Supplementary text
(Note: References to Figure numbers refer to those in the main text. Citations are numbered and listed separately in this text)
Note 1: “Perfusion response as a surrogate tracker of CSD”
Cerebral blood flow changes coupled to spreading depolarization may be studied as surrogate markers to image depolarization events in real time using laser speckle flowmetry (LSF imaging) (Dunn et al., 2001; Strong et al., 2006). This method has yielded valuable information on occurrence of depolarizations and their hemodynamics following experimental occlusion of the proximal middle cerebral artery (MCAO), and on the capacity of depolarizations to reduce perfusion further in border zones of the ischemic focus (Shin et al., 2006; Strong et al., 2007). However, the extent of the imaging field in the cat and in some rodent MCAO models of stroke is necessarily limited by anatomical and technical factors, so that with proximal occlusion it is not possible to expose the entire core and penumbra surgically and encompass the entire perimeter of the lesion in a single surface image. Thus apparent radial (centrifugal) as well as circumferential (anteroposterior or the reverse) spread of depolarizations in the imaged segments of penumbral gyri have been seen after permanent MCAO in cats, but not fully analyzed or reported to date.
Are spreading changes in perfusion reliable surrogates for spreading depolarization? Several authors have imaged serially a metabolic (NADH fluorescence change (Higuchi et al., 2002; Strong et al., 1996) or perfusion (Dunn et al., 2001) correlate of CSD, principally as a means of tracking its propagation. That the imaged variable follows – rather than leads – the depolarization has been amply confirmed in both lissencephalic (rodents) and gyrencephalic (cats) species (Shin et al., 2006; Strong et al., 2007). The sensitivity of the perfusion transient as a surrogate for depolarization depends on the constancy of the coupling. In normally perfused tissue the associated hyperemia appears from a large volume of publications extending back to the first description by Leão (Leao, 1944 ) to be highly robust. In contrast, once tissue becomes progressively more ischemic, the perfusion response to depolarization changes, also progressively, to biphasic reduction/increase, to monophasic transient decrease, and finally to more sustained transient, or permanent, reduction (Strong et al., 2007). Is there an intermediate level of basal perfusion in which hyperemic and hypoperfusion responses to depolarization are balanced, resulting in no detectable perfusion response? Given the frequency of the biphasic response, it seems to us highly unlikely that such a balance would be achieved often enough to give rise to significant false negative data. As an example, Supplementary video 2 suggests abrupt transition from hypo- to hyperperfusion during radial spread of a depolarization as it moves from suprasylvian to marginal gyrus. In any event, the large number of perfusion responses available for analysis from the cat experiments provides a solid basis for conclusions on the spatial and temporal properties of at least those depolarizations detected by perfusion imaging.
Note 2: ”Spatial patterns of CSD spread after MCAo in cats”
This note is on retrospective analyses of spatial patterns of spread of depolarizations and inter-depolarization intervals following middle cerebral artery occlusion (MCAo) in cats in the laboratories in Cologne and London, respectively (including details of anesthesia and surgical preparations).
Cologne
Details of anesthesia and surgical preparation: General anesthesia was induced with ketamine hydrochloride (25mg/kg, i.m.) in female cats weighing 2.6-5.0 kg. The left femoral vein and artery were cannulated to administer drugs and to measure arterial pressure and arterial blood gases. The animals were tracheotomized and immobilized with pancuronium bromide (0.2 mg/kg, i.v.). Artificial ventilation was started, and anesthesia was changed to halothane (0.6-1.2%) in a 70% nitrous oxide/30% oxygen gas mixture. Ringer’s solution containing gallamine triethiodide (5mg/kg/h) was infused intravenously (3ml/h) for immobilization throughout the experiment. Artificial ventilation was controlled, keeping arterial and expiratory gases within normal physiological ranges. Deep body temperature was maintained at 37°C using a heating blanket feedback controlled by a rectal temperature probe. The right MCA was exposed transorbitally in all experiments. A device for remote occlusion was implanted at the proximal portion of the MCA between the right optic nerve and the MCA bifurcation) (Ohta et al., 1997). The device was completely fixed with gelfoam and rapid-drying glue. Finally, the orbit was sealed with dental cement to avoid CSF leakage. The scalp and calvarium were removed and the dura was opened over the right cerebral hemisphere so as to expose the majority of the ectosylvian (EG), suprasylvian (SG) and marginal (MG) gyri. Intracortical microelectrodes and microdialysis probes (surface brain layers: 2 mm probes) were inserted as required. A mineral oil pool was established above the exposed cortex, and an electric miniature heater inserted in the pool to control brain surface temperature at around 37 °C. After preparation of the animals, a bolus of α-chloralose (60mg/kg/15min, i.v.) was administered, halothane was switched off, and artificial ventilation was continued, with 70% nitrous oxide/30% oxygen. To maintain α-chloralose anesthesia throughout the experiment, continuous α-chloralose infusion (5mg/kg/h) was started following the bolus. Proximal MCA occlusion (MCAo) was performed by remote occlusion. Observation time with LSF was 16.9±5.7 (mean ± SD) hours. Coupling of DC and CBF change was investigated by the combination of LSF and depth microelectrodes.
