of TRPV4 Channels Does Not Mediate Inversion of Neurovascular Coupling After SAH

Fig. 1

TRPV4 inhibition does not alter the amplitude or frequency of spontaneous Ca²⁺ events in astrocyte endfeet after SAH. (a) Spontaneous Ca²⁺ oscillations recorded from 1.2 × 1.2-μm regions of interest placed on distinct astrocyte endfeet in a brain slice from one SAH animal in the absence (upper trace) and presence (lower trace) of the TRPV4 antagonist HC-067047 (10 μM). (b) Summary data of endfoot Ca²⁺ levels in the absence and presence of HC-067047 (10 μM) obtained from brain slices of SAH model rats. The term peak represents the average maximum Ca²⁺ concentration measured during individual spontaneous Ca²⁺ oscillations. (c) Summary data of the frequency of spontaneous Ca²⁺ oscillations ± HC-067047 (10 μM) obtained from 4-min recordings using brain slices from SAH model rats. For panels (b, c), recordings were made from 13 endfeet in four brain slices from four animals. NS, not statistically significant (P > 0.05), using paired Student’s t-test

TRPV4 Channels Do Not Mediate SAH-Induced Inversion of Neurovascular Coupling

To examine the impact of TRPV4 channels on SAH-induced inversion of neurovascular coupling, electrical field simulation (EFS) was used to induce neuronal action potentials in brain slices from SAH animals. Consistent with our previous studies [12, 13], EFS resulted in an increase in endfoot Ca²⁺ from a resting level of 169.6 ± 8.6 nM to a peak of 348.9 ± 33.0 nM (n = 5) (Fig. 2a, c). Associated with this EFS-induced increase in Ca²⁺, parenchymal arterioles encased by these endfeet constricted by 21.2 ± 4.3 % (Fig. 2a, b). This vasoconstriction in response to neuronal activation after SAH represents an inversion of the physiological vasodilation observed in brain slices from healthy control animals [8, 12, 25]. Incubation of brain slices with the TRPV4 channel antagonist, HC-067047 (10 μM), did not alter EFS-evoked increases in endfoot Ca²⁺ (peak Ca²⁺ after EFS: 330 ± 30.5 nM) or the magnitude of ensuing vasoconstriction (18.6 ± 3.1 % decrease in diameter) (Fig. 2a–c). These findings indicate that TRPV4 channels do not alter EFS-induced increases in endfoot Ca²⁺ or SAH-induced inversion of neurovascular coupling.

Fig. 2

TRPV4 channel inhibition does not influence SAH-induced inversion of neurovascular coupling. Parenchymal arteriolar diameter and astrocyte endfoot Ca²⁺ concentration were simultaneously measured using two-photon imaging and infrared–differential interference contrast (IR-DIC) microscopy. (a) IR-DIC images in the absence (upper panels) and in the presence (lower panels) of the TRPV4 antagonist HC-067047 (10 μM) obtained from brain slices of SAH model rats. Red dashes outline the intraluminal diameter of parenchymal arterioles using IR-DIC microscopy. Overlapping pseudocolor-mapped endfoot Ca²⁺ levels were obtained using the fluorescent Ca²⁺ indicator Fluo-4 and two-photon imaging. Scale bars, 10 μm. (b, c) Summary of EFS-evoked changes in arteriolar diameter (b) and astrocytic endfoot Ca²⁺ (c) obtained from SAH (n = 5 brain slices from three animals) model rats in the presence and absence of HC-067047. NS not statistically significant (P > 0.05), –HC-067047 vs + HC-067047, using paired Student’s t-test. **P < 0.001, before vs after EFS, using ANOVA followed by Tukey test

Discussion

Neurovascular coupling is an important physiological process enabling increased local blood flow to metabolically active regions of the brain. Recent evidence indicates that subarachnoid blood causes a fundamental change in NVC that could lead to pathological decreases in blood flow, rather than the “normal” physiological increases in blood flow associated with localized, task-dependent increases in neuronal activity. Altered Ca²⁺ signaling in astrocyte endfeet, in the form of high-amplitude spontaneous Ca²⁺ oscillations, are responsible for this SAH-induced inversion of neurovascular coupling. In this present study, we hypothesized that enhanced Ca²⁺ entry via TRPV4 channels contributes to the increased amplitude of spontaneous Ca²⁺ events observed in astrocyte endfeet after SAH. However, this does not appear to be the case, because pharmacological inhibition of TRPV4 channels did not alter endfoot Ca²⁺ signaling or the inversion of neurovascular coupling in brain slices from SAH model animals.

