The absence of AQP4/TRPV4 complex substantially reduces acute cytotoxic edema following ischemic injury

Abstract

Introduction: Astrocytic Aquaporin 4 (AQP4) and Transient receptor potential vanilloid 4 (TRPV4) channels form a functional complex that likely influences cell volume regulation, the development of brain edema, and the severity of the ischemic injury. However, it remains to be fully elucidated whether blocking these channels can serve as a therapeutic approach to alleviate the consequences of having a stroke.

Methods and results: In this study, we used in vivo magnetic resonance imaging (MRI) to quantify the extent of brain lesions one day (D1) and seven days (D7) after permanent middle cerebral artery occlusion (pMCAO) in AQP4 or TRPV4 knockouts and mice with simultaneous deletion of both channels. Our results showed that deletion of AQP4 or TRPV4 channels alone leads to a significant worsening of ischemic brain injury at both time points, whereas their simultaneous deletion results in a smaller brain lesion at D1 but equal tissue damage at D7 when compared with controls. Immunohistochemical analysis 7 days after pMCAO confirmed the MRI data, as the brain lesion was significantly greater in AQP4 or TRPV4 knockouts than in controls and double knockouts. For a closer inspection of the TRPV4 and AQP4 channel complex in the development of brain edema, we applied a real-time iontophoretic method in situ to determine ECS diffusion parameters, namely volume fraction (α) and tortuosity (λ). Changes in these parameters reflect alterations in cell volume, and tissue structure during exposure of acute brain slices to models of ischemic conditions in situ, such as oxygen-glucose deprivation (OGD), hypoosmotic stress, or hyperkalemia. The decrease in α was comparable in double knockouts and controls when exposed to hypoosmotic stress or hyperkalemia. However, during OGD, there was no decrease in α in the double knockouts as observed in the controls, which suggests less swelling of the cellular components of the brain.

Conclusion: Although simultaneous deletion of AQP4 and TRPV4 did not improve the overall outcome of ischemic brain injury, our data indicate that the interplay between AQP4 and TRPV4 channels plays a critical role during neuronal and non-neuronal swelling in the acute phase of ischemic injury.

Keywords: AQP4; ECS diffusion; MRI; TRPV4; brain edema; cerebral ischemia.

FIGURE 1

Quantification of the ischemic lesion volume using T₂-weighted magnetic resonance imaging of the mouse brain 1 day (D1) and 7 days (D7) after pMCAO. (A) Bar graph showing an average volume of the ischemic lesion in the AQP4^–/–, TRPV4^–/–, and AQP4^–/–/TRPV4^–/– mice compared to the Ctrl at D1 after pMCAO. Note that mice lacking AQP4 or TRPV4 channels showed significantly higher lesion volume, whereas the volume of the lesions in the mice lacking both channels was significantly reduced when compared to the single knockouts. (B) Representative T₂-weighted images of the brains of the Ctrl, AQP4^–/–, TRPV4^–/–, and AQP4^–/–/TRPV4^–/– mice at D1 after pMCAO. The lesion is visible as a mild hyperintense area in the left hemisphere (yellow arrowheads). (C) Bar graph showing an average volume of the ischemic lesion in the AQP4^–/–, TRPV4^–/–, and AQP4^–/–/TRPV4^–/– mice compared to the Ctrl at D7 after pMCAO. Note that at D7 after pMCAO the lesion volume remains larger in the AQP4^–/– as well as TRPV4^–/– mice compared to the Ctrl, but in the AQP4^–/–/TRPV4^–/–, the lesion size is comparable to Ctrl. (D) Representative T₂-weighted images of the brains of the Ctrl, AQP4^–/–, TRPV4^–/–, and AQP4^–/–/TRPV4^–/– mice at D7 after pMCAO. The lesion is visible as a mild hyperintense area in the left hemisphere (yellow arrowheads). Data are presented as mean + SEM. Asterisks indicate significant differences between the AQP4^–/–/TRPV4^–/– mice and the Ctrl (*p < 0.05, **p < 0.01, ***p < 0.001). Ctrl, control; AQP4^–/–, AQP4-deficient mice; TRPV4^–/–, TRPV4-deficient mice; AQP4^–/–/TRPV4^–/–, AQP4- and TRPV4-deficient mice; pMCAO, permanent middle cerebral artery occlusion.

FIGURE 2

Immunohistochemical staining of the brains 7 days (D7) after pMCAO. Staining with antibodies against GFAP (A) and Iba1 (B) showed that the extent of the damage in Aqp4^–/–/Trpv4^–/– mice is comparable to the Ctrl. Conversely, significantly greater damage was observed in the Aqp4^–/–/or Trpv4^–/– compared to the Ctrl and the Aqp4^–/–/Trpv4^–/– mice. The white dotted lines show the original size of the tissue slices in the places, where due to the extent of the ischemic damage some parts of the slices fell out. Details of the pictures showing GFAP or Iba1 expression are shown at right in white frames. Ctrl, control; AQP4^–/–, AQP4-deficient mice; TRPV4^–/–, TRPV4-deficient mice; AQP4^–/–/TRPV4^–/–, AQP4- and TRPV4-deficient mice; GFAP, glial fibrillary acidic protein; Iba1, ionized Ca²⁺-binding adapter molecule.

