A JOURNAL OF NEUROLOGY
Biphasic direct current shift, haemoglobin
desaturation and neurovascular uncoupling in
cortical spreading depression
Joshua C. Chang,1,2,?Lydia L. Shook,1,?Jonathan Biag,3,?Elaine N. Nguyen,1Arthur W. Toga,3
Andrew C. Charles1and Kevin C. Brennan1,3
1 Headache Research and Treatment Program, Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
2 Department of Biomathematics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
3 Laboratory of Neuro Imaging, Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
?These authors contributed equally to this work.
Correspondence to: K. C. Brennan,
635 Charles E. Young Drive South,
Neuroscience Research Building 1,
Cortical spreading depression is a propagating wave of depolarization that plays important roles in migraine, stroke, subar-
achnoid haemorrhage and brain injury. Cortical spreading depression is associated with profound vascular changes that may be
a significant factor in the clinical response to cortical spreading depression events. We used a combination of optical intrinsic
signal imaging, electro-physiology, potassium sensitive electrodes and spectroscopy to investigate neurovascular changes asso-
ciated with cortical spreading depression in the mouse. We identified two distinct phases of altered neurovascular function, one
during the propagating cortical spreading depression wave and a second much longer phase after passage of the wave. The
direct current shift associated with the cortical spreading depression wave was accompanied by marked arterial constriction and
desaturation of cortical haemoglobin. After recovery from the initial cortical spreading depression wave, we observed a second
phase of prolonged, negative direct current shift, arterial constriction and haemoglobin desaturation, lasting at least an hour.
Persistent disruption of neurovascular coupling was demonstrated by a loss of coherence between electro-physiological activity
and perfusion. Extracellular potassium concentration increased during the cortical spreading depression wave, but recovered and
remained at baseline after passage of the wave, consistent with different mechanisms underlying the first and second phases of
neurovascular dysfunction. These findings indicate that cortical spreading depression is associated with a multiphasic alteration
in neurovascular function, including a novel second direct current shift accompanied by arterial constriction and decrease in
tissue oxygen supply, that is temporally and mechanistically distinct from the initial propagated cortical spreading depression
wave. Vascular/metabolic uncoupling with cortical spreading depression may have important clinical consequences, and the
different phases of dysfunction may represent separate therapeutic targets in the disorders where cortical spreading depression
Keywords: spreading depression; haemoglobin; neurovascular coupling; migraine; stroke
doi:10.1093/brain/awp338Brain 2010: 133; 996–1012 |
Received August 27, 2009. Revised November 23, 2009. Accepted December 13, 2009
? The Author (2010). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: firstname.lastname@example.org
Abbreviations: CSD=cortical spreading depression; DC=direct current; [Hbtot]=total haemoglobin; K+=potassium ion;
OIS=optical intrinsic signal; SatO2=haemoglobin oxygen saturation
Cortical spreading depression (CSD) is a massive spreading depo-
larization of neurons and glia (Leao, 1944; Somjen, 2001). CSD is
thought to underlie the migraine aura, and functional imaging has
shown propagated events consistent with CSD in migraine patients
(Olesen et al., 1981; Hadjikhani et al., 2001). Related depo-
larizations occur in humans around infarcts, cortical trauma and
subarachnoid haemorrhage (Mayevsky et al., 1996; Strong et al.,
2002; Dohmen et al., 2008). CSD, once considered an experimen-
tal curiosity, is increasingly recognized as an element of human
disease and a target for treatment.
Large changes in perfusion (blood flow, blood volume or arterial
diameter) accompany the electro-cortical activity of CSD. Most
prominent is a brief increase in perfusion, followed by a
long-lasting hypoperfusion (Lauritzen et al., 1982; Busija et al.,
2008). These changes were initially interpreted as reflecting
normal neurovascular coupling, with perfusion increases to meet
the needs of depolarization, and decreases afterward during the
relative quiescence (‘depression’) of cortical activity. However,
paradoxical reductions in perfusion, opposite to what would be
expected from normal neurovascular coupling, may occur dur-
ing the CSD wave in multiple species (Ayata et al., 2004;
Tomita et al., 2005; Osada et al., 2006), including humans
(Dreier et al., 2009). Changes in arterial carbon dioxide reactivity
(Lauritzen et al., 1982, 1984; Scheckenbach et al., 2006) and in
physiologically induced blood volume responses (Guiou et al.,
2005; Piilgaard and Lauritzen, 2009) show that long-lasting
changes in neurovascular coupling can follow the CSD wave.
The CSD perfusion response varies with the physiological state
of the organism: decreasing oxygenation or blood pressure,
increasing extracellular potassium ion (K+) concentration, or inhi-
biting nitric oxide synthesis can enhance CSD-associated perfusion
decreases (Dreier et al., 1998; Sukhotinsky et al., 2008).
Peri-infarct depolarizations, which are electro-physiologically indis-
tinguishable from CSD (Cze ´h et al., 1993) show a more prominent
constrictive vascular response than CSD in normal tissue (Shin
et al., 2006; Strong et al., 2007). The perfusion response is also
variable across species; mice appear to have a more pronounced
hypoperfusion and less prominent hyperaemia than other animals
(Ayata et al., 2004; Brennan et al., 2007). Finally, the perfusion
response varies with the number of CSD events. In animals under-
going repetitive CSD, the perfusion response is attenuated, despite
similar cortical depolarization (Brennan et al., 2007). These data
suggest that the coupling of neural activity to blood flow during
CSD functions on a physiological gradient or ‘sliding scale’ and
that the conditions in which CSD occurs may thus have profound
effects on the tissue outcome.
CSD is clearly an event outside the bounds of normal physiol-
ogy, but the extent to which it is pathological has been debated
(Nedergaard and Hansen, 1988; Gorji, 2001; Somjen, 2001, 2006).
Recent evidence suggests that under certain circumstances it can be
harmful. CSD has been directly recorded from humans with brain
injury and stroke (Mayevsky et al., 1996; Strong et al., 2002;
Dohmen et al., 2008) and in animal models, CSD around infarct
is correlated with decreased perfusion and increased stroke size
(Mies et al., 1993; Strong et al., 2007). It is also possible that a
deleterious effect of CSD could be involved in the higher rate of
stroke in patients with migraine with aura (Kruit et al., 2004;
Etminan et al., 2005; Scher et al., 2009; Schu ¨rks et al., 2009).
In this study, we use optical intrinsic signal (OIS) imaging, opti-
cal spectroscopy, electro-physiology and K+sensitive electrodes to
investigate both short and longer term physiological changes
during CSD in mouse. We show a significant biphasic change in
direct current (DC) field potential, perfusion and haemoglobin sat-
uration: an initial phase occurring during the CSD wave and a
second prolonged phase that begins after recovery from the first
phase. Though they involve similar changes—negative DC shift,
arterial constriction, haemoglobin desaturation and altered neuro-
vascular coupling—we show that these phases of CSD-associated
disruption are mechanistically distinct.
Male and female C57Bl/6J mice (45 total mice, weight 22–35g) and
one male Sprague–Dawley rat (weight 350g) were used for experi-
ments, in accordance with the University of California, Los Angeles
Animal Research Committee Guidelines. Temperature was maintained
at 37.0?0.7?C with a rectal temperature probe and homoeothermic
blanket. Anaesthesia was induced with isoflurane (5%) in a 2:1 nitro-
gen:oxygen mixture and adjusted (1.0–1.6%) to maintain a respiratory
rate of ?80–120, heart rate of ?440–530 beats/min and field poten-
tial burst-suppression with an interburst interval of 2–7s. In selected
experiments, heart rate, pulse oxygenation and respiratory rate were
monitored using a pulse oximeter (three animals with Nonin 8600V,
Nonin Inc., Minneapolis, MN, USA and four animals with MouseOx,
Starr Life Sciences Corp., Oakmont, PA, USA). Blood pressure was also
monitored in seven experiments by cannulating the femoral artery
(Blood Pressure Display Unit, Stoelting, Wood Dale, IL, USA).
Arterial blood gas samples were not taken due to the duration of
the experiments. In two animals each, anaesthesia was changed to a
combination of low dose isoflurane (0.6–0.8%) and chlorprothixene
(3.3mg/kg); or urethane (0.75g/kg).
Mice were placed in a stereotaxic frame (Kopf Instruments, Tujunga,
CA, USA). The skull was exposed and a rectangular section of the
parietal bone (1mm from the sagittal suture, temporal ridge, lambdoi-
dal suture and coronal suture; Fig. 1A) was thinned to transparency.
Burrholes were placed (i) 0.5mm, antero-medial to lambda; (ii) 0.5mm
from the temporal ridge mid-way between bregma and lambda; and
(iii) 0.5mm anterior to the occipital suture. A glass recording electrode
(0.5MX resistance) filled with artificial CSF (in mM: 125 NaCl, 3 KCl,
1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 25 NaHCO3, 11 glucose; all chem-
icals from Sigma-Aldrich, St Louis, MO, USA) was inserted into the
first burrhole and advanced 550mm into the cortex. In the second
Biphasic neurovascular dysfunction in spreading depressionBrain 2010: 133; 996–1012 |
burrhole, either a bipolar tungsten microelectrode (80kX resistance,
300mm tip diameters, spacing 200mm) was placed and advanced
550mm, or a 34 gauge fused silica micropipette filled with 1M KCl
and attached to a pneumatic pico-pump (PV830, World Precision
Instruments, Sarasota, FL, USA) was placed at the surface of the
cortex. A silver–silver chloride ground wire was inserted into the cer-
ebellum in the third burrhole. A thin film of silicone oil covered the
skull to reduce specular artefacts and preserve bone transparency.
