Cortical blood flow changes during spreading depression in cats RICHARD
D. PIPER,
GEOFFREY
A. LAMBERT,
AND
JOHN
W. DUCKWORTH
Institute of Neurological Sciences, The Prince Henry and Prince of Wales Hospitals, and School of Medicine, University of New South Wales, Little Bay, Sydney 2036, Australia
PIPER, RICHARD D., GEOFFREY DUCKWORTH. Cortical blood flow
A. LAMBERT, AND JOHN W. changes during spreading depression in cats. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H96-H102, 1991.-Changes in cortical blood flow and cerebrovascular activity occurring during and after cortical spreading depression (CSD) were studied in ar-chloralose-urethan-anesthetized cats. CSD was induced by superficial cortical pinprick, and laser-Doppler velocimetry (LDV) was used to measure cerebral blood flow (CBF&. CSD resulted in a wave of cortical hyperemia during which there was a 215 * 48% peak increase in cortical blood flow that lasted for 2.7 t 0.4 min. This hyperemic phase was followed by prolonged cortical oligemia, with a reduction in flow of 20 t 4% at 1 h and 28 t 4% at 2 h. After CSD, cerebrovascular reactivity to the inhalation of CO, was abolished and did not fully recover for at least 10 h. Spontaneous vasomotor activity in the cerebral microcirculation was significantly decreased after CSD, and autoregulation of cortical blood flow in response to hypotension was preserved. The abnormal cerebrovascular reactivity seen after CSD in the gyrencephalic cortex of the cat has possible significance for human migraine with aura.
METHODS
H96
the American
Animal preparation. Male and female cats weighing 2.2 kg t 0.8 kg (mean t SD) were anesthetized with intraperitoneal injections of cu-chloralose (40 mg/kg) and urethan (500 mg/kg, n = 15) or a-chloralose (60 mg/kg) alone (n = 28). Supplementary doses of anesthetic were administered intravenously (20 mg/kg every 4-6 h) so as to maintain a stable level of anesthesia, as assessed by absence of tachycardia, hypertension, pupillary dilatation, or withdrawal to painful stimuli. Experiments during which cerebrovascular reactivity was being assessed lasted for up to 16 h. After intubation, the animals were ventilated with a mixture of 30% oxygen and air to maintain an end-expiratory CO, of 4.0-4.5%. Right femoral arterial and venous catheters were inserted to monitor blood pressure and administer drugs. A heating blanket was used to maintain rectal temperature at 3738OC. At the end of the experiment the animals were killed with an intravenous bolus of potassium chloride. Craniotomies were performed using a 6-mm dental velocimetry; migraine; autoregulation; cerebra - burr at low speed. The inner and outer diploe were laser-Doppler vascular reactivity removed in layers, exposing an underlying translucent layer of periosteum adherent to the dura. While drilling was in progress, the surgical field was bathed in normal CIRCUMSTANTIAL EVIDENCE suggests that cortica 1 saline to minimize the possibility of thermal injury to spreading depression (CSD) may cause focal neurologica 1 the underlying cortex. CBF was monitored with a single channel laserchanges (15, 19), such as scintillating scotomas and the Doppler perfusion monitor (Laserflo BPM403, TSI) and cortical “spreading oligemia” (24) seen during the aura pencil probe (P-431, TSI, diam of 1.5 mm). The laserof migraine. CSD has been shown in a number of species to result in a phase of cortical hyperemia (11, 13, 14, 17). Doppler probe was placed perpendicular to the cortical surface away from large pial and dural vessels, in gentle In the lissencephalic cortex of the rat, this initial hyperemit phase is followed by cortical oligemia, decreased contact with the dura or periosteum. Adequate probe pulsatile flow cortical vasodilatation during arterial hypercapnia, and placement was assessed by demonstrating synchronous with heart rate or vasomotor activity. Stapreservation of autoregulation (10). The hypothesis that ble flow recordings were usually obtained between 12 and CSD causes the spreading oligemia seen during migraine 25 units of laser Doppler flow (TSI calibrated units of with aura, assumes that CSD results in delayed cortical flow). Using this model, we have previously measured oligemia in other species, including humans. In the only baseline flows after death in 13 animals and demonsuch study, Wahl et al. (30) demonstrated dilatation of strated that the flowmeter readings closely approximate the pial vessels 15-120 min after CSD in the cat, in zero (0.15 t 0.16). To ensure stability of recordings, the contrast to pial constriction in the rat. To characterize animal was placed in a David Kopf head holder and the the delayed effects of CSD in species other than the rat, probe positioned with a stereotactic electrode manipuwe recorded cerebral blood flow (CBF) and cerebrovaslator. Blood flow was measured in the suprasylvian gyrus cular reactivity, occurring after CSD in the gyrencephalic of the parietal cortex or the posteromedial composite cortex of the cat. Blood flow measurements by laser- gyrus (visual cortex). The dura and periosteum were left Doppler velocimetry (CBFLn) allowed cortical flow to be intact unless otherwise indicated. monitored continuously in discrete areas of cerebral corCSD was initiated 5-20 mm away from the site of tex and the time course of changes in cortical cerebrolaser-Doppler probe by the rapid insertion and removal vascular reactivity to be determined. (l-2 mm depth) of a 26- to 30-gauge needle, avoiding the 0363-6135/91
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BLOOD
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local pial vessels. This method of initiating CSD was chosen because it was simple, effective, and analogous to experimental situations where electrodes (250-500 pm) are inserted into the cortex. To avoid any possible carryover effect, only one wave of CSD was studied in each hemisphere. If there was evidence of subdural blood at the end of the experiment, the animal was excluded from the study. In all experiments, expired Cog, blood pressure, heart rate, and CBFLn were recorded on-line using an analog-to-digital card (Labmaster DMA, Scientific Solutions) in an IBM-compatible computer. Arterial blood samples were analyzed with a Radiometer ABL300 blood gas machine and a Radiometer KNA2 sodiumpotassium analyzer. Evidence of spreading depression. Three criteria were used to confirm the development of CSD. First, DC potentials were recorded in eight animals using paired calomel electrodes with a tip diameter of 100-200 pm (26) or micropipettes filled with normal saline with a tip diameter of 2-10 pm (22). Recordings were made 0.25-l mm below the cortical surface. Second, electroencephalogram (EEG) was monitored in 10 animals using bipolar silver ball electrodes placed extradurally on the supra-sylvian gyrus ipsilateral to the site of cortical pinprick. Maximum peak to peak EEG amplitude was calculated during each 60-s period following the initiation of CSD. A 40% reduction in peak to peak amplitude was selected to mark the beginning and end of the period of EEG attenuation. EEG and DC potential recordings were made with a Narco Biosystems biopotential amplifier (type 7189) or Dagan 8700 amplifier Finally, to demonstrate that the blood flow disturbance after cortical pinprick propagated at a rate typical of CSD, the distance between the site of cortical pinprick and the laser-Doppler probe was correlated with the interval between pinprick and the first positive deflection in blood flow. The degree of correlation between these two variables was used as evidence of propagation, and the mean velocity was calculated using linear regression. Blood flow measurements. The immediate effects of CSD on CBFLn were studied in two groups of animals. In the first group the laser-Doppler probe was placed directly on the cortex. In the second group, to minimize the possibility of surgical trauma, blood flow was measured with the dura and periosteum intact. Cortical blood flow was measured during a 5-min control period and for 25 min after pinprick. The delayed effect of CSD on CBFLn was examined in a separate group of animals. CBFLn was measured over a 2-h control period in the intact animal. CSD was then precipitated with cortical pinprick and flow monitored for a further 2 h. The a priori hypothesis was that over the 2-h control period there would be no significant change in CBF LD due to the effects of anesthesia alone but that after CSD there would be a significant decrease in CBFLD. Average flow over a 5-min interval was calculated at the beginning of each 2-h recording period and at 60 and 120 min. Comparisons were made between the initial and latter two readings using a paired t test in both the control and CSD groups. As this hypothesis involved four comparisons, significant results were inter-
SPREADING
DEPRESSION
H97
