A Critical Role for Astrocytes in Hypercapnic Vasodilation in Brain

Abstract

Cerebral blood flow (CBF) is controlled by arterial blood pressure, arterial CO₂, arterial O₂, and brain activity and is largely constant in the awake state. Although small changes in arterial CO₂ are particularly potent to change CBF (1 mmHg variation in arterial CO₂ changes CBF by 3%-4%), the coupling mechanism is incompletely understood. We tested the hypothesis that astrocytic prostaglandin E₂ (PgE₂) plays a key role for cerebrovascular CO₂ reactivity, and that preserved synthesis of glutathione is essential for the full development of this response. We combined two-photon imaging microscopy in brain slices with in vivo work in rats and C57BL/6J mice to examine the hemodynamic responses to CO₂ and somatosensory stimulation before and after inhibition of astrocytic glutathione and PgE₂ synthesis. We demonstrate that hypercapnia (increased CO₂) evokes an increase in astrocyte [Ca²⁺]_i and stimulates COX-1 activity. The enzyme downstream of COX-1 that synthesizes PgE₂ (microsomal prostaglandin E synthase-1) depends critically for its vasodilator activity on the level of glutathione in the brain. We show that, when glutathione levels are reduced, astrocyte calcium-evoked release of PgE₂ is decreased and vasodilation triggered by increased astrocyte [Ca²⁺]_iin vitro and by hypercapnia in vivo is inhibited. Astrocyte synthetic pathways, dependent on glutathione, are involved in cerebrovascular reactivity to CO₂ Reductions in glutathione levels in aging, stroke, or schizophrenia could lead to dysfunctional regulation of CBF and subsequent neuronal damage.SIGNIFICANCE STATEMENT Neuronal activity leads to the generation of CO₂, which has previously been shown to evoke cerebral blood flow (CBF) increases via the release of the vasodilator PgE₂ We demonstrate that hypercapnia (increased CO₂) evokes increases in astrocyte calcium signaling, which in turn stimulates COX-1 activity and generates downstream PgE₂ production. We demonstrate that astrocyte calcium-evoked production of the vasodilator PgE₂ is critically dependent on brain levels of the antioxidant glutathione. These data suggest a novel role for astrocytes in the regulation of CO₂-evoked CBF responses. Furthermore, these results suggest that depleted glutathione levels, which occur in aging and stroke, will give rise to dysfunctional CBF regulation and may result in subsequent neuronal damage.

Keywords: astrocyte; calcium; cerebral blood flow; glutathione; hypercapnia.

Figure 1.

Astrocyte [Ca²⁺]_i transients are evoked by CO₂ in vivo. A, Example still images of mouse cortical layer II/III from 2PLSM. OGB is used as a calcium indicator (Ai–Aiii) and sulforhodamine 101 (SR101, Aiv, average image for whole recording) is used to stain astrocytes. Color scale refers to images Ai–Aiii. White arrows indicate astrocytes that show a Ca²⁺ response to CO₂ of at least twice its baseline Ca²⁺ fluctuation. In this case, CO₂ stimulus begins at t = 0 s and is applied for 36 s. Aiii, Recovery of immediate CO₂ induced Ca²⁺ transient. Scale bars, 40 μm. Bi, Biii, Further example images of mouse cortical layer II/III from 2PLSM showing example ROI placement. Merge images showing OGB and SR101 (Bi, Biii). Red ROI1 indicates astrocyte endfoot. Red RO12 indicates astrocyte soma (layer II: n = 181, 8 mice). Green ROI indicates neuron soma (layer II: n = 153, 8 mice). Blue ROI indicates neuropil (layer II: n = 104, 8 mice). Scale bar, 20 μm. Example time series (Bii, Biv) of [Ca²⁺]_i response in astrocyte and neuron soma ROIs (as indicated in Bi, Biii). Blue box represents time during which expired CO₂ level is increased. C, Mean Ca²⁺ response in ROIs. Colors represent ROIs located as shown in Bi. D, Percentage of ROIs for each cell type that showed a Ca²⁺ response with and without a hypercapnia stimulus. For no hypercapnia (control), n = 170 astrocyte somas, n = 148 neuronal soma, and n = 96 neuropil ROIs, n = 8 mice. Colors represent description in B. E, Delay from hypercapnia start time to start of Ca²⁺ response in ROI. F, Duration of Ca²⁺ response in each ROI in response to CO₂ stimulus. E, F, Box plots represent the mean (small square). Edges of the box represent 25% and 75% of data. End lines indicate maximum and minimum values. Data are mean ± SEM. **p < 0.01. ***p < 0.001.

Figure 2.