The animal rested under anaesthesia for at least 1h after electrode
placement. To ensure a constant anaesthetic environment, depth was
adjusted to maintain burst-suppression activity (2–7s interburst inter-
val) prior to each CSD induction. For tetanic stimulation, a stimulus
isolation unit (Model 2100, A-M Systems, Carlsborg, WA, USA)
generated a bipolar stimulus train at 200Hz, with a 200ms pulse
width, for 50s. Stimulus began at 50mA and was increased every
100s in fixed steps to a maximum of 1.6mA until CSD was elicited.
For KCl stimulation, increasing volumes of 1M KCl were ejected with
progressively increasing pressure (4–40psi, 0.11–1.1ml), at 25s
intervals, until CSD was achieved. Each animal rested for 30–90min
preceding each of three CSD inductions. At the conclusion of each
experiment, the animal was euthanized with anaesthetic overdose
and nitrogen asphyxia.
Middle cerebral artery infarct preparation
In two animals, after thin skull preparation, the skin and muscles cov-
ering the temporal bone were dissected. The suprasylvian branch of
the middle cerebral artery leading into the parietal bone window was
cauterized through a burrhole and the animal was allowed to rest for
1h before imaging.
Anaesthetic induction and skull exposure were performed as above.
In four animals, a suspended preparation rather than a stereotaxic
frame was used; the skull was affixed with cyanoacrylate glue to a
circular frame and suspended from this frame in order to eliminate
pressure from ear or zygoma bars. A cisternal puncture was per-
formed and a layer of cyanoacrylate glue was placed over the
exposed skull to minimize movement along suture planes. A dental
cement well was built up around the craniotomy region. The craniot-
omy was performed with steel or diamond burrs (Meisinger USA,
Centennial, CO, USA) under irrigation with chilled saline. The well
was either filled with artificial CSF or 3% agarose (Sigma-Aldrich)
dissolved in artificial CSF. The preparation was sealed with a coverslip.
Pipettes for induction of CSD and recording electrodes were carefully
advanced under the coverslip. Rat craniotomy was similar but did not
require suspended preparations; for details see Brennan et al. (2007).
In two animals, the contralateral hindpaw was stimulated (50Hz
square wave pulse trains through bipolar needle electrodes in the
proximal and distal walking pads, pulse duration 1ms, train duration
1s, amplitude 0.2–0.4mA) and evoked maps were recorded under
617nm LED light (10 trials, baseline subtracted and divided, trials
We found that both arterial and parenchymal responses to CSD
were changed by craniotomy (n=10) in the absence of any apparent
tissue or vascular injury associated with the craniotomy preparation.
Arterial constriction was either significantly attenuated or absent, and
the parenchymal response was characterized by much reduced hypo-
perfusion and more prominent hyperperfusion, quite similar to that
seen in rat craniotomy preparations (Fig. 2A and B). Although
CSD-associated arterial constriction was not always eliminated (4/10
experiments), when it occurred it only occurred with the first CSD (this
is contrast to what is observed in the thin skull preparation, in which
Figure 1 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 530–610nm, fitting the second derivative absorbance allowed us to minimize the contribution due to
Brain 2010: 133; 996–1012J. C. Chang et al.
multiple CSD events can be elicited without loss of constriction)
(Brennan et al., 2007; unpublished data). Despite the disruption of
CSD-associated arterial changes, we were able to obtain evoked fore-
paw and hindpaw OIS maps with this preparation (Fig. 2D). While
craniotomy markedly reduced the hypoperfusion associated with the
CSD wave, there was no apparent effect on the second phase of
sustained hypoperfusion that occurred after the CSD wave (n=3/3
long-term craniotomy experiments) (Fig. 2C). We concluded that our
craniotomy preparations were affecting CSD-associated surface vessel
reactivity and thus all further experiments were conducted using thin
Potassium concentration sensor preparations
Electrodes were constructed according to standard protocols (Lux and
Neher, 1973). Briefly, pulled capillary tubing (?20mm tip diameter)
was silanized, the tip filled with K+sensitive resin (Fluka Cocktail B,
Sigma-Aldrich) and the remainder filled with 100mM KCl, in contact
with an Ag/AgCl electrode. The electrode and an artificial CSF-filled
reference electrode were placed in a burrhole immediately adjacent to
a region of thinned skull. Because of space constraints, field potentials
were not recorded in these experiments. The K+electrode was cali-
brated prior to each experiment, and a response of 554mV/decade
was verified, or the electrode was discarded.
Figure 2 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 617nm LED light before and during 50Hz 1s contralateral hind-paw
stimulation (see Methods section for details). Light (617nm) 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
Biphasic neurovascular dysfunction in spreading depressionBrain 2010: 133; 996–1012 |
The cortex was illuminated with broad-spectrum white light (5500K;
400–800nm spectral range; Phillips Lumileds). Reflected light was
collected with a 4? microscope lens (UPlanSApo, Olympus, Melville,
NY, USA). A beamsplitter (half silvered glass, Edmund Optics,
Barrington, NJ, USA) passed one half of the reflected light to the
camera (902H2 Ultimate, Watec, Tsuruoka, Japan). The other half
was directed to the spectroscope probe (Fig. 1A). The camera field
of view was 3.2mm?2.4mm with a pixel size of 5mm. 8-bit images
were acquired at 1–2Hz, concurrent with electro-physiology, by a
custom LabView Virtual Instrument (National Instruments, Austin,
Field potentials were acquired continuously, amplified with a band pass
of 0–1kHz (A-M Systems 3000), digitized (PCI 6251, National
Instruments) and recorded simultaneously with optical data. Stability
of the DC record over the course of our experiments was crucial, so
experiments with40.2mV drift over 10min of baseline recording were
A fibre optic probe (R400-7-UV/VIS, Ocean Optics; Dunedin, FL,
USA) (225mm diameter), parfocal with the OIS camera (Fig. 1A),
was used to collect reflected light over a small spectral region of inter-
est on the cortical surface. X–Y translators allowed movement of the
region of interest to desired locations on the cortical surface. Regions
of interest were located at distances 0.5–3.0mm from the stimulating
electrode. A spectrometer (USB400-UV-VIS, Ocean Optics) collected
the probe output at 1Hz with a 1s integration time and 5nm boxcar
smoothing. One hundred percent oxygenated haemoglobin spectrum
was obtained by bubbling a 0.5% by volume solution of mouse blood
in pH 7.4 buffered saline with oxygen gas for 20min. Deoxygenated
haemoglobin spectrum was obtained by reducing the previous solution
with sodium hydrosulphite (10mg/ml; Sigma-Aldrich). Sodium hydro-
sulphite had no appreciable effect on absorbance within our wave-
The emittance spectrograph of the incident light, ?(?), was sampled
off a thin 20% Intralipid (Baxter Healthcare, Deerfield, IL, USA) emul-
sion spread over the surface of the imaging window. Reflectance, R(?),
was calculated as the proportion of the light reflected into the detector
at a given wavelength ?. For in vivo preparations, reflectance was
typically within the range of 15–75% for wavelength between 530
and 610nm, placing our measurements within the range of linearity
needed for application of the Beer–Lambert law. Designating reflected
light intensity as I(?), the absorbance was calculated as follows:
A(?)=?log R(?)=log ?(?)?log I(?).
Relative quantities of haemoglobin moieties were determined using a
second-derivative form of the Beer–Lambert law:
d2A ? ð Þ
d?2?HbO2? ð Þ þ Hb?
d?2?Hb?? ð Þð1Þ
where ?Hb?ð?Þ and ?HbO2ð?Þ are the experimentally determined
pathlength-normalized optical attenuation coefficients of unbound
haemoglobin (Hb?) and oxygen-bound haemoglobin (HbO2).
The second derivative method (Merrick and Pardue, 1986; Myers
et al., 2005) exploits the curvature of the haemoglobin spectra to
minimize contributions due to other absorbers and pathlength-
independent scatter. Second derivative spectra were calculated using
penalized fifth degree polynomial splines (Ripley, 2007) and fit to
the model specified by equation (1) at 397 equally spaced points
between 530and610nm. This
of [Hb?] and [HbO2], from which haemoglobin oxygen saturation
([Hb?]+[HbO2]=[Hbtot]) were calculated. Assuming constant haema-
tocrit, [Hbtot] measures the total blood volume within penetrating
depth of the light source. The half-width of the 95% confidence
interval for SatO2was ?3% (parametric bootstrap).
procedure allowed estimation
Validation of spectroscopic measures
To determine normoxic cortical SatO2in our thin skull preparation, we
sampled spectroscopic data from 14 mice preceding burrhole and elec-
trode placement. Mean SatO2was 66.6% (CI: 63.9–68.5). Pre-CSD
values were slightly lower—mean SatO2was 65% (CI: 63–67). Note
that tissue haemoglobin saturation is not equivalent to pulse oxygen-
ation, which measures only arterial haemoglobin saturation; our values
integrate arterial, capillary and venous haemoglobin saturation, and
are in agreement with other published measures of tissue haemoglobin
saturation (Benaron et al., 2004).