preted using the Bonferroni procedure. If supplementary anesthesia was required during the recording period, the animal was not included in the data analysis. Only animals demonstrating a normal increase (X00%) in CBFLD during arterial hypercapnia (see Assessment of cerebrovascular reactivity) and evidence of active autoregulation as assessedby a gain factor BO.65 (see below) were included in the study. Assessment of cerebrovascular reactivity. Autoregulation of cortical blood flow was assessedby bleeding and subsequently reinfusing the animals to achieve a 20% step reduction in mean arterial blood pressure, while maintaining the systolic blood pressure ~80 mmHg. The integrity of autoregulation was assessedby calculating the gain factor (Gf) Gf
= 1 - Kwww/P)l
where (AF/F)/( AP/P) is the slope of normalized pressure-flow curve at a given point (P,F), as described by Norris et al. (23). If Gf is 1, cortical flow is maintained independent of systemic blood pressure; however, if GF is 0, autoregulation is absent and CBF passively follows blood pressure. Measurements were made before CSD and 30-60 min after the induction of CSD. Cortical vasodilatation following arterial hypercapnia was assessedby adding a gas mixture of 8% COa, 30% 02, and air to the ventilatory circuit for 5 min. This standard stimulus resulted in an arterial PCO~ of -60 mmHg (see Table 3). CBFLn was measured before and after 5 min of CO, inhalation, and the change in flow was calculated. The effect of hypercapnia on CBFLD was assessed at intervals up to 16 h after CSD. Similar observations were made on cortex where CSD had not been induced to examine the effect of prolonged anesthesia on COZ responsiveness. Assessment of vasomotor activity. Spontaneous vasomotor activity in the cortical microcirculation was assessed before and after CSD. Vasomotor activity was defined as spontaneous fluctuation in CBFLD (X0%) independent of heart rate and ventilation. As an index of the amplitude of vasomotor activity, a 2-min stationary period of the laser-Doppler signal was analyzed (120150 data points). The mean flow was calculated, and all data were expressed as percent deviation from the mean. The root mean square value of this transformed data was then used as an index of signal variability (CBFRMs). A similar analysis was performed on the blood pressure signal to obtain an index of variability (BPRMS). Statistical analysis. Values are means t SE unless otherwise stated. Statistical significance was tested using either a paired or unpaired t test as indicated. Each hemisphere was regarded as a separate cortical preparation when the number of measurements was greater than the number of animals quoted. The baseline was calculated from the mean flow of the preceding 5 min where flows are expressed as percentage change from baseline. RESULTS
In all animals (n = 43) cortical pinprick resulted in a wave of cortical hyperemia after the initial attempt. Evidence of CSD. In eight animals, cortical interstitial
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BLOOD
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DC potential was measured during the wave of hyperemia. These animals displayed DC changes typical of CSD. The mean maximum DC potential shift was -10.7 t 1.0 mV (n = 25, range -4.0 to -20 mV). In four animals the recording electrode was positioned underneath the laser-Doppler probe. In these animals the DC potential shift coincided with the onset of cortical hyperemia (Fig. 1). The surface EEG was recorded in 10 animals. The mean latency from cortical pinprick, to the onset of a 40% reduction in the peak to peak amplitude was 3 t 0.3 min. This degree of attenuation of the EEG persisted for a mean of 8.6 t 2 min, with recovery of normal peak to peak amplitudes (>90% of control) at a mean latency of 22 t 6 min. The correlation between the distance from the site of cortical pinprick to the laserDoppler probe, and the interval between pinprick and the first positive deflection in blood flow was examined in 15 cu-chloralose-urethan-anesthetized cats. Linear regression demonstrated that these two variables were correlated (r2 = 0.73, P C 0.001, n = 19) and that the mean velocity of propagation (3.1 t 0.4 mm/min) was typical of CSD (6). Changes in cortical blood flow. In 14 cu-chloralose-anesthetized animals, blood flow measurements were made with the dura and periosteum intact. In this group of animals CSD was associated with a 215 t 48% increase in CBFLD (Fig. 10). This period of intense hyperemia lasted for a mean duration of 2.5 t 0.2 min. In 11 of these animals a period of more prolonged, but less intense hyperemia (Fig. 1A ), followed this initial peak in flow (mean duration 5.8 t 1.1 min). At 25 min after CSD, CBFLn was decreased by 10 t 13.6%, but this result did not reach statistical significance (P = 0.4, unpaired t test) when compared with the change in CBFLD in the contralateral cortex (Fig. 1C). In the cat, the dura is thin and it is likely that blood flow measurements largely 0
A
Change
in
Flow
0 (4 0
B
in
Flow
( %)
D
Change
in 0 (4 0
20
25
o
(mV)
-16
-. . . . . . . . . . . . . . . . . . . I
'
501
Change
15
represent flow in the underlying cortex. To test this hypothesis similar observations were made in 15 urethan-cr-chloralose-anesthetized animals in which the dura had been removed and the cortex bathed in warmed Merlis artificial cerebrospinal fluid. In these animals, CSD was associated with a wave of cortical hyperemia that resulted in a mean 201 t 34% increase in CBDLn. These results suggest that it is possible to detect CSD in the cat using a minimally invasive preparation and that it is unlikely that the flow disturbance originates in the dural vessels. In 11 animals anesthetized with ac-chloralose alone the more prolonged effect of CSD on CBF was examined. CBFLn was measured through a small cranial window with the dura and periosteum intact. Biochemical data and systemic variables for these animals were within normal limits (Table 1). After CSD, CBFLn was decreased by 20 t 4% at 60 min and 28 t 4% at 120 min compared with initial flow (Table 2). These reductions in CBF JJ) were statistically significant when compared with CBFLn at the beginning of the 2-h recording period (P c 0.01 at 60 min and P c 0.01 at 120 min, paired t test after Bonferroni correction, n = 11, Table 2). During the 2-h control recording period, there was no significant change in CBF Ln at 60 and 120 min, assessed using a paired t test (P > 0.05). Changes in cerebrovascular reactivity. CSD was followed by abolition of cortical cerebrovascular reactivity to COa. Cerebrovascular reactivity to hypercapnia was tested before and after CSD in seven cu-chloralose-anesthetized animals. Before CSD, cortical blood flow increased with hypercapnia by 185 t 32% arterial PCO~ [(Paoo,) before = 32 t 0.9 mmHg, hypercapnic Pace, = 60 + 2 mmHg]. This response was significantly decreased when repeated ~60 min after CSD (3.0 t 2.0%, P < 0.01, data (Table 3) n = 7, paired t test). The biochemical
200
-24
C
10
DEPRESSION
300
DC Potential Change
5
SPREADING
I
25
,
I
1
1
1
- . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*.. . .-. 9.. _ -s-e .... s . ..a-\. *.*. .... ,.. ..-* ... -. . - 0 *..a.,. -.*. e.. I... a s - @.-.‘. -. ........* .‘.w_ _ c ..‘\.. w- e.-25 - . . . x.. . . . . . . . . . . . . . . . * . . * . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..a
. . . . . . . . C-J -
FLOW
Flow
. . . . . . . . . (-1 0
5
10
15
Time
(minutes)
20
FIG. 1. Values are means t 95% confidence interval (CI). A and B: typical trace showing blood flow changes occurring after cortical spreading depression (CSD). CSD was initiated by pinprick (vertical line) 10 mm from the site of Doppler probe. DC potential was recorded from an electrode placed under probe. C and D: mean changes in cerebral blood flow using laser-Doppler velocimetry (CBF& occurring after CSD. After CSD, blood flow in contralateral cortex was unchanged (C). Traces were averaged from end of the 5-min control period (vertical line) before initiation of CSD (n = 5). After CSD, ipsilateral transient cortical hyperemia is followed by a gradual decrease in cortical CBFLn (D). Traces were averaged from beginning of hyperemic phase (n = 14). Percentage changes in CBFr,D & 95% confidence intervals compared with control are shown.
FLOW
25
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BLOOD
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SPREADING 300
1. Physiological variables in animals in which CBFLII was measured after CSD TABLE
2 2 u
120 min
Control 17.6k2.3
CW.D %A CBFLh MEco,, % Mean BP, mmHg
18.0k2.1
2.6kO.l 109*5
1
CSD initiated
-5 100
2. Effect of CSF on CBFLL) and systemic variables
CBF’LD %A CBFLu MEco,, % Mean BP, mmHg
I
is z
TABLE
60 min
I
- 200 VX
Values are means t SE; n = 11 cats. Biochemical variables and blood pressure were measured just before the 4-h recording period. The change in cortical blood flow was measured after the inhalation of 8% CO, for 5 min before inducing CSD. Pace,, Pao,, arterial PCO~ and Po2, respectively.
