Collection and analysis of OIS data. (A) Preparation schematic. Broad spectrum white LED light illuminated the thin skull mouse preparation. Reflected light was split between the camera and spectroscope. (B) Determination of haemoglobin content by the Beer–Lambert law. Second derivatives of measured absorbences were fit as a linear combination of empirically derived oxyhaemoglobin (HbO₂) and deoxyhaemoglobin (Hb⁻) second-derivative absorbance standards. Conventional absorbences are on top; second derivative traces are on bottom. Haemoglobin saturation, and total haemoglobin were estimated from this fit. Since the haemoglobin spectrum displays high curvature in the region between 530–610 nm, fitting the second derivative absorbance allowed us to minimize the contribution due to background absorbers.

Craniotomy alters vascular characteristics of CSD. (A) Images show changes in OIS reflectance under white light in mouse craniotomy preparation during passage of a CSD wave. OIS signal increases (blood volume decreases) in regions outside of the craniotomy window but decreases (blood volume increases) in region of exposed cortex. White labels are time in seconds. (B) OIS traces of (1) mouse region of interest outside craniotomy, (2) mouse region of interest inside craniotomy and rat region of interest inside craniotomy (images not shown). In the same mouse preparation, OIS traces show significant differences based on whether the cortex is exposed or not. Interestingly, the mouse craniotomy trace, though longer in duration, has a similar shape to rat craniotomy trace. Signal changes were too small outside rat craniotomy to allow measurement. Y-axis: OIS change in reflectance from baseline. (C) Contrasting arterial diameter changes in thin skull (above) and craniotomy (below) preparations in a separate experiment. Arterial constriction is attenuated in craniotomy region, but not outside it. (D) Hypoperfusion following passage of the CSD wave is preserved in craniotomy preparations even when the hypoperfusion during the wave is lost. This suggests that the mechanisms of hypoperfusion during the wave and following its passage are different (see Discussion section). (E) Despite attenuated CSD-associated arterial reactivity, evoked OIS maps are intact in craniotomy preparations. Panels show craniotomy preparation under 617 nm LED light before and during 50 Hz 1 s contralateral hind-paw stimulation (see Methods section for details). Light (617 nm) was used to optimize mapping, but maps under white light are similar. Note the difference in colour scale from the panels in A. CSD associated changes are at least two orders of magnitude larger than sensory mapping changes.

CSD causes haemoglobin saturation (SatO₂) to drop to levels seen in ischaemic cortex. (A) Measuring SatO₂ in superior branch middle cerebral artery infarct. Spectral regions of interest (circles) shown dispersed throughout middle cerebral artery infarct preparation window, with superior division middle cerebral artery cauterized just outside the imaging window (bottom centre). A sharp border is visible in OIS images (dotted line). SatO₂ was lower than 27% in the region >500 μm proximal to the OIS border (closest to the cauterized middle cerebral artery; solid white circles) and was <40% within 500 μm of the border (dotted circles). Saturation was >40% (but not necessarily normal) in regions of interest >500 μm distal to the border. (B and C) Comparing SatO₂ in CSD to SatO₂ in middle cerebral artery infarct. Scatterplot shows that following middle cerebral artery infarct, SatO₂ depends linearly upon distance from ischaemic core. Desaturation in regions closest to the infarct locus is the most severe. Boxplots of observed minimum SatO₂ values in CSD are overlaid on scatterplots of middle cerebral artery infarct SatO₂, to gauge the severity of the CSD related desaturation relative to middle cerebral artery infarct related desaturation. (B) Results from long-term recordings where the animal was given 90 min to recover completely between CSD inductions. Note that both the first desaturation, associated with the CSD wave (1) and the second desaturation, after passage of the wave (2), were in an ischaemic range. (C) CSD-associated desaturations for repetitive CSD separated by 30 min intervals (incomplete recovery; see Fig. 7). The saturation drop was smaller with subsequent CSDs, though still in an ischaemic range. This was not the case with complete recovery, where no difference in saturation drop between CSD episodes was seen. (D) Upon nitrogen asphyxia, SatO₂ dropped to zero while [Hb_tot] increased, showing that SatO₂ and [Hb_tot], which generally move in the same direction, were effectively distinguished by our spectroscopic techniques. (E) Magnitude of changes in the pial artery diameter (PAD), [Hb_tot] and SatO₂ (rectified normalized traces averaged over 50 s) for each of three time periods: pre-CSD baseline (BL), DC shift (DC) and end of experiment (END). During DC shift, arterial diameter and SatO₂ (both unlikely to be affected by cell swelling—see Methods section) undergo overall changes of ∼10% whereas [Hb_tot] change (likely to be affected by cell swelling) is significantly attenuated at ∼2%. This shows that relative spectroscopic measures such as SatO₂ may be more reliable indicators of perfusion than absolute measures like [Hb_tot] during cortical events involving significant tissue swelling. Vertical scale is in arbitrary units. (F) [Hb_tot] gives insight into optical pathlength changes caused by tissue swelling. The ratio of [Hb_tot] to arterial diameter gives an approximation of mean optical pathlength. This ratio peaks during the CSD wave and is slightly attenuated after passage of the wave, before returning to baseline. Change in pathlength is indicative of change in refractive properties of the tissue, including changes in cellular volume and thus light scatter. The duration of these changes agrees with invasive measures of tissue swelling (see Methods). Dotted lines show 95% confidence intervals. Vertical scale is in arbitrary units.