Superior branch middle cerebral artery infarct provided scaling for
CSD-induced SatO2changes. SatO2dropped notably in post-infarct
cortical tissue. Relative to a visible sharp border (Fig. 3A), the region
closest to the cauterized vessel (4500mm from the border) ranged in
saturation from 13.8% (CI: 10.6–17.0) to 26.6% (CI: 22.5–30.8), the
region surrounding the border (?500mm) ranged from 26.6%
(CI: 22.5–30.8) to 40.4% (CI: 37.7–43.1). Additional scaling was
performed with nitrogen asphyxia at the end of each experiment
(Fig. 3D). SatO2reached zero upon nitrogen asphyxia in all experi-
ments, confirming the validity of our measures. As SatO2decreased,
[Hbtot] increased sharply, revealing a physiological response to hypoxia
and confirming that SatO2 and [Hbtot] (normally closely associated)
were being measured independently.
CSD is associated with significant cellular swelling, which can alter
spectroscopic measures by changing the pathlength of light through
tissue (Kohl et al., 1998). In contrast to SatO2, a ratio measure that
intrinsically normalizes for pathlength changes, spectroscopic blood
volume ([Hbtot]) is susceptible to these changes (Fig. 3E). Though
this makes for inaccurate moiety measurements, it also provides
an indicator of tissue swelling if it can be referenced to a measure
unaffected by such changes. Pial arteries provide such a measure
because they rest on the cortical surface. The ratio of [Hbtot] to pial
artery diameter gives an approximation of mean optical pathlength
which corresponds to tissue swelling. While this ratio was stable at
baseline before CSD, the onset of CSD coincided with a marked
increase (Fig. 3F). The duration of these changes corresponded to
reported durations oftissue swelling
invasive methods (Hansen and Olsen, 1980; Mazel et al., 2002;
Takano et al., 2007), as well as different spectroscopic methods
(Kohl et al., 1998).
during CSD using more
Arterial diameter measurements
ImageJ (NIH; Bethesda, MD, USA) was used for all image analysis. To
measure arterial diameter, a square 225?225mm section of the OIS
imaging field was chosen according to the following criteria: (i) the
section contained only one artery of at least a 5 pixel diameter and (ii)
the section was within 1mm of the centre of the spectroscopic region
of interest. If no section of the image satisfied these conditions, the
experiment was rejected for analysis. Vessel discrimination was per-
formed by applying the IsoData threshold algorithm (Velasco, 1980).
Brain 2010: 133; 996–1012J. C. Chang et al.
This technique gave a binary map distinguishing the artery from back-
ground tissue and enabled a pixel count of cross-sectional arterial area,
proportional to arterial diameter. Threshold vessel diameters were in
close agreement with manual vessel diameter measurements (?2
pixels on average).
Time series analysis
For OIS analysis, the mean pixel value (OIS reflectance) was calculated
within a square surrounding our spectroscopic region of interest. CSD
can be characterized in terms of its two distinct phases: the first during
the spreading CSD wave and the second following its passage. Phase
duration was determined using cross-validated cubic spline-smoothed
traces (Ripley, 2007). In general, the time series were characterized by
a sharp deflection, followed by a much longer second deflection
before final recovery. First derivatives were used to identify local
extrema (minima/maxima). First phase amplitude (associated with
passage of the CSD wave) was measured between the first and
second extremum and duration was measured between the first and
For measurement of long-term changes, a physiological measure
was defined as having returned to baseline if its spline-interpolated
mean returned to the baseline median and remained within baseline
interquartile range. To account for unobserved recovery, the Kaplan–
Meier estimator (Armitage et al., 2002) was used to estimate the
empirical cumulative distribution function of the time elapsed until
return to baseline.
To quantify recovery of oscillatory activity after CSD, wideband OIS
and field potential data were high pass filtered at 0.5Hz and rectified
(Igor Pro 6, Wavemetrics, Oswego, OR, USA). Onset of EEG and OIS
activity after CSD was defined as the time point after which at least
3 bursts within a 50s period exceeded the root mean square (RMS)
value of a 50s pre-CSD baseline.
Figure 3 CSD causes haemoglobin saturation (SatO2) to drop to levels seen in ischaemic cortex. (A) Measuring SatO2in 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). SatO2was lower than 27% in the region4500mm proximal to the OIS border (closest to the cauterized middle
cerebral artery; solid white circles) and was540% within 500mm of the border (dotted circles). Saturation was440% (but not necessarily
normal) in regions of interest4500mm distal to the border. (B and C) Comparing SatO2in CSD to SatO2in middle cerebral artery infarct.
Scatterplot shows that following middle cerebral artery infarct, SatO2depends linearly upon distance from ischaemic core. Desaturation in
regions closest to the infarct locus is the most severe. Boxplots of observed minimum SatO2values 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 90min 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 30min 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, SatO2dropped to zero while [Hbtot]
increased, showing that SatO2and [Hbtot], which generally move in the same direction, were effectively distinguished by our spectroscopic
techniques. (E) Magnitude of changes in the pial artery diameter (PAD), [Hbtot] and SatO2(rectified normalized traces averaged over 50s)
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 [Hbtot] 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 [Hbtot] during cortical events involving significant tissue swelling.
Vertical scale is in arbitrary units. (F) [Hbtot] gives insight into optical pathlength changes caused by tissue swelling. The ratio of [Hbtot] 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 neurovascular dysfunction in spreading depressionBrain 2010: 133; 996–1012 |
Response kinetics for OIS and long-term changes in all measures
were modelled as first order linear time–invariate systems (Liptak,
2003), with time constant (?) defined as the time taken to rise to
63% or fall to 37% of maximum.
Wavelet coherence analysis for
Neurovascular coupling was determined based on the degree by which
oscillations in EEG activity corresponded with similar responses in the
OIS signal. The wavelet transform coherence (Torrence and Compo,
1998) finds regions in time–frequency space where two signals are
correlated. The Wavelet Coherence Toolkitfor MATLAB (Grinsted
et al., 2004) was used to calculate the continuous wavelet transform,
cross-wavelet transform and wavelet transform coherence.
In addition to the time–frequency representation of wavelet coher-
ence, a frequency-averaged coherence was calculated. Fisher trans-
formed (Fisher, 1915) coherence values were weighted by the
continuous wavelet transform spectral power. This approach is similar
to other approaches in literature (Porges et al., 1980) and measures
how well the OIS signal responds to EEG activity across all frequencies.
We present bias-adjusted estimates using non-parametric bootstraps
with 10000 replicates. Confidence intervals for correlations were com-
puted using the Fisher transformation. All confidence intervals for
these estimates are given as 95% bootstrap bias-corrected and accel-
erated (BCa) intervals (Efron and Tibshirani, 1986) unless otherwise
stated. Multiple group-wise comparisons and repeated measurement
comparisons were performed within a generalized linear regression
framework using generalized linear mixed effects modelling (Pinheiro
and Bates, 1999). Subject variability was controlled for by treating it as
a random effect (Laird and Ware, 1982). Post hoc pairwise multiple
comparisons were completed using Tukey’s honestly significant differ-
ences test while holding family wise type-I error to under ?=0.05.
Inferences on repeated measures over continuous time were made
by using bootstrap repeated measures spline regression (Tibshirani
and Knight, 1999). To determine continuous confidence bands, 1200
bootstrap resamplings of subjects were used, controlling for subject
wise effects. All statistical inference was performed using the
R Language and Environment for Statistical Computing (http://
Physiological variables are not affected
Continuous monitoring of blood pressure, heart rate, respiration
rate and pulse oximetry revealed spontaneous fluctuations within a
physiological range. CSD however had no noticeable effect on
observed in cortex did not have systemic correlates (Table 1).
Biphasic neurovascular response to CSD
Following CSD induction, we observed changes in all measures—
direct and alternating current field potential, cortical haemoglobin
saturation, spectroscopic blood volume [Hbtot], pial arterial diam-
eter and OIS—over two distinct phases (Fig. 4A). The first centred
around the acute spreading changes of CSD and lasted at most a
few minutes (Fig. 4A and Table 2). The second occurred after a
brief recovery following the passage of the wave, and was many
times longer (lasting at least 1h for all measures).
To estimate temporal alignment of changes, we placed the
spectroscopic region of interest directly over the field potential
electrode (n=6 animals, 15 CSDs). To the limits of our temporal
resolution, constrained by spectroscopy to 1Hz, spectroscopic, OIS
and field potential changes were concurrent (Fig. 5A). Arterial
constriction also appeared to coincide with CSD onset, but
because arteries were sampled outside the spectral region of inter-
est this could not be confirmed.
In order to verify that the biphasic changes we observed were
not due to anaesthetic depth or type of anaesthesia, we used
either an isoflurane/chlorprothixene combination (n=2 animals)
or urethane anaesthesia (n=2 animals). Both resulted in markedly
different EEG records (continuous activity rather than burst-
suppression) as compared with the standard isoflurane regime.
Similar biphasic changes in DC field potential, spectroscopic
measurements and OIS changes were observed with isoflurane/
chlorprothixene and urethane, despite different EEG activity
(Supplementary Fig. S2).
Neurovascular response during the CSD wave
There was a rapid and significant desaturation of cortical haemo-
globin coincident with the DC shift of the propagating CSD wave
(Figs 4 and 5, Table 2). This desaturation was sizable, involving a
halving of pre-CSD baseline values [pre-CSD baseline SatO265%
(CI: 63–67); minimum SatO2 35% (CI: 30–39); n=21 CSDs].
Putting the desaturation into physiological perspective, SatO2
during passage of the CSD wave dropped to levels seen after
middle cerebral artery infarct (infarct was performed in separate
experiments) (Fig. 3B and C). In 14 of 21 trials, SatO2dropped
to540% (equivalent to levels within 500mm of the infarct border
in stroke experiments) and the time spent below this threshold
ranged from2 to 86s(interquartile
In half of these trials (7/14), SatO2dropped to527% (equivalent
to levels4500mm inside the infarct border). Time spent under this
threshold ranged from 1 to 39s (interquartile range: 6.5–25s).