0 min
I
‘4 I
7.33t0.01 33t1.8 184k13 -7.6k0.8 17.5kO.7 99.320.2 145t1.0 3.020.1 115t5.2 188t16.6 0.82t0.03
PH Pm,, mmHg Pao,, mmHg Base excess HCO:], mM %O, saturation Na, mM K, mM Blood pressure, mmHg %A in CBFLD with CO2 Gain factor
H99
DEPRESSION
17.1t2.0 -2t4 2.7tO.l 116t5
18.222.2 +6t6 2.8t0.2 118&3
14.3t1.8* -2Ot4 2.8tO.l 113t6
12.7&1.3? -28t4 2.8k0.2 113t5
0
I
-100
-100
I
200 Time
I
I
500
800 (minutes)
Post-CSD
1100
FIG. 2. Reduction in hypercapnic vasodilatation after CSD. Hypercapnia was induced after inhalation of a mixture of 8% C02-30% oxygen and air for 5 min. Response before CSD is plotted, followed by mean response over periods of 200 min after CSD. Mean response in animals in which CSD had not been induced is also plotted over periods of 200 min after commencement of cortical recordings (*P < 0.001; * P < 0.05; unpaired t test). Values are means t SE. 400
CSD
2.8tO.l 115*6
Values are means & SE. After a 2-h control period, CSD was initiated with cortical pinprick, and CBF LD was measured for a further 2 h. CBFLD is expressed in flowmeter (TSI) units of flow. CBFLD, mean expired CO, (MEcon) and blood pressure were followed serially. %A made at 0 min were CBFLD, percent change in CBF LD. Measurements compared with those at 60 and 120 min using a paired t test. * P = 0.001, t P = 0.002. Significant at the P < 0.001 level after the Bonferroni procedure.
TABLE 3. Physiological variables in animals tested for cortical responsivenessto hypercapnia
PH Pac02, h,,
mmHg mm&
HCOs, mM %02 saturation Na, mM K, mM Blood pressure,
mmHg
Control
B%CO,
7.32t0.01 3220.9 181klO 16kO.6 99t0.2 144k1.2 3.05kO.l 119&7
7.12kO.01 6022 193k9.3 19t0.6 98tO. 1 145kl.O 2.8kO.07 123*8
Values are means t SE; n = 7 cats. Biochemical variables were measured on arterial blood before and then after inhalation of 8% CO, for 5 min.
collected from this group of animals were within the physiological range for the cat (18). The time course of change in cerebrovascular reactivity to the inhalation of 8% COz was followed serially in 12 cu-chloralose-anesthetized animals, including the above 7 (Fig. 2). Cortical blood flow was measured through the intact dura and periosteum. The mean increase in CBF after the inhalation of CO2 was plotted for each succes-
.r(c
-100
I
0
I
I
I
I
I
4
8
12
16
Time(minutes) 3. Decrease in spontaneous vasomotor activity after CSD. Data from a cat in which vasomotor activity was prominent. CSD was initiated by pinprick (vertical line) 6 mm from site of laser-Doppler probe. FIG.
sive ZOO-min interval after the initiation of CSD. These data suggest that cerebrovascular reactivity to the inhalation of CO2 is impaired for as long as 12-h after the initiation of CSD. The control group in Fig. 2 consists of data from 20 hemispheres in which CSD had not been induced. This group demonstrates that cerebrovascular responsiveness to CO2 is preserved despite prolonged periods of recording under general anesthesia. Changes in vasomotor activity. In 13 cu-chloralose-anesthetized animals, the effect of CSD on spontaneous vasomotor activity was observed. Data from a typical animal are presented in Fig. 3. In 6 of 13 animals, spontaneous vasomotor activity, defined as spontaneous fluctuations in CBF LD (X0%) independent of changes in heart rate and respiration, was observed. The mean frequency of such fluctuations was 6.4 t 0.2 per min. CBFRMs signal was recorded before CSD (17.4 t 2.5%) and again 30-60 min post-CSD (5.27 t 0.6%). The mean percent change in CBF RMSwas 66 k 5% (P c 0.005). In the seven remaining animals, where spontaneous vaso-
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HlOO
BLOOD
FLOW
CHANGES
DURING
motor activity was not evident, there was a 25 t 4% reduction in CBF RMS after CSD (before, 11.5 t 2.7 TSI units of flow; after, 8.3 t 1.9 TSI units of flow; P = 0.36). These data suggest that variability in CBFLD is generally decreased by CSD and that spontaneous vasomotor activity is particularly affected. Linear regression between the percentage change in CBFRMs and the pre-CSD CBFRMs value demonstrated that these two variables were linearly related in animals displaying spontaneous vasomotor activity but not in the other animals (Fig. 4). Spontaneous vasomotor activity was thus selectively affected by CSD. To assess whether this was due to changes in either BP RMS or the mean BP, these two variables were assessed before and after CSD in the group of animals (n = 6) with spontaneous vasomotor activity. However, neither the mean BP (before, 107 t 6.7 mmHg; after, 108 t 9.5 mmHg; paired t test, P = 0.72) nor BPRMs (before, 5.2 t 1.2 mmHg; after, 5.6 t 1.0 mmHg; paired t test, P = 0.31) was affected by CSD. Autoregulation was unaffected when assessed by venesection before and then 30-60 min after CSD (n = 7, Table 4). DISCUSSION
Laser-Doppler blood flow measurement. The area sampled by laser-Doppler velocimetry has been estimated to be approximately 1 mm3 (9, 28). A number of studies 100
FIG. 4. Spontaneous vasomotor activity is reduced by CSD in proportion to its control amplitude. Linear regression between pre-CSD root mean square CBF (CBF lIMS) and percent change in CBFHMs after CSD is plotted for 7 animals with and 6 animals without preexisting vasomotor activity. 0, vasomotor activity present; r2 = 0.81, slope = = 32 t 8.6, P < 0.05. l , vasomotor activity 1.95 k 0.47, intercept = 20.5 t 8.6, P > 0.05. absent, r’! = 0.07, slope = 0.42 ~fr 0.65, intercept
TABLE
4. Effect of CSD on autoregulation Control BP, mmHg
Pre-CSD Post-CSD
109t3.5 llOt3.6
%A BP
25.2-el.5 21.2t1.0
%A CBFLD
Factor
Gain
2.7t1.6 1.5t1.3
0.80t0.04 0.86t0.03
cats. Autoregulation was assessed by in a step reduction in blood pressure; Post-CSD measurements were made
CORTICAL
SPREADING
DEPRESSION
have demonstrated that flow measured by laser-Doppler correlates well with absolute flow measured with established techniques [intestinal mucosa (1); spinal cord (16); rabbit cerebral cortex (5)]. Dirnagl et al. (3) in a study of the rat cerebral cortex, compared CBF measured by the [ 14C]iodoantipyrine technique with laser-Doppler flow measurements and found that CBFLD correlated well with relative changes in flow but poorly with absolute flow. For this reason blood flow changes in this study are not expressed in absolute units of flow but rather as percentage change in flow from baseline. Du ring this series of experiments, the laser-Doppler probe was held in position with a stereotactic holder to minimize the effect of probe movement on blood flow readings. Additionally, the use of urethan and/or cu-chloralose, both of which are anesthetic agents with intermediate to long half-lives, aided in obtaining stable baseline CBFLD recordings. We have demonstrated that this model provides stable baseline flows for periods of up to 2 h. It is likely that changes in baseline flow due to probe movement would be random and result in an increased experimental error causing failure to reject a null hypothesis, rather than the demonstration of statistically significant changes in flow. Laser-Doppler velocimetry and detection of CSD. Other workers (6, 17) have suggested that CSD is difficult to induce in the gyrencephalic cortex. Our results suggest that this is no t the ca.se with urethan-cr-chloralose anesthesia in the cat. It seems unlikely that the an .imals studied were susceptible to CSD on account of either surgical trauma or metabolic perturbation. Using a minimally invasive technique, which involved measuring CBFLD through the intact dura and periosteum, it was still possible to induce CSD in all animals in which there was physiological cerebrovascular reactivity before the study. We have demonstrated that this preparation does not result in marked changes in biochemical variables or alterations in cerebrovascular responsiveness to hypercapnia. The ease with which CSD is induced in this preparation is of interest because of the common use of the cu-chloralose-anesthetized cat as a cortical experimental model .. Blood flow changes associated with CSD. A wave of cortical hyperemia in association with CSD was first suggested by Lego (14) who demonstrated a brief period of pial dilatation in the rabbit. Subsequently, a number of authors using a variety of techniques have confirmed that CSD results in a phase of cortical hyperemia (11, 13,17). Hansen et al. (7) examined this hyperemic phase using the [“Cl iodoantipyrine technique and demonstrated that the cortical hyperemia accompanying CSD starts during the normalization of interstitial K+ concentrations, rather than during the period of maximal increase in interstitial K+. These observations would suggest that the hyperemic phase is due to increased local oxygen demand as a result of the metabolic activity required to reconstitute membrane ionic gradients. A phase of cortical oligemia after CSD was first described by Lauritzen et al. (13) in the cortex of the rat. In this study cortical oligemia was evident 5 min after the passage of a wave of CSD and persisted for at least 60 min. Cortical oligemia, persisting for as long as 200
