Requisite ischemia for spreading depolarization occurrence after subarachnoid hemorrhage in rodents

Introduction

Spreading depolarizations (SD) originate in an apparently spontaneous fashion in the human brain after ischemic, hemorrhagic and traumatic injury. ¹ Associated with massive transmembrane ion and water shifts, SDs impose severe metabolic mismatch by increasing the energy demand and reducing blood supply in already critically compromised brain tissue. ² As a result, SDs expand the injury volume and worsen neurological outcomes.

We have recently shown that in the setting of focal cerebral ischemia, SD waves are triggered when the supply-demand mismatch in moderately ischemic peri-infarct tissue is transiently worsened either due to increased demand or reduced supply. ³ While this provided a mechanism for SD occurrence in ischemic stroke, mechanisms triggering recurrent SDs days to weeks after subarachnoid hemorrhage (SAH) have been unclear. Previous studies have elegantly demonstrated that elevations in extracellular K⁺ concentrations ([K⁺]_e), when combined with reduced NO availability, evoked an SD event coupled to severe vasoconstriction (i.e. inverse coupling, spreading ischemia) that may form the basis of spontaneous SD occurrence after SAH. ⁴ Here, we examined spontaneous SD occurrence after experimental SAH in rodents. Our data implicate tissue ischemia as a critical condition for spontaneous SDs to develop after SAH.

Methods

All experimental procedures were carried out in accordance with the ARRIVE guidelines, and the Guide for Care and Use of Laboratory Animals (NIH Publication No. 85-23, 1996), and were approved by the institutional review board (MGH Subcommittee on Research Animal Care [SRAC]). We used male C57BL6J mice (3–4 months age, 25–30 g) or Sprague-Dawley rats (3–4 months age, 300–400 g) in all experiments (Charles River Laboratories, Wilmington, MA, USA). Arterial pH, pO₂, pCO₂, and blood pressure were monitored via a femoral artery catheter and maintained within normal limits (Supplemental Table 1). The rectal temperature was kept at 37 ℃ using a thermostatic heating pad. Technical failure was the only exclusion criterion, and none was excluded. Where relevant, animals were randomly assigned to experimental groups. Experiments were not carried out in a blinded fashion.

Results

Twelve hours after pcSAH, large amounts of blood covered the dorsal cortical surface and pial vasculature (Supplemental Figure 1). Gross examination over time revealed persistent albeit reduced presence of blood 3 days after pcSAH, with complete clearance at 7 days. In mice with cmSAH, blood accumulated predominantly in the pre-pontine cistern extending around the circle of Willis, although a thin layer of blood tracked along the perivascular space surrounding the arteries as far as the dorsal cortical surface (Supplemental Figure 2). In contrast to pcSAH, however, blood cleared much faster after cmSAH, becoming undetectable within 24 h.

We monitored spontaneous SD occurrence after pcSAH bilaterally in mice for a cumulative total of 72 h (n = 4 mice each between 3 and 9 h, 12 and 18 h, and 72 and 78 h after pcSAH), but did not detect a single spontaneous SD (Figure 1(a)). In three more mice, we monitored SDs 12–18 h after cmSAH (total 18 h of recordings), and once again did not detect any spontaneous SD. This complete absence of spontaneous SDs was in striking contrast to focal cerebral ischemia where numerous SDs consistently occur with high frequency in all species studied to date.^9,16–21 At the end of each experiment, we confirmed the integrity and sensitivity of electrophysiological recordings by inducing and successfully detecting an SD at each recording site in all mice using pinprick (Figure 1(a), dashed arrows). OPEN IN VIEWER