The similarity in magnitude to middle cerebral artery infarct sug-
gests that the desaturation associated with the CSD wave is phys-
The desaturation was not artefactual to stimulation. There was
no significant difference between desaturation in CSD elicited
by KCl ejection (n=12) or tetanic electrical stimulation (n=31).
Desaturation could be observed over the entire exposed region
Table 1 CSD has no effect on heart rate, respiratory rate,
pulse oximetry and blood pressure
Heart rate (b.p.m.)
Respiratory rate (breaths/min)
Pulse oximetry (% saturation)
Mean arterial pressure (mmHg) 69.9–99.8 70.2–99.5 70.6–99.1
97.9–99.0 97.8–99.1 97.8–99.1
Brain 2010: 133; 996–1012J. C. Chang et al.
and we detected no relationship between depth of desaturation
and distance from the CSD stimulus (r=0.07; CI: ?0.28 to +0.38).
Finally, if the animal was allowed to recover completely, there
was no significant difference in desaturation in the same
animal across multiple CSD inductions (this changed with incom-
plete CSD recovery; see below).
accompanied the haemoglobin desaturation (Figs 4A and 5). The
amplitude of desaturation during the CSD wave was positively
associated with the amplitudes of changes in haemodynamic vari-
decrease (r=0.69; CI: 0.40–0.83) and OIS increase (r=0.69;
Figure 4 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. [Hbtot], pial arterial diameter and OIS
reflectance are normalized to pre-CSD baseline. Inset of 20s 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=5s; vertical scale bar=0.5mV. (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.
Table 2 Amplitude and duration of CSD-associated measures
First phase (CSD wave)Second phase
Amplitude (95% CI)Duration (95% CI)Amplitude (95% CI) Duration (lower 95% CL)
?18.5mV (?19.7 to ?16.9)
?6.52mV (?7.16 to ?5.88)
a[Hbtot] value is likely to be affected by CSD-associated cellular swelling (see Methods section, Fig. 3E and F).
n=7 animals (21 CSD events). OIS mean pixel intensity maximum, pial arterial diameter minimum, haemoglobin saturation (SatO2) and spectroscopic blood volume
([Hbtot]) minimum are reported as percentage of pre-CSD baseline. Second phase durations are defined as the median times to persistent recovery from CSD induction,
determined by the Kaplan–Meier Estimator, with first phase duration subtracted; lower tail 95% confidence limit is given.
Biphasic neurovascular dysfunction in spreading depressionBrain 2010: 133; 996–1012 |
CI: 0.45–0.84). The amplitude of desaturation was also positively
associated with the duration (r=0.43; CI: 0.14–0.64), but not
the amplitude of the DC shift (r=0.25; CI: ?0.03 to +0.49).
Overall, both vascular supply (arterial diameter, [Hbtot], OIS reflec-
tance) and metabolic demand (approximated by DC shift) are
likely to contribute to haemoglobin desaturation during the CSD
Neurovascular response after the CSD wave
Continuous recording allowed investigation of DC field potential
both during and after an individual CSD event. After recovery
from the classic CSD-associated DC shift, there was a second
sustained DC shift (Fig. 4A, Table 2 and Supplementary Fig. S2).
Although smaller than the initial DC shift, this second change
non-CSD changes we have encountered (n4100 mice; data
The second phase of sustained DC shift was accompanied by
a second phase of reduction in SatO2. Although the SatO2drop
with the CSD wave was significantly larger (average difference
in saturation 4.0; lower CI: 0.1), SatO2 in the second phase
also dropped to ischaemic levels: all 14 trials that dropped below
SatO240% during the CSD wave also dropped below this level
in the second phase. Moreover, SatO2was depressed far longer
in the second phase, with time under SatO240%, ranging from
15s to 31min (interquartile range: 40s to 12.5min) (Fig. 4
and Table 2).
The magnitude of the second SatO2 drop was significantly
correlated with the magnitude of SatO2 drop during the CSD
wave (r=0.78; CI: 0.44–0.93). The minimum SatO2 during
each of these phases was also correlated (r=0.65; CI: 0.22–
0.87). This suggests that second phase saturation changes
might represent a response to the propagated wave of CSD.
Arterial diameter and DC field potential changes in general
paralleled SatO2 (Fig. 4 and Table 2), with smaller but long--
lasting changes in both variables compared to the CSD wave.
The only exception was [Hbtot], where the decrease was greater
in the second phase than the first. However, this was likely arte-
factual, due to optical pathlength changes caused by tissue swell-
ing during passage of the CSD wave (Fig. 3E and F, Table 2,
Methods section). There was also some variability in the arterial
response. Immediately after passage of the CSD wave, arterial
diameter often underwent a small constriction and dilation
(Fig. 4 and Supplementary Fig. S1) before the long-lasting
second constriction. However these changes did not appear to
affect the local perfusion state of the tissue as OIS, SatO2and
[Hbtot] remained relatively unchanged.
The second DC shift appeared to have different kinetics from
second phase haemodynamic changes (Fig. 5A). Duration of DC
field potential changes was similar to arterial diameter, [Hbtot],
SatO2, and OIS duration (Fig. 4A and Table 2). However minimum
levels were achieved much faster (Fig. 4A): mean time constant (?)
of the second DC shift was 65s (CI: 34–163s; n=9), compared
with: 7min (CI: 3–12min), 4min (CI: 2–8min), 5min (CI: 3–7min)
and 3.4min (CI: 2.5–5.5min), for arterial diameter, [Hbtot], SatO2
and OIS, respectively.
Neurovascular uncoupling following
passage of the CSD wave
Return of EEG oscillations precedes return of OIS
The baseline EEG record (alternating current field potential; high
passed at 0.5Hz) consisted of burst-suppression activity with a
mean burst frequency of 0.14Hz (CI: 0.12–0.16Hz; n=9). This
activity was attenuated during the first DC shift, as is classically
the case for EEG during CSD (Leao, 1944). However, it returned
to baseline level more quickly than other measures (Fig. 6A and
B). BurstingEEG activitybegan
50.2–186.3s) after the end of the first DC shift, and reached
equal amplitude to baseline bursts 7min (CI: 5–13min) after
the end of the first DC shift. In contrast, oscillations in whole
field OIS reflectance (which normally correlates with EEG during
anaverage of 99s(CI:
Figure 5 Haemoglobin desaturation during CSD wave is due to
coincident arterial constriction and cortical depolarization. (A)
Field potential (FP), SatO2, [Hbtot], 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 depolar-
ization with a paradoxical reduction in blood supply. Field
potential is shift from baseline in mV. OIS, [Hbtot] and arterial
diameter are normalized to pre-CSD baseline. SatO2is 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) imme-
diately 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.
Brain 2010: 133; 996–1012J. C. Chang et al.
burst-suppression), did not return to baseline amplitude for 17min
(CI: 15–19min) after the end of the first DC shift (Fig. 6A). During
the second phase of neurovascular dysfunction, burst frequency
significantly increased to an average of 0.25Hz (CI: 0.22–0.27Hz;
n=9) at the second DC nadir (typically 18–24min after the end of
the first DC shift). Then it gradually decreased, reaching an
average of 0.18Hz (CI: 0.16–0.21Hz), but not regaining its
original frequency by the end of the recording period.
Uncoupling of EEG and OIS activity
The beginning of the second SatO2 drop coincided with the
resumption of bursting EEG activity, vasoconstriction, decreased
blood volume and increased OIS (Fig. 6A). This pattern was similar
to that seen during the CSD wave, with a paradoxical decrease in
perfusion in the face of metabolic demand.
Because burst-suppression EEG is generally a pan-cortical
phenomenon (Swank and Watson, 1949; Steriade et al., 1994),
it reliably generates whole field changes in OIS signal, with
decreased reflectance in response to each burst. We used the
EEG/OIS interaction as an index of neurovascular coupling, quan-
tifying it with wavelet coherence (Fig. 6C, Supplementary Fig. S3).
The recovery of other measures (SatO2, [Hbtot], arterial diameter)
closely followed the resumption of coherent EEG and OIS activity
(Figs 4 and 6). As EEG burst amplitude had been at baseline levels
for several minutes prior (Fig. 6A), the increasing coherence was
probably brought about by the return of OIS response. This in
turn suggested that the recovery depended on intact functional
Once neurovascular coupling returned, it was quantitatively sim-
ilar to baseline. We computed the time constant (?) of OIS
changes in response to spontaneous EEG activity before CSD,
sub-CSD-threshold tetanic stimulation and spontaneous EEG
bursts during the second phase after recovery of coherence.
Differences in ? were minimal (mean pre-CSD spontaneous ?
2.1s, pre-CSD induced ? 2.0s, second phase ? 1.9s; n=10 exper-
iments), suggesting that mechanisms of coupling were not
deranged by CSD in the long term.
Uncoupling of DC and EEG activity
Comparing DC and EEG changes, there was no significant corre-
lation between second DC shift duration or amplitude and time to
recovery of EEG bursting. Likewise we found no significant
Figure 6 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 7min after CSD (bursts of this amplitude and
duration normally are associated with an OIS response, but are not here), showed some recovery at 24min, and largely returned by
34min. Vertical scale bar: 2% OIS change, 1.2mV 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
Biphasic neurovascular dysfunction in spreading depression Brain 2010: 133; 996–1012 |
correlation between latency or amplitude of the second DC nadir,
and recovery of EEG bursts. Finally, we found no correlation
between DC level (either at the second DC nadir or at the end of
the experiment) and burst rate (n=10 animals, 10 CSDs). These data
suggest that different mechanisms may be involved in DC and alter-
nating current electro-physiological activity in the wake of CSD.
