Impaired endothelium-mediated cerebrovascular reactivity promotes anxiety and respiration disorders in mice

Abstract

Carbon dioxide (CO₂), the major product of metabolism, has a strong impact on cerebral blood vessels, a phenomenon known as cerebrovascular reactivity. Several vascular risk factors such as hypertension or diabetes dampen this response, making cerebrovascular reactivity a useful diagnostic marker for incipient vascular pathology, but its functional relevance, if any, is still unclear. Here, we found that GPR4, an endothelial H⁺ receptor, and endothelial Gα_q/11 proteins mediate the CO₂/H⁺ effect on cerebrovascular reactivity in mice. CO₂/H⁺ leads to constriction of vessels in the brainstem area that controls respiration. The consequential washout of CO₂, if cerebrovascular reactivity is impaired, reduces respiration. In contrast, CO₂ dilates vessels in other brain areas such as the amygdala. Hence, an impaired cerebrovascular reactivity amplifies the CO₂ effect on anxiety. Even at atmospheric CO₂ concentrations, impaired cerebrovascular reactivity caused longer apneic episodes and more anxiety, indicating that cerebrovascular reactivity is essential for normal brain function. The site-specific reactivity of vessels to CO₂ is reflected by regional differences in their gene expression and the release of vasoactive factors from endothelial cells. Our data suggest the central nervous system (CNS) endothelium as a target to treat respiratory and affective disorders associated with vascular diseases.

Keywords: anxiety; brain endothelial cells; endothelial dysfunction; hypercapnia; respiration.

Fig. 1.

GPR4 mediates CO₂-induced perfusion increase in the cortex. (A) Representative images taken before (air) and during CO₂ stimulation, and quantification of laser speckle imaging measuring cortical perfusion of P2yr2^beKO and control mice during artificial ventilation with 20% CO₂. An exemplary region of analysis is indicated by the white dotted box. (B) Areas under the curves shown in A; n = 19 to 20 mice per group. (C) Representative images taken before (air) and during CO₂ stimulation, and quantification of laser speckle imaging of Gpr4^−/− and control mice during artificial ventilation with 20% CO₂. An exemplary region of analysis is indicated by the white dotted box. (D) Areas under the curves shown in C. Student’s t test, **P < 0.01; n = 20 mice per group. (E) Representative images taken before (air) and during CO₂ stimulation, and quantification of laser speckle imaging of Gpr68^−/− and control mice during artificial ventilation with 20% CO₂. An exemplary region of analysis is indicated by the white dotted box. (F) Areas under the curves shown in E; n = 12 to 14 mice per group. (G) In situ hybridization of Gpr4 mRNA shows coexpression with the endothelial cell marker Pecam and colocalization with the basement membrane protein collagen IV. (Scale bar, 10 µm.) The figure is a magnified image of SI Appendix, Fig. S1E. (H) PGF_1α as a surrogate for prostacyclin release of PFBECs of Gpr4^−/− and control mice after 30-min stimulation with 5% (control) or 15% CO₂. *P < 0.05 (2-way ANOVA with Bonferroni posttest); n = 12 per group. (I) Assessment of NO release by measurement of nitrate concentrations in the supernatant of PFBECs of Gpr4^−/− and control mice after 30-min stimulation with 5% (control) or 15% CO₂. *P < 0.05 (2-way ANOVA with Bonferroni posttest); n = 12 per group. Data are means ± SEM. Color scales in A, C, and E indicate arbitrary units of laser speckle images. (Scale bars in A, C, and E, 1 mm.).

Fig. 2.

Endothelial Gα_q/11 signaling mediates CO₂-induced perfusion increase in the cortex. (A) Representative images of laser speckle recordings of Gα_q/11^beKO and control mice ventilated with different CO₂ concentrations taken before (air) and during CO₂ stimulation. Color scale indicates arbitrary units. An exemplary region of analysis is indicated by the white dotted box. (Scale bars, 1 mm.) (B and D) Quantification of laser speckle imaging measuring cortical perfusion of Gα_q/11^beKO and control mice during artificial ventilation with 10% CO₂ (B) or 20% CO₂ (D). (C and E) Areas under the curves shown in B or D, respectively. Mann–Whitney U test, *P < 0.05; n = 11 to 13 mice per group. (F and H) Quantification of laser speckle imaging measuring cortical perfusion of Gα_q/11^beKO and control mice during and after short apneic periods of 2 (F) or 3 (H) seconds. *P < 0.05 (RM-ANOVA with Bonferroni posttest); n = 6 to 7 mice per group. (G and I) Areas under the curves shown in F or H, respectively. Mann–Whitney U test, *P < 0.05; n = 6 to 7 mice per group. (J) Representative images of arterial spin labeling (ASL)-MRI of Gα_q/11^beKO and control mice before (air) and during stimulation with 10% CO₂. (K) Quantification of ASL-MRI perfusion measurements in the cortex of unstimulated Gα_q/11^beKO and control mice; n = 14 to 15 mice per group. (L) Difference in ASL-MRI perfusion measurements in the cortex of CO₂-exposed and unexposed Gα_q/11^beKO and control mice. Student’s t test, **P < 0.01; n = 14 to 15 mice per group. (M) Representative images of stained arterioles in acute cortical brain slices of Gα_q/11^beKO and control mice before and during stimulation with CO₂. (N) Representative traces of diameter measurements in acute cortical brain slices of Gα_q/11^beKO and control mice during stimulation with CO₂. (O) Change in arteriolar diameters after stimulation with CO₂ in acute cortical brain slices of Gα_q/11^beKO and control mice (1 arteriole per animal, mean of 3 different sites of each vessel; n = 5 to 8 mice per group). Mann–Whitney U test, *P < 0.05. (P) Baseline diameters of the measured arterioles in acute cortical brain slices of Gα_q/11^beKO and control mice; n = 5 to 8 mice per group. Data are means ± SEM.