Spatial features of propagation of PIDs in the gyrencephalic (cat) brain: Mean arterial pressure was 100-120 mm Hg, higher than in London experiments. In the course of focal ischemia with observation times typically of 17 hours after transorbital middle cerebral artery occlusion, CBFIND wave propagation often changed during the course of an experiment from a radial pattern (initiation at the border of the core and movement outwards into the periphery of the ischemic territory) to a circumferential pattern with propagation around the core, examples of which are given in Figures 4 and 5 respectively and in supplementary videos 2 and 3. In the case of radial propagation, waves therefore spread perpendicular to - but in circumferential propagation, parallel to - the three gyri. Analysis of all events revealed that 34 out of 82 observed CBFIND waves propagated radially, and 48/82 circumferentially. We observed monogyral waves remaining in one gyrus throughout the full course seen in the field of view by LSF both for radial (10/82) and for circumferential propagation (33/82) but also polygyral waves crossing from the suprasylvian gyrus to the marginal gyrus as radial waves (24/82), or moving circumferentially at once (15/82) in a broad front on these two gyri around the ischemic focus. In case of radial polygyral propagation, crossing the sulcus between suprasylvian and marginal gyri led to an apparent delay of wave movement before it reached the marginal gyrus (see Video 2) since LSF images only the brain surface.
Periodicity was seen in one Cologne experiment (#03) in which 2 clusters occurred, of 3 waves each. In the first, intervals were 20 and 18 minutes (consistent with completion of circumferential propagation of 2 cycles around a lesion of constant size), and in the second, 33 and 40 minutes: this is compatible with lesion enlargement before and/or during the second cluster, but brain swelling vitiated attempts to verify this by measuring the CBFIND-thresholded lesion area. These findings thus resemble those in rat dMCAo (CBFIND waves cycling periodically around the ischemic focus). Waves usually propagated repetitively in one direction (e.g. clockwise, as is true for most waves in Figure 5), however, in some instances (e.g. waves 12 and 13 in Figure 5), waves took instead the opposite, counter clockwise route. These results are assessed in the main Discussion text.
London
Details of anesthesia and surgical preparation: Methods were similar to those described above for Cologne, with these differences. (1) Anesthesia was induced with metetomidate/halothane, but maintained with α-chloralose, as in Cologne. Mean arterial pressure typically lay in the range 70-90 mm Hg, somewhat lower than in Cologne. The right hemisphere was exposed over the area indicated in Figure 1A and protected with mineral oil. Duration of observation after MCAo was confined to 4 hours.
Spatial features of propagation of PIDs in the gyrencephalic (cat) brain:We revisited earlier laser speckle imaging data from 11 permanent MCAO experiments (4 hour duration) carried out in cats, and whose hemodynamic data have previously been reported (Strong et al., 2007). There, the emphasis was on characterizing the vascular response to a depolarization at different distances from the core infarct. Here, we recorded one or more transient, spreading changes in laser speckle perfusion signal in 9 of the experiments and sought to identify their source, direction of spread, and their timing in relation to other events in the same experiment. Of a total of 73 events, 33 were seen to originate near the edge of the imaged field and were judged to have originated elsewhere and propagated into the field, 27 clearly originated within the field, and 13 could not be assigned to either category and were not considered further. Two types of propagation patterns termed as “colliding” and “splitting” events need to be specifically described: (a) “Colliding” events: on 4 occasions in 3 experiments, events judged to have originated elsewhere appeared at anterior and posterior extremes of the (imaged) marginal gyrus (“outer penumbra”) either simultaneously or with 1- 2 minutes of one another, spread towards each other and collided at varying points on the marginal gyrus, with, as expected, no further propagation beyond the collision point. This mode of spread was circumferential, as described for the studies in rats. Colliding events are capable of interpretation as having arisen from “splitting” of a single event at the opposite edge of an approximately circular cortical lesion, as demonstrated in the rat dMCAO lesion described in the main text. Events originating outside the imaged field, and not identified as colliding as above, usually but not always spread for the full exposed length of the gyrus. Of 25 such events, 15 spread anteroposterior and 10 posteroanterior. (b) “Splitting” events: Of 27 events clearly originating within the field, 7 appeared to arise from a single focus, most commonly at the extreme lateral edge of the marginal gyrus (and thus perhaps in fact arising within the marginal sulcus), and then spread both anterior (“clockwise”) and posterior (“anticlockwise”) on the gyrus. A further 13 originated similarly but spread only clockwise, while 5 spread anticlockwise. Of these 18 that clearly spread in one or other direction (but not both) circumferentially, 4 also clearly displayed a radial mode of spread on the gyrus soon after initiation. We speculate that a new event arising at the core margin may be constrained to spread only in one circumferential direction by persisting non-excitability in the other direction, resulting from a previous event (Please see also Discussion in main text.).