Attenuation of arterial response when
CSD is elicited before full recovery
Our results suggested a prolonged uncoupling of cortical supply
and demand in the wake of a single CSD, with a prominent deficit
in vascular response. CSD is often repetitive, even in humans
(Dreier et al., 2009), and close spacing of CSD episodes can
significantly alter subsequent events. For example, continuous
repetitive CSD enhances dissociation between vascular and
electro-cortical activity (Shibata et al., 1990; Brennan et al.,
2007). We examined whether repetitive CSD, spaced 30min
apart, would elicit further dissociation between haemodynamic
and electro-physiological measures.
In contrast to CSD elicited after full recovery, CSD elicited at
30min intervals was associated with an attenuated haemodynamic
response. We found significantly larger and faster desaturation in
the first CSD wave when compared to the two subsequent waves
(Figs 7 and 3C). The vascular response paralleled the haemoglobin
desaturation: vessel constriction amplitude and rate of constriction
were more pronounced in the first wave than in subsequent waves
(Fig. 7). In contrast, there was no significant difference in the
amplitude or kinetics of the first (CSD wave-associated) DC
shift. Though we intentionally did not observe full recovery in
these experiments, we saw the beginnings of second phase
dysfunction in DC and EEG field potential, spectroscopic measures,
pial artery diameter and OIS in each experiment. These data
strongly suggest that distinct mechanisms mediate the response
of surface vessels and underlying cortex. They also confirm that
the vascular response is more susceptible to derangement than the
Extracellular potassium is not involved
in the vascular response after passage of
the CSD wave
We measured extracellular K+concentration in response to CSD
(n=5 animals) to evaluate its effect on arterial diameter (Fig. 8).
We observed a mean pre-CSD baseline extracellular K+concen-
tration of 5.89mM (CI: 4.82–7.38mM) which increased to a
Figure 7 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 30min 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. SatO2values 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 30min 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.
Brain 2010: 133; 996–1012 J. C. Chang et al.
mean of 32.1mM (CI: 26.1–37.2mM) at peak, coincident with
the first DC shift. After passage of the CSD wave, extracellular
K+concentration returned to a baseline value of 6.22mM (CI:
4.9–8.1mM). With repetitive CSD, the duration of extracellular
K+concentration elevation increased (Fig. 8B). Comparing the
kinetics of extracellular K+concentration to arterial diameter,
extracellular K+concentration time to peak was longer, at an
average of 67s (CI: 56–84s) to reach peak, compared to 13.8s
(CI: 8–35s) for arterial constriction. Duration of extracellular
K+concentration elevation was 7min (CI: 6–9min), compared to
43.5s (CI: 39.6–47.6s) for arterial constriction. Arteries constricted
monotonically with the onset of the CSD wave—there was no
dilation in response to lower levels of extracellular K+concentra-
tion elevation, and arteries ended constriction and began to
re-dilate well before extracellular K+concentration returned to
baseline. Finally, there was no extracellular K+concentration
change accompanying the arterial constriction that followed the
passage of the CSD wave (Fig. 8C).
Biphasic DC shift associated with CSD
Following recovery from the classic DC shift associated with the
CSD wave, our field potential recordings reveal a significant and
long-lasting second DC shift in cortical potential, whose duration
was similar to the haemodynamic changes (Fig. 4 and Table 2). To
our knowledge a long-term DC shift of this nature has not been
The incidence, amplitude, duration and kinetics of the second
DC shift were highly replicable—we observed it on every
long-term recording (n=14) and observed its beginning in every
recording (n=43). Interestingly, though its duration was similar
to that of haemodynamic changes, its onset was significantly
faster (Fig. 4).
Several physiological processes can cause DC shifts, among
them hyper- and hypoventilation, hypo- and hypercapnoea,
administration of anaesthetic agents, and disruption of the blood
brain barrier (Nita et al., 2004). However, respiratory rate was
unaffected by CSD (Table 1) and no changes to anaesthetic
parameters were made during recordings. Moreover, hypoventila-
tion, isoflurane anaesthesia and blood brain barrier disruption all
cause positive, not negative, DC shifts and of much smaller ampli-
tude (usually 51mV) than we observed (Nita et al., 2004). The
possibility of electrode compromise is made unlikely by the fact
that the majority of DC recordings returned to baseline potential,
and that further recording of DC shifts and EEG activity was
Large extracellular negative DC shifts are associated with
neuronal depolarization, both in vitro and in vivo, during spread-
ing depression but also during seizure activity and stroke
Figure 8 Extracellular K+does not affect perfusion after passage of the CSD wave. (A) Extracellular K+concentration [K+] increases to
over 30mM 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.
Biphasic neurovascular dysfunction in spreading depression Brain 2010: 133; 996–1012 |
(Marshall, 1959; Wadman et al., 1992; Somjen, 2001; Canals
et al., 2005). They are also associated with glial depolarization;
in fact, Sugaya et al. (1975) found that the DC shift of CSD
correlated betterwith astrocytic
Without current source density analysis or intracellular recordings,
we could not conclusively determine the origin of the second DC
shift. We find it reasonable to propose that our electrodes
detected a cortical (neuronal, glial or vascular) depolarization
during the second DC shift, given that (i) other sources of DC
shift were ruled out and (ii) the same electrode, in the same loca-
tion, detected negative CSD wave-associated DC shifts (known to
involve cortical depolarization) both before and after the second
DC shift. A complementary explanation, based on the findings of
Makarova et al. (2008) is that increases in extracellular resistivity,
likely to be due to tissue swelling, play a role in the second DC
shift. A tissue resistivity increase correlates temporally with the
known duration of tissue swelling in CSD and contributes to the
large size of the first DC shift. However, though our spectroscopic
measures agree with other measures of tissue swelling in the
literature, they do not show pathlength changes consistent with
swelling after passage of the CSD wave (there are mild but
significant decreases in pathlength after the wave-associated
increase) (Fig. 3F). This does not rule out either swelling or resis-
tance changes as mechanisms for the DC shift, as our technique
might lack the resolution to detect subtle changes.
Biphasic disruption of neurovascular
function associated with CSD
We observed two large desaturations of cortical haemoglobin, the
first during the CSD wave and the second, after a brief recovery,
in its aftermath. Both desaturations were within an ischaemic
range, and appeared to be due to a paradoxical combination of
increased tissue demand and reduced arterial supply, opposite to
what would be expected from conventional neurovascular cou-
pling. Despite some similarities, the first and second phases of
haemoglobin desaturation clearly involve distinct mechanisms, as
evidenced by their different relations to the CSD wave, different
spatio-temporal characteristics, different relation to extracellular K+
concentration and differential sensitivity to craniotomy.
Arterial constriction (van Harreveld and Stamm, 1952; Osada
et al., 2006; Brennan et al., 2007; Chuquet et al., 2007), blood
flow or volume decrease (Fabricius et al., 1995; Sonn et al., 1996;
Reuter et al., 1998; Ba et al., 2002; Tomita et al., 2005) and
hypoxia or haemoglobin desaturation (Marshall, 1959; Mayevsky
et al., 1980; LaManna et al., 1989; Haselgrove et al., 1990;
Takano et al., 2007; Piilgaard and Lauritzen, 2009) all occur in
multiple species, including humans (Dreier et al., 2009), during
CSD. The CSD-associated arterial constriction in mouse is larger
and more consistent than in other species (Ayata et al., 2004;
Brennan et al., 2007), but tissue hypoxia has also been reported
with CSD even in species where there is prominent arterial dilation
or hyperperfusion (Marshall, 1959; Mayevsky et al., 1980;
LaManna et al., 1989; Haselgrove et al., 1990; Piilgaard and
Lauritzen, 2009), probably because metabolic demand outstrips
vascular supply. Moreover, the transition from a dilatory to a
constrictive response to the CSD wave has been demonstrated,
under conditions of metabolic challenge, in animals that normally
have a predominantly dilatory response (Dreier et al., 1998;
Sukhotinsky et al., 2008). Manipulations that favour this response
in experimental animals—elevations in extracellular K+, depletion
of nitric oxide, hypotension, ischaemia and hypoxia (Duckrow,
1993; Dreier et al., 1998; Shin et al., 2006; Sukhotinsky et al.,
2008)—are all consistent with either increases in metabolic
demand or reduction in vascular supply of metabolic substrates.
In this regard, the mouse may have a decreased metabolic/perfu-
sion reserve compared to larger animals. However it should be
emphasized that neurovascular coupling is likely to function on a
gradient in all species and can actively contribute to pathology
when deranged. Moreover, all species studied thus far can
undergo constrictive neurovascular coupling during the CSD wave.
While the response to the CSD wave in mouse may or may not
be different from other species, long-lasting hypoperfusion follow-
ing the wave is a consistent finding in all model systems (Lauritzen
et al., 1982; Busija et al., 2008) and in humans after CSD-like
events (Olesen et al., 1981; Lauritzen et al., 1983, 1984).
We observed a sustained vasoconstriction and hypoperfusion
which began after a brief recovery from the initial wave of CSD.
The vascular changes occur in the face of a second sustained DC
shift and recovery of spontaneous electro-cortical activity, and are
associated with a second nadir in haemoglobin saturation, consis-
tent with a prolonged derangement of neurovascular coupling. As
with the CSD wave itself, the result is paradoxically reduced
perfusion in the setting of increased parenchymal demand.