Astrocyte [Ca²⁺]_i signals evoke COX-1- and GSH-dependent vasodilations in vitro. A, 2PLSM imaging: example Ca²⁺ and arteriole diameter changes in response to tACPD with and without BSO. Images represent overlay of pseudo-colored Ca²⁺ changes and transmitted light images. Dotted line indicates initial vessel diameter. Scale bar, 10 μm. B, Mean time course of increase in astrocyte [Ca²⁺]_i in response to tACPD. Colored box represents time of tACPD application. Control, n = 56 from 26 rats; BSO, n = 39 from 18 rats. C, Mean tACPD-evoked increase in astrocyte [Ca²⁺]_i. tACPD, n = 56 from 26 rats; tACPD + SC560, n = 12 from 7 rats; tACPD + BSO, n = 39 from 18 rats. D, Mean tACPD-evoked PgE₂ release, measured by ELISA. Within a group, each experiment (n) uses tissue from a different rat (i.e., control, n = 8 from 8 rats). E, Mean tissue GSH concentration; data from 4 rats for each group. F, Mean time course of tACPD-evoked change in lumen diameter. Colored box represents time of tACPD application. Control, n = 31 slices from 26 rats; BSO, n = 21 slices from 18 rats. G, Mean changes in lumen diameter evoked by tACPD and clonidine. tACPD, n = 31 slices from 26 rats; SC560 + tACPD, n = 7 slices from 7 rats; BSO + tACPD, n = 21 slices from 18 rats; clonidine, n = 8 slices from 8 rats; BSO + clonidine, n = 8 slices from 7 rats. H, Mean changes in lumen diameter evoked by PgE₂ and NE. PgE₂, n = 5 slices from 4 rats; BSO + PgE₂, n = 3 slices from 3 rats; NE, n = 14 slices from 11 rats; BSO + NE, n = 8 slices from 7 rats. Data are mean ± SEM. **p < 0.01. ***p < 0.001. n, number of experiments conducted or, for calcium measurements, number of astrocyte ROIs analyzed.

Figure 3.

Astrocytes express mPGES-1 and contain high levels of GSH. A, Immunohistochemistry showing astrocytic expression of GSH-dependent mPGES-1 in the CA3 of the hippocampus. Astrocyte marker, GFAP (red), mPGES-1 (green), and merge (yellow). Scale bar, 20 μm. B, MCB-loaded hippocampal-neocortical slices. Astrocytes (identified by SR101, red, white arrowheads) contain higher levels of GSH (as indicated by MCB staining, green) than neurons (white arrows). Merge (yellow). Scale bar, 20 μm.

Figure 4.

Astrocyte [Ca²⁺]_i transient-evoked vasodilations are GSH dependent in vitro. A, Mean IP₃-evoked increases in astrocyte [Ca²⁺]_i. Control, n = 21 from 6 rats; +BSO, n = 11 from 4 rats. B, Mean time course of increase in astrocyte [Ca²⁺]_i. Dotted line indicates time of photolysis of caged IP₃. n as described in A. C, Mean lumen diameter change in response to uncaging of IP_3. Uncage IP₃, n = 11 slices from 6 rats; +BSO, n = 6 slices from 4 rats. Data are mean ± SEM. **p < 0.01. n, number of experiments conducted or, for calcium measurements, number of astrocyte ROIs analyzed.

Figure 5.

CO₂ evoked CBF responses in vivo are GSH dependent. A, Mean traces of local CBF response to hypercapnia, measured by laser speckle contrast imaging, in vehicle (DMSO)- (blue) and SC560- (red) injected animals. n = 7 rats for each group. Colored box represents time of CO₂ application. Data shown as fractional change with baseline of 0 (baseline taken during 60 s prechallenge) and a pretreatment peak of 1 (black dotted line on graph). B, Mean AUC of CBF response to hypercapnia in the presence of vehicle (DMSO) or SC560 (normalized to pretreatment maxima for each animal). n = 7 rats for each group. C, Tissue GSH levels 24 h after injection of BSO or saline into the barrel cortex (n = 7 rats). D, Mean trace of local CBF response to hypercapnia, measured by laser Doppler flowmetry, in saline- (blue) and BSO- (red) injected rats. n = 6 rats in each group. E, Mean values of AUC of CBF response to hypercapnia. n = 6 rats in each group. F, G, Neural activity. Power in frequency bands. F, During baseline (Base) and in response to hypercapnia (HCN) for saline- (blue) and BSO- (red) treated animals. n = 3 rats. G, Hypercapnia (HCN)/baseline (Base). Treatment with BSO does not change the effect of hypercapnia on neural activity. n = 3 rats. Data are mean ± SEM. * p < 0.05.

Figure 6.

CBF responses to whisker pad stimulation in vivo are independent of GSH. A, Mean time course of local CBF response to whisker pad stimulation, measured by laser speckle contrast imaging, in vehicle (DMSO)- (blue) and SC560- (red) injected rats. Colored box represents time of stimulation. Dotted black line indicates pretreatment peak of 1. B, Mean AUC of the CBF response to whisker pad stimulation. n = 7 rats for each group. C, Mean neural response (LFP) magnitude to whisker pad stimulus (summed over total 16 s length of stimulus). Responses are normalized to the first pulse response for each rat. n = 4 DMSO-treated rats; n = 3 SC560-treated rats. D, Mean AUC of the whisker pad stimulation-evoked CBF response in saline- (blue) and BSO- (red) injected rats. n = 10 rats for each group. E, Mean neural response (LFP) magnitude to whisker pad stimulation (summed over total 16 s length of stimulus). Responses are normalized to the first pulse response for each rat. n = 3 rats in each group. Data are mean ± SEM.

Figure 7.

Increases in astrocytic [Ca²⁺]_i may lead to GSH-dependent, PgE₂-mediated vasodilation. Schematic diagram depicting how CO₂-evoked increases in astrocytic [Ca²⁺]_i may lead to PgE₂-mediated vasodilation. As a result of elevated [Ca²⁺]_i, PLA₂ is activated. PLA₂ generates AA from the plasma membrane. AA can be processed locally by COX enzymes to produce AA derivatives, such as prostaglandin H₂ (PgH₂). PgE₂ is produced from PgH₂ by the enzyme PGEs, which requires GSH as a cofactor (Jakobsson et al., 1999; Murakami et al., 2000; Tanioka et al., 2000). PgE₂ is released from astrocyte endfeet, which are apposed to the smooth muscle layer surrounding arterioles, resulting in activation of K⁺ channels, a decrease in Ca²⁺ entry into the smooth muscle cell and vasodilation.