Journal: Frontiers in Molecular Neuroscience

Article Title: TRPV4 Blockade Preserves the Blood–Brain Barrier by Inhibiting Stress Fiber Formation in a Rat Model of Intracerebral Hemorrhage

doi: 10.3389/fnmol.2018.00097

Figure Lengend Snippet: Time course and spatial expression of TRPV4 after ICH. (A) Representative bands and quantitative analyses of TRPV4 expression around the lesion sites are shown. Relative densities of each protein were normalized to the sham group. (B) Representative images of immunofluorescence staining for TRPV4 (red) in the perihematomal area (white ∗ ) 24 h after ICH are shown. Scale bar: 200 μm. (C) Representative images of immunofluorescence staining for TRPV4, vWF (green) and GFAP (green) in the perihematomal area indicated by the white arrow 24 h after ICH are shown. Scale bar: 20 μm, n = 6 rats per group. Data are presented as the means ± standard errors of the means. ∗ p < 0.05; ∗∗ p < 0.01 compared with the sham group.

Article Snippet: Three unique 27-mer TRPV4 siRNA duplexes (OriGene, Rockville, MD, United States) were mixed to enhance the gene silencing efficacy.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: TRPV4 Blockade Preserves the Blood–Brain Barrier by Inhibiting Stress Fiber Formation in a Rat Model of Intracerebral Hemorrhage

doi: 10.3389/fnmol.2018.00097

Figure Lengend Snippet: The effects of TRPV4 siRNA on neurological deficits and BBB integrity after ICH. (A) Representative bands and quantitative analysis of the inhibitory effect of the TRPV4 siRNA are shown. Relative densities of each protein were normalized to the sham group, n = 5 rats per group. (B) A modified Garcia test and (C) corner Turn test were performed on the sham, vehicle, scrambled siRNA and TRPV4 siRNA groups 24 h after the operation. (D) The Evans blue extravasation evaluation was performed on the sham, vehicle, scrambled siRNA, and TRPV4 siRNA groups 24 h after the operation, n = 6 rats per group. Data are presented as the means ± standard errors of the means. ∗∗ p < 0.01 and ∗ p < 0.05 compared with the sham group; # p < 0.05 compared with the vehicle group; @ p < 0.05 compared with the scrambled siRNA group.

Article Snippet: Three unique 27-mer TRPV4 siRNA duplexes (OriGene, Rockville, MD, United States) were mixed to enhance the gene silencing efficacy.

Techniques: Modification

Journal: Frontiers in Molecular Neuroscience

Article Title: TRPV4 Blockade Preserves the Blood–Brain Barrier by Inhibiting Stress Fiber Formation in a Rat Model of Intracerebral Hemorrhage

doi: 10.3389/fnmol.2018.00097

Figure Lengend Snippet: The effects of TRPV4 siRNA on the expression of adherens/tight junction proteins and activation of the PKCα/RhoA/MLC2 pathway after ICH. (A) Representative bands and quantitative analysis of the expression levels of VE-Cadherin, Occludin, and Claudin-5 in the sham, vehicle, scramble siRNA, and TRPV4 siRNA groups 24 h after the operation are shown. (B) Representative bands and quantitative analysis of the activation of the PKCα/RhoA/MLC2 pathway in the sham, vehicle, scramble siRNA, and TRPV4 siRNA groups 24 h after the operation are shown, n = 5 rats per group. Data are presented as the means ± standard errors of the means. ∗∗ p < 0.01 compared with the sham group; # p < 0.05 compared with the vehicle group; @ p < 0.05 compared with the scrambled siRNA group.

Article Snippet: Three unique 27-mer TRPV4 siRNA duplexes (OriGene, Rockville, MD, United States) were mixed to enhance the gene silencing efficacy.

