerebrum and increased in 1 to 5 d after FPI (Fig. 1A). Expressions of TRPV4 were also observed in cultured astrocytes and bEnd.3 cells, and the level of TRPV4 in cultured astrocytes was higher than that in bEnd.3 cells (Fig. 1B). Additionally, expressions of TRPV4 were increased in cultured astrocytes but not bEnd.3 cells in 1 to 6 h after FPI (Fig. 1C).

Fig. 1. Expressions of TRPV4 in the Mouse Cerebrum and Cultured Cells

(A) Expression of TRPV4 in the mouse cerebrum. In 1 to 5 d after FPI, brain tissues were collected. Results are shown as the mean ±standard error of the mean (S.E.M.), n = 4 mice. ** p < 0.01 vs. sham; one-way ANOVA and Dunnett’s test. (B) Expressions of TRPV4 in cultured astrocytes and bEnd.3 cells. Results are presented as the mean ± S.E.M. of data from four different protein samples. ** p < 0.01 vs. bEnd.3 cell; Student's t-test. (C) Expressions of TRPV4 in cultured cells after TBI. In 1 to 6 h after FPI, cultured cells were collected. Results are presented as the mean ± S.E.M. of data from four different protein samples. * p < 0.05, ** p < 0.01 vs. control; one-way ANOVA and Dunnett’s test.

TRPV4 Antagonists Reduced TBI-Induced Brain Water Content

Brain edema results from an increase in the brain water content. We previously demonstrated that brain water content increased in the mouse cerebrum after FPI.^19,20) Similarly, brain water content was found to be significantly increased in the injured cerebrum 3 d after FPI compared to that in the sham groups (Figs. 2A, B). To investigate the involvement of TRPV4 in TBI-induced increase in brain water content, the effects of the two pharmacological TRPV4 antagonists were examined. Repeated i.c.v. administration of HC-067047 (2, 20, or 200 nmol per day), a specific TRPV4 antagonist, from 3 h to 3 d after FPI reduced the FPI-induced increase in brain water content significantly (Fig. 2A). Similarly, i.c.v. administration of RN-1734 (2, 20, or 200 nmol per day), another specific TRPV4 antagonist, also significantly reduced the FPI-induced increase in brain water content (Fig. 2B). Administration of HC-067047 or RN-1734 did not affect the brain water content in the non-injured cerebrum (Figs. 2A, B).

Fig. 2. Effects of the TRPV4 Antagonists on TBI-Induced Increase in Brain Water Content

(A) Effects of HC-067047. Repeated i.c.v. administrations of HC-067047 were performed at the indicated dosages from 3 h to 3 d after FPI. At 3 d after FPI, the effects of HC-067047 were investigated by determining the brain water content. Results are shown as the mean ±S.E.M., n = 6 mice. ** p < 0.01 vs. sham (vehicle), ^## p < 0.01 vs. FPI (vehicle); one-way ANOVA and Tukey’s test. (B) Effects of RN-1734. Repeated i.c.v. administrations of RN-1734 were performed at the indicated dosages from 3 h to 3 d after FPI. At 3 d after FPI, the effects of RN-1734 were investigated by determining the brain water content. Results are presented as the mean ± S.E.M., n = 6 mice. ** p < 0.01 vs. sham (vehicle), ^## p < 0.01 vs. FPI (vehicle); one-way ANOVA and Tukey’s test.

TRPV4 Antagonists Alleviated TBI-Induced BBB Disruption

As vasogenic edema is caused by BBB disruption, we investigated the effects of the TRPV4 antagonists on BBB disruption assessed based on EB extravasation into the cerebral parenchyma. We previously showed that EB extravasation was prominently accelerated in the injured cerebrum after FPI.^19,21) In this study, we confirmed that EB extravasation increased remarkably in the injured cerebrum 3 d after FPI (Figs. 3A–C). Repeated i.c.v. administration of HC-067047 (2, 20, or 200 nmol per day) from 3 h to 3 d after FPI significantly reduced the FPI-induced EB extravasation (Figs. 3A, B). Similarly, i.c.v. administration of RN-1734 (2, 20, or 200 nmol per day) also alleviated FPI-induced EB extravasation (Figs. 3A, C). Administration of HC-067047 or RN-1734 did not affect EB extravasation in the non-injured cerebrum (Figs. 3B, C).