These data clearly suggested that the presence of dense subarachnoid blood was not sufficient to trigger an SD, even in the highly susceptible mouse cortex. To further confirm this, we tested whether pial application of normal or acutely hemolyzed arterial blood triggered an SD. Topical application of whole non-hemolyzed arterial blood did not trigger SD in any of the experiments. In contrast, acutely hemolyzed undiluted blood triggered an SD 67% of the time (Figure 1(b), left panel). However, this effect was rapidly lost upon stepwise dilution using normal saline, with a concentration–response relationship. We measured the extracellular (i.e. plasma) K⁺ concentration ([K⁺]_e) in undiluted hemolyzed blood and found it to be 57.3 ± 0.3 mM (hemoglobin 1.96 ± 0.07 mM; n = 3 independent samples). In separate experiments, we determined the concentration–response relationship between topical KCl solution and SD induction, and found that the ability of hemolyzed blood to trigger SD correlated well with [K⁺]_e (Figure 1(b), right panel). These data suggested that K⁺ is the main SD trigger in hemolyzed blood, and other substances (e.g. ions, small molecules, peptides) do not significantly contribute. We next measured CSF [K⁺]_e after pcSAH in mice but did not detect an increase at any time point (3.7 ± 0.6, 3.4 ± 0.1, and 3.5 ± 0.6 mM, in naïve, and 12 and 72 h after pcSAH, respectively; n = 3, 3, and 5), as previously reported.^22–25 This was not surprising because K⁺ released into the CSF during gradual lysis of subarachnoid blood is likely taken up by astrocytes and neurons and/or otherwise diluted via diffusion and bulk CSF flow. Altogether these data strongly argued against high CSF [K⁺]_e as the trigger for spontaneous SDs observed after SAH in human brain.

Next, we tested whether smaller local increases in [K⁺]_e or cellular changes after SAH somehow enhance the tissue susceptibility to SD; this might predispose to SDs after SAH under the right conditions. We found no evidence for enhanced SD susceptibility at any of the tested time points after pcSAH or cmSAH in mice, and cmSAH in rats (Figure 2). To the contrary, SD susceptibility was significantly reduced at 12 h after pcSAH or cmSAH in mice, as evident in lower KCl-induced SD frequencies and SD propagation speeds, and normalized within 72 h. Cumulative depolarization durations during continuous topical KCl were also shorter (naïve 366 ± 70 s, 12 h after pcSal 325 ± 101 s, 12 h after pcSAH 228 ± 62 s, 72 h after pcSAH 392 ± 74 s; p < 0.05 12 h after pcSAH vs. all other groups). Similarly, in the rat cmSAH double injection model, electrical SD threshold was significantly elevated at 5 days, and SD propagation speed was reduced. SD duration or amplitude did not show a consistent change (Supplemental Table 2). We selected the 5-day time point in the rat because cmSAH double injection model in this species typically leads to slower onset and longer lasting abnormalities than single injection SAH models in mice. Selected time points, taken together, covered the period of frequent spontaneous SD occurrence after SAH in human brain. These data suggested that the presence of dense subarachnoid blood did not enhance intrinsic SD susceptibility in cortex. OPEN IN VIEWER

Presence of SAH has been proposed as a mechanism of inverse coupling, where the normally vasodilator, hyperemic response to SD is transformed into a vasoconstrictive hypoperfusion response. Therefore, we tested whether the CBF response to SD is altered after SAH. As a prerequisite for proper interpretation of acute CBF changes during SD, we first measured resting CBF using laser speckle flowmetry and found it to be about 25% and 15% lower than baseline at 12 and 72 h after pcSAH, respectively, in mice; this mild hypoperfusion completely normalized within 7 days (Figure 3(a)). However, we did not observe the typical “spreading ischemia” during SD previously described in the setting of elevated [K⁺]_e combined with nitric oxide synthase inhibition to mimic SAH.²⁷There were only minor, albeit statistically significant, differences in CBF changes during SD at 12 and 72 h after pcSAH in mice (Figure 3(b)). These changes were largely explained by the reduced resting CBF, which normalized within 7 days. In rats, CBF changes during SD were nearly identical between cmSAH and cmSal (Figure 3(c)). All CBF responses to SD were within previously reported limits for these species.^2,10,12,28 Altogether, the data did not support the notion that SAH alone radically transforms the cerebral blood flow impact of SDs. OPEN IN VIEWER

The absence of spontaneous SDs and the normal CBF response to SD after SAH in rodents contrasted sharply with the observations in human brain, where numerous spontaneous SDs and vasoconstrictive (i.e. inverse) hemodynamic response to SDs have been uniformly observed. To explain this paradox, we reasoned that the absence of spontaneous SD events after SAH in rodent brain might be due to milder delayed cerebral ischemia compared with human brain. Hence, we hypothesized that tissue ischemia is a prerequisite for the occurrence of spontaneous SDs and abnormal hemodynamic response to SDs after SAH.