Our finding of persistent haemoglobin desaturation after the
CSD wave complements the recent work of Piilgaard and
Lauritzen (2009), who report a long-lasting decrease in pO2,
accompanied reduced blood flow and increased cerebral metabolic
rate of O2, following the wave. That the authors found these
changes in rat, a species with a different response to the acute
CSD wave than mouse, is strong evidence that significant, meta-
bolically relevant changes in perfusion response following CSD are
generalized. Moreover, their finding of increased cerebral meta-
bolic rate of O2after passage of the CSD wave is consistent with
an increase in cortical metabolic demand that is not met by
We also found that arterial response could vary in the face of a
consistent electro-physiological response. When repetitive CSD
was elicited at intervals shorter than required for complete recov-
ery, the rate and amplitude of arterial constriction during the CSD
wave decreased, but no such decrement occurred in DC shift
parameters (Fig. 7). This confirms that the vascular and parench-
ymal compartments are separable in CSD (Brennan et al., 2007)
and further suggests that the ‘weak link’ in CSD-associated neu-
rovascular uncoupling lies in the vascular response. Given the
smaller size of desaturation with equally sized DC shifts, it also
shows conclusivelythat arterial
CSD-associated desaturation in mouse.
Another indication of the potential variability of the vascular
response was the finding that apparently atraumatic craniotomies
resulted in substantial changes to the vascular and parenchymal
CSD signal in mouse (Fig. 2). Our findings are consistent with
those of Ayata and Moskowitz (2006), who reported arterial
Brain 2010: 133; 996–1012J. C. Chang et al.
dilation rather than constriction in a closed craniotomy preparation
of N-methyl-D-aspartic acid-induced mouse CSD. While we
cannot rule out the possibility of subtle trauma or unrecorded
CSD in our preparations, our results suggest that alterations in
the perivascular environment caused by craniotomy can result in
markedly different vascular responses. The finding of a ‘normal’
mouse neurovascular response outside the craniotomy in the same
experiments that showed altered craniotomy response (Fig. 2A
and B), provides strong evidence that the changes we observed
were not due to generalized dysfunction like that generated by
The craniotomy preparation also provides important evidence
that the first and second neurovascular derangements are mech-
anistically distinct. Long-term craniotomy recordings showed intact
hypoperfusion after passage of the CSD wave, even when the
perfusion response associated with the wave itself was reversed
(Fig. 2C). The finding that evoked OIS maps are apparently
normal in craniotomy (Fig. 2D) suggests that the parenchymal
dilation response is intact, at least to physiological stimuli. This
difference in parenchymal and surface vessel response may be
due to the different innervation of pial and penetrating arteries
(Cipolla et al., 2004).
Finally, our craniotomy findings may resolve a discrepancy in the
literature regarding the vascular response to CSD. Takano et al.
(2007) measured subsurface arterial response using two-photon
microscopy, and unexpectedly found that parenchymal arterioles
dilated mildly rather than constricted during the CSD wave in
mouse. We also saw evidence of parenchymal vessel dilation
during the CSD wave in craniotomy (decrease in OIS signal
consistent with increased blood volume). It is possible that the
craniotomy necessary to perform two-photon imaging caused a
change in the parenchymal arterial response to the CSD wave,
such that a normally constrictive event became dilatory. Taken
together with our finding of apparently normal dilation during
cortical mapping, these data suggest an impairment of parenchy-
mal as well as surface artery constriction during the CSD wave in
craniotomy. Future studies of vascular reactivity in different
species would benefit from a comparison of thin skull versus
craniotomy methods, not only to determine whether differences
occur, but also to investigate possible mechanisms (mechanical,
vasoactive mediators) contributing to the difference.
Potassium concentration and
mechanisms of arterial behaviour
Extracellular K+levels closely follow the initial DC shift of CSD
(Brinley et al., 1960; Vyskocil et al., 1972; Hansen et al., 1980).
K+is also highly vasoactive (Golding et al., 2000; Horiuchi et al.,
2002), causing constriction via L-type voltage-gated calcium chan-
nels (Nelson et al., 1990) and dilation through inwardly rectifying
K+channels (Knot et al., 1996), in addition to modulation through
indirect mechanisms (Windmuller et al., 2005). It could thus
account for the large changes in arterial diameter both during
and following CSD, perhaps by overwhelming normal mechanisms
of neurovascular coupling (Ayata et al., 2004). We found that
cortical surface arteries underwent a monophasic constriction
and return to baseline during the period of extracellular K+
concentration elevation, which corresponded with the CSD
wave. Thus our data are consistent with increasing extracellular
K+concentration as a possible driver of arterial constriction.
However, the recovery from the initial constriction appears to be
mediated by other mechanisms. Moreover, in contrast to the CSD
wave, we were able to rule out any role of extracellular K+con-
centration elevations in the sustained arterial changes in the
second phase, as extracellular K+concentration had returned to
normal levels before the second desaturation began (Fig. 8). This
highlights the fact that the two phases of vascular change with
CSD have different mechanisms of action.
Spontaneous vasomotion, OIS responses to evoked neuronal
activity, and arterial responses to pH, extracellular K+concentra-
tion, adenosine, papaverine, bradykinin and hypercapnoea are all
attenuated for at least an hour after CSD in different species
(Wahl et al., 1987; Piper et al., 1991; Lacombe et al., 1992;
Florence et al., 1994; Seitz et al., 2004; Guiou et al., 2005;
Scheckenbach et al., 2006). The depletion or exhaustion of a
vasodilatoris onepossible mechanism
response. Nitric oxide depletion occurs in the wake of the CSD
wave and decreased cerebrovascular reactivity after the wave can
be rescued by treatment with nitric oxide donors or cyclic GMP
agonists (Fabricius et al., 1995; Scheckenbach et al., 2006). It is
also possible that the increased release of a vasoconstrictor
contributes to the disruption. Shibata et al. (1992) found that
long-lasting arterial constriction after CSD in rabbits was inhibited
by indomethacin, suggesting a role for prostanoids in this
We found that there was delayed clearance of extracellular
on repetitive CSD with incomplete recovery (Fig. 8C).
This may be due to the ischaemic levels of hypoperfusion
we encountered: Hansen et al. (1981) observed reduced extracel-
lular K+clearance in post-ischaemic rat brain. Impaired astrocyte
function may also play a role: astrocytic aquaporin mutations
(Padmawar et al., 2005) and selective poisoning of astrocytes
(Lian and Stringer, 2004) reduce extracellular K+clearance. As
astrocytic K+transport depends on intact membrane potential
(Kucheryavykh et al., 2007), the second DC shift we observed
might be a source of astrocytic dysfunction.
for this attenuated
Relation of second DC shift
to haemodynamic and
A long-lasting cortical depolarization could have effects on vascu-
lar reactivity: cerebral vessels can respond directly to depolariza-
tion, as well as to mediators released (or depleted) during
depolarization (Bai et al., 2004). We ruled out a role for K+, but
to our knowledge the effect of persistent DC shift on other mech-
anisms has not been investigated.
We hypothesized that the EEG and DC traces would bear some
relation to each other, with a presumed depolarization favouring
higher frequency EEG bursts and a longer time to burst recovery.
However, we found no significant correlations in these variables,
suggesting that the DC and EEG portions of the field potential
Biphasic neurovascular dysfunction in spreading depressionBrain 2010: 133; 996–1012 |
might not be mechanistically linked in the wake of CSD. This could
be due to a predominant glial, rather than neuronal, contribution
to the DC shift (Sugaya et al., 1975). Glial swelling might also
contribute to cortical resistance changes (Makarova et al., 2008),
which could result in DC changes uncorrelated with EEG. Further
investigation of glial roles in the post-CSD state is warranted.
Whatever the mechanism of the second DC shift, its occurrence
coincided with changes in neuronal activity. Early in the course of
the second depolarization, EEG activity was disorganized and often
paroxysmal in appearance (Fig. 4 and Supplementary Fig. S2). We
could not conclusively demonstrate spike-wave discharges or other
clearly epileptic phenomena, but the appearance of the traces was
suggestive of increased excitability. Seizure-like activity has been
noted in the setting of CSD since the time of Leao (1944) and van
Harreveld (1953), and recently Fabricius et al. (2008) have
reported association of seizures and CSD in injured human brain,
so an increase in cortical excitability after passage of the CSD
wave is tenable. The idea of an increase in excitability following
CSD is supported by the work of Kru ¨ger et al. (1996) and Piilgaard
and Lauritzen (2009), who showed a reduction in presumed
GABAergic activity in the wake of CSD. Even after other measures
recovered, EEG activity was characterized by burst-suppression at
a higher frequency than prior to CSD (Fig. 4), again suggestive of
a possible increase in excitability.
Physiological and clinical significance
Our results have potential clinical significance at multiple levels.
We show that there are two separate phases of neurovascular
dysfunction associated with CSD that involve distinct mechanisms.
Consideration of the physiological consequences of CSD must
include not only the initial propagated wave, but also the sus-
tained changes that occur after the wave. Given both the first
and second DC shift and desaturation, our results support studies
in humans indicating that CSD may not be a benign phenomenon,
especially if the cortex is already compromised. The sustained
derangement of neurovascular coupling following CSD may rep-
resent a distinct therapeutic target in migraine, stroke, subarach-
noid haemorrhage, brain trauma and other disorders where CSD
The National Institutes of Health (T32 GM008185 to J.C.C., R01
MH52083 to A.W.T., K08 NS059072 to K.C.B.); Larry L. Hillblom
Foundation (to A.C.C. and K.C.B.).