Fig. 3. Effects of the TRPV4 Antagonists on TBI-Induced BBB Disruption

(A) Representative images of Evans blue extravasation into brain tissue after FPI. Vehicle, HC-067047 (20 nmol per day), or RN-1734 (20 nmol per day) was administrated. (B) Effects of HC-067047. Repeated i.c.v. administrations of HC-067047 were performed at the indicated dosages from 3 h to 3 d after FPI. At 3 d after FPI, the effects of HC-067047 were investigated by evaluating the Evans blue extravasation into brain tissue. Results are shown as the mean ± S.E.M., n = 6 mice. ** p < 0.01 vs. sham (vehicle), ^# p < 0.05, ^## p < 0.01 vs. FPI (vehicle); one-way ANOVA and Tukey’s test. (C) Effects of RN-1734. Repeated i.c.v. administrations of RN-1734 were performed at the indicated dosages from 3 h to 3 d after FPI. At 3 d after FPI, the effects of RN-1734 were investigated by evaluating the Evans blue extravasation into brain tissue. Results are shown as the mean ± S.E.M., n = 6 mice. ** p < 0.01 vs. sham (vehicle), ^# p < 0.05, ^## p < 0.01 vs. FPI (vehicle); one-way ANOVA and Tukey’s test.

TRPV4 Antagonists Decreased the TBI-Induced Increase in mRNA Levels of Vascular Permeability Accelerating Factors in the Mouse Cerebrum

Vascular permeability accelerating factors such as MMP-9, VEGF-A, and ET-1, are known to cause BBB disruption after brain damage.^1,23) We previously showed that an MMP-9 inhibitor, a VEGF neutralizing antibody, and ET receptor antagonists reduced brain edema and alleviated BBB disruption in TBI mice.^19,20) Thus, we focused on MMP-9, VEGF-A, and ET-1 to investigate the mechanism of TRPV4 antagonist action in alleviating BBB disruption. Three days after FPI, the mRNA levels of MMP-9, VEGF-A, and preproET-1 were found to be increased in the injured cerebrum (Fig. 4). Repeated i.c.v. administration of HC-067047 (20 nmol per day) or RN-1734 (20 nmol per day) from 3 h to 3 d after FPI significantly decreased the FPI-induced increase in MMP-9, VEGF-A, and preproET-1 mRNA levels (Fig. 4). Administration of HC-067047 or RN-1734 did not affect the corresponding mRNA levels in the non-injured cerebrum (Fig. 4).

Fig. 4. Effects of the TRPV4 Antagonists on TBI-Induced Increase in mRNA Levels of MMP-9, VEGF-A, and ET-1 in the Mouse Cerebrum

Repeated i.c.v. administrations of HC-067047 (20 nmol per day) or RN-1734 (20 nmol per day) were performed from 3 h to 3 d after FPI. At 3 d after FPI, the effects of HC-067047 on the mRNA levels of MMP-9, VEGF-A, and prepro ET-1 (PPET-1) were investigated by real-time PCR analysis. Results are shown as the mean ± S.E.M., n = 8 mice. ** p < 0.01 vs. sham (vehicle), ^# p < 0.05, ^## p < 0.01 vs. FPI (vehicle); one-way ANOVA and Tukey’s test.

TRPV4 Antagonists Decreased the Increase in mRNA Levels of Vascular Permeability Accelerating Factors in Cultured Astrocytes Subjected to in Vitro TBI

Several studies have shown that MMP-9, VEGF-A, and ET-1 are expressed in astrocytes and brain microvascular endothelial cells (BMECs).^{24–26,19,20)} Thus, the effects of TRPV4 antagonists on MMP-9, VEGF-A, and ET-1 were investigated in astrocytes and bEnd.3 cells subjected to TBI. The mRNA levels of MMP-9, VEGF-A, and preproET-1 were significantly increased in cultured astrocytes subjected to in vitro TBI (Fig. 5A). However, they were not significantly increased in bEnd.3 cells subjected to in vitro TBI (Fig. 5B). When cultured astrocytes subjected to in vitro TBI were incubated with HC-067047 (10 or 100 µM) for 3 h, the mRNA levels of MMP-9, VEGF-A, and preproET-1 were reduced (Fig. 5C). Similarly, treatment with RN-1734 (10 or 100 µM) also reduced their levels in cultured astrocytes subjected to in vitro TBI (Fig. 5D).

Fig. 5. Effects of the TRPV4 Antagonists on mRNA Levels of MMP-9, VEGF-A, and ET-1 in Astrocytes Subjected to in Vitro TBI

(A) Analyses of MMP-9, VEGF-A, and prepro ET-1 (PPET-1) mRNA levels in astrocytes. Results are shown as the mean ± S.E.M. of data from eight different mRNA samples. * p < 0.05, ** p < 0.01 vs. control; one-way ANOVA and Dunnett’s test. (B) Analyses of MMP-9, VEGF-A, and PPET-1 mRNA levels in bEnd.3 cells. Results are shown as the mean ± S.E.M. of data from eight different mRNA samples. (C) Effects of HC-067047 on the mRNA levels of MMP-9, VEGF-A, and PPET-1 in astrocytes. Cell were treated with HC-067047 at the indicated concentrations for 3 h, and the effects were investigated by real-time PCR analysis. Results are shown as the mean ± S.E.M. of data from eight different mRNA samples. ** p < 0.01 vs. control, ^# p < 0.05, ^## p < 0.01 vs. FPI (vehicle); one-way ANOVA and Tukey’s test. (D) Effects of RN-1734 on mRNA levels of MMP-9, VEGF-A, and PPET-1 in astrocytes. Cells were RN-1734 at the indicated concentrations for 3 h, and the effects were investigated by real-time PCR analysis. Results are shown as the mean ± S.E.M. of data from eight different mRNA preparations. ** p < 0.01 vs. control, ^# p < 0.05 vs. FPI (vehicle); one-way ANOVA and Tukey’s test.

