Supply-demand mismatch transients in susceptible peri-infarct hot zones explain the origins of spreading injury depolarizations

Abstract

Peri-infarct depolarizations (PIDs) are seemingly spontaneous spreading depression-like waves that negatively impact tissue outcome in both experimental and human stroke. Factors triggering PIDs are unknown. Here, we show that somatosensory activation of peri-infarct cortex triggers PIDs when the activated cortex is within a critical range of ischemia. We show that the mechanism involves increased oxygen utilization within the activated cortex, worsening the supply-demand mismatch. We support the concept by clinical data showing that mismatch predisposes stroke patients to PIDs as well. Conversely, transient worsening of mismatch by episodic hypoxemia or hypotension also reproducibly triggers PIDs. Therefore, PIDs are triggered upon supply-demand mismatch transients in metastable peri-infarct hot zones due to increased demand or reduced supply. Based on the data, we propose that minimizing sensory stimulation and hypoxic or hypotensive transients in stroke and brain injury would reduce PID incidence and their adverse impact on outcome.

Figure 1. Somatosensory stimulation triggers PIDs in stroke

a. Laser speckle imaging field was positioned over the right hemisphere as shown overlaid on a mouse skull (left panel). Distal middle cerebral artery occlusion (MCAO) induced a focal reduction in cerebral blood flow (CBF) in dorsolateral cortex (middle panel). Blue pixels indicate regions with less than 30% residual CBF after MCAO. We detected PIDs using the propagating large amplitude CBF transients on high spatiotemporal resolution full-field images (Movies S1–S4), as shown in the representative CBF tracing (right panel) from ischemic penumbra (yellow square). Global CBF fluctuations also occurred (*), often linked to systemic transients; these were distinguished from PIDs by lack of propagation on movies. b. In control (i.e., non-stimulated) animals, PIDs spontaneously occurred in apparently random fashion throughout the MCAO (green circles). Each horizontal line represents one animal, and the start and end of each line indicate the time span of imaging. c. Light tactile stimulation of the contralesional forepaw and shoulder (red and orange, respectively) triggered a PID within 5 minutes (yellow circles) with high incidence (p<0.001 vs. spontaneous PID rate). Inset shows the latency between forepaw or shoulder stimulation onset and PID onset. In contrast, contralesional whisker pad or hindpaw stimulation (light and dark blue, respectively), and ipsilesional stimulation to activate the non-ischemic contralesional hemisphere (black) did not differ from the spontaneous PID rate in non-stimulated mice. Importantly, somatosensory stimulation did not alter systemic blood pressure (ΔBP=−1±1 mmHg). Tactile stimulation sites are shown in Figure S1. Electrophysiological confirmation of extracellular slow potential shifts characteristic of PIDs is shown in Figure S2. Entire timelines from all experiments can be found in Figure S4. d. Because tactile stimulation was delivered for 5 minutes, we quantified the spontaneous PID occurrence rate in non-stimulated mice as average PID incidence within each 5-minute period (i.e., the chance that a PID will occur within any 5-minute period). Spontaneous PIDs occurred throughout the MCAO at an average 5-minute incidence of 12% (dark green) without a strong temporal predilection (light green). Contralesional forepaw or shoulder stimulation dramatically increased PID incidence during the 5-minute stimulation period (p<0.001 vs. spontaneous PID rate; χ² test). In contrast, contralesional whisker pad or hindpaw stimulations, or ipsilesional stimulation (7 forepaw, 4 shoulder, 3 hindpaw), did not differ from the spontaneous PID rate in non-stimulated mice. 95% confidence intervals are indicated above the bars.

Figure 2. Somatosensory stimulation triggers PIDs within an activated hot zone at a narrow critical range of ischemia