To this end, we induced focal cerebral ischemia at different time points after SAH and examined the peri-infarct SDs and stroke outcome. Occlusion of the middle cerebral artery using an intraluminal filament consistently yielded 4.3 ± 1.2 peri-infarct SDs per hour in pcSal (i.e. control) cohort (pooled data from 12 h, 72 h, and 7 days; n = 5, 8, 2, respectively), which did not differ from naïve mice without any subarachnoid injection (3.4 ± 0.9 peri-infarct SDs/h; n = 5). In the pcSAH cohort, peri-infarct SD frequency decreased at 12 h (∼50% reduction; Figure 4). Identical data were found when cumulative peri-infarct SD duration was calculated instead of peri-infarct SD frequency (348 ± 148, 251 ± 129, 492 ± 117*, 304 ± 130 s depolarization per hour of MCAO per animal; *p < 0.05 vs. pcSAH 12 h). These data were consistent with reduced susceptibility to KCl-induced SDs 12 h after pcSAH as described above (Figure 2). Surprisingly, however, there was a marked increase in peri-infarct SD occurrence when ischemia was induced 72 h after pcSAH (∼80% increase, p < 0.05). We have not detected any increase in SD susceptibility when induced by topical KCl at 72 h after pcSAH. This effect appeared to be directly related to SAH severity when mice were dichotomized based on the amount of residual subarachnoid blood on postmortem examination (Figure 4(e)). At 7 days, peri-infarct SD frequency was normalized. These data suggested that SAH facilitated peri-infarct SDs in a delayed fashion around 72 h. OPEN IN VIEWER

We next examined the tissue and neurological outcomes after fMCAO to specifically test whether SAH sensitized the brain to focal ischemic injury. Transient fMCAO for 30 or 60 min yielded significantly larger infarct volumes and worse neurological deficits at 24 h when induced 12 or 72 h after pcSAH, respectively, compared with pcSal (Figure 5(a)). Outcomes did not differ between 12 and 72 h; therefore, focal ischemic outcomes corresponded to subarachnoid blood content rather than peri-infarct SD frequency. Consistent with this, infarcts were also larger when fMCAO was induced 12 but not 24 h after cmSAH compared with cmSal (Figure 5(a)); in this model, subarachnoid blood completely cleared within 24 h. To test whether cerebrovascular dysfunction could explain the worse focal ischemic outcomes after SAH, we next employed laser speckle flow mapping of the perfusion defect during acute distal middle cerebral artery occlusion (dMCAO). The area of cortical perfusion defect almost doubled when dMCAO was induced 12 h after pcSAH, remained significantly larger at 72 h, and normalized at 7 days (Figure 5(b)). The time course of the perfusion defect mirrored resting perfusion changes after pcSAH (Figure 3(a)), rather than the peri-infarct SD frequency (Figure 4). These data implicated cerebral hemodynamic dysfunction as a major mechanism for larger infarcts and worse neurological deficits after fMCAO superimposed on SAH. OPEN IN VIEWER