Biphasic tissue response to CSD. (A) Physiological measurements over a long time scale following CSD induction by tetanic stimulation in a representative experiment. Note the distinct biphasic response in each measure. Additional phases [most prominent on the pial artery diameter (PAD) trace and driven by arterial constriction and dilation] could sometimes be seen superimposed on the long second phase (arrow on pial artery diameter trace; Supplementary Fig. S1). Small arrows above OIS trace indicate thresholding steps, which cause decreases in OIS signal due to cortical activation. Similar changes can be seen on other traces. Large arrow under OIS trace indicates when CSD wave starts. Field potential is in millivolts, cortical haemoglobin saturation (SatO₂) in percent. [Hb_tot], pial arterial diameter and OIS reflectance are normalized to pre-CSD baseline. Inset of 20 s samples of field potential at selected time points to show: (1) baseline burst-suppression, (2) return of spontaneous activity after DC shift, (3) return of irregular bursting activity with intervals of quiescence and (4) burst-suppression at recovered baseline. Horizontal scale bar = 5 s; vertical scale bar = 0.5 mV. (B) Empirical distribution function [1-Survival(t)] plots for total CSD recovery time. Recovery time is total time (in min) elapsed from the beginning of the DC shift—including both phases of neurovascular dysfunction. To account for experiments where recovery was not observed, the Kaplan–Meier estimator was used. Dashed dark lines show boundaries for 95% confidence intervals of the empirical cumulative distribution function. Dashed grey lines intersect the expected median recovery times.

Haemoglobin desaturation during CSD wave is due to coincident arterial constriction and cortical depolarization. (A) Field potential (FP), SatO₂, [Hb_tot], pial artery diameter (PAD) and OIS reflectance changes during the CSD wave. The size of the haemoglobin desaturation is likely to be due to the simultaneous occurrence of a metabolically expensive depolarization with a paradoxical reduction in blood supply. Field potential is shift from baseline in mV. OIS, [Hb_tot] and arterial diameter are normalized to pre-CSD baseline. SatO₂ is percent oxyhaemoglobin. Labelled time points correspond to images in panel B. (B) Raw OIS images (above) and binarized images used for measurement of arterial diameter (below): (1) immediately before CSD, (2) at maximal constriction during the CSD wave and (3) at maximal dilation. Circle: spectroscopic and intrinsic signal region of interest. Square: section of surface artery selected for analysis. (C) Raw tissue absorbance at time points indicated in (A). Experimentally derived absorbance traces for oxyhaemoglobin (solid) and deoxyhaemoglobin (dotted) are included for reference. Note that absorbance is dominated by oxyhaemoglobin at baseline and during the brief recovery after passage of the CSD wave, but is closer to the pure deoxyhaemoglobin spectrum during passage of the wave.