Techniques: Expressing, Activation Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: TRPV4 Blockade Preserves the Blood–Brain Barrier by Inhibiting Stress Fiber Formation in a Rat Model of Intracerebral Hemorrhage

doi: 10.3389/fnmol.2018.00097

Figure Lengend Snippet: The effects of HC-067047, TRPV4 siRNA, and GSK1016790A on the formation of F-actin stress fibers after ICH. (A) Representative images of phalloidin fluorescence around the lesion sites in the sham, vehicle, HC-067047, TRPV4 siRNA, and GSK1016790A groups captured 24 h after the operation. Scale bar: 50 μm. (B) Representative bands and quantitative analysis of the expression levels of polymerized F-actin, soluble G-actin, and total β-actin in the sham, vehicle, HC-067047, TRPV4 siRNA, and GSK1016790A groups 24 h after the operation are shown. The ratio of F-actin to G-actin was quantified as a measure of stress fiber formation, n = 5 rats per group. Data are presented as the means ± standard errors of the means. ∗∗ p < 0.01 compared with the sham group; ∗ p < 0.05 compared with the sham group; # p < 0.05 compared with the vehicle group; @ p < 0.05 compared with the scrambled siRNA.

Article Snippet: Three unique 27-mer TRPV4 siRNA duplexes (OriGene, Rockville, MD, United States) were mixed to enhance the gene silencing efficacy.

Techniques: Fluorescence, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: TRPV4 Blockade Preserves the Blood–Brain Barrier by Inhibiting Stress Fiber Formation in a Rat Model of Intracerebral Hemorrhage

doi: 10.3389/fnmol.2018.00097

Figure Lengend Snippet: Schematic of the effect of TRPV4 on BBB integrity after ICH.

Article Snippet: Three unique 27-mer TRPV4 siRNA duplexes (OriGene, Rockville, MD, United States) were mixed to enhance the gene silencing efficacy.

Techniques:

Journal: The Journal of allergy and clinical immunology

Article Title: TRPV4-expressing Macrophages and Keratinocytes Contribute Differentially to Allergic and Non-allergic Chronic Itch

doi: 10.1016/j.jaci.2017.05.051

Figure Lengend Snippet: TRPV4 expression is elevated in skin biopsies of CIP patients and TRPV4 function is required for generating mouse models of both allergic and non-allergic chronic itch. A, The bar charts show the averaged itch numerical rating scale (NRS) scores and mRNA transcripts for TRPV4 and TRPV3 in CIP patients and health controls.. *p<0.05, **p<0.01, Student’s t test; n=8. n.s. not significant versus control group. B, Spontaneous scratches in Trpv4+/+ and Trpv4−/− mice after 7 days of AEW treatment. ***p<0.001, Student’s t test; n=7.. C, Spontaneous scratches in Trpv4+/+ mice after 7 days of AEW treatment were reduced by HC067 (20 mg/kg, i.p or topical application) compared with vehicle. *p<0.05, Student’s t test; n=8–9. D, Spontaneous scratches after the treatment with the 1:1 mixture of acetone and ether followed by 0.9% NaCl, 0.45% NaCl, or distilled water, respectively. **p<0.01, ANOVA; n=5–7. E, Spontaneous scratches in Trpv4+/+ and Trpv4−/− mice after SADBE treatment. *p<0.05, Student’s t test; n=6. F, SADBE-induced spontaneous scratches in Trpv4+/+ mice after treatment with HC067 (20 mg/kg, i.p or topical application) or vehicle. *p<0.05, Student’s t test; n=5–6.

Article Snippet: Adult male and female C57BL/6J mice (Jackson Laboratories), Trpv4 eGFP (Mutant Mouse Regional Resource Centers, MMRRC), Trpv4 − / − , 11 Kit W-sh/W-sh (Jackson Laboratories), Htr7 − / − (Jackson Laboratories), Htr2a − / − (a kind gift from Dr Jay Gingrich at the Columbia University) mice were used for the study.