DISCUSSION

Vasogenic edema is a potentially fatal condition that can cause unexpected death or irreversible neuronal dysfunction resulting from the BBB disruption after brain injury; however, potential therapeutic drugs have not yet been developed. In the present study, TRPV4 antagonists reduced the brain water content and BBB disruption in TBI model mice. Thus, TRPV4 antagonists could be candidate therapeutic drugs for vasogenic edema. TBI-induced BBB disruption is caused not only by direct physical impact, but also by various endogenous causal factors. MMP-9, VEGF-A, and ET-1 are well-known endogenous causal factors of BBB disruption, and increased MMP-9, VEGF-A, and ET-1 expression is observed in TBI animal models as well as patients with TBI.^27–31) We confirmed that the mRNA levels of these factors were also increased in our mouse model of FPI and also showed that TRPV4 antagonists reversed this increase. Several studies have suggested that MMP-9 inhibition can reduce brain edema and alleviate BBB disruption in animal models of TBI.^19,32) Additionally, a VEGF-neutralizing antibody attenuated brain edema and ameliorated BBB disruption in mice with TBI.¹⁹⁾ BQ788, a selective ET_B receptor antagonist, and bosentan, a non-selective ET receptor antagonist, also alleviated TBI-induced vasogenic edema in mice.^19–21) These observations implied that the decrease in MMP-9, VEGF-A, and ET-1 expression may be a mechanism by which TRPV4 antagonists alleviate vasogenic edema.

As we previously found that the expression of MMP-9, VEGF-A, and ET-1 was observed in astrocytes and BMECs after TBI,^19,20) we focused on these cells in this study as well. Interestingly, the level of TRPV4 was higher in cultured astrocytes than in bEnd.3 cells, and the level of TRPV4 was increased in cultured astrocytes but not bEnd.3 cells after in vitro TBI. Moreover, MMP-9, VEGF-A, and ET-1 increased in astrocytes but not in bEnd.3 cells after in vitro TBI. TRPV4 antagonists reduced the increased MMP-9, VEGF-A, and ET-1 in astrocytes after in vitro TBI. TRPV4 is a calcium permeable channel that accelerates intracellular calcium signaling, and several studies have indicated that calcium signaling is involved in the expression of MMP-9, VEGF-A, and ET-1.^33–35) Therefore, increased TRPV4 expression may be responsible for the increase in MMP-9, VEGF-A, and ET-1 in astrocytes after TBI.

Knockdown or pharmacological inhibition of TRPV4 is known to alleviate neuronal damage and brain edema in several animal models of cerebral ischemia.^12,13,15) Similarly, the beneficial effects of TRPV4 knockdown or TRPV4 antagonist administration on brain edema were also confirmed in animal models of TBI. In mice subjected to TBI in the form of a stab wound injury, TRPV4 knockout reduced brain edema.¹⁰⁾ Administration of a TRPV4 antagonist also attenuated brain edema in a TBI rat model.⁹⁾ Additionally, Wang et al. have suggested that TRPV4 could be a target therapeutic candidate for the treatment of TBI.³⁶⁾ In this study, we confirmed that TBI-induced vasogenic edema was alleviated by the pharmacological TRPV4 antagonists HC-067047 and RN-1734. These results suggest that inhibition of TRPV4 function could be beneficial for treating vasogenic edema as well as other forms of brain damage. Although we administered TRPV4 antagonists intracerebroventricularly for TBI model mice in this study, oral or systemic administrations are appropriate for clinical use. Liao et al. indicated that intravenous administration of HC-067047 prevented shockwave-induced BBB disruption in rats.³⁷⁾ Additionally, Lu et al. suggested that intraperitoneal administration of RN-1734 reduced brain edema after TBI by weight-drop device in rats.⁹⁾ Thus, systemic administration of TRPV4 antagonists may reduce TBI-induced vasogenic edema, and it should be examined in future. Nevertheless, Chen et al. have indicated that activation of TRPV4 promotes neuronal recovery after ischemic damage via angiogenesis and neurogenesis.³⁸⁾ Although more detailed studies on the beneficial or harmful effects of TRPV4 inhibition on brain injury should be conducted, pharmacological inhibition of TRPV4 may be a novel therapeutic strategy for vasogenic edema in the future.

Acknowledgments

This work was supported by a Grant-in-Aid for Young Scientists (Grant No. 20K16016) from the Japan Society for the Promotion of Science.

Conflict of Interest

The authors declare no conflict of interest.

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