Fig. 3.

Impaired vascular reactivity to CO₂ in the amygdala leads to increased fear responses. (A) Representative images of stained arterioles in acute amygdala slices of Gα_q/11^beKO and control mice before and during stimulation with CO₂. (B) Representative traces of diameter measurements in acute amygdala slices of Gα_q/11^beKO and control mice during stimulation with CO₂. (C) Change in arteriolar diameters after stimulation with CO₂ in acute amygdala slices of Gα_q/11^beKO and control mice (1 arteriole per animal, mean of 3 different sites of each vessel, n = 17 mice per group). Student’s t test, *P < 0.05. (D) Baseline diameters of the measured arterioles in acute amygdala slices of Gα_q/11^beKO and control mice; n = 17 mice per group. (E) Representative track reports of Gα_q/11^beKO and control mice exposed to normal air or CO₂. (F) Quantification of freezing behavior during a 10-min normal air or 10% CO₂ exposure in Gα_q/11^beKO and control mice. *P < 0.05, ***P < 0.001 (2-way ANOVA with Bonferroni posttest); n = 16 to 17 mice per group. (G) Representative track reports of Gα_q/11^beKO and control mice during a 10-min open field test and quantification of the time mice spent in the inner zone of the open field arena. Student’s t test, *P < 0.05; n = 13 to 16 mice per group. (H) Representative track reports of Gα_q/11^beKO and control mice during a 5-min elevated plus maze test and quantification of the time mice spent in the closed arm of the maze. Student’s t test, *P < 0.05; n = 13 to 16 mice per group. Data are means ± SEM.

Fig. 4.

Impaired vascular reactivity to CO₂ leads to respiratory changes. (A) Representative images of stained arterioles in acute RTN slices of Gα_q/11^beKO and control mice before and during stimulation with CO₂. (B) Representative traces of diameter measurements in acute RTN slices of Gα_q/11^beKO and control mice during stimulation with CO₂. (C) Change in arteriolar diameters after stimulation with CO₂ in acute RTN slices of Gα_q/11^beKO and control mice (1 arteriole per animal, mean of 3 different sites of each vessel). Student’s t test, **P < 0.01; n = 14 to 16 mice per group. (D) Baseline diameters of the measured arterioles in acute RTN slices of Gα_q/11^beKO and control mice; n = 14 to 16 mice per group. (E) Representative respiratory flow traces of Gα_q/11^beKO and control mice exposed to different concentrations of CO₂ and the quantification thereof, recorded by head-out plethysmography. *P < 0.05 (RM-ANOVA with Bonferroni posttest). (F) Respiratory flow of Gα_q/11^beKO and control mice exposed to different CO₂ concentrations, as recorded by whole-body plethysmography. *P < 0.05 (RM-ANOVA with Bonferroni posttest); n = 7 to 9 mice per group. (G) Representative apnea phases of Gα_q/11^beKO and control mice recorded by whole-body plethysmography during the inactive period and quantification of the number of apneic phases; n = 9 to 10 mice per group. (H) Mean duration of all recorded apnea within 1 h during the inactive period of the day for each mouse in Gα_q/11^beKO and control mice. Student’s t test, *P < 0.05; n = 9 to 10 mice per group. Data are means ± SEM.

Fig. 5.

Release of vasoactive substances differs between subcortical-telencephalic and brainstem endothelial cells. (A) Scheme of the brain areas that were used for the preparation of area-specific primary endothelial cells. (B) Assessment of NO release by measuring nitrate concentrations in the supernatant of SCT or brainstem endothelial cells after 20-min stimulation with 5% (control) or 15% CO₂. ***P < 0.001 (2-way ANOVA with Bonferroni posttest); n = 4 to 6 per group, 2 independent experiments. (C) PGF_1α as a surrogate for prostacyclin release from SCT or brainstem endothelial cells after 20-min stimulation with 5% (control) or 15% CO₂. **P < 0.01, ***P < 0.001 (2-way ANOVA with Bonferroni posttest); n = 5 to 6 per group, 2 independent experiments. (D) PGE₂ release from SCT or brainstem endothelial cells after 20-min stimulation with 5% (control) or 15% CO₂. ***P < 0.001 (2-way ANOVA with Bonferroni posttest); n = 5 to 6 per group, 2 independent experiments. (E) TXB₂ as a surrogate for TXA₂ release from SCT or brainstem endothelial cells after 20-min stimulation with 5% (control) or 15% CO₂. P < 0.05 for treatment condition in 2-way ANOVA; n = 3 to 5 per group, 2 independent experiments. (F) PGF_2α release from SCT or brainstem endothelial cells after 20-min stimulation with 5% (control) or 15% CO₂; n = 5 to 6 per group, 2 independent experiments. Absolute values of prostanoids released by endothelial cells are shown in SI Appendix, Fig. S10B. (G) Representative traces of diameter measurements in acute cortical and RTN brain slices of C57BL/6 mice during stimulation with 1 µM iloprost and quantification thereof (1 arteriole per animal, mean of 3 different sites of each vessel, n = 3 mice per group). Student’s t test, *P < 0.05. Data are means ± SEM.