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HlOl
min in the rat cortex, has subsequently been confirmed intended experimental model. We have demonstrated by other workers (4, 21). In this study we have demonthat the abnormalities in cerebrovascular reactivity to strated a similar phase of cortical oligemia in the cat, CO2 induced by CSD are more prolonged than those but the latency to onset is longer than that reported in demonstrated thus far in the rat and that they are the rat (Table 2, Fig. 1). associated with decreased spontaneous vasomotor activity. It has previously been suggested that delayed hypoWahl et al. (30) have demonstrated constriction of pial vessels (25-62 pm) in the rat after CSD and dilatation perfusion seen after CSD is a result of impairment of the in pial vessels (66-257 pm) in the cat. This pial dilatation metabolism flow couple, rather than a primary reduction in cortical metabolism (12, 21). The primary alterations was observed in 80% of vessels in the post-CSD period (15-120 min). Our observation that CSD results in de- in cerebrovascular reactivity seen after CSD in the cat creased CBFLn in the cat demonstrates that while the may be the basis of this abnormal coupling between blood flow and metabolism. pial vessels dilate, the resistance vessels constrict. This observation would suggest that the pial dilatation may We have demonstrated that the changes in cortical blood flow seen after CSD in the lissencephalic cortex, be a compensatory mechanism secondary to increased resistance in small diameter arterioles (400 pm). This also occur in the cat; an animal with a gyrencephalic hypothesis is attractive because in the cat 45% of vas- cortex that morphological .ly resembles more closely the cular resistance derives from vessels less than 100 pm in human cortex. If CSD occurs in the human cortex, it diameter (29), and it is likely that small diameter arte- may well be a pathological entity of clinical importance rioles (~100 pm) would be more intimately affected by during the aura of migraine following head injury or the parenchymal metabolic disturbance seen during CSD neurosurgery. than larger pial vessels. Changes in cerebrovascular reactivity. Lauritzen et al. The authors thank Professor J. W. Lance for advice and critical evaluation of the manuscript. We also thank M. Hellier for technical (10) have demonstrated in the rat that CSD is associated assistance and Dr. Karen Bythe for statistical advice. with blunted vascular reactivity to COZ assessed 30-60 This work was supported by the National Health and Medical min after CSD. Wahl et al. (30) using a cranial window Research Council of Australia, the Basser Trust, the J. A. Perini Family preparation in the cat, looked at cerebrovascular reactivTrust, and Warren and Cheryl Anderson. Address for reprint requests: R. D. Piper, Dept. of Neurology, ity following CSD. After examining the effects of direct application of K+, H’, adenosine, and bradykinin to the Clinical Sciences Bldg., Prince Henry Hospital, Anzac Pde, Little Bay 2036, Sydney, Australia. pial vessels (66-257 pm), they concluded that cerebroReceived 14 August 1990; accepted in final form 12 March 1991. vascular reactivity to a wide range of chemical stimuli was severely impaired. In the cat, we have demonstrated that after CSD CO2 reactivity, a physiological index of REFERENCES cerebrovascular responsiveness, is decreased for up to 12 1. AHN, H., J. LINDHAGEN, G. E. NILSSON, E. G. SALERUD, M. h. JODAL, AND 0. LUNDGREN. Evaluation of laser Doppler flowmetry Changes in vasomotor activity. Vasomotor activity has in the assessment of intestinal blood flow in cat. Gastroenterology 88: 951-957, 1985. been described in cerebral arterioles using a variety of 2. AUER, L. M., AND B. GALLHOFER. Rhythmic activity of pial vessels techniques. Spontaneous fluctuations in cortical vasoin vivo. Eur. Neurol. 20: 448-468, 1981. motor tone are well described in vivo using videoangiog3. DIRNAGL, U., B. KAPLAN, M. JACEWICZ, AND W. PULSINELLI. raphy and the cranial window technique (2, 8). It has Continuous measurement of cerebral cortical blood flow by laserDoppler flowmetry in a rat stroke model. J. Cereb. Blood Flow also been demonstrated in vitro in arterioles taken from Metab. 9: 589-596, 1989. branches of the posterior cerebral arteries of sponta4. DUCKROW, R. B. Regional cerebral blood flow during cortical neously hypertensive rats (25). This phenomenon has spreading depression in conscious rats (Abstract). J. Cereb. Blood recently been demonstrated using laser-Doppler velociFlow Metab. 9, Suppl. 1: S510, 1989. metry in the rat cortex (3) and also in the feline spinal 5. EYRE, J. A., T. J. ESSEX, P. A. FLECKNELL, P. H. BARTHOLOMEW, AND J. I. SINCLAIR. A comparison of measurements of cerebral cord (20). Electrophysiological studies of the cerebrovasblood flow in the rabbit using laser Doppler spectroscopy and cular smooth muscle of the rat have demonstrated flucradionuclide labelled microspheres. Clin. Phys. Physiol. Meas. 9: tuations in membrane potential in small distal arterioles 65-74, 1988. (lo-50 pm) that often result in rhythmic constriction of 6. HANSEN, A. J., AND M. LAURITZEN. Spreading depression of Leao. In: Basic Mechanisms of Headache, edited by J. Olesen and L. the vessel, particularly in more distal resistance vessels Edvinsson. Amsterdam: Elsevier, 1988, p. 99-107. (27). The observation that vasomotor activity is reduced 7. HANSEN, A. J., B. QUISTORFF, AND A. GJEDDE. Relationship after CSD, suggests that the phenomenon results in a between local changes in cortical blood flow and extracellular K+ generalized decrease in cerebrovascular reactivity affectduring spreading depression. Acta Physiol. Stand. 109: l-6, 1980. ing the more distal resistance vessels. 8. HUNDLEY, W. G., G. J. RENALDO, J. E. LEVASSEUR, AND H. A. KONTOS. Vasomotion in cerebral microcirculation of awake rabConclusion. This study demonstrates that CSD is easbits. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): H67-H71, 1988. ily and noninvasively detected using LDV. It emphasizes 9. KEIL, J. W., G. L. REIDEL, S. R. DIRESTA, AND A. P. SHEPERD. that in the cu-chloralose-urethan-anesthetized cat, cortiGastric mucosal flow measured by laser-Doppler velocimetry. Am. cal micropuncture with a 26- to 30-gauge needle almost J. Physiol. 249 (Gastrointest. Liver Physiol. 12): G539-G545, 1985. 10. LAURITZEN, M. Long-lasting reduction of cortical blood flow of the uniformly results in CSD, an observation not widely brain after spreading depression with preserved autoregulation and recognized in this species. These observations have imimpaired CO, response. J. Cereb. Blood Flow Metab. 4: 546-554, portant implications for experimental work on the cat 1984. cortex, since it is possible that CSD induced during 11. LAURITZEN, M. Regional cerebral blood flow during cortical spreadnrenaration mav have a long-lasting influence on an ing depression in rat brain: increased reactive hyperperfusion in Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (128.205.114.091) on December 1, 2018. Copyright © 1991 the American Physiological Society. All rights reserved.
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low-flow states. Acta Neurol. &and. 75: l-8, 1987. 12. LAURITZEN, M., AND N. H. DIEMER. Uncoupling of cerebral blood flow and metabolism after single episode of cortical spreading depression in the rat brain. Brain Res. 370: 405-408, 1986. 13. LAURITZEN, M., M. B. JORGENSEN, N. H. DIEMER, A. GJEDDE, AND A. J. HANSEN. Persistent oligemia of rat cerebral cortex in the wake of spreading depression. Ann. Neurol. 12: 469-474, 1982. 14. LEAo, A. A. P. Pial circulation and spreading depression of activity and the cerebral cortex. J. Neurophysiol. 7: 391-396, 1944. 15. LEAo, A. A. P., AND R. S. MORRISON. Propagation of cortical spreading depression. J. Neurophysiol. 8: 33-45, 1945. 16. LINDSBERG, P. J., J. T. O’NEILL, I. A. PAAKKARI, J. M. HALLENBECK, AND G. FEUERSTEIN. Validation of laser-Doppler flowmetry in measurement of spinal cord blood flow. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H674-H680, 1989. 17. MARSHALL, W. H. Spreading cortical depression of Leao. Physiol. Rev. 39: 239-279, 1959. 18. MIDDLETON, D. J., J. E. ILKIW, AND A. D. WATSON. Arterial and venous blood gas tensions in clinically healthy cats. Am. J. Vet. Res. 42: 1609-1611,198l. 19. MILNER, B. Note a possible correspondence between the migraine scotoma and spreading depression. Electroencephalogr. Clin. Neurophysiol. 10: 705, 1958. 20. MIZUTANI, M., T. YAMAMURO, AND J. SHIKATA. Vasomotion in normal and injured spinal cord. Exp. Neurol. 101: 256-266, 1988. 21. MRAOVITCH, S., Y. CALANDO, AND J. SEYLAZ. Long-lasting cerebral blood flow and metabolic changes within the limbic and brainstem regions following cortical spreading depression in the rat (Abstract). J. Cereb. Blood Flow Metab. 9, Suppl. 1: S508, 1989.