Techniques: Expressing

Journal: The Journal of allergy and clinical immunology

Article Title: TRPV4-expressing Macrophages and Keratinocytes Contribute Differentially to Allergic and Non-allergic Chronic Itch

doi: 10.1016/j.jaci.2017.05.051

Figure Lengend Snippet: TRPV4 is functionally expressed by mouse skin-resident cells. A, Double labeling reveals the co-localization of TRPV4-eGFP with K14 (left panel) and F4/80 (right panel) in skin sections from the Trpv4eGFP mice. Bar=50 μm. HF: hair follicle. Inset shows the magnification of boxed area. B, RT-PCR shows the correlation between TRPV4 and GFP in sorted GFP+ and GFP− ear skin single-cell suspensions. C, Flow cytometry analysis illustrates that TRPV4-eGFP is expressed in CD11b-positive skin-resident cells. D, Representative traces showing the GSK101-elicited large [Ca2+]i responses in freshly dissociated ear skin single-cell suspensions from Trpv4+/+ (left) but not Trpv4−/− (middle) mice. Arrows indicate the time points of GSK101 applications. Ionomycin (Ion) was used as positive control. Summarized data from in the right showing the percentages of GSK101-responsive skin cells isolated from the Trpv4+/+ and Trpv4−/− mice. ***p<0.001, Student’s t test; n=5–8. E, Time course (left) and representative current-voltage curves (middle) elicited by voltage ramps in the absence or presence of 0.3 μM GSK101 in the TRPV4-eGFP+ myeloid cells from the ear skin single-cell suspensions. HC067 at 5 μM markedly inhibited the GSK101-activated currents. Bar charts in the right illustrate summarized data. ***p<0.001, ANOVA; n=5.

Article Snippet: Adult male and female C57BL/6J mice (Jackson Laboratories), Trpv4 eGFP (Mutant Mouse Regional Resource Centers, MMRRC), Trpv4 − / − , 11 Kit W-sh/W-sh (Jackson Laboratories), Htr7 − / − (Jackson Laboratories), Htr2a − / − (a kind gift from Dr Jay Gingrich at the Columbia University) mice were used for the study.

Techniques: Labeling, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Positive Control, Isolation

Journal: The Journal of allergy and clinical immunology

Article Title: TRPV4-expressing Macrophages and Keratinocytes Contribute Differentially to Allergic and Non-allergic Chronic Itch

doi: 10.1016/j.jaci.2017.05.051

Figure Lengend Snippet: TRPV4 channels expressed by macrophages and keratinocytes contribute differentially to allergic and non-allergic chronic itch. A, Representative images showing the TRPV4-eGFP+ cells in the skin of Trpv4eGFP mice treated with vehicle control and AEW. B, The epidermal thickness was significantly increased by AEW and SADBE treatments compared with their respective vehicle controls. ***p<0.001, Student’s t test; n=6–8. C, The number of TRPV4-eGFP+ dermal macrophages increased significantly following AEW or SADBE treatment. ***p<0.001, Student’s t-test; n=6–8. D, Relative TRPV4 mRNA in the skin of AEW- or SADBE-treated mice, *p<0.05, Student’s f test; n=6–8. E, Spontaneous scratching in the K14-Cre+ and K14-Cre− mice after AEW treatment, *p<0.05, Student’s f test; n=5. F, Spontaneous scratching in the Cx3cr1-Cre+ and Cx3cr1-Cre− mice after AEW treatment. Student’s t test; n=5. n.s. not significant. G, Spontaneous scratching in the K14-Cre+ and K14-Cre− mice after SADBE treatment. Student’s t test; n=8–9. n.s. not significant. H, Spontaneous scratching in the Cx3cr1-Cre+ and Cx3cr1-Cre− mice after SADBE treatment. **p<0.01, Student’s t test; n=9.

Article Snippet: Adult male and female C57BL/6J mice (Jackson Laboratories), Trpv4 eGFP (Mutant Mouse Regional Resource Centers, MMRRC), Trpv4 − / − , 11 Kit W-sh/W-sh (Jackson Laboratories), Htr7 − / − (Jackson Laboratories), Htr2a − / − (a kind gift from Dr Jay Gingrich at the Columbia University) mice were used for the study.