Discussion

Our findings challenge the notion that subarachnoid blood breakdown products can directly trigger SDs by way of elevated [K⁺]_e or scavenging of NO by hemoglobin. Instead, we propose that focal cerebral ischemia is a prerequisite for SD occurrence after SAH, and that interaction between focal ischemia and SAH creates a vicious cycle (Figure 6). The absence of spontaneous SDs after SAH in mice was likely because delayed cerebral ischemia is typically milder in rodents than human. ²⁹ By superimposing focal ischemia with SAH to better simulate the human condition, we found that the presence of SAH significantly worsens ischemic outcome by disrupting collateral perfusion (12 and 72 h) and facilitating peri-infarct SDs (72 h) in mice. After SAH, focal ischemia can develop via a number of mechanisms such as microvascular plugging due to in situ thrombosis or microembolization. ³⁰ Regardless of the mechanisms precipitating ischemia, SAH-induced vascular dysfunction^31–34 diminishes collateral flow and expands the volume of ischemic tissue. We posit that focal ischemia in turn triggers peri-infarct SDs, facilitated by SAH in a delayed fashion possibly via blood breakdown products or vascular dysfunction. Peri-infarct SDs impose additional metabolic burden, exacerbate vascular dysfunction, and expand the perfusion defect,^2,15,18 particularly when they occur with high frequency and in clusters.^18,35–37 Such a vicious cycle may explain the occurrence and timing of delayed ischemic neurological deficits and infarction (Figure 6). OPEN IN VIEWER

Peri-infarct SD occurrence showed a biphasic time course after SAH. An early decrease at 12 h was followed by a marked increase at 72 h and normalization at 7 days. The timing of increased peri-infarct SD frequency at 72 h implicates erythrocyte breakdown products, which start by 16–32 h and peak at 7 days. ³⁸ Faster clearance of SAH in rodents compared with human may also explain the normalization of peri-infarct SD frequency at 7 days in our model. The release of intracellular K⁺ from ruptured erythrocytes has always been considered a prime candidate as a trigger for SD. ³⁹ Although we did not detect an increase in [K⁺] in the CSF collected from the cisterna magna, or spontaneous SDs to suggest that [K⁺]_e ever exceeded the SD threshold (∼12 mM), small and/or focal elevations in [K⁺]_e around the microvasculature and even in the parenchyma ⁴⁰ might still facilitate peri-infarct SD occurrence. Not consistent with this notion, however, is the lack of an increase in KCl- or electrically induced SD susceptibility at any time point after SAH in mice or rats. Alternatively, increased peri-infarct SD frequency at 72 h may simply reflect larger volumes of ischemic tissue, since larger infarcts naturally trigger more peri-infarct SDs. ²¹ However, the volume of ischemic tissue was equally large at 12 h, yet peri-infarct SD frequency was decreased rather than increased. Mechanisms of reduced SD susceptibility and peri-infarct SD frequency at 12 h after pcSAH and cmSAH in mice, and 5 days after cmSAH in rats, were also not clear. A similar trend has recently been reported 1 day after endovascular perforation model of SAH in the rat. ⁴¹ We have previously shown that mild hypoperfusion induced by bilateral carotid stenosis reduces KCl-induced SD frequency; however, this was consistently associated with significantly prolonged SD durations, which we did not detect after SAH (Supplemental Table 2). ⁴² Astrocytic and microglial activation may be an alternative mechanism that can suppress SD susceptibility, ⁴³ although their time course in our SAH models remains to be determined. Last, it should be noted that acute SAH likely triggers one or more SDs within seconds to minutes after aneurysmal rupture, perhaps due to global ischemia during the intracranial pressure rise. Acute experimental SAH does indeed elevate local [K⁺]_e in cats, which starts within seconds and lasts a few minutes.^44,45 The mechanism may involve local pressure build up or acute perfusion drop. These acute changes, however, cannot explain the delayed occurrence of recurrent SDs for many days after SAH.

In summary, we show that elevated [K⁺]_e by itself is not sufficient to explain SD occurrence after SAH. Our data suggest that focal cerebral ischemia is a critical determinant of SD occurrence, and reciprocal interactions between focal cerebral ischemia and SAH synergize to create a vicious cycle and facilitate injury, leading to the syndrome of delayed cerebral ischemia (DCI). Similar ischemic triggers may underlie SD occurrence in traumatic brain injury and intracerebral hemorrhage as well. Based on these findings, we speculate that antithrombotic therapies, when safe to implement after SAH (e.g. aneurysm clipping), might prevent microvascular occlusion and peri-infarct SD occurrence after SAH to break the vicious cycle.