CORTICAL
SPREADING
DEPRESSION
22. NEDERGAARD, M., AND A. J. HANSEN. Spreading depression is not associated with neuronal injury in the normal brain. Brain Res. 449: 395-398,1988. 23. NORRIS, C. P., G. E. BARNES, E. E. SMITH, AND H. J. GRANGER. Autoregulation of superior mesenteric flow in fasted and fed dogs. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H174-H177, 1979. 24. OLESEN, J., B. LARSEN, AND M. LAURITZEN. Focal hyperemia followed by spreading oligemia and impaired activation of rCBF in classic migraine. Ann. Neurol. 9: 344-352, 1981. 25. OSOL, G., AND W. HALPERN. Spontaneous vasomotion in pressurized cerebral arteries from genetically hypertensive rats. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): H28-H33, 1988. 26. SHITABA, M., B. SEIGFRIED, AND J. P. HUSTON. Miniature calomel electrode for recording DC potential changes accompanying spreading depression. Physiol. Behav. 18: 1171-1174, 1977. 27. SILVERBERG, G. D. Ionic channels and control of cerebrovascular diameter. A review of cerebrovascular physiology. In: Neurotransmission and Cerebrovascular Function, edited by J. Seylaz and R. Sercombe. Amsterdam: Excerpta Med., 1989, p. 33-50. 28. STERN, M. D., P. D. BOWEN, R. PARMA, R. W. OSGOOD, R. L. BOWMAN, AND J. H. STEIN. Measurement of renal cortical and medullary blood flow by laser-Doppler spectroscopy in the rat. Am. J. Physiol. 236 (Renal Fluid Electrolyte Physiol. 5): F80-F87, 1979. 29. TAMAKI, K., AND D. D. HEISTAD. Response of cerebral arteries to sympathetic stimulation during acute hypertension. Hypertension Dallas 8: 911-917, 1986. 30. WAHL, M., M. LAURITZEN, AND L. SCHILLING. Change of cerebrovascular reactivity after cortical spreading depression in cats and rats. Brain Res. 411: 72-80, 1987.
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D. PIPER,
GEOFFREY
A. LAMBERT,
AND
JOHN
W. DUCKWORTH
Institute of Neurological Sciences, The Prince Henry and Prince of Wales Hospitals, and School of Medicine, University of New South Wales, Little Bay, Sydney 2036, Australia
PIPER, RICHARD D., GEOFFREY DUCKWORTH. Cortical blood flow
A. LAMBERT, AND JOHN W. changes during spreading depression in cats. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H96-H102, 1991.-Changes in cortical blood flow and cerebrovascular activity occurring during and after cortical spreading depression (CSD) were studied in ar-chloralose-urethan-anesthetized cats. CSD was induced by superficial cortical pinprick, and laser-Doppler velocimetry (LDV) was used to measure cerebral blood flow (CBF&. CSD resulted in a wave of cortical hyperemia during which there was a 215 * 48% peak increase in cortical blood flow that lasted for 2.7 t 0.4 min. This hyperemic phase was followed by prolonged cortical oligemia, with a reduction in flow of 20 t 4% at 1 h and 28 t 4% at 2 h. After CSD, cerebrovascular reactivity to the inhalation of CO, was abolished and did not fully recover for at least 10 h. Spontaneous vasomotor activity in the cerebral microcirculation was significantly decreased after CSD, and autoregulation of cortical blood flow in response to hypotension was preserved. The abnormal cerebrovascular reactivity seen after CSD in the gyrencephalic cortex of the cat has possible significance for human migraine with aura.
METHODS
H96
the American
Animal preparation. Male and female cats weighing 2.2 kg t 0.8 kg (mean t SD) were anesthetized with intraperitoneal injections of cu-chloralose (40 mg/kg) and urethan (500 mg/kg, n = 15) or a-chloralose (60 mg/kg) alone (n = 28). Supplementary doses of anesthetic were administered intravenously (20 mg/kg every 4-6 h) so as to maintain a stable level of anesthesia, as assessed by absence of tachycardia, hypertension, pupillary dilatation, or withdrawal to painful stimuli. Experiments during which cerebrovascular reactivity was being assessed lasted for up to 16 h. After intubation, the animals were ventilated with a mixture of 30% oxygen and air to maintain an end-expiratory CO, of 4.0-4.5%. Right femoral arterial and venous catheters were inserted to monitor blood pressure and administer drugs. A heating blanket was used to maintain rectal temperature at 3738OC. At the end of the experiment the animals were killed with an intravenous bolus of potassium chloride. Craniotomies were performed using a 6-mm dental velocimetry; migraine; autoregulation; cerebra - burr at low speed. The inner and outer diploe were laser-Doppler vascular reactivity removed in layers, exposing an underlying translucent layer of periosteum adherent to the dura. While drilling was in progress, the surgical field was bathed in normal CIRCUMSTANTIAL EVIDENCE suggests that cortica 1 saline to minimize the possibility of thermal injury to spreading depression (CSD) may cause focal neurologica 1 the underlying cortex. CBF was monitored with a single channel laserchanges (15, 19), such as scintillating scotomas and the Doppler perfusion monitor (Laserflo BPM403, TSI) and cortical “spreading oligemia” (24) seen during the aura pencil probe (P-431, TSI, diam of 1.5 mm). The laserof migraine. CSD has been shown in a number of species to result in a phase of cortical hyperemia (11, 13, 14, 17). Doppler probe was placed perpendicular to the cortical surface away from large pial and dural vessels, in gentle In the lissencephalic cortex of the rat, this initial hyperemit phase is followed by cortical oligemia, decreased contact with the dura or periosteum. Adequate probe pulsatile flow cortical vasodilatation during arterial hypercapnia, and placement was assessed by demonstrating synchronous with heart rate or vasomotor activity. Stapreservation of autoregulation (10). The hypothesis that ble flow recordings were usually obtained between 12 and CSD causes the spreading oligemia seen during migraine 25 units of laser Doppler flow (TSI calibrated units of with aura, assumes that CSD results in delayed cortical flow). Using this model, we have previously measured oligemia in other species, including humans. In the only baseline flows after death in 13 animals and demonsuch study, Wahl et al. (30) demonstrated dilatation of strated that the flowmeter readings closely approximate the pial vessels 15-120 min after CSD in the cat, in zero (0.15 t 0.16). To ensure stability of recordings, the contrast to pial constriction in the rat. To characterize animal was placed in a David Kopf head holder and the the delayed effects of CSD in species other than the rat, probe positioned with a stereotactic electrode manipuwe recorded cerebral blood flow (CBF) and cerebrovaslator. Blood flow was measured in the suprasylvian gyrus cular reactivity, occurring after CSD in the gyrencephalic of the parietal cortex or the posteromedial composite cortex of the cat. Blood flow measurements by laser- gyrus (visual cortex). The dura and periosteum were left Doppler velocimetry (CBFLn) allowed cortical flow to be intact unless otherwise indicated. monitored continuously in discrete areas of cerebral corCSD was initiated 5-20 mm away from the site of tex and the time course of changes in cortical cerebrolaser-Doppler probe by the rapid insertion and removal vascular reactivity to be determined. (l-2 mm depth) of a 26- to 30-gauge needle, avoiding the 0363-6135/91
$1.50
Copyright
0 1991
Physiological
Society
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BLOOD
FLOW
CHANGES
DURING
CORTICAL
local pial vessels. This method of initiating CSD was chosen because it was simple, effective, and analogous to experimental situations where electrodes (250-500 pm) are inserted into the cortex. To avoid any possible carryover effect, only one wave of CSD was studied in each hemisphere. If there was evidence of subdural blood at the end of the experiment, the animal was excluded from the study. In all experiments, expired Cog, blood pressure, heart rate, and CBFLn were recorded on-line using an analog-to-digital card (Labmaster DMA, Scientific Solutions) in an IBM-compatible computer. Arterial blood samples were analyzed with a Radiometer ABL300 blood gas machine and a Radiometer KNA2 sodiumpotassium analyzer. Evidence of spreading depression. Three criteria were used to confirm the development of CSD. First, DC potentials were recorded in eight animals using paired calomel electrodes with a tip diameter of 100-200 pm (26) or micropipettes filled with normal saline with a tip diameter of 2-10 pm (22). Recordings were made 0.25-l mm below the cortical surface. Second, electroencephalogram (EEG) was monitored in 10 animals using bipolar silver ball electrodes placed extradurally on the supra-sylvian gyrus ipsilateral to the site of cortical pinprick. Maximum peak to peak EEG amplitude was calculated during each 60-s period following the initiation of CSD. A 40% reduction in peak to peak amplitude was selected to mark the beginning and end of the period of EEG attenuation. EEG and DC potential recordings were made with a Narco Biosystems biopotential amplifier (type 7189) or Dagan 8700 amplifier Finally, to demonstrate that the blood flow disturbance after cortical pinprick propagated at a rate typical of CSD, the distance between the site of cortical pinprick and the laser-Doppler probe was correlated with the interval between pinprick and the first positive deflection in blood flow. The degree of correlation between these two variables was used as evidence of propagation, and the mean velocity was calculated using linear regression. Blood flow measurements. The immediate effects of CSD on CBFLn were studied in two groups of animals. In the first group the laser-Doppler probe was placed directly on the cortex. In the second group, to minimize the possibility of surgical trauma, blood flow was measured with the dura and periosteum intact. Cortical blood flow was measured during a 5-min control period and for 25 min after pinprick. The delayed effect of CSD on CBFLn was examined in a separate group of animals. CBFLn was measured over a 2-h control period in the intact animal. CSD was then precipitated with cortical pinprick and flow monitored for a further 2 h. The a priori hypothesis was that over the 2-h control period there would be no significant change in CBF LD due to the effects of anesthesia alone but that after CSD there would be a significant decrease in CBFLD. Average flow over a 5-min interval was calculated at the beginning of each 2-h recording period and at 60 and 120 min. Comparisons were made between the initial and latter two readings using a paired t test in both the control and CSD groups. As this hypothesis involved four comparisons, significant results were inter-