Techniques:

Journal: The Journal of allergy and clinical immunology

Article Title: TRPV4-expressing Macrophages and Keratinocytes Contribute Differentially to Allergic and Non-allergic Chronic Itch

doi: 10.1016/j.jaci.2017.05.051

Figure Lengend Snippet: 5-HT signaling is required for TRPV4-dependent chronic itch. A, Percentages of DRG neurons responding to skin superfusates from normal Trpv4+/+, AEW Trpv4+/+, and AEW Trpv4−/− mice. ***p<0.001, ANOVA. B, Percentages of 5-HT-responsive DRG neurons responding to the AEW skin superfusates from Trpv4+/+ and Trpv4−/− mice. ***p<0.001, Student’s t test. C, Schematic drawing of the 5-HT synthesis pathway. D and E, Spontaneous itching in AEW- (Fig 4, D) and SADBE- (Fig 4, E) treated mice with pCPA or pCPA+5-HTP. *p<0.05, ***p<0.001, ANOVA; n=8–10. F, Spontaneous scratching in the Htr2a+/+ and Htr2a−/− mice after SADBE treatment. *p<0.05, Student’s t test; n=5. G, Spontaneous scratching in mice treated with vehicle, Ketanserin, or Htr7 antagonist SB269970 (SB269) after SADBE treatment. *p<0.05, ANOVA; n=6. H, Spontaneous scratching in Htr7+/+ and Htr7−/− mice after SADBE treatment. Student’s t test; n=6. I, Spontaneous scratching in Htr2a+/+ and Htr2a−/− mice after AEW treatment. Student’s t test; n=9. J, Spontaneous scratching after AEW treatment in mice treated with vehicle, Htr7 antagonist SB269970 (SB269), or Ketanserin. **p<0.01, ANOVA; n=6–7. K, Spontaneous scratching after AEW treatment in the Htr7+/+ and Htr7−/− mice. ***p<0.001, Student’s t test; n=10–11. n.s. not significant versus vehicle group.

Article Snippet: Adult male and female C57BL/6J mice (Jackson Laboratories), Trpv4 eGFP (Mutant Mouse Regional Resource Centers, MMRRC), Trpv4 − / − , 11 Kit W-sh/W-sh (Jackson Laboratories), Htr7 − / − (Jackson Laboratories), Htr2a − / − (a kind gift from Dr Jay Gingrich at the Columbia University) mice were used for the study.

Techniques:

Journal: The Journal of allergy and clinical immunology

Article Title: TRPV4-expressing Macrophages and Keratinocytes Contribute Differentially to Allergic and Non-allergic Chronic Itch

doi: 10.1016/j.jaci.2017.05.051

Figure Lengend Snippet: Platelets but not mast cells are required for TRPV4-dependent chronic itch. A, Immunofluorescent staining shows that TRPV4-eGFP was not colocalized with streptavidin, a mast cell marker. Bar=50 μm. B and C, Spontaneous scratching induced by AEW (Fig 5, B) or SADBE (Fig 5, C) treatment was not significantly affected in the sash mice. Student’s t test; n=6 −7. n.s. not significant versus wt control group. D, Platelet count in Pf4-Cre mice and Pf4-Cre+ mice 6 days after the DTX treatment. **p<0.01, Student’s t test; n=7–8. E and F, Spontaneous scratching responses induced by AEW (Fig 5, E) or SADBE (Fig 5, F) in the Pf4-Cre− mice (n=5 for AEW and SADBE) mice and the Pf4-Cre+ mice (n=6 for AEW and SADBE). *p<0.05, Student’s t test. G and H, Spontaneous scratching responses induced by AEW (Fig 5, G) or SADBE (Fig 5, H) in the absence or presence of eptifibatide or clopidogrel. *p<0.05, ANOVA; n=6.

Article Snippet: Adult male and female C57BL/6J mice (Jackson Laboratories), Trpv4 eGFP (Mutant Mouse Regional Resource Centers, MMRRC), Trpv4 − / − , 11 Kit W-sh/W-sh (Jackson Laboratories), Htr7 − / − (Jackson Laboratories), Htr2a − / − (a kind gift from Dr Jay Gingrich at the Columbia University) mice were used for the study.

Techniques: Staining, Marker

Journal: Experimental Biology and Medicine

Article Title: Roles of TRPV4 and piezo channels in stretch-evoked Ca 2+ response in chondrocytes

doi: 10.1177/1535370219892601

Figure Lengend Snippet: Primers for the target gene.

Article Snippet: siRNAs against mouse TRPV4, Piezo1, Piezo2, and an siRNA negative control (NC) were designed and chemically synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China).

Techniques: Sequencing

Journal: Experimental Biology and Medicine

Article Title: Roles of TRPV4 and piezo channels in stretch-evoked Ca 2+ response in chondrocytes

doi: 10.1177/1535370219892601

Figure Lengend Snippet: Effect of CTS on TRPV4/Piezo1/Piezo2 protein levels in chondrocytes. (a) Expression of TRPV4/Piezo1/Piezo2 channels in articular chondrocytes following exposure to CTS for 8 h. The fold change in the expression of (b) TRPV4/(c) Piezo1/(d) Piezo2 channels based on densitometric analysis of Western blot using Image-Pro Plus by comparing to the housekeeping protein α-tubulin. Data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05, stretched vs. non-stretched control.

Article Snippet: siRNAs against mouse TRPV4, Piezo1, Piezo2, and an siRNA negative control (NC) were designed and chemically synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China).

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: Experimental Biology and Medicine

Article Title: Roles of TRPV4 and piezo channels in stretch-evoked Ca 2+ response in chondrocytes

doi: 10.1177/1535370219892601

Figure Lengend Snippet: Quantitative analysis of (a) TRPV4/(b) Piezo1/(c) Piezo2 mRNA levels by real-time PCR in untreated cells (naive) and cells treated with control siRNA or TRPV4/Piezo1/Piezo2 siRNA, respectively. Representative Western blot image for (d) TRPV4/(e) Piezo1/(f) Piezo2 channel protein expression. Quantitative analysis based on densitometry using Image-Pro Plus by comparing to the housekeeping protein α-tubulin showing the specific knockdown of the expression of (g) TRPV4/(h) Piezo1/(i) Piezo2. Data are presented as percentage of naive cells and the mean ± standard deviation of three independent experiments. *P < 0.05, TRPV4, Piezo1, or Piezo2 siRNA vs. control siRNA.

Article Snippet: siRNAs against mouse TRPV4, Piezo1, Piezo2, and an siRNA negative control (NC) were designed and chemically synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China).

Techniques: Real-time Polymerase Chain Reaction, Control, Western Blot, Expressing, Knockdown, Standard Deviation

Journal: Experimental Biology and Medicine

Article Title: Roles of TRPV4 and piezo channels in stretch-evoked Ca 2+ response in chondrocytes

doi: 10.1177/1535370219892601

Figure Lengend Snippet: [Ca2+]i oscillation evoked by CTS stimulation at different strain magnitudes of (a) 3%, (b) 8%, (c) 13%, or (d) 18% in control siRNA-treated chondrocytes (control), TRPV4-KD cells (TRPV4-KD), Piezo1-KD cells (Piezo1-KD), and Piezo2-KD cells (Piezo2-KD). TRPV4: transient receptor potential vanilloid 4; CTS: cyclic tensile strains; KD: knockdown. (A color version of this figure is available in the online journal.)

Article Snippet: siRNAs against mouse TRPV4, Piezo1, Piezo2, and an siRNA negative control (NC) were designed and chemically synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China).

Techniques: Control, Knockdown

Journal: Experimental Biology and Medicine

Article Title: Roles of TRPV4 and piezo channels in stretch-evoked Ca 2+ response in chondrocytes

doi: 10.1177/1535370219892601

Figure Lengend Snippet: [Ca 2+ ]i oscillation for Control, TRPV4-KD, Piezo1-KD, and Piezo2-KD stimulated with 3% CTS stimulation (mean ± standard deviation).

Article Snippet: siRNAs against mouse TRPV4, Piezo1, Piezo2, and an siRNA negative control (NC) were designed and chemically synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China).

Techniques: Control

Journal: Experimental Biology and Medicine

Article Title: Roles of TRPV4 and piezo channels in stretch-evoked Ca 2+ response in chondrocytes

doi: 10.1177/1535370219892601

Figure Lengend Snippet: [Ca 2+ ]i oscillation for control, TRPV4-KD, Piezo1-KD, and Piezo2-KD stimulated with 8% CTS stimulation (mean ± standard deviation).

Article Snippet: siRNAs against mouse TRPV4, Piezo1, Piezo2, and an siRNA negative control (NC) were designed and chemically synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China).

Techniques: Control

Journal: Experimental Biology and Medicine

Article Title: Roles of TRPV4 and piezo channels in stretch-evoked Ca 2+ response in chondrocytes

doi: 10.1177/1535370219892601

Figure Lengend Snippet: [Ca 2+ ] i oscillation for control, TRPV4-KD, Piezo1-KD, and Piezo2-KD stimulated with 13% CTS stimulation (mean ± standard deviation).

Article Snippet: siRNAs against mouse TRPV4, Piezo1, Piezo2, and an siRNA negative control (NC) were designed and chemically synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China).

Techniques: Control

Journal: Experimental Biology and Medicine

Article Title: Roles of TRPV4 and piezo channels in stretch-evoked Ca 2+ response in chondrocytes

doi: 10.1177/1535370219892601

Figure Lengend Snippet: [Ca 2+ ] i oscillation for control, TRPV4-KD, Piezo1-KD, and Piezo2-KD stimulated with 18% CTS stimulation (mean ± standard deviation).

Article Snippet: siRNAs against mouse TRPV4, Piezo1, Piezo2, and an siRNA negative control (NC) were designed and chemically synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China).

Techniques: Control

Journal: Fluids and Barriers of the CNS

Article Title: Lysophosphatidic acid as a CSF lipid in posthemorrhagic hydrocephalus that drives CSF accumulation via TRPV4-induced hyperactivation of NKCC1

doi: 10.1186/s12987-022-00361-9

Figure Lengend Snippet: LPA is a novel endogenous agonist of luminal membraneous TRPV4. A Representative current traces obtained from uninjected or TRPV4-expressing oocytes during a 200 ms step protocol (upper panel, control solution; lower panel, LPA-containing solution). B Averaged I/V curves from TRPV4–expressing oocytes in control solution, n = 9 (black), during application of LPA without, n = 8 (orange) or with TRPV-4 inhibition, n = 8 (+RN, purple). Uninjected oocytes treated with LPA are shown in grey, n = 12. C TRPV4-mediated current activity (at V m = − 85 mV) summarized in control solution (black) and after exposure to LPA without (orange) or with TRPV4 inhibition (purple). D Transcript abundance (in TPM) of members of the transient receptor potential vanilloid family obtained from RNA sequencing of rat choroid plexus; TRPV1, 0.4; TRPV2, 0.5; TRPV4, 58.5; TRPV6, 0.06. E Transcript abundance of all filtered ion channels. F Representative immunolabeling of TRPV4 in rat choroid plexus. Nuclei staining by DAPI. Scale bar = 5 μm. Statistical evaluation with one-way ANOVA with Dunnett’s post hoc test **P < 0.01; ***P < 0.001

Article Snippet: Blockage was done with 10% swine serum diluted in PBS + 1%Tween-20 (PBST), and the sections were immunolabeled with primary rabbit anti-NKCC1 (Abcam #AB59791, 1:400) and mouse anti-TRPV4 (BD BioSciences #AB53079, 1:500) overnight at 4 °C, and Alexa Fluor TM 488 and Alexa Fluor TM 647 (ThermoFisher Scientific 1:500) for 2 h at room temperature.

Techniques: Expressing, Inhibition, Activity Assay, RNA Sequencing Assay, Immunolabeling, Staining

Journal: Fluids and Barriers of the CNS

Article Title: Lysophosphatidic acid as a CSF lipid in posthemorrhagic hydrocephalus that drives CSF accumulation via TRPV4-induced hyperactivation of NKCC1

doi: 10.1186/s12987-022-00361-9

Figure Lengend Snippet: TRPV4 activity modulates ICP and CSF production in vivo. A Schematic illustrating the cranial window (shown as a dotted circle) into which the ICP probe is positioned, LV; lateral ventricle. B ICP as a function of time upon infusion of control solution, n = 5 (black) or TRPV4 inhibitor-containing solution, n = 5 (RN; purple) shown as 5 min average values normalized to the baseline. Inset, initial ICP, n = 10. C Summarized changes in ICP with control solution (black) and TRPV4 inhibitor-containing solution after 1.5 h. D Schematic of the VCP method used to determine the CSF production rate. E Representative time course of the dextran ratio (outflow/inflow) with a mock solution change with control solution (indicated with a grey bar). Inset, average CSF production rate, n = 7. F CSF production as a function of time. Data normalized to the last four samples before solution change to either the control solution, n = 7 (black), the TRPV4 activator GSK, n = 6 (green), or the TRPV4 inhibitor RN, n = 6 (purple). G Summarized CSF production rates in % of control after exposure to vehicle (black), TRPV4 activation (green) and TRPV4 inhibition (purple). H Correctly targeted dye delivery in mid-saggital sections of a rat brain. I A representative image of a rat after injection of IRDye 800CW carboxylate dye (superimposed pseudo-color). The square placed in line with lambda indicates the area of dye content quantification. J Representative images obtained at t = 0.5 (t 0.5 ) and t = 5 (t 5 ) min in control solution (left) or upon TRPV4 inhibition (RN; right). K The dye intensity normalized to that obtained in the first image and plotted as a function of time representing flow rate for control, n = 5 (black), TRPV4 activation, n = 6 (GSK, green) and TRPV4 inhibition, n = 6 (RN, purple). L Quantification of the dye intensity (flow rate) determined from linear regression in K over the 5 min. time window from control (black), TRPV4 activation (green) and TRPV4 inhibition (purple). Statistical evaluation with Student’s t- test ( C ) or one-way ANOVA with Dunnett’s post hoc test ( F and K ) *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Blockage was done with 10% swine serum diluted in PBS + 1%Tween-20 (PBST), and the sections were immunolabeled with primary rabbit anti-NKCC1 (Abcam #AB59791, 1:400) and mouse anti-TRPV4 (BD BioSciences #AB53079, 1:500) overnight at 4 °C, and Alexa Fluor TM 488 and Alexa Fluor TM 647 (ThermoFisher Scientific 1:500) for 2 h at room temperature.

Techniques: Activity Assay, In Vivo, Activation Assay, Inhibition, Injection

Journal: Fluids and Barriers of the CNS

Article Title: Lysophosphatidic acid as a CSF lipid in posthemorrhagic hydrocephalus that drives CSF accumulation via TRPV4-induced hyperactivation of NKCC1

doi: 10.1186/s12987-022-00361-9

Figure Lengend Snippet: TRPV4 co-localizes with—and regulates—NKCC1. A Representative immunolabeling in rat choroid plexus of TRPV4 (red) and NKCC1 (green). DAPI is used for nuclei staining. Scale bar = 10 μm. B Proximity ligation assayed interaction complex between NKCC1 and TRPV4 shown as red speckles. Inset: Absence of primary antibodies against TRPV4 and NKCC1. Scale bar = 10 μm. C Efflux of 86 Rb + from choroid plexus (inset) in control settings without, n = 5 (black) or with NKCC1 inhibition by bumetanide (BUM), n = 5 (grey), or upon TRPV4 activation by GSK without, n = 5 (green) or with BUM, n = 5 (light green). GSK-mediated 86 Rb + efflux obtained with inclusion of the TRPV4 inhibitor RN, n = 5 (dotted, purple line). Y-axis is the natural logarithm of the amount of left in the choroid plexus at time t (A t ) divided by the amount at time 0 (A 0 ). D NKCC1-mediated efflux rate constants for 86 Rb + in control, n = 5 (black) or upon TRPV4 activation, n = 5 (GSK; green). Statistical evaluation with Student’s t- test. ***P < 0.001