Supplementary material is available at Brain online.
Armitage P, Berry G, Matthews J. Statistical methods in medical research.
Blackwell Science Malden, MA, 2002.
Ayata C, Shin H, Salomone S, Ozdemir-Gursoy Y, Boas D, Dunn AK,
et al. Pronounced hypoperfusion during spreading depression in mouse
cortex. J Cereb Blood Flow Metab 2004; 24: 1172–82.
Ayata C, Moskowitz MA. Cortical spreading depression confounds con-
centration-dependent pial arteriolar dilation during N-methyl-D-aspar-
tate superfusion. Am J Physiol Heart Circ Physiol 2006; 290:
Ba A, Guiou M, Pouratian N, Muthialu A, Rex D, Cannestra A, et al.
Multiwavelength optical intrinsic signal imaging of cortical spreading
depression. J Neurophysiol 2002; 88: 2726–35.
Bai N, Moien-Afshari F, Washio H, Min A, Laher I. Pharmacology
of the mouse-isolated cerebral artery. Vasc Pharmacol 2004; 41:
Benaron DA, Parachikov IH, Friedland S, Soetikno R, Brock-Utne J, van
der Starre PJA, et al. Continuous, noninvasive, and localized microvas-
cular tissue oximetry using visible light spectroscopy. Anesthesiology
2004; 100: 1469–75.
Brennan KC, Beltra ´n-Parrazal L, Lo ´pez-Valde ´s HE, Theriot J, Toga AW,
Charles AC. Distinct vascular conduction with cortical spreading
depression. J Neurophysiol 2007; 97: 4143–51.
Brinley FJ, Kandel ER, Marshall WH. Potassium outflux from rabbit cortex
during spreading depression. J Neurophysiol 1960; 23: 246–56.
Busija DW, Bari F, Domoki F, Horiguchi T, Shimizu K. Mechanisms
involved in the cerebrovascular dilator effects of cortical spreading
depression. Prog Neurobiol 2008; 86: 379–95.
Canals S, Makarova I, Lo ´pez-Aguado L, Largo C, Ibarz JM, Herreras O.
Longitudinal depolarization gradients along the somatodendritic axis of
CA1 pyramidal cells: a novel feature of spreading depression. J
Neurophysiol 2005; 94: 943–51.
Chuquet J, Hollender L, Nimchinsky E. High-resolution in vivo imaging of
the neurovascular unit during spreading depression. J Neurosci 2007;
Cipolla MJ, Li R, Vitullo L. Perivascular innervation of penetrat-
ing brain parenchymal arterioles. J Cardiovasc Pharmacol 2004; 44:
Cze ´h G, Aitken PG, Somjen GG. Membrane currents in CA1 pyramidal
cells during spreading depression (SD) and SD-like hypoxic depolariza-
tion. Brain Res 1993; 632: 195–208.
Dohmen C, Sakowitz OW, Fabricius M, Bosche B, Reithmeier T,
Ernestus R, et al. Spreading depolarizations occur in human ischemic
stroke with high incidence. Ann Neurol 2008; 63: 720–8.
Dreier JP, Major S, Manning A, Woitzik J, Drenckhahn C, Steinbrink J,
et al. Cortical spreading ischaemia is a novel process involved in
ischaemic damage in patients with aneurysmal subarachnoid haemor-
rhage. Brain 2009; 132: 1866–81.
Dreier J, Korner K, Ebert N, Gorner A, Rubin I, Back T, et al. Nitric oxide
scavenging by hemoglobin or nitric oxide synthase inhibition by N-
nitro-L-arginine induces cortical spreading ischemia when K+ is
increased in the subarachnoid space. J Cereb Blood Flow Metab
1998; 18: 978–90.
Duckrow R. A brief hypoperfusion precedes spreading depression if nitric
oxide synthesis is inhibited. Brain Res 1993; 618: 190–5.
Efron B, Tibshirani R. Bootstrap methods for standard errors, confidence
intervals, and other measures of statistical accuracy. Stat Sci 1986; 1:
Etminan M, Takkouche B, Isorna F, Samii A. Risk of ischaemic stroke in
people with migraine: systematic review and meta-analysis of observa-
tional studies. Br Med J 2005; 330: 63–6.
Fabricius M, Akgoren N, Lauritzen M. Arginine-nitric oxide pathway and
cerebrovascular regulation in cortical spreading depression. Am J
Physiol Heart Circ Physiol 1995; 269: 23–9.
Fabricius M, Fuhr S, Willumsen L, Dreier J, Bhatia R, Boutelle M, et al.
Association of seizures with cortical spreading depression and peri-
infarct depolarisations in the acutely injured human brain. Clin
Neurophysiol 2008; 119: 1973–84.
Fisher RA. Frequency distribution of the values of the correlation coeffi-
cients in samples from and indefinitely large population. Biometrika
1915; 10: 507–18.
Brain 2010: 133; 996–1012J. C. Chang et al.
Florence G, Bonvento G, Charbonne R, Seylaz J. Spreading depression
reversibly impairs autoregulation of cortical blood flow. Am J Physiol
Regul Integr Comp Physiol 1994; 266: R1136–40.
Golding EM, Steenberg ML, Johnson TD, Bryan RM. The effects of
potassium on the rat middle cerebral artery. Brain Res 2000; 880:
Gorji A. Spreading depression: a review of the clinical relevance. Brain
Res Rev 2001; 38: 33–60.
Grinsted A, Moore JC, Jevrejeva S. Application of the cross wavelet
transform andwavelet coherence
Nonlinear Proc Geophys 2004; 11: 561–6.
Guiou M, Sheth S, Nemoto M, Walker M, Pouratian N, Ba A, et al.
Cortical spreading depression produces long-term disruption of activ-
ity-related changes in cerebral blood volume and neurovascular cou-
pling. J Biomed Opt 2005; 10: 11004.
Hadjikhani N, Sanchez Del Rio M, Wu O, Schwartz D, Bakker D, Fischl B,
etal. Mechanismsof migraine
MRI in human visual cortex. Proc Natl Acad Sci USA 2001; 98:
Hansen AJ, Gjedde A, Siemkowicz E. Extracellular potassium and blood
flow in the post-ischemic rat brain. Pflu ¨gers Arch 1981; 389: 1–7.
Hansen A, Olsen C. Brain extracellular space during spreading depression
and ischemia. Acta Physiol Scand 1980; 108: 355–65.
Hansen A, Quistorff B, Gjedde A. Relationship between local changes in
cortical blood flow and extracellular K+ during spreading depression.
Acta Physiol Scand 1980; 109: 1–6.
Haselgrove JC, Bashford CL, Barlow CH, Quistorff B, Chance B,
Mayevsky A. Time resolved 3-dimensional recording of redox ratio
during spreading depression in gerbil brain. Brain Res 1990; 506:
Horiuchi T, Dietrich H, Hongo K, Dacey R. Mechanism of extracellular
K+-induced local and conducted responses in cerebral penetrating
arterioles. Stroke 2002; 33: 2692–9.
Knot HJ, Zimmermann PA, Nelson MT. Extracellular K(+)-induced hyper-
polarizations and dilatations of rat coronary and cerebral arteries
involve inward rectifier K(+) channels. J Physiol 1996; 492 (Pt 2):
Kohl M, Lindauer U, Dirnagl U, Villringer A. Separation of changes in
light scattering and chromophore concentrations during cortical
spreading depression in rats. Opt Lett 1998; 23: 555–7.
Kruit M, van Buchem M, Hofman P, Bakkers J, Terwindt G, Ferrari M,
et al. Migraine as a risk factor for subclinical brain lesions. JAMA 2004;
Kru ¨ger H, Luhmann HJ, Heinemann U. Repetitive spreading depression
causes selective suppression of GABAergic function. Neuroreport 1996;
Kucheryavykh YV, Kucheryavykh LY, Nichols CG, Maldonado HM,
Baksi K, Reichenbach A, et al. Downregulation of Kir4.1 inward recti-
fying potassium channel subunits by RNAi impairs potassium transfer
and glutamate uptake by cultured cortical astrocytes. Glia 2007; 55:
Lacombe P, Sercombe R, Correze J, Springhetti V, Seylaz J. Spreading
depression induces prolonged reduction of cortical blood flow reactiv-
ity in the rat. Exp Neurol 1992; 117: 278–86.
Laird N, Ware J. Random-effects models for longitudinal data. Biometrics
1982; 38: 963–74.
LaManna J, McCracken K, Patil M, Prohaska O. Stimulus-activated
changes in brain tissue temperature in the anesthetized rat. Metab
Brain Dis 1989; 4: 225–37.
Lauritzen M. Long-lasting reduction of cortical blood flow of the brain
after spreading depression with preserved autoregulation and impaired
CO2 response. J Cereb Blood Flow Metab 1984; 4: 546–54.
Lauritzen M, Balslev Jorgensen M, Diemer N, Gjedde A, Hansen A.
Persistent oligemia of rat cerebral cortex in the wake of spreading
depression. Ann Neurol 1982; 12: 469–74.
Lauritzen M, Olsen T, Lassen N, Paulson O. Changes in regional cerebral
blood flow during the course of classic migraine attacks. Ann Neurol
1983; 13: 633–41.
aura revealedby functional
Leao A. Spreading depression of activity in the cerebral cortex. J
Neurophysiol 1944; 7: 359–90.
Lian X, Stringer J. Astrocytes contribute to regulation of extracellular
calcium and potassium in the rat cerebral cortex during spreading
depression. Brain Res 2004; 1012: 177–84.