SPREADING
DEPRESSION
H97
preted using the Bonferroni procedure. If supplementary anesthesia was required during the recording period, the animal was not included in the data analysis. Only animals demonstrating a normal increase (X00%) in CBFLD during arterial hypercapnia (see Assessment of cerebrovascular reactivity) and evidence of active autoregulation as assessedby a gain factor BO.65 (see below) were included in the study. Assessment of cerebrovascular reactivity. Autoregulation of cortical blood flow was assessedby bleeding and subsequently reinfusing the animals to achieve a 20% step reduction in mean arterial blood pressure, while maintaining the systolic blood pressure ~80 mmHg. The integrity of autoregulation was assessedby calculating the gain factor (Gf) Gf
= 1 - Kwww/P)l
where (AF/F)/( AP/P) is the slope of normalized pressure-flow curve at a given point (P,F), as described by Norris et al. (23). If Gf is 1, cortical flow is maintained independent of systemic blood pressure; however, if GF is 0, autoregulation is absent and CBF passively follows blood pressure. Measurements were made before CSD and 30-60 min after the induction of CSD. Cortical vasodilatation following arterial hypercapnia was assessedby adding a gas mixture of 8% COa, 30% 02, and air to the ventilatory circuit for 5 min. This standard stimulus resulted in an arterial PCO~ of -60 mmHg (see Table 3). CBFLn was measured before and after 5 min of CO, inhalation, and the change in flow was calculated. The effect of hypercapnia on CBFLD was assessed at intervals up to 16 h after CSD. Similar observations were made on cortex where CSD had not been induced to examine the effect of prolonged anesthesia on COZ responsiveness. Assessment of vasomotor activity. Spontaneous vasomotor activity in the cortical microcirculation was assessed before and after CSD. Vasomotor activity was defined as spontaneous fluctuation in CBFLD (X0%) independent of heart rate and ventilation. As an index of the amplitude of vasomotor activity, a 2-min stationary period of the laser-Doppler signal was analyzed (120150 data points). The mean flow was calculated, and all data were expressed as percent deviation from the mean. The root mean square value of this transformed data was then used as an index of signal variability (CBFRMs). A similar analysis was performed on the blood pressure signal to obtain an index of variability (BPRMS). Statistical analysis. Values are means t SE unless otherwise stated. Statistical significance was tested using either a paired or unpaired t test as indicated. Each hemisphere was regarded as a separate cortical preparation when the number of measurements was greater than the number of animals quoted. The baseline was calculated from the mean flow of the preceding 5 min where flows are expressed as percentage change from baseline. RESULTS
In all animals (n = 43) cortical pinprick resulted in a wave of cortical hyperemia after the initial attempt. Evidence of CSD. In eight animals, cortical interstitial
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H98
BLOOD
FLOW
CHANGES
DURING
CORTICAL
DC potential was measured during the wave of hyperemia. These animals displayed DC changes typical of CSD. The mean maximum DC potential shift was -10.7 t 1.0 mV (n = 25, range -4.0 to -20 mV). In four animals the recording electrode was positioned underneath the laser-Doppler probe. In these animals the DC potential shift coincided with the onset of cortical hyperemia (Fig. 1). The surface EEG was recorded in 10 animals. The mean latency from cortical pinprick, to the onset of a 40% reduction in the peak to peak amplitude was 3 t 0.3 min. This degree of attenuation of the EEG persisted for a mean of 8.6 t 2 min, with recovery of normal peak to peak amplitudes (>90% of control) at a mean latency of 22 t 6 min. The correlation between the distance from the site of cortical pinprick to the laserDoppler probe, and the interval between pinprick and the first positive deflection in blood flow was examined in 15 cu-chloralose-urethan-anesthetized cats. Linear regression demonstrated that these two variables were correlated (r2 = 0.73, P C 0.001, n = 19) and that the mean velocity of propagation (3.1 t 0.4 mm/min) was typical of CSD (6). Changes in cortical blood flow. In 14 cu-chloralose-anesthetized animals, blood flow measurements were made with the dura and periosteum intact. In this group of animals CSD was associated with a 215 t 48% increase in CBFLD (Fig. 10). This period of intense hyperemia lasted for a mean duration of 2.5 t 0.2 min. In 11 of these animals a period of more prolonged, but less intense hyperemia (Fig. 1A ), followed this initial peak in flow (mean duration 5.8 t 1.1 min). At 25 min after CSD, CBFLn was decreased by 10 t 13.6%, but this result did not reach statistical significance (P = 0.4, unpaired t test) when compared with the change in CBFLD in the contralateral cortex (Fig. 1C). In the cat, the dura is thin and it is likely that blood flow measurements largely 0
A
Change
in
Flow
0 (4 0
B
in
Flow
( %)
D
Change
in 0 (4 0
20
25
o
(mV)
-16
-. . . . . . . . . . . . . . . . . . . I
'
501
Change
15
represent flow in the underlying cortex. To test this hypothesis similar observations were made in 15 urethan-cr-chloralose-anesthetized animals in which the dura had been removed and the cortex bathed in warmed Merlis artificial cerebrospinal fluid. In these animals, CSD was associated with a wave of cortical hyperemia that resulted in a mean 201 t 34% increase in CBDLn. These results suggest that it is possible to detect CSD in the cat using a minimally invasive preparation and that it is unlikely that the flow disturbance originates in the dural vessels. In 11 animals anesthetized with ac-chloralose alone the more prolonged effect of CSD on CBF was examined. CBFLn was measured through a small cranial window with the dura and periosteum intact. Biochemical data and systemic variables for these animals were within normal limits (Table 1). After CSD, CBFLn was decreased by 20 t 4% at 60 min and 28 t 4% at 120 min compared with initial flow (Table 2). These reductions in CBF JJ) were statistically significant when compared with CBFLn at the beginning of the 2-h recording period (P c 0.01 at 60 min and P c 0.01 at 120 min, paired t test after Bonferroni correction, n = 11, Table 2). During the 2-h control recording period, there was no significant change in CBF Ln at 60 and 120 min, assessed using a paired t test (P > 0.05). Changes in cerebrovascular reactivity. CSD was followed by abolition of cortical cerebrovascular reactivity to COa. Cerebrovascular reactivity to hypercapnia was tested before and after CSD in seven cu-chloralose-anesthetized animals. Before CSD, cortical blood flow increased with hypercapnia by 185 t 32% arterial PCO~ [(Paoo,) before = 32 t 0.9 mmHg, hypercapnic Pace, = 60 + 2 mmHg]. This response was significantly decreased when repeated ~60 min after CSD (3.0 t 2.0%, P < 0.01, data (Table 3) n = 7, paired t test). The biochemical
200
-24
C
10
DEPRESSION
300
DC Potential Change
5
SPREADING
I
25
,
I
1
1
1
- . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*.. . .-. 9.. _ -s-e .... s . ..a-\. *.*. .... ,.. ..-* ... -. . - 0 *..a.,. -.*. e.. I... a s - @.-.‘. -. ........* .‘.w_ _ c ..‘\.. w- e.-25 - . . . x.. . . . . . . . . . . . . . . . * . . * . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..a
. . . . . . . . C-J -
FLOW
Flow
. . . . . . . . . (-1 0
5
10
15
Time
(minutes)
20
FIG. 1. Values are means t 95% confidence interval (CI). A and B: typical trace showing blood flow changes occurring after cortical spreading depression (CSD). CSD was initiated by pinprick (vertical line) 10 mm from the site of Doppler probe. DC potential was recorded from an electrode placed under probe. C and D: mean changes in cerebral blood flow using laser-Doppler velocimetry (CBF& occurring after CSD. After CSD, blood flow in contralateral cortex was unchanged (C). Traces were averaged from end of the 5-min control period (vertical line) before initiation of CSD (n = 5). After CSD, ipsilateral transient cortical hyperemia is followed by a gradual decrease in cortical CBFLn (D). Traces were averaged from beginning of hyperemic phase (n = 14). Percentage changes in CBFr,D & 95% confidence intervals compared with control are shown.