a. The location of the perfusion defect (blue) after MCAO in relation to the primary somatosensory cortex (S1), and PID origin and propagation patterns. Because of the dorsal cortical position of the imaging field (gray shaded area), lateral boundary of the perfusion defect and temporal cortex were poorly visualized. PIDs originated either outside the imaging field (pattern I) or within (pattern II). PIDs originating outside the imaging field often emerged anteriorly and propagated along the medial border of the perfusion defect (pattern I; Movie S1); the mirror wave propagating posteriorly along the lateral border likely collided with and was annihilated by the wave propagating along the medial border, and therefore, it was often not directly visualized (dashed lines; see Movie S2). PIDs that originated within the imaging field (pattern II) usually did so within the S1 cortex and propagated centrifugally (solid green lines; Movies S3 and S4). b. Spontaneous PIDs, and PIDs that coincided with forepaw, whisker, hindpaw or ipsilesional stimulation predominantly originated outside the imaging field (pattern I), while shoulder (and upper forelimb) stimulation gave rise to PIDs almost exclusively from a medial focus (pattern II; p<0.001; χ² test). c. Laser speckle contrast image showing the locations of primary (S1) forepaw (Fp, red), shoulder (Sh, orange), whisker (Wh, light blue) and hindpaw (Hp, dark blue) sensory cortices mapped using evoked potentials (referenced to bregma; see Figure S1 for details). Shoulder and upper forearm stimulation-induced PIDs (yellow circles) that originated within the imaging field (n=15) did so exclusively within their S1 representations (shoulder, see Figure S1, orange outlined region). In 9 additional attempts, shoulder stimulation was continued for up to 20 minutes until a PID occurred. In 5 of these, a PID occurred (56%), 4 of which also originated from within the stimulated S1. Hence, altogether a total of 19 PIDs originated within the shoulder and upper forearm S1 during shoulder and upper forearm stimulation. d. Residual CBF values within the stimulated whisker (Wh), forepaw (Fp), shoulder (Sh) and hindpaw (Hp) S1 cortex are shown as a measure of ischemic severity at the onset of stimulation. Only shoulder and upper forearm stimulation triggered PIDs within its S1. We divided the shoulder stimulation attempts based on whether a PID was triggered within the activated S1 (filled bar) or not (empty bars). We further subdivided the latter into 2 groups based on residual CBF above or below the mean residual CBF in the PID (+) group. PIDs were triggered only when residual CBF in activated S1 was within a narrow range (p<0.001 vs. all other groups; one-way ANOVA followed by Holm-Sidak’s multiple comparisons test). Surface artifacts precluded CBF quantification in one shoulder and one forepaw attempt.

Figure 3. Systemic hypotension dynamically shifts the hot zone within which tactile stimulation triggers PIDs

a. In hypotensive mice that did not receive tactile stimulation (i.e., controls), the spontaneous PID rate after dMCAO did not differ from the normotensive group shown in Figure 1. In contrast to normotensive mice, however, light tactile stimulation of the contralesional hindpaw (dark blue) triggered PIDs within 5 minutes (yellow circles) with high incidence, while shoulder and upper forelimb stimulation (orange) did not consistently trigger a PID over the spontaneous rate. b. PID occurrence rates (i.e., the chance that a PID will occur within a 5-minute period) are shown in non-stimulated (i.e., spontaneous PIDs) and stimulated mice after dMCAO. Spontaneous PIDs occurred throughout the dMCAO at a rate of 15% (dark green). Contralesional hindpaw stimulation significantly increased PID incidence during the 5-minute stimulation period (p=0.001 vs. spontaneous PID rate; χ² test). 95% confidence intervals are also shown. c. All but one hindpaw stimulation-induced PIDs originated from the hindpaw S1, shown superimposed on the S1 map (see Figures 2 and S1 for details). d. Residual CBF values within the stimulated shoulder (Sh) and hindpaw (Hp) S1 cortex are shown as a measure of ischemic severity at the onset of stimulation. We divided the shoulder stimulation attempts based on whether a PID was triggered within the activated S1 (filled bar) or not (empty bars). We further subdivided the latter into 2 groups based on residual CBF above or below the mean residual CBF in the PID (+) group. PIDs were triggered only when residual CBF in activated S1 was within a narrow range (p<0.001 vs. all other Hp groups; one-way ANOVA followed by Holm-Sidak’s multiple comparisons test).

Figure 4. Somatosensory stimulation triggers PIDs by worsening the supply-demand mismatch in activated cortex

