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Spreading depression (SD) is a slowly propagating wave of transient neuronal and glial depolarization that develops after stroke, trauma and subarachnoid hemorrhage. In compromised tissue, repetitive SD-like injury depolarizations reduce tissue viability by worsening the mismatch between blood flow and metabolism. Although the mechanism remains unknown, SDs show delayed electrophysiological recovery within the ischemic penumbra. Here, we tested the hypothesis that the recovery rate of SD can be varied by modulating tissue perfusion pressure and oxygenation. Systemic blood pressure and arterial pO2 were simultaneously manipulated in anesthetized rats under full physiologic monitoring. We found that arterial hypotension doubled the SD duration, whereas hypertension reduced it by a third compared with normoxic normotensive rats. Hyperoxia failed to shorten the prolonged SD durations in hypotensive rats, despite restoring tissue pO2. Indeed, varying arterial pO2 (40 to 400mmHg) alone did not significantly influence SD duration, whereas blood pressure (40 to 160mmHg) was inversely related to SD duration in compromised tissue. These data suggest that cerebral perfusion pressure is a critical determinant of SD duration independent of tissue oxygenation over a wide range of arterial pO2 levels, and that hypotension may be detrimental in stroke and subarachnoid hemorrhage, where SD-like injury depolarizations have been observed.
Propagating depolarizations akin to SD are triggered within injured brain tissue and are accompanied by loss of membrane resistance and massive redistribution of ions across membranes. Although initiation and propagation of SD have been extensively studied both in vivo and in vitro, recovery mechanisms such as reestablishing the ionic gradients and restoration of the resting membrane potential are less well investigated (Somjen, 2001). In particular, the clearance of elevated extracellular K+ concentration ([K+]e) of >40mmol/L, is believed to be an energy-dependent process via Na+/K+ ATPase activation. However, glial spatial buffering, passive diffusion, and vascular clearance may also play a role. Injury depolarizations, such as those in focal ischemic penumbra, often show prolonged DC shifts, possibly due to hypoxic energy deficit (i.e., reduced Na+/K+ ATPase activity), decreased tissue perfusion pressure, or reduced volume of the extracellular compartment for passive diffusion.
We showed earlier that mild systemic hypotension prolonged the SD duration by as much as 135% in otherwise normal anesthetized rats. As hypoxemia was much less effective, tissue perfusion pressure was implicated as a critical factor for SD recovery rather than tissue oxygenation (Sukhotinsky et al, 2008). We now present additional evidence implicating perfusion pressure as the more important determinant to SD recovery than the state of tissue oxygenation, through an undetermined mechanism. Modulation of the electrophysiological recovery by perfusion pressure correlated with that of the hyperemic response to SD, which may limit glucose delivery or K+ clearance mechanisms via blood vessels.
National and institutional guidelines for animal care and use for research purposes were strictly followed, and study protocol was approved by the institutional review board.
Rats (Sprague-Dawley, male, 200 to 450g) were anesthetized with urethane (1.3 to 1.5g/kg, intraperitoneal), and intubated via a tracheostomy for mechanical ventilation (70% N2O/30% O2 during preparation, room air during the experiment; SAR-830, CWE, PA, USA). Femoral vein and artery were cannulated for drug infusions, continuous mean arterial blood pressure (MABP) recording (PowerLab, ADInstruments, MA, USA), