Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury

Abstract

Currently, the role of transient receptor potential vanilloid type 4 (TRPV4), a nonselective cation channel in the pathology of spinal cord injury (SCI), is not recognized. Herein, we report the expression and contribution of TRPV4 in the pathology of scarring and endothelial and secondary damage after SCI. TRPV4 expression increased during the inflammatory phase in female rats after SCI and was expressed primarily by cells at endothelial-microglial junctions. Two-photon microscopy of intracellular-free Ca²⁺ levels revealed a biphasic increase at similar time points after SCI. Expression of TRPV4 at the injury epicenter, but not intracellular-free Ca²⁺, progressively increases with the severity of the injury. Activation of TRPV4 with specific agonist altered the organization of endothelial cells, affected tight junctions in the hCMEC/D3 BBB cell line in vitro, and increases the scarring in rat spinal cord as well as induced endothelial damage. By contrast, suppression of TRPV4 with a specific antagonist or in female Trpv4 KO mouse attenuated inflammatory cytokines and chemokines, prevented the degradation of tight junction proteins, and preserve blood-spinal cord barrier integrity, thereby attenuate the scarring after SCI. Likewise, secondary damage was reduced, and behavioral outcomes were improved in Trpv4 KO mice after SCI. These results suggest that increased TRPV4 expression disrupts endothelial cell organization during the early inflammatory phase of SCI, resulting in tissue damage, vascular destabilization, blood-spinal cord barrier breakdown, and scarring. Thus, TRPV4 inhibition/knockdown represents a promising therapeutic strategy to stabilize/protect endothelial cells, attenuate nociception and secondary damage, and reduce scarring after SCI.SIGNIFICANCE STATEMENT TRPV4, a calcium-permeable nonselective cation channel, is widely expressed in both excitable and nonexcitable cells. Spinal cord injury (SCI) majorly caused by trauma/accidents is associated with changes in osmolarity, mechanical injury, and shear stress. After SCI, TRPV4 was increased and were found to be linked with the severity of injury at the epicenter at the time points that were reported to be critical for repair/treatment. Activation of TRPV4 was damaging to endothelial cells that form the blood-spinal cord barrier and thus contributes to scarring (glial and fibrotic). Importantly, inhibition/knockdown of TRPV4 prevented these effects. Thus, the manipulation of TRPV4 signaling might lead to new therapeutic strategies or combinatorial therapies to protect endothelial cells and enhance repair after SCI.

Keywords: TRPV4; blood–spinal cord barrier; endothelial cell; scarring; spinal cord injury; two-photon microscopy.

Figure 1.

TRPV4 expression increased during the inflammatory/acute phase of SCI. A, Schematic showing SCI instrument and method. B, Schematic illustration showing TPM imaging after SCI. Representative immunohistochemistry images performed on longitudinal sections at epicenter of the damage for TRPV4 (C), and NF and RECA-1 (D) at 3 h, 8 h, and 1, 3, 5, and 7 d after moderate static compression (35 g/5 min) SCI (Carl Zeiss microscope, 3 fields/slide, n = 3/time point). SCI rat sectional slice labeled with 10 μm SCa1-IREF for 40 min. In situ Ca²⁺ levels were determined in the transverse spinal cord using TPM at similar time points after SCI at the epicenter of the damage. E, Two-photon excited fluorescence was collected using 750 nm excitation and emission windows at 400–430 nm (Ch1) and 500–600 nm (Ch2). Total RNA was prepared from the epicenter of the damage collected 3 h, 8 h, and 1, 3, 5, 7, 14, 21, and 28 d after SCI to determine the expression of TRPV4 (F), IL-6 (H), and Pacsin-3 (I) (n = 4–6/time point, performed in triplicates). G, Quantification of in situ Ca²⁺ levels. GAPDH was used as internal controls for qRT-PCR. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with sham group. ^$p < 0.05, ^$$$p < 0.001 compared with HPI-3. ^#p < 0.05 compared with HPI-8. ^@p < 0.05, ^@@p < 0.01, ^@@@p < 0.001 compared with DPI-1. ^∧p < 0.05, ^∧∧p < 0.01 compared with DPI-3. ⁿp < 0.05, ⁿⁿp < 0.01 compared with DPI-5. ^gp < 0.05, ^gggp < 0.01 compared with DPI-7. ^xp < 0.05, ^xxxp < 0.01 compared with DPI-21 by one-way ANOVA Tukey's post test.

Figure 2.

TRPV4 expression is linked with injury severity and inflammation. Schematic showing method for injury-dependent expression and impounder of different weight (A,B). H&E staining was performed on the longitudinal section after mild (20 g/5 min), moderate (35 g/5 min), and severe (50 g/5 min) injury (C). Total RNA was prepared from spinal cord tissues at the rostral, epicenter, and caudal of the damage collected at 1 d after SCI (mild, moderate, and severe compression) to determine the expression of TRPV4 (D), IL-6 (E), Pacsin3 (F), and occludin (G). RNA expression was determined at the epicenter at 1 d after SCI (mild, moderate, and severe) (n = 2 or 3/group performed in triplicates). TRPV4 (I) and NF and RECA-1 (L) IHC was performed after mild (20 g), moderate (35 g), and severe (50 g) injury (Carl Zeiss microscope, 3 fields/slide, n = 3/time point). In situ Ca²⁺ levels were determined using TPM at 8 h after SCI (mild, moderate, and severe) at the epicenter of the damage (J). Quantification of in situ Ca²⁺ levels (H). Colocalization of TRPV4 with CD-31, an endothelial marker, and Iba-1, a microglial marker (K). Total RNA was prepared from spinal cord tissues after spinal cord hemisection to determine the expression of TRPV4 (M), IL-6 (N), and Pacisn3 (O) (n = 4 or 5/group). GAPDH was used as internal controls for qRT-PCR. Data are mean ± SEM performed in triplicates. *p < 0.05, **p < 0.01, ***p < 0.001 compared with sham group. ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001 compared with 20 g injury group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with 35 g injury group by one-way ANOVA Tukey's post test.

Figure 3.

TRPV4 activation causes endothelial remodeling/damage, whereas TRPV4 inhibition protects SCI-induced endothelial damage. TRPV4 and CD-31 (A) immunocytochemistry was performed on fixed hCMEC/D3 BBB cell line as described in Materials and Methods. Quantification of TRPV4 and CD-31 fluorescence (B,C). TRPV4 and vWF immunocytochemistry was performed on fixed HUVECs (D) and its quantification (E,F). GSK1016790A (50 pmol, 10 μl) was injected into the spinal cord of the rats as mentioned in Materials and Methods. Samples from sham, GSK1016790A, vehicle (injury), or GSK1016790A (injury) were prepared 28 d after injection/injury. Representative images of laminin (magenta) and RECA-1 (G; green), GFAP (H; green), Iba-1 (H; red), and NFs (I; red). J–L, Bar charts represent the fluorescence IMV for corresponding protein as per randomly selected field area at the injury epicenter (3 fields/slide, n = 3/group). Samples from sham or injured untreated (injury) or after RN-1734 (5 mg/kg) treatment were prepared 1 d after moderate injury (35 g/5 min). Representative transverse section of TRPV4 (M) and ZO-1 (N) (3 fields/slide, n = 3) and fluorescence IMV for corresponding protein (P,Q). Total RNA from sham, vehicle (injury), or RN-1734-treated (5 mg/kg, i.p.) samples was prepared DPI-1 after injury. RT-PCR results are showing relative expression levels of TRPV4 (S), Pacsin3 (T), inducible nitric oxide synthase (U), IL-6 (V), and occludin (W). In situ Ca²⁺ levels were determined in the transverse spinal cord using TPM at 8 h after SCI at the epicenter of the damage (O). Quantification of in situ Ca²⁺ levels (R). Data are mean ± SEM (n = 2 or 3/group performed in triplicates). *p < 0.05, **p < 0.01, compared with sham group. ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001 compared with GSK group. ^#p < 0.05 (B,C,E). ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with sham group. *p < 0.05, **p < 0.01, compared with injury group (P–W). *p < 0.05, **p < 0.01, ***p < 0.001 compared with sham group. ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001 compared with GSK group (J–L) by one-way ANOVA Tukey's post test.

Figure 4.

Reduced endothelial damage and inflammation after SCI in TRPV4 KO mice. Representative images of H&E staining at DPI-1 after SCI (A). Representative whole spinal cords show Evans blue dye extravasation of the spinal cord 1 d after SCI (B) and its quantification (C). Total RNA from sham (black bar), wild (red bar), or TRPV4 KO (blue bar) samples was prepared DPI-1 after the injury as described in Materials and Methods. RT-PCR results show relative expression levels of occludin (D), VEGF (E), IL-1β (F), IL-6 (G), TNF-α (H), macrophage-1 antigen (Mac-1) (I), chemokine (C-C motif) ligand 2 (CCL-2) (J), and chemokine (C-C motif) ligand 3 (CCL-3) (K) after injury (n = 2 or 3/group performed in triplicates). GAPDH was used as internal controls for qRT-PCR. TUNEL assay was performed at DPI-1 (L). Quantification of TUNEL-positive cells (M). SCI-induced mouse sectional slice labeled with 10 μm SCa1-IREF for 40 min. In situ Ca²⁺ levels were determined in the longitudinal spinal cord using TPM at 8 h after SCI at the epicenter of the damage. Two-photon excited fluorescence was collected using 750 nm excitation and emission windows at 400–430 nm (Ch1) and 500–600 nm and its quantification (Ch2) (N,O). Representative images for ZO-1 (magenta) and occludin (Q; green) and fluorescence IMV for corresponding protein (P). ^###p < 0.001 compared with sham group. *p < 0.05, **p < 0.01, ***p < 0.001 compared with WT-injury group (D–K,O) by one-way ANOVA Tukey's post test. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT-injury group (C,M,P) by unpaired t test.

Figure 5.

TRPV4 KO mice exhibit less fibrotic and glial scarring after SCI. Samples from WT (injury) or TRPV4 KO (injury) were prepared at DPI-28 as described in Materials and Methods. Representative merges images (longitudinal) for Collagen IV (A; white), laminin (B; magenta), GFAP (C; green), NF (C; magenta), Iba-1 (D; magenta), CD-206 (E; green), CD-68 (F, green), and TGF-β1 (F; magenta) at DPI-28. Bar charts represent the fluorescence IMV for Collagen IV (G), Iba-1 (H), laminin (I), CD-206 (J), GFAP and NF (K), and TGF-β1 and CD-68 (L) as per randomly selected field area at the injury epicenter (3 fields/slide, n = 3/group). M, Western blots of NFs, Iba-1, Connexin-43, GFAP, and CD-31 expression at 28 d after injury. Quantification of immunoblot for NF (N), Iba-1 (O), Connexin-43 (P), GFAP (Q), and CD-31 (R) was performed using ImageJ. Actin was used as internal controls for Western blot (n = 2 or 3/group). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT-injury group by unpaired t test.

Figure 6.

TRPV4 KO mice display reduced hyperalgesia, improved functional recovery, enhanced neuroprotection, and endothelial preservation after SCI. A, Representative images of H&E staining at DPI-28 after SCI. B, Functional recovery was assessed in open-field testing by using the 9-point BMS locomotor test at 1, 3, 7, 14, 21, and 28 d after SCI (n = 10/group). C, Nociception was evaluated using hotplate test. D, SCI-induced hypersensitivity (decrease in reaction latency time) was assessed at pre-SCI (basal), 1, 3, 7, 14, 21, and 28 d after SCI (n = 6/group). Samples from WT (injury) or TRPV4 KO (injury) were prepared at DPI-28 as described in Materials and Methods. Representative sections of angiopoietin-1 (ANG-1, white), NT-3 (red), and BDNF (green) in WT (E) and KO (F). Representative section of vWF (G; green), neural/glial antigen NG-2 (H; green), α-SMA (H; red), Connexin-43 (I; green), Tuj-1 (I; magenta); growth cone (green; J), and NeuN (magenta, J). Bar charts represent the IMV (fluorescence) for corresponding protein as per randomly selected field area at the injury epicenter (3 fields/slide, n = 3/group). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT-injury group.