a. Representative tracings obtained by multispectral reflectance imaging show that tactile stimulation of the shoulder area (horizontal orange bar) decreases oxyHb concentration within the stimulated S1 (red) but not outside (blue). A PID originated within the activated S1 (arrow, yellow circle). Grey shades indicate the approximate time segments (pre-stimulation baseline and pre-PID, respectively) averaged to calculate the oxygenation changes (ΔoxyHb and ΔsatHb) shown in Figure 4b. b. Average oxyHb and satHb changes during tactile stimulation of the shoulder area are shown as box-whisker plots (line, median; +, mean; box, 25–75% range; whiskers, min-max range) from within the stimulated S1 (red) and from comparably hypoxic penumbra outside the stimulated S1 (blue). Tactile shoulder stimulation decreased oxyHb and satHb by approximately 35–40% in activated S1, whereas no net change occurred in penumbra outside this region (p<0.01; paired t-test). c. Light tactile stimulation of the contralesional forepaw (red lines) for 5 minutes often induced a PID during the stimulation (yellow circles). In 3 experiments, we instituted normobaric hyperoxia (light blue shade) during the second half of the experiment and repeated the forepaw stimulation. In 1 experiment each, we either did not institute hyperoxia (time control) or instituted it during the entire experiment. Importantly, in this experimental cohort, normoxic ventilation was carried out with 70%N₂/30%O₂ mixture; hyperoxia was instituted by switching the gas mixture to 100% O₂. By doing this, we avoided the confounding effects of changes in N₂O concentrations when switching from normoxia to NBO, and showed that stimulation-induced PID triggering was not influenced by N₂O in other cohorts. d. Contralesional forepaw stimulation triggered a PID half of the time under normoxic conditions. Hyperoxia markedly reduced the PID occurrence rate during forepaw stimulation (p<0.05; χ² test). 95% confidence intervals are indicated above the bars.

Figure 5. Topical tetrodotoxin (TTX) application diminishes functional activation of the cortex and prevents shoulder stimulation-induced PIDs

a. Somatosensory evoked potentials (SSEPs) were recorded using glass micropipettes from barrel cortex during electrical stimulation of whisker pad in a separate group of normal (i.e. non-ischemic) mice to confirm the efficacy of selected concentration of TTX (1 μM) to suppress SSEPs. Topical application of TTX progressively abolished whisker barrel SSEPs within 15 minutes, as shown in this representative experiment. Average SSEP tracings from successive 2-minute periods are shown in different colors. Barrel cortex was chosen because of its large size. b. Average SSEP amplitude is shown as a function of time after topical TTX application (n=5). c. Light tactile stimulation of the contralesional shoulder for 5 minutes (orange lines) induced a PID at a high rate during the stimulation (yellow circles) in the presence of a small cranial window and topical vehicle application (n=4 mice). Topical TTX application markedly diminished PID induction rate during shoulder stimulation (n=6 mice). The two PIDs that occurred during shoulder stimulation under TTX originated outside the shoulder S1, and thus were most likely spontaneous. Systemic ketamine (120 mg/kg, intraperitoneal, 10 minutes prior to stimulation) treatment completely prevented PID occurrence during shoulder stimulation (n=3 mice). Isoflurane concentration was reduced to 0.3 % after ketamine administration. d. Contralesional shoulder stimulation triggered a PID nearly 80% of the time in the presence of an open cranial window over shoulder S1 and topical vehicle application. Topical TTX markedly reduced and systemic ketamine completely prevented PID occurrence during shoulder stimulation (p<0.05; χ² test). 95% confidence intervals are indicated above the bars.

Figure 6. Hypoxic and hypotensive transients consistently trigger PIDs

a. Representative CBF tracing showing 5 PIDs (yellow circles) triggered by transient hypoxic episodes induced by reducing the fraction of O₂ in ventilation gas mixture (blue lines). No spontaneous PID occurred in this experiment. b. Summary timeline of transient hypoxia (blue lines) and hypotension (purple lines) experiments. Each horizontal line represents one animal, and the start and end of each line indicate the time span of imaging for up to 95 minutes. Insets show the latency between hypoxia or hypotension onset and PID onset. c. Hypoxia (blue bar) or hypotension (purple bar) triggered PIDs at a markedly higher rate than spontaneous occurrence (green bar, data from control experiments in Figure 1d shown here for comparison; p<0.05; χ² test). 95% confidence intervals are indicated above the bars. d. Summary of arterial pO₂ and BP at baseline and during hypoxia (left) or hypotension (right), dichotomized based on whether a PID occurred (red) or not (blue) during each attempt. Individual experiments and averages at baseline and during hypoxic or hypotensive trial are shown for each group. The arterial pO₂ level dropped to a significantly lower level during hypoxia in the group that developed a PID compared to the group that did not. In contrast, neither baseline pO₂ nor the magnitude of pO₂ drop differed between the groups. In hypotension attempts, both baseline and hypotensive BP levels differed between the PID and no PID groups; however, once again final BP alone could predict PID occurrence. Prediction success was 87.5% (90% for occurrence of a PID and 83.3% for absence of a PID) for final pO₂, and 89.4% (94.4% for occurrence of a PID and 72.7% for absence of a PID) for final BP. A binary logistic regression analysis was conducted to predict the onset of a PID using baseline pO₂, hypoxic pO₂, the magnitude of pO₂ drop, baseline BP, hypotensive BP, and the magnitude of BP drop, as predictors. e. The time course of resting BP during 10 minutes preceding spontaneous PIDs (n=32; BP could not be measured reliably during 1 spontaneous PID). There was on average a 10% drop in resting BP 2 minutes before the occurrence of a spontaneous PID. One-way ANOVA for repeated measures. f. Depiction of tissue pO₂ probe (ptiO2) and 6-contact platinum subdural ECoG recording strip implanted over peri-infarct tissue guided by laser speckle imaging (see Methods). g. Representative tissue pO₂ tracing showing the transient dip in O₂ availability preceding a PID in human cortex. Grey shade indicates the 5-minute segment averaged to calculate the pre-PID oxygenation shown in Figure 6h. Baseline levels were selected when there was no PID occurrence within a 2 hour period (see Methods). h. Mean tissue pO₂ levels prior to PID were significantly lower compared with baseline levels (Wilcoxon Signed Rank Test).

