Lung Purinoceptor Activation Triggers Ventilator-Induced Brain Injury

Abstract

Objectives: Mechanical ventilation can cause ventilator-induced brain injury via afferent vagal signaling and hippocampal neurotransmitter imbalances. The triggering mechanisms for vagal signaling during mechanical ventilation are unknown. The objective of this study was to assess whether pulmonary transient receptor potential vanilloid type-4 (TRPV4) mechanoreceptors and vagal afferent purinergic receptors (P2X) act as triggers of ventilator-induced brain injury.

Design: Controlled, human in vitro and ex vivo studies, as well as murine in vivo laboratory studies.

Setting: Research laboratory.

Subjects: Wild-type, TRPV4-deficient C57BL/6J mice, 8-10 weeks old. Human postmortem lung tissue and human lung epithelial cell line BEAS-2B.

Intervention: Mice subjected to mechanical ventilation were studied using functional MRI to assess hippocampal activity. The effects of lidocaine (a nonselective ion-channel inhibitor), P2X-purinoceptor antagonist (iso-PPADS), or genetic TRPV4 deficiency on hippocampal dopamine-dependent pro-apoptotic signaling were studied in mechanically ventilated mice. Human lung epithelial cells (BEAS-2B) were used to study the effects of mechanical stretch on TRPV4 and P2X expression and activation. TRPV4 levels were measured in postmortem lung tissue from ventilated and nonventilated patients.

Measurements and main results: Hippocampus functional MRI analysis revealed considerable changes in response to the increase in tidal volume during mechanical ventilation. Intratracheal lidocaine, iso-PPADS, and TRPV4 genetic deficiency protected mice against ventilationinduced hippocampal pro-apoptotic signaling. Mechanical stretch in both, BEAS-2B cells and ventilated wild-type mice, resulted in TRPV4 activation and reduced Trpv4 and P2x expression. Intratracheal replenishment of adenosine triphosphate in Trpv4 mice abrogated the protective effect of TRPV4 deficiency. Autopsy lung tissue from ventilated patients showed decreased lung TRPV4 levels compared with nonventilated CONCLUSIONS:: TRPV4 mechanosensors and purinergic receptors are involved in the mechanisms of ventilator-induced brain injury. Inhibition of this neural signaling, either using nonspecific or specific inhibitors targeting the TRPV4/adenosine triphosphate/P2X signaling axis, may represent a novel strategy to prevent or treat ventilator-induced brain injury.

Figure 1.

Hippocampal brain network response upon elevation in tidal volume revealed by using independent component analysis (ICA) on functional MRI data. A, Network activity calculated by correlations of signal time courses in ICA group analysis of four mice shows great overlaps with the anatomical structure of the hippocampus. Gray shadings behind mouse brains (left) indicate the range of coronal brain slices (right). B, Signal time courses of independent components (ICs) extracted from the hippocampal network in four single subjects (see right for the spatial distribution of respective IC).

Figure 2.

Effects of lidocaine over ventilator-induced brain injury (VIBI). A–D, Representative Western blots and densitometric quantification showing the beneficial effects of intratracheal but not IV lidocaine over the prosurvival protein kinase B (AKT)/glycogen synthase kinase-3β (GSK3β) pathway and the apoptotic pathway (n = 8). The changes triggered by mechanical ventilation in B, phosphorylation levels of AKT at its serine 473; C, phosphorylation levels of GSK3β at its serine 9; and D, cleaved poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) were significantly mitigated by treatment with intratracheal lidocaine (7 mg/kg) but not IV lidocaine (7 mg/kg). *p < 0.05 in parametric post hoc test versus the sham group. **p < 0.05 in nonparametric post hoc test versus the sham group. E, Survival study (n = 20) shows improved survival to mechanical ventilation in the intratracheal lidocaine group. *p < 0.05 log rank (Mantel-Cox). it-Lido = lidocaine at a dose of 7 mL/kg intratracheally, iv-Lido = lidocaine at a dose of 7 mL/kg IV.

Figure 3.

Effects of mechanical ventilation and mechanical cyclic stretch over transient receptor potential vanilloid 4 (TRPV4) phosphorylation. A, Representative Western blots and densitometric quantification showing mechanical ventilation–related increased TRPV4 activation/phosphorylation (serine 824 (serine 824 [pSer824]) in mouse lungs (n = 6). B, Representative Western blots and densitometric quantification showing stretch-induced activation/phosphorylation of TRPV4 at its serine 824 (pSer824) in human epithelial cell line BEAS-2B (n = 5). *p < 0.05 in parametric post hoc test versus the sham group. C, TRPV4 immunohistochemistry of lung sections from lung autopsies obtained from nonventilated (n = 7) and mechanically ventilated patients (n = 10). Immunoreactivity in the ventilated patients is lower compared with the nonventilated patients. The intensity of immunoreaction was semiquantitatively analyzed. **p < 0.05 in Mann-Whitney U test. it-Lido = lidocaine at a dose of 7 mL/kg intratracheally, Lido = lidocaine.

Figure 4.

Transient receptor potential vanilloid 4 (TRPV4) deficiency–related protection against ventilator-induced brain injury (VIBI) is lost after adding intratracheal exogenous adenosine triphosphate (ATP). Trpv4 knockout mice were significantly protected against VIBI. Addition of intratracheal ATP (100 μg/kg) prior to ventilation (Vent.) of Trpv4 knockout mice leads to restoration of acute VIBI (n =7). A, Representative Western blots. B, Densitometry analysis of phosphorylation levels of protein kinase B (AKT) at its serine 473. C, Densitometry analysis of phosphorylation levels of glycogen synthase kinase-3β (GSK3β) at its serine 9. D, Densitometry analysis of cleaved poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1). **p < 0.05 in nonparametric post hoc test versus the sham group.

Figure 5.

triggering mechanisms within the lung. The mechanical stretch–caused ventilation (Vent.) cause phosphorylation/activation of transient receptor potential vanilloid 4 (TRPV4) channels. Increased activation of TRPV4 leads to release of adenosine triphosphate (ATP) to the alveoli that in turn activates the P2X purinergic channels present in the lung tissue including the sensory nerves. The increased influx of sodium ion (Na⁺) and calcium ion (Ca⁺) would trigger lung sensory neurons that would carry on the signal to the brain through the afferent vagus nerve triggering VIBI. *p < 0.05 in parametric post hoc test versus the sham group. **p < 0.05 in nonparametric post hoc test versus the sham group.