FLOW
25
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BLOOD
FLOW
CHANGES
DURING
CORTICAL
SPREADING 300
1. Physiological variables in animals in which CBFLII was measured after CSD TABLE
2 2 u
120 min
Control 17.6k2.3
CW.D %A CBFLh MEco,, % Mean BP, mmHg
18.0k2.1
2.6kO.l 109*5
1
CSD initiated
-5 100
2. Effect of CSF on CBFLL) and systemic variables
CBF’LD %A CBFLu MEco,, % Mean BP, mmHg
I
is z
TABLE
60 min
I
- 200 VX
Values are means t SE; n = 11 cats. Biochemical variables and blood pressure were measured just before the 4-h recording period. The change in cortical blood flow was measured after the inhalation of 8% CO, for 5 min before inducing CSD. Pace,, Pao,, arterial PCO~ and Po2, respectively.
0 min
I
‘4 I
7.33t0.01 33t1.8 184k13 -7.6k0.8 17.5kO.7 99.320.2 145t1.0 3.020.1 115t5.2 188t16.6 0.82t0.03
PH Pm,, mmHg Pao,, mmHg Base excess HCO:], mM %O, saturation Na, mM K, mM Blood pressure, mmHg %A in CBFLD with CO2 Gain factor
H99
DEPRESSION
17.1t2.0 -2t4 2.7tO.l 116t5
18.222.2 +6t6 2.8t0.2 118&3
14.3t1.8* -2Ot4 2.8tO.l 113t6
12.7&1.3? -28t4 2.8k0.2 113t5
0
I
-100
-100
I
200 Time
I
I
500
800 (minutes)
Post-CSD
1100
FIG. 2. Reduction in hypercapnic vasodilatation after CSD. Hypercapnia was induced after inhalation of a mixture of 8% C02-30% oxygen and air for 5 min. Response before CSD is plotted, followed by mean response over periods of 200 min after CSD. Mean response in animals in which CSD had not been induced is also plotted over periods of 200 min after commencement of cortical recordings (*P < 0.001; * P < 0.05; unpaired t test). Values are means t SE. 400
CSD
2.8tO.l 115*6
Values are means & SE. After a 2-h control period, CSD was initiated with cortical pinprick, and CBF LD was measured for a further 2 h. CBFLD is expressed in flowmeter (TSI) units of flow. CBFLD, mean expired CO, (MEcon) and blood pressure were followed serially. %A made at 0 min were CBFLD, percent change in CBF LD. Measurements compared with those at 60 and 120 min using a paired t test. * P = 0.001, t P = 0.002. Significant at the P < 0.001 level after the Bonferroni procedure.
TABLE 3. Physiological variables in animals tested for cortical responsivenessto hypercapnia
PH Pac02, h,,
mmHg mm&
HCOs, mM %02 saturation Na, mM K, mM Blood pressure,
mmHg
Control
B%CO,
7.32t0.01 3220.9 181klO 16kO.6 99t0.2 144k1.2 3.05kO.l 119&7
7.12kO.01 6022 193k9.3 19t0.6 98tO. 1 145kl.O 2.8kO.07 123*8
Values are means t SE; n = 7 cats. Biochemical variables were measured on arterial blood before and then after inhalation of 8% CO, for 5 min.
collected from this group of animals were within the physiological range for the cat (18). The time course of change in cerebrovascular reactivity to the inhalation of 8% COz was followed serially in 12 cu-chloralose-anesthetized animals, including the above 7 (Fig. 2). Cortical blood flow was measured through the intact dura and periosteum. The mean increase in CBF after the inhalation of CO2 was plotted for each succes-
.r(c
-100
I
0
I
I
I
I
I
4
8
12
16
Time(minutes) 3. Decrease in spontaneous vasomotor activity after CSD. Data from a cat in which vasomotor activity was prominent. CSD was initiated by pinprick (vertical line) 6 mm from site of laser-Doppler probe. FIG.
sive ZOO-min interval after the initiation of CSD. These data suggest that cerebrovascular reactivity to the inhalation of CO2 is impaired for as long as 12-h after the initiation of CSD. The control group in Fig. 2 consists of data from 20 hemispheres in which CSD had not been induced. This group demonstrates that cerebrovascular responsiveness to CO2 is preserved despite prolonged periods of recording under general anesthesia. Changes in vasomotor activity. In 13 cu-chloralose-anesthetized animals, the effect of CSD on spontaneous vasomotor activity was observed. Data from a typical animal are presented in Fig. 3. In 6 of 13 animals, spontaneous vasomotor activity, defined as spontaneous fluctuations in CBF LD (X0%) independent of changes in heart rate and respiration, was observed. The mean frequency of such fluctuations was 6.4 t 0.2 per min. CBFRMs signal was recorded before CSD (17.4 t 2.5%) and again 30-60 min post-CSD (5.27 t 0.6%). The mean percent change in CBF RMSwas 66 k 5% (P c 0.005). In the seven remaining animals, where spontaneous vaso-
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HlOO
BLOOD
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motor activity was not evident, there was a 25 t 4% reduction in CBF RMS after CSD (before, 11.5 t 2.7 TSI units of flow; after, 8.3 t 1.9 TSI units of flow; P = 0.36). These data suggest that variability in CBFLD is generally decreased by CSD and that spontaneous vasomotor activity is particularly affected. Linear regression between the percentage change in CBFRMs and the pre-CSD CBFRMs value demonstrated that these two variables were linearly related in animals displaying spontaneous vasomotor activity but not in the other animals (Fig. 4). Spontaneous vasomotor activity was thus selectively affected by CSD. To assess whether this was due to changes in either BP RMS or the mean BP, these two variables were assessed before and after CSD in the group of animals (n = 6) with spontaneous vasomotor activity. However, neither the mean BP (before, 107 t 6.7 mmHg; after, 108 t 9.5 mmHg; paired t test, P = 0.72) nor BPRMs (before, 5.2 t 1.2 mmHg; after, 5.6 t 1.0 mmHg; paired t test, P = 0.31) was affected by CSD. Autoregulation was unaffected when assessed by venesection before and then 30-60 min after CSD (n = 7, Table 4). DISCUSSION
Laser-Doppler blood flow measurement. The area sampled by laser-Doppler velocimetry has been estimated to be approximately 1 mm3 (9, 28). A number of studies 100
FIG. 4. Spontaneous vasomotor activity is reduced by CSD in proportion to its control amplitude. Linear regression between pre-CSD root mean square CBF (CBF lIMS) and percent change in CBFHMs after CSD is plotted for 7 animals with and 6 animals without preexisting vasomotor activity. 0, vasomotor activity present; r2 = 0.81, slope = = 32 t 8.6, P < 0.05. l , vasomotor activity 1.95 k 0.47, intercept = 20.5 t 8.6, P > 0.05. absent, r’! = 0.07, slope = 0.42 ~fr 0.65, intercept
TABLE
4. Effect of CSD on autoregulation Control BP, mmHg
Pre-CSD Post-CSD
109t3.5 llOt3.6
%A BP
25.2-el.5 21.2t1.0
%A CBFLD
Factor
Gain
2.7t1.6 1.5t1.3
0.80t0.04 0.86t0.03
cats. Autoregulation was assessed by in a step reduction in blood pressure; Post-CSD measurements were made
CORTICAL
SPREADING
DEPRESSION
have demonstrated that flow measured by laser-Doppler correlates well with absolute flow measured with established techniques [intestinal mucosa (1); spinal cord (16); rabbit cerebral cortex (5)]. Dirnagl et al. (3) in a study of the rat cerebral cortex, compared CBF measured by the [ 14C]iodoantipyrine technique with laser-Doppler flow measurements and found that CBFLD correlated well with relative changes in flow but poorly with absolute flow. For this reason blood flow changes in this study are not expressed in absolute units of flow but rather as percentage change in flow from baseline. Du ring this series of experiments, the laser-Doppler probe was held in position with a stereotactic holder to minimize the effect of probe movement on blood flow readings. Additionally, the use of urethan and/or cu-chloralose, both of which are anesthetic agents with intermediate to long half-lives, aided in obtaining stable baseline CBFLD recordings. We have demonstrated that this model provides stable baseline flows for periods of up to 2 h. It is likely that changes in baseline flow due to probe movement would be random and result in an increased experimental error causing failure to reject a null hypothesis, rather than the demonstration of statistically significant changes in flow. Laser-Doppler velocimetry and detection of CSD. Other workers (6, 17) have suggested that CSD is difficult to induce in the gyrencephalic cortex. Our results suggest that this is no t the ca.se with urethan-cr-chloralose anesthesia in the cat. It seems unlikely that the an .imals studied were susceptible to CSD on account of either surgical trauma or metabolic perturbation. Using a minimally invasive