Figure 7. Tactile stimulation-induced PIDs worsen ischemic tissue perfusion and outcome

a. Average cumulative induced and spontaneous PID frequency in mice receiving tactile stimulation starting 10 minutes after MCAO (red, n=14), and spontaneous PID frequency in non-stimulated controls (green, n=17). None of the mice in these cohorts were subjected to transient hypoxia or hypotension as part of the experimental protocol. b. Area of perfusion defect with ≤30% residual CBF increased in proportion with PID occurrence in the stimulated group over time. Two-way ANOVA for repeated measures. c. Representative laser speckle flowmetry images showing the expansion of perfusion defect (blue pixels with ≤30% residual CBF) between 10 and 60 minutes after MCAO in a mouse that received tactile stimulation, and the stable perfusion defect in a mouse without tactile stimulation. d. Infarct volumes measured using TTC staining were larger in the group receiving forepaw but not whisker stimulation starting 10 minutes after MCAO (n=6, 4 and 4 in control, forepaw and whisker stimulation groups; p<0.05; t-test). These experiments were performed in a separate cohort of mice without endotracheal intubation or mechanical ventilation in order to minimize surgical morbidity and mortality. The experimental protocol was otherwise identical to other tactile stimulation experiments above. There were a total of 2.25±0.25 PIDs in the forepaw-stimulated group, whereas no spontaneous or induced PID was detected in non-stimulated and whisker stimulated cohorts, respectively. e. Infarct volumes measured 72 hours after stroke onset using H&E-stained coronal brain sections were larger in the group receiving forepaw stimulation starting 10 minutes after dMCAO (n=9 each; t-test). In this cohort, we initially studied n=5 mice per group. The data showed higher than expected coefficient of variation. We then performed a power analysis, determined n=10 mice per group as the appropriate sample size, and added 5 more mice in each group. One mouse was excluded in each group based on a priori criteria (1 in non-stimulated group due to total absence of an infarct; 1 in stimulated group due to clip-related trauma precluding reliable infarct measurement). Therefore, final sample sizes were n=9 per group. As with the 24-hour assessment group above, these experiments were performed in a separate cohort of mice without endotracheal intubation or mechanical ventilation in order to minimize surgical morbidity and mortality. In this experimental cohort, inhalation gas included 70%N₂/30%O₂ mixture. By doing this, we also reproduced the effect of stimulation-induced PIDs on tissue outcome in the absence of N₂O. The experimental protocol was otherwise identical to other tactile stimulation experiments above. Representative sections show the infarct at each slice level in one non-stimulated and forepaw and shoulder stimulated mouse each.

Figure 8. The concept of peri-infarct hot zones susceptible to PID initiation

a. Three-dimensional rendering of a hypothetical CBF defect upon focal arterial occlusion, depicting the conceptual framework consisting of the severely ischemic depolarized core (red), the penumbra (yellow), and the metastable hot zone surrounding it (lavender). The narrow critical CBF range defining the hot zone is also shown from Figure 2d (yellow bar, residual CBF in shoulder S1 where a PID is triggered upon shoulder stimulation). b. Two-dimensional projection of the perfusion defect. In the metastable hot zone, increased demand during functional activation of the tissue worsens the supply-demand mismatch to trigger a PID. Because penumbra is electrophysiologically silent, by definition it cannot be activated upon somatosensory stimulation; therefore, it is only susceptible to reduced O₂ supply during hypoxic and hypotensive transients to trigger a PID.