Article Snippet: Blockage was done with 10% swine serum diluted in PBS + 1%Tween-20 (PBST), and the sections were immunolabeled with primary rabbit anti-NKCC1 (Abcam #AB59791, 1:400) and mouse anti-TRPV4 (BD BioSciences #AB53079, 1:500) overnight at 4 °C, and Alexa Fluor TM 488 and Alexa Fluor TM 647 (ThermoFisher Scientific 1:500) for 2 h at room temperature.

Techniques: Immunolabeling, Staining, Ligation, Inhibition, Activation Assay

Journal: Fluids and Barriers of the CNS

Article Title: Lysophosphatidic acid as a CSF lipid in posthemorrhagic hydrocephalus that drives CSF accumulation via TRPV4-induced hyperactivation of NKCC1

doi: 10.1186/s12987-022-00361-9

Figure Lengend Snippet: TRPV4-mediated Ca 2+ fluctuations activate NKCC1 in a WNK-SPAK-dependent manner. A Efflux of 86 Rb + from choroid plexus in control solution, n = 4 (black), in Ca 2+ -free solution, n = 4 (grey), upon TRPV4-activation by GSK with, n = 4 (green) or without Ca 2+ , n = 4 (light green). B GSK-sensitive efflux rate constants for 86 Rb + with (green) or without (light green) Ca 2+ present. C Schematic of the hypothesized LPA-induced TRPV4-mediated Ca 2+ influx regulating WNK-SPAK-mediated phosphorylation of NKKC1. D Transcript abundance of filtered kinases in rat choroid plexus with rank of SPAK and WNK kinases depicted. E Network analysis depicting published protein-protein associations amongst those illustrated in Panel A. F 86 Rb + efflux from choroid plexus in control settings without, n = 6 (black) or with SPAK inhibition, n = 6 (grey), or upon TRPV4 activation by GSK without, n = 6 (green) or with, n = 6 (light green) SPAK inhibition. Y-axis is the natural logarithm of the amount of left in the choroid plexus at time t (A t ) divided by the amount at time 0 (A 0 ). G GSK-mediated efflux rate constants for 86 Rb + without (green) or with SPAK-inhibition (light green). H 86 Rb + efflux in control settings without, n = 5 (black) or with, n = 5 (grey) WNK inhibition, or upon TRPV4 activation by GSK without, n = 5 (green) or with, n = 5 (light green) WNK inhibition. I GSK-mediated efflux rate constants for 86 Rb + without (green) or with (light green) WNK-inhibition. J 86 Rb + efflux in control settings without, n = 5 (black) or with, n = 5 (grey) NKCC1 inhibition by bumetanide (BUM), or upon application of LPA without, n = 5 (orange) or with, n = 5 (black/orange) NKCC1 inhibition (BUM). K BUM-sensitive efflux rate constants for 86 Rb + in control setting (black) or upon LPA application (orange). Statistical evaluation with Student’s t- test. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Blockage was done with 10% swine serum diluted in PBS + 1%Tween-20 (PBST), and the sections were immunolabeled with primary rabbit anti-NKCC1 (Abcam #AB59791, 1:400) and mouse anti-TRPV4 (BD BioSciences #AB53079, 1:500) overnight at 4 °C, and Alexa Fluor TM 488 and Alexa Fluor TM 647 (ThermoFisher Scientific 1:500) for 2 h at room temperature.

Techniques: Activation Assay, Inhibition