FIGURE 3

The effect of oxygen-glucose deprivation (OGD) on the ECS diffusion parameters and extracellular potassium concentration [K⁺]_o in situ. (A) Representative diffusion curves with the corresponding values of the ECS volume fraction (α) and tortuosity (λ) recorded in the Ctrl, AQP4^–/–, TRPV4^–/–, and AQP4^–/–/TRPV4^–/– mice. Averaged data of α (B) and λ (C) measured in the Ctrl, AQP4^–/–, TRPV4^–/–, and AQP4^–/–/TRPV4^–/– mice at resting conditions (22–24°C), 33°C and at 5-min intervals during and after (washout) OGD. Asterisks indicate significant differences between Ctrl and AQP4^–/–, TRPV4^–/–, or AQP4^–/–/TRPV4^–/– mice (*p < 0.05, **p < 0.01, ***p < 0.001). (D) Values of [K⁺]_o recorded in 1-min intervals in the Ctrl, AQP4^–/–, TRPV4^–/–, and AQP4^–/–/TRPV4^–/– mice. Data are presented as mean ± SEM. Asterisks indicate significant differences between AQP4^–/–/TRPV4^–/– mice and AQP4^–/–, TRPV4^–/–, or Ctrl (*p < 0.05, **p < 0.01, ***p < 0.001). Ctrl, control; AQP4^–/–, AQP4-deficient mice; TRPV4^–/–, TRPV4-deficient mice; AQP4^–/–/TRPV4^–/–, AQP4- and TRPV4-deficient mice; OGD, oxygen-glucose deprivation; n, number of slices; ECS, extracellular space.

FIGURE 4

The effect of hypoosmotic stress (A,B) and hyperkalemia (C) on the ECS diffusion parameters in situ. Representative diffusion curves with the corresponding values of the ECS volume fraction (α) and tortuosity (λ) recorded in the Ctrl, AQP4^–/–, TRPV4^–/–, and AQP4^–/–/TRPV4^–/– mice (upper panels). Averaged data of α (middle panels) and λ (bottom panels) measured in the Ctrl, AQP4^–/–, TRPV4^–/–, and AQP4^–/–/TRPV4^–/– mice at resting conditions (22–24°C), 33°C and at 5-min intervals during and after (washout) applications of aCSF_H–50, aCSF_H–100, and aCSF_K+ solutions. Data are expressed as mean ± SEM. No significant differences were detected between the AQP4^–/–/TRPV4^–/– and control animals. Ctrl, control; AQP4^–/–, AQP4-deficient mice; TRPV4^–/–, TRPV4-deficient mice; AQP4^–/–/TRPV4^–/–, AQP4- and TRPV4-deficient mice; aCSF_H–50, 250 mOsmol hypotonic solution; aCSF_H–100, 200 mOsmol hypotonic solution; aCSF_K+, 50 mM K⁺ solution.

FIGURE 5

Scheme of the main mechanisms involved in astrocyte volume changes and volume regulation in the acute post-ischemic phase, effect of AQP4 and TRPV4 channel deletion. Proposed mechanisms explaining the changes in the lesion size observed in the Ctrl (A), AQP4^–/– (B), TRPV4^–/– (C), and AQP4^–/–/TRPV4^–/– (D) mice. We propose that the absence of AQP4 in single (B) or double knock-outs (D) leads to slowed swelling that is insufficient to trigger regulatory volume decrease (RVD). Such a decrease in RVD presumably occurs due to low activity of TRPV4 channels, which is unable to activate SACs in panel (B), or lack of TRPV4 channels in panel (D). Water entry into the cell occurs via ion/glutamate transporters, while its efflux occurs besides ion channels by simple diffusion. Deletion of TRPV4 results in the absence of RVD activated by cell membrane stretch. Both slower swelling and limited RVD in double knock-outs, lead to less edema in the acute post-ischemic phase compared to control. Panels (B,D) both display limited swelling as well as RVD however in AQP4^–/– (B) functional TRPV4 channels in neurons prolong neuronal activity with further neurotoxic impact on nervous tissue. A, astrocyte; N, neuron; Ctrl, control; AQP4^–/–, AQP4-deficient mice; TRPV4^–/–, TRPV4-deficient mice; AQP4^–/–/TRPV4^–/–, AQP4- and TRPV4-deficient mice; RVD, regulatory volume decrease; NKCC, Na⁺/K^+/Cl^– co-transporter; SAC, stretch activated ion channel; EAAT, excitatory amino acid transporter; AQP4, aquaporin-4 channel; TRPV4, Transient receptor potential cation channel subfamily V member 4.