High-resolution in vivo imaging of the neurovascular unit during spreading depression

Abstract

Spreading depression (SD) is a propagating wave of neuronal depolarization and ionic shifts, seen in stroke and migraine. In vitro, SD is associated with astrocytic [Ca2+] waves, but it is unclear what role they play and whether they influence cerebral blood flow, which is altered in SD. Here we show that SD in vivo is associated with [Ca2+] waves in astrocytes and neurons and with constriction of intracortical arterioles severe enough to result in arrest of capillary perfusion. The vasoconstriction is correlated with fast astrocytic [Ca2+] waves and is inhibited when they are reduced. [Ca2+] waves appear in neurons before astrocytes, and inhibition of astrocytic [Ca2+] waves does not depress SD propagation. This suggests that astrocytes do not drive SD propagation but are responsible for the hemodynamic failure seen deep in the cortex. Similar waves occur in anoxic depolarizations (AD), supporting the notion that SD and AD are related processes.

Figure 1.

SD-associated [Ca²⁺] waves in astrocytes in vivo. Astrocytes were labeled with a combination of Fluo-4 (a) and SR-101 (b; merged in c). Infusion of FITC dextran labeled the vasculature (d). Scale bar, 50 μm. An SD wave is shown in e; the wave front is indicated by the black line. Time stamps are in seconds. Scale bar, 100 μm. The change in astrocytic [Ca²⁺] is plotted in f (mean ± SEM over 13 astrocytes). g, Spontaneous waves before and after the needle prick differed from both slow and fast SD-associated waves amplitude, duration, and rise time.

Figure 2.

SD-associated [Ca²⁺] waves in neurons in vivo. Astrocytes and neurons were labeled with OGB-1 (a), and astrocytes were also labeled with SR-101 (b). Green and red channels are merged in c; note that neurons are labeled only with OGB-1, whereas astrocytes are double labeled as in Figure 1, permitting their identification. Scale bar, 100 μm. Astrocytic [Ca²⁺] waves plotted by velocity of propagation showed a broad distribution with slow (<10 μm/s) and faster (>15 μm/s) waves (d), whereas the neuronal distribution included no waves slower than 15 μm/s (e). Arrowheads indicate the mean velocity of waves associated with vasoconstrictions. Line-scan experiments of nearby astrocytes (f; A1, A2) and neurons (N1, N2) showed that the [Ca²⁺] wave usually occurred first in neurons. Scale bar, 20 μm. g, Raw line-scan data for the four cells indicated in f. h, Same data low-pass filtered to show onsets of the relatively slow waves. i, Summary data for 20 neuron–astrocyte pairs. The lag is expressed as the time when the neuronal transient was detected minus the time when the astrocytic transient was detected (in seconds). Note that the majority of lags were <500 ms in either direction.

Figure 3.

Vascular dynamics in SD. Penetrating intracortical arteries constricted dramatically during SD (a; numbers indicate time points marked in b). a, b, Cerebral vessels were imaged by the intravascular introduction of FITC dextran, enabling measurement of changes in vascular diameter as well as in capillary perfusion. Scale bar, 20 μm. b, Correlation of DC potential with vasoconstriction, and astrocytic and neuronal [Ca²⁺] waves. Note the slow astrocytic [Ca²⁺] wave colliding with the fast wave in c that was not associated with vascular change. In this example, a DC potential was recorded with a subdural electrode. c, Slow astrocytic [Ca²⁺] waves (arrowhead) were not associated with vasoconstriction, whereas fast ones (arrow) were. This example was taken from a different animal from that shown in a and b. d, Pial arterioles showed a biphasic response, with initial constriction followed by a long-lasting dilation. Intracortical arterioles (n = 29 from 15 animals) showed only a constriction that returned to baseline. As with intracortical arterioles, the onset of pial vasoconstriction was spatiotemporally correlated with the calcium wave. e, Peak diameter change varied with vessel location and caliber. Pial arteries showed constriction and dilation that were inversely related to caliber, whereas small intracortical arteries notably lacked a considerable dilatory response, in contrast to comparably sized pial arterioles (n = 6 from 4 animals). Constriction of large pial arteries (50–100 μm; n = 5 from 4 animals) was significantly smaller than that in other arteries, and intracortical small arteriolar dilation differed significantly from dilation of small (10–25 μm) and medium (25–50 μm) pial arteries (n = 8 from 3 animals). *p < 0.05; **p < 0.001.

Figure 4.

Capillary blood flow dynamics in SD. Capillary blood flow slowed dramatically (a) and occasionally ceased completely (b, c) in SD. a, Line scans taken at the time points indicated by the arrowheads. Note the rebound increase in blood flow after SD (2 rightmost scans). Velocity (b) and flux (c) were fairly stable in baseline and then, in this example, dropped to 0 during SD. DC recording (intracortical electrode 300 μm from imaging site) is shown in d. e, Distribution of duration of capillary flow cessation in cases in which flow stopped completely. Duration of blood flow stop was negatively correlated with the initial blood cell velocity (f) and flux (g).

Figure 5.

Thapsigargin selectively reduced astrocytic [Ca²⁺] waves and blunted SD-associated vasoconstriction. Fast astrocytic [Ca²⁺] waves were reduced in amplitude (a; *p ≪ 0.001), duration (b; *p = 0.007), and rise time (c; *p ≪ 0.001) in the presence of thapsigargin (n = 50 astrocytes from 3 animals). Neuronal [Ca²⁺] waves were unaffected (n = 36 neurons from 3 animals). In contrast, TTX had no effect on astrocytic waves but increased the duration of neuronal [Ca²⁺] waves (n = 19 astrocytes and n = 15 neurons from 3 animals). Tetanus toxin had no effect on astrocytic or neuronal [Ca²⁺] wave amplitude but prolonged neuronal [Ca²⁺] waves and slowed the rise time of astrocytic and neuronal [Ca²⁺] waves (n = 49 astrocytes and n = 56 neurons from 3 animals). C, Control; Th, thapsigargin; TnT, tetanus toxin. d, e, Astrocytic (red) and neuronal (blue) [Ca²⁺] waves and vasoconstriction (green) in the absence (d) and presence (e) of thapsigargin. f, g, SD-associated vasoconstriction was not mediated by neuronal depolarization or direct vascular depolarization by K⁺. f, KCl application via a micropipette (filled with SR-101; dotted lines) constricted arterioles (arrow) in both the presence and absence of thapsigargin, in contrast to SD, whose vasoconstriction was reduced by thapsigargin. Scale bar, 20 μm. g, Summary data of SD and KCl vasoconstriction in the absence (n = 26 arteries from 12 animals and n = 9 arteries from 3 animals, respectively) and presence (n = 12 arteries from 4 animals and n = 8 arteries from 3 animals, respectively) of thapsigargin (*p = 0.009). h, SD-associated vasoconstriction (thick line) is inhibited by topically applied MAFP (thin line). i, Summary data of SD-associated vasoconstriction in the absence and presence of MAFP. (*p ≪ 0.0001; n = 29 arterioles in 15 animals and n = 10 arterioles in 3 animals, respectively)

Figure 6.

Astrocytic (a, c) and neuronal (b, d) [Ca²⁺] waves in AD (a, b) and SD (c, d). AD-associated [Ca²⁺] waves in both astrocytes and neurons had a greater amplitude (e; *p ≪ 0.0001) and duration (f; *p ≪ 0.0001, respectively) and a slower rise time (*p ≪ 0.0001) than SD-associated [Ca²⁺] waves (n = 31 astrocytes and n = 25 neurons from 3 animals).