Radial versus circumferential spread: Essentially all events judged to have arisen outside the image field spread in a circumferential fashion around the edge of the core lesion (“core” was recognizable as having low levels of perfusion with no change in perfusion during transit of a spreading event nearby: in several cases the lateral suprasylvian gyrus behaved in this way, such that events could be seen to spread only along the medial, penumbral strip of the gyrus). The pattern of spread of events arising inside the image field was harder to define. Although events could be seen to spread forwards or backwards, or both (“circumferential”), there was also medial spread (“radial”) that varied in prominence. It is possible that the proximity of collateral input from the anterior cerebral artery, and variations in this, may account for such heterogeneity in this area (Please see also DISCUSSION in main text).
Periodicity: Primary analysis of the London experiments had shown occasional sequences of spreading laser speckle or DC-electrode-detected events that appeared to show periodicity (recurrence of 3 or more events at intervals that varied by not more ~10-15%). Results from the experiments with small focal lesions described above prompted review of the timing of such sequences in relation to the spatial patterns of spread, in a search for evidence of 360⁰ circumferential spread (cycling) of a depolarization around the entire core infarct. The present review identified relevant findings in 2 of 9 informative experiments. In the first, a cluster of events appeared at the low anterior extreme of the ascending limb of the suprasylvian gyrus at consecutive intervals of 22, 21, 26, 33, 34, 20, 23 and 16 minutes. The steady increase to a period of 34 minutes is most readily interpreted as repeat cycling of a single event around a core lesion of increasing cortical area: this pattern was fully documented in the rat dMCAO experiments (main text, Fig 2A,B). The later, shorter periods can be explained as new events arising from new foci at the edge of the core lesion, or as indicating reduction in lesion area: the first explanation seems more probable. A further feature of this experiment was two additional events appearing posteriorly and spreading forwards (interval= 32 min), with collisions with two of the above anterior events at an interval of 30 min. In a second experiment, transient changes in DC-potential characteristic of spreading depolarization were recorded with a surface electrode at consecutive intervals of 34, 35, 28, 26, 27, 21, 24, and 28 minutes (laser speckle imaging was technically unsatisfactory in this experiment).
Note 3: “Estimation of lesion size in relation to circumferential CSD in cat MCAo”
This comparison was undertaken by reviewing an image of a core lesion (permanent MCAo in one cat) projected onto the brain surface, available from Fig. 4 B (time point MCAO3, left hemisphere) of a PET study (Heiss et al., 1994)9. The image of oxygen extraction fraction shows a highly demarcated area with essentially zero CMRO2, taken to represent core. Without reference to periodicity data above, we estimated the mean diameter as 22mm, hence “mean” lesion perimeter = 72.3 mm. For a lesion of the size indicated, a depolarization tracking the perimeter for one complete cycle would require to transit 4 sulci (marginal and suprasylvian, each twice). Taking sulcal depth as typically 6 mm, the perimeter to be traversed now amounts to 120mm. At the typical depolarization speed of spread of 3mm/min, cycle time would be 40 minutes. Although this crude calculation involves important assumptions and the errors are significant, the value calculated for cycle time lies well within the range of values recorded in the 2 series reported here (main text Figure 6).
References in supplementary text
Dunn AK, Bolay H, Moskowitz MA, Boas DA. Dynamic imaging of cerebral blood flow using laser speckle. J Cereb Blood Flow Metab 2001; 21: 195-201.
Heiss WD, Graf R, Wienhard K, Lottgen J, Saito R, Fujita T, et al. Dynamic penumbra demonstrated by sequential multitracer PET after middle cerebral artery occlusion in cats. Journal of Cerebral Blood Flow & Metabolism 1994; 14: 892-902.