Neurovascular uncoupling after passage of the CSD wave. (A) Electro-physiological activity returns well before the haemodynamic signal and recovery begins only after the haemodynamic response is re-established. Four representative long-term experiments (coloured) and average trace (black). Upper traces show OIS reflectance, lower traces show haemoglobin saturation, beginning after the end of the first DC shift. H-shaped bars mark the time period when significant EEG activity (defined as discharges at 1–2 root mean square of baseline discharge amplitude) returns, and straight bars mark the time period when OIS oscillations (1–2 root mean square) return, for each experiment. In the period after the first DC shift, bursting activity returns well before oscillations in OIS signal. Note that the second desaturation peaks after significant EEG bursting activity returned, and recovery of saturation occurs only after significant oscillations return in the OIS (and saturation) signal. (B) OIS oscillatory response (orange; corresponding to orange trace in A) to EEG bursts (black) shows changes in neurovascular coupling. OIS activity was impaired 7 min after CSD (bursts of this amplitude and duration normally are associated with an OIS response, but are not here), showed some recovery at 24 min, and largely returned by 34 min. Vertical scale bar: 2% OIS change, 1.2 mV field potential. The small regular upward deflections during suppression periods (between bursts) are respiratory artefact. (C) EEG-power-weighted coherence across all long-term experiments (n = 8 animals, 17 CSDs). Dashed line represents the 95% confidence boundary. Please see Methods section and Supplementary Fig. S3 for how coherence was calculated.

Reduced haemodynamic response but preserved electro-physiological response when CSD is elicited at short intervals. (A) Haemoglobin saturation, arterial diameter and DC field potential for three CSD waves elicited at 30 min intervals (i.e. before complete recovery from the prior event). The magnitude and rate of desaturation and vascular constriction decreases with subsequent CSDs, but the size and kinetics of the DC shift do not change. SatO₂ values are shown in percent saturation. Pial artery diameter measurements are normalized to pre-CSD baseline. Note that baseline saturation is similar in all three experiments, while in Fig. 4 saturation 30 min after CSD is significantly lower than baseline. This is because the tetanic electrical stimulation used to induce CSD causes a blood volume, arterial diameter and saturation increase over the whole field. The fact that the saturation response is attenuated despite this ‘normalization’ to pre-CSD baseline values points to a specific change in vascular reactivity. (B) Tukey box and whisker plots showing trends in magnitude (top row) and rate (bottom row) of the change of haemoglobin saturation, pial artery diameter and DC field potential across each experiment. While there was a decrement in the size and speed of haemoglobin desaturation and arterial constriction across each subsequent CSD, there was no such change in DC shift measures; n = 20 animals, 60 CSDs. Tukey 95% family-wise contrasts: magnitude of desaturation was 8.44 (^†CI: 4.60–12.29) greater in first CSD versus second, 4.89 (^†CI: 1.03–8.74) greater in second versus third; rate of desaturation was 0.37/s (^†CI: 0.02–0.72/s) greater in first versus second, 0.18/sec (^†CI: 0.17–0.53/s) greater is second versus third. Arterial constriction was 13% greater in first versus second (^†CI: 3.26–23.23%), 2.99% (CI: −6.99 to 12.98%) greater in second versus third; rate of constriction was 1.44%/s (^†CI: 0.37–2.42%/s) greater in first CSD versus second, 0.22%/s (CI: −0.86 to 1.29%/s) greater in second versus third. ^†Denotes statistically significant difference in means.

Extracellular K⁺ does not affect perfusion after passage of the CSD wave. (A) Extracellular K⁺ concentration [K⁺] increases to over 30 mM while surface vessels constrict and OIS signal increases during the CSD wave. (B) After passage of the CSD wave, extracellular K⁺ concentration levels returns to baseline even though other measures are disturbed, showing that extracellular K⁺ concentration is not involved in the second phase of CSD-associated neurovascular derangement. (C) During CSD waves with incomplete recovery, the rise time of extracellular K⁺ concentration increases, as does the time to recover to baseline values.