technique, which involved measuring CBFLD through the intact dura and periosteum, it was still possible to induce CSD in all animals in which there was physiological cerebrovascular reactivity before the study. We have demonstrated that this preparation does not result in marked changes in biochemical variables or alterations in cerebrovascular responsiveness to hypercapnia. The ease with which CSD is induced in this preparation is of interest because of the common use of the cu-chloralose-anesthetized cat as a cortical experimental model .. Blood flow changes associated with CSD. A wave of cortical hyperemia in association with CSD was first suggested by Lego (14) who demonstrated a brief period of pial dilatation in the rabbit. Subsequently, a number of authors using a variety of techniques have confirmed that CSD results in a phase of cortical hyperemia (11, 13,17). Hansen et al. (7) examined this hyperemic phase using the [“Cl iodoantipyrine technique and demonstrated that the cortical hyperemia accompanying CSD starts during the normalization of interstitial K+ concentrations, rather than during the period of maximal increase in interstitial K+. These observations would suggest that the hyperemic phase is due to increased local oxygen demand as a result of the metabolic activity required to reconstitute membrane ionic gradients. A phase of cortical oligemia after CSD was first described by Lauritzen et al. (13) in the cortex of the rat. In this study cortical oligemia was evident 5 min after the passage of a wave of CSD and persisted for at least 60 min. Cortical oligemia, persisting for as long as 200
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HlOl
min in the rat cortex, has subsequently been confirmed intended experimental model. We have demonstrated by other workers (4, 21). In this study we have demonthat the abnormalities in cerebrovascular reactivity to strated a similar phase of cortical oligemia in the cat, CO2 induced by CSD are more prolonged than those but the latency to onset is longer than that reported in demonstrated thus far in the rat and that they are the rat (Table 2, Fig. 1). associated with decreased spontaneous vasomotor activity. It has previously been suggested that delayed hypoWahl et al. (30) have demonstrated constriction of pial vessels (25-62 pm) in the rat after CSD and dilatation perfusion seen after CSD is a result of impairment of the in pial vessels (66-257 pm) in the cat. This pial dilatation metabolism flow couple, rather than a primary reduction in cortical metabolism (12, 21). The primary alterations was observed in 80% of vessels in the post-CSD period (15-120 min). Our observation that CSD results in de- in cerebrovascular reactivity seen after CSD in the cat creased CBFLn in the cat demonstrates that while the may be the basis of this abnormal coupling between blood flow and metabolism. pial vessels dilate, the resistance vessels constrict. This observation would suggest that the pial dilatation may We have demonstrated that the changes in cortical blood flow seen after CSD in the lissencephalic cortex, be a compensatory mechanism secondary to increased resistance in small diameter arterioles (400 pm). This also occur in the cat; an animal with a gyrencephalic hypothesis is attractive because in the cat 45% of vas- cortex that morphological .ly resembles more closely the cular resistance derives from vessels less than 100 pm in human cortex. If CSD occurs in the human cortex, it diameter (29), and it is likely that small diameter arte- may well be a pathological entity of clinical importance rioles (~100 pm) would be more intimately affected by during the aura of migraine following head injury or the parenchymal metabolic disturbance seen during CSD neurosurgery. than larger pial vessels. Changes in cerebrovascular reactivity. Lauritzen et al. The authors thank Professor J. W. Lance for advice and critical evaluation of the manuscript. We also thank M. Hellier for technical (10) have demonstrated in the rat that CSD is associated assistance and Dr. Karen Bythe for statistical advice. with blunted vascular reactivity to COZ assessed 30-60 This work was supported by the National Health and Medical min after CSD. Wahl et al. (30) using a cranial window Research Council of Australia, the Basser Trust, the J. A. Perini Family preparation in the cat, looked at cerebrovascular reactivTrust, and Warren and Cheryl Anderson. Address for reprint requests: R. D. Piper, Dept. of Neurology, ity following CSD. After examining the effects of direct application of K+, H’, adenosine, and bradykinin to the Clinical Sciences Bldg., Prince Henry Hospital, Anzac Pde, Little Bay 2036, Sydney, Australia. pial vessels (66-257 pm), they concluded that cerebroReceived 14 August 1990; accepted in final form 12 March 1991. vascular reactivity to a wide range of chemical stimuli was severely impaired. In the cat, we have demonstrated that after CSD CO2 reactivity, a physiological index of REFERENCES cerebrovascular responsiveness, is decreased for up to 12 1. AHN, H., J. LINDHAGEN, G. E. NILSSON, E. G. SALERUD, M. h. JODAL, AND 0. LUNDGREN. Evaluation of laser Doppler flowmetry Changes in vasomotor activity. Vasomotor activity has in the assessment of intestinal blood flow in cat. Gastroenterology 88: 951-957, 1985. been described in cerebral arterioles using a variety of 2. AUER, L. M., AND B. GALLHOFER. Rhythmic activity of pial vessels techniques. Spontaneous fluctuations in cortical vasoin vivo. Eur. Neurol. 20: 448-468, 1981. motor tone are well described in vivo using videoangiog3. DIRNAGL, U., B. KAPLAN, M. JACEWICZ, AND W. PULSINELLI. raphy and the cranial window technique (2, 8). It has Continuous measurement of cerebral cortical blood flow by laserDoppler flowmetry in a rat stroke model. J. Cereb. Blood Flow also been demonstrated in vitro in arterioles taken from Metab. 9: 589-596, 1989. branches of the posterior cerebral arteries of sponta4. DUCKROW, R. B. Regional cerebral blood flow during cortical neously hypertensive rats (25). This phenomenon has spreading depression in conscious rats (Abstract). J. Cereb. Blood recently been demonstrated using laser-Doppler velociFlow Metab. 9, Suppl. 1: S510, 1989. metry in the rat cortex (3) and also in the feline spinal 5. EYRE, J. A., T. J. ESSEX, P. A. FLECKNELL, P. H. BARTHOLOMEW, AND J. I. SINCLAIR. 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22. NEDERGAARD, M., AND A. J. HANSEN. Spreading depression is not associated with neuronal injury in the normal brain. Brain Res. 449: 395-398,1988. 23. NORRIS, C. P., G. E. BARNES, E. E. SMITH, AND H. J. GRANGER. Autoregulation of superior mesenteric flow in fasted and fed dogs. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H174-H177, 1979. 24. OLESEN, J., B. LARSEN, AND M. LAURITZEN. Focal hyperemia followed by spreading oligemia and impaired activation of rCBF in classic migraine. Ann. Neurol. 9: 344-352, 1981. 25. OSOL, G., AND W. HALPERN. Spontaneous vasomotion in pressurized cerebral arteries from genetically hypertensive rats. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): H28-H33, 1988. 26. SHITABA, M., B. SEIGFRIED, AND J. P. HUSTON. Miniature calomel electrode for recording DC potential changes accompanying spreading depression. Physiol. Behav. 18: 1171-1174, 1977. 27. SILVERBERG, G. D. Ionic channels and control of cerebrovascular diameter. A review of cerebrovascular physiology. In: Neurotransmission and Cerebrovascular Function, edited by J. Seylaz and R. Sercombe. Amsterdam: Excerpta Med., 1989, p. 33-50. 28. STERN, M. D., P. D. BOWEN, R. PARMA, R. W. OSGOOD, R. L. BOWMAN, AND J. H. STEIN. Measurement of renal cortical and medullary blood flow by laser-Doppler spectroscopy in the rat. Am. J. Physiol. 236 (Renal Fluid Electrolyte Physiol. 5): F80-F87, 1979. 29. TAMAKI, K., AND D. D. HEISTAD. Response of cerebral arteries to sympathetic stimulation during acute hypertension. Hypertension Dallas 8: 911-917, 1986. 30. WAHL, M., M. LAURITZEN, AND L. SCHILLING. Change of cerebrovascular reactivity after cortical spreading depression in cats and rats. Brain Res. 411: 72-80, 1987.
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