Liptak BG. Instrument Engineers’ Handbook: Process control and optimi-
zation. CRC Press: Boca Raton, FL; 2003.
Lux HD, Neher E. The equilibration time course of (K +) 0 in cat cortex.
Exp Brain Res 1973; 17: 190–205.
Makarova J, Go ´mez-Gala ´n M, Herreras O. Variations in tissue resistivity
and in the extension of activated neuron domains shape the voltage
signal during spreading depression in the CA1 in vivo. Eur J Neurosci
2008; 27: 444–56.
Marshall W. Spreading cortical depression of Leao. Physiol Rev 1959; 39:
Mayevsky A, Lebourdais S, Chance B. The interrelation between brain
PO2 and NADH oxidation-reduction state in the Gerbil. J Neurosci Res
1980; 5: 173–82.
Mayevsky A, Doron A, Manor T, Meilin S, Nili Zarchin, Ouaknine G.
Cortical spreading depression recorded from the human brain using a
multiparametric monitoring system. Brain Res 1996; 740: 268–74.
Mazel T, Richter F, Vargova L, Sykova E. Changes in extracellular space
volume and geometry induced by cortical spreading depression in
immature and adult rats. Physiol Res 2002; 51: 85–94.
Merrick MF, Pardue HL. Evaluation of absorption and first- and second-
derivative spectra for simultaneous quantification of bilirubin and
hemoglobin. Clin Chem 1986; 32: 598–602.
Mies G, Iijima T, Hossmann KA. Correlation between peri-infarct DC
shifts and ischaemic neuronal damage in rat. Neuroreport 1993; 4:
Myers D, Anderson L, Seifert R, Ortner J, Cooper C, Beilman G, et al.
Noninvasive method for measuring local hemoglobin oxygen satura-
tion in tissue using wide gap second derivative near-infrared spectro-
scopy. J Biomed Opt 2005; 10: 034017.
Nedergaard M, Hansen AJ. Spreading depression is not associated with
neuronal injury in the normal brain. Brain Res 1988; 449: 395–8.
Nelson MT, Patlak JB, Worley JF, Standen NB. Calcium channels, potas-
sium channels, and voltage dependence of arterial smooth muscle
tone. Am J Physiol 1990; 259: C3–18.
Nita D, Vanhatalo S, Lafortune F, Voipio J, Kaila K, Amzica F.
Nonneuronal origin of CO2-related DC EEG shifts: an in vivo study
in the cat. J Neurophysiol 2004; 92: 1011–22.
Olesen J, Larsen B, Lauritzen M. Focal hyperemia followed by spreading
oligemia and impaired activation of rcbf in classic migraine. Ann
Neurol 1981; 9: 344–52.
Osada T, Tomita M, Suzuki N. Spindle-shaped constriction and propa-
gated dilation of arterioles during cortical spreading depression.
Neuroreport 2006; 17: 1365.
Padmawar P, Yao X, Bloch O, Manley G, Verkman A. K+ waves in brain
cortex visualized using a long-wavelength K+-sensing fluorescent indi-
cator. Nat Meth 2005; 2: 825–7.
Piilgaard H, Lauritzen M. Persistent increase in oxygen consumption and
impaired neurovascular coupling after spreading depression in rat neo-
cortex. J Cereb Blood Flow Metab 2009; 29: 1517–27.
Pinheiro JC, Bates DM. Lme and nlme: mixed-effects methods and
classes for S and S-plus. Madison: Bell Labs, Lucent Technologies
and University of Wisconsin; 1999.
Piper RD, Lambert GA, Duckworth JW. Cortical blood flow changes
during spreading depression in cats. Am J Physiol Heart Circ Physiol
1991; 261: 96–102.
Porges S, Bohrer R, Cheung M, Drasgow F, McCabe P, Keren G. New
time-series statistic for detecting rhythmic co-occurrence in the fre-
quency domain: the weighted coherence and its application to psy-
chophysiological research. Psychol Bull 1980; 88: 580–87.
Reuter U, Weber J, Gold L, Arnold G, Wolf T, Dreier J, et al. Perivascular
nerves contribute tocortical
hyperemia in rats. Am J Physiol Heart Circ Physiol 1998; 274:
Biphasic neurovascular dysfunction in spreading depressionBrain 2010: 133; 996–1012 |
Ripley B. pspline: penalized smoothing splines. R package version 1.0–12 Download full-text
ScheckenbachKEL, DreierJP, Dirnagl
rats: restoration by nitric oxide or cGMP. Exp Neurol 2006; 202:
Scher A, Gudmundsson L, Sigurdsson S, Ghambaryan A, Aspelund T,
Eiriksdottir G, et al. Migraine headache in middle age and late-life
brain infarcts. JAMA 2009; 301: 2563.
Schu ¨rks M, Rist PM, Bigal ME, Buring JE, Lipton RB, Kurth T. Migraine
and cardiovascular disease: systematic review and meta-analysis. Br
Med J 2009; 339: b3914.
Seitz I, Dirnagl U, Lindauer U. Impaired vascular reactivity of isolated rat
middle cerebral artery after cortical spreading depression in vivo. J
Cereb Blood Flow Metab 2004; 24: 526–30.
Shibata M, Leffler CW, Busija DW. Cerebral hemodynamics during cor-
tical spreading depression in rabbits. Brain Res 1990; 530: 267.
Shibata M, Leffler C, Busija D. Pial arteriolar constriction following cor-
tical spreading depression is mediated by prostanoids. Brain Res 1992;
Shin HK, Dunn AK, Jones PB, Boas DA, Moskowitz MA, Ayata C.
Vasoconstrictive neurovascular coupling during focal ischemic depolar-
izations. J Cereb. Blood Flow Metab 2006; 26: 1018–30.
Somjen G. Mechanisms of spreading depression and hypoxic spreading
depression-like depolarization. Physiol Rev 2001; 81: 1065–96.
Somjen GG. Is spreading depression bad for you? Focus on ‘‘repetitive
normoxic spreading depression-like events result in cell damage in
juvenile hippocampal slice cultures’’. J Neurophysiol 2006; 95: 16–7.
Sonn J, Dekel N, Kadusi R, Mayevsky A. Responses to cortical spreading
depression of the normoxic and ischemic brain. Israel J Med Sci 1996;
Steriade M, Amzica F, Contreras D. Cortical and thalamic cellular corre-
lates of electroencephalographic burst-suppression. Electroencephalogr
Clin Neurophysiol 1994; 90: 1–16.
Strong AJ, Anderson PJ, Watts HR, Virley DJ, Lloyd A, Irving EA, et al.
Peri-infarct depolarizations lead to loss of perfusion in ischaemic gyr-
encephalic cerebral cortex. Brain 2007; 130: 995.
Strong A, Fabricius M, Boutelle M, Hibbins S, Hopwood S, Jones R, et al.
Spreading and synchronous depressions of cortical activity in acutely
injured human brain. Stroke 2002; 33: 2738–43.
Sugaya E, Takato M, Noda Y. Neuronal and glial activity during spread-
ing depression in cerebral cortex of cat. J Neurophysiol 1975; 38:
Sukhotinsky I, Dilekoz E, Moskowitz M, Ayata C. Hypoxia and hypoten-
sion transform the blood flow response to cortical spreading depres-
sion from hyperemia into hypoperfusion in the rat. J Cereb Blood Flow
Metab 2008; 28: 1369.
Swank R, Watson C. Effects of barbiturates and ether on spontaneous
electrical activity of dog brain. J Neurophysiol 1949; 12: 137–60.
Takano T, Tian G, Peng W, Lou N, Lovatt D, Hansen A, et al. Cortical
spreading depression causes and coincides with tissue hypoxia. Nat
Neurosci 2007; 10: 754.
Tibshirani R, Knight K. Model search by bootstrap ‘‘Bumping’’. J Comput
Graph Stat 1999; 671–86.
Tomita M, Schiszler I, Tomita Y, Tanahashi N, Takeda H, Osada T, et al.
Initial oligemia with capillary flow stop followed by hyperemia during
K+-induced cortical spreading depression in rats. J Cereb Blood Flow
Metab 2005; 25: 742–7.
Torrence C, Compo G. A practical guide to wavelet analysis. Bull Am
Meteorol Soc 1998; 79: 61–78.
Velasco RD. Thresholding using the ISODATA clustering algorithm. IEEE
Trans Sys Man Cybernet 1980; 10: 771.
van Harreveld A, Stamm JS. Cerebral asphyxiation and spreading cortical
depression. Am J Physiol 1953; 173: 171–5.
van Harreveld A, Stamm J. Vascular concomitants of cortical spreading
depression. J Neurophysiol 1952; 16: 487–96.
Vyskocil F, Kritz N, Bures J. Potassium-selective microelectrodes used for
measuring the extracellular brain potassium during spreading depres-
sion and anoxic depolarization in rats. Brain Res 1972; 39: 255–9.
Wadman WJ, Juta AJ, Kamphuis W, Somjen GG. Current source density
of sustained potential shifts associated with electrographic seizures and
with spreading depression in rat hippocampus. Brain Res 1992; 570:
Wahl M, Lauritzen M, Schilling L. Change of cerebrovascular reactivity
after cortical spreading depression in cats and rats. Brain Res 1987;
WindmullerO, LindauerU, Foddis
Heinemann U, et al. Ion changes in spreading ischaemia induce rat
middle cerebral artery constriction in the absence of NO. Brain 2005;
M,Einhaupl K, DirnaglU,
Brain 2010: 133; 996–1012J. C. Chang et al.