Higuchi T, Takeda Y, Hashimoto M, Nagano O, Hirakawa M. Dynamic changes in cortical NADH fluorescence and direct current potential in rat focal ischemia: relationship between propagation of recurrent depolarization and growth of the ischemic core. J Cereb Blood Flow Metab 2002; 22: 71-9.
Leao AAP. Pial circulation and spreading depression of activity in cerebral cortex. J Neurophysiol 1944 7: 391 – 396
Ohta K, Graf R, Rosner G, Heiss WD. Profiles Of Cortical Tissue Depolarization In Cat Focal Cerebral Ischemia In Relation to Calcium Ion Homeostasis and Nitric Oxide Production. Journal of Cerebral Blood Flow & Metabolism 1997; 17: 1170-1181.
Shin HK, Dunn AK, Jones PB, Boas DA, Moskowitz MA, Ayata C. Vasoconstrictive neurovascular coupling during focal ischemic depolarizations. J Cereb Blood Flow Metab 2006; 26: 1018-30.
Strong AJ, Anderson PJ, Watts HR, Virley DJ, Lloyd A, Irving EA, et al. Peri-infarct depolarizations lead to loss of perfusion in ischaemic gyrencephalic cerebral cortex. Brain 2007; 130: 995-1008.
Strong AJ, Bezzina EL, Anderson PJ, Boutelle MG, Hopwood SE, Dunn AK. Evaluation of laser speckle flowmetry for imaging cortical perfusion in experimental stroke studies: quantitation of perfusion and detection of peri-infarct depolarisations. J Cereb Blood Flow Metab 2006; 26: 645-53.
Strong AJ, Harland SP, Meldrum BS, Whittington DJ. The Use Of In Vivo Fluorescence Image Sequences to Indicate the Occurrence and Propagation Of Transient Focal Depolarizations In Cerebral Ischemia. Journal of Cerebral Blood Flow & Metabolism 1996; 16: 367-377.
Table 1 Arterial blood pressure and blood gases
preischemic control / 3 hours after occlusionRats / MABP / Mean / 121.2 / 122.2
SD / 6.6 / 16.5
ptiO2 / Mean / 104.3 / 106.6
SD / 16.4 / 30.8
ptiCO2 / Mean / 40.1 / 39.6
SD / 13.8 / 6.7
pH / Mean / 7.37 / 7.4
SD / 0.13 / 0.05
Cats / MABP / Mean / 128.7 / 121.9
SD / 9.9 / 18.3
ptiO2 / Mean / 138.0 / 128.6
SD / 26.0 / 22.2
ptiCO2 / Mean / 32.5 / 32.2
SD / 4.9 / 5.8
pH / Mean / 7.31 / 7.30
SD / 0.04 / 0.01
Video legends
In all videos, the multicolor lookup table is coded for indicative perfusion (CBFIND; ml/100g/min): the red-blue sequences use a lookup table indicating arithmetic difference in perfusion from pre-injection (red = increase, blue = decrease).
Video 1
Sequence of laser speckle images acquired in a rat (0.8-1.2% isofluorane anesthesia), starting 5.5 minutes following tandem occlusion of distal middle cerebral and ipsilateral common carotid arteries. The wave cycles anticlockwise 7 times around the core lesion (see also figure 1 in main text).
Video 2
Laser speckle image sequence showing the first spreading event in the territory imaged, 49.5 minutes after permanent middle cerebral artery (MCA) occlusion in a cat. A wave of hypoperfusion appears on the low suprasylvian gyrus (nearest the occluded MCA input) and spreads radially before disappearing into the marginal sulcus. On re-emerging onto the marginal gyrus, the vascular response to the event has reverted to the more normal hyperemia (see also figure 3 in main text).
Video 3
Two speckle sequences ~7.5 and 11 hours after middle cerebral artery occlusion in a cat. In both sequences, a depolarization event appears on the marginal gyrus (MG) at the posterior margin of the field and spreads forwards. The posterior half of the suprasylvian gyrus (SG) is not affected as it is already fully depolarized. The first event is entirely hyperemic until it approaches the anterior SG, when the leading edge becomes hypoperfusion, but is still followed by hyperemia, indicating a biphasic response in this area of cortex. In the second event shown, some 4 hours later, the primary change in the event as it spreads is hypoperfusion, and the subsequent hyperemia is much less prominent, indicating deterioration in the vascular response to depolarization (see also figure 4 in main text).