Astrocytes exhibit a diverse array of Ca²⁺ signaling events, including nerve-evoked propagating Ca²⁺ transients [8, 13, 25], spontaneous intracellular and intercellular propagating Ca²⁺ waves [7, 22], and nonpropagating spontaneous Ca²⁺ oscillations that can occur in either the cell body or in cell processes such as endfeet wrapping around intracerebral blood vessels [13, 17, 20]. Our recent evidence demonstrates that a marked elevation in the amplitude of spontaneous Ca²⁺ oscillations in endfeet rather than changes in nerve-evoked astrocyte Ca²⁺ signaling underlie inversion of neurovascular coupling after SAH [12]. In brain slices from SAH animals, high-amplitude Ca²⁺ oscillations in endfeet lead to increased K⁺ efflux into the perivascular space via increased activity of large-conductance Ca²⁺-activated K⁺ (BK) channels. This increase in basal extracellular K⁺ when summed with nerve-evoked astrocyte K⁺ efflux elevates extracellular K⁺ in the microenvironment surrounding parenchymal arterioles above the constriction threshold, leading to a polarity change in the neurovascular response from vasodilation to vasoconstriction.

In astrocytes from healthy animals, spontaneous Ca²⁺ oscillations reflect the release of Ca²⁺ stored in endoplasmic reticulum through activation of IP₃-sensitive Ca²⁺ release channels (i.e., IP₃ receptors) and occur independently from neuronal activity or mGluR activation [17, 20]. The activity of IP₃ receptors is bimodally regulated by cytoplasmic Ca²⁺, with moderate increases in Ca²⁺ leading to an increase in IP₃ receptor activation [9]. Thus, although requiring release of Ca²⁺ from intracellular stores, the amplitude of these spontaneous Ca²⁺ events in endfeet can be modulated by Ca²⁺ entering the cell through the plasma membrane. For example, Ca²⁺-permeable TRPV4 channels are present on astrocyte processes [3, 4], and activation of these channels caused an increase in the amplitude of spontaneous Ca²⁺ events in endfeet of brain slices prepared from healthy mice [6]. In addition, TRPV4 expression and function is upregulated in hippocampal astrocytes after cerebral ischemia [4]. However, our present data indicates that Ca²⁺ entry via TRPV4 channels does not contribute to the increase in amplitude of endfoot Ca²⁺ events occurring in SAH model animals. The present study does not exclude the possibility that Ca²⁺ entry through other TRP family members is upregulated in astrocytes after SAH. Although evidence suggests that normal native astrocytes do not express voltage-dependent Ca²⁺ channels, it is also possible that expression of these channels is upregulated in astrocytes after SAH. Alternatively, an increase in IP₃ levels or other Ca²⁺-independent mechanisms leading to enhanced IP₃ receptor activity may underlie the increase in the amplitude of spontaneous Ca²⁺ events in endfeet after SAH. Altered astrocyte Ca²⁺ signaling has been reported to occur in brain pathologies other than SAH, such as Alzheimer’s disease, ischemia/hypoxia, and epilepsy [2, 4, 5, 23]. Further, reactive astrogliosis and microglia activation have also been associated with multiple forms of brain injuries, including SAH [16, 21]. It has been postulated that Ca²⁺ oscillations in activated astrocytes may result in gliotransmitter release starting a cascade of events leading to excitotoxicity and brain damage [1]. Presently, the relationship between induction of reactive astrogliosis and the enhancement of astrocyte Ca²⁺ signaling is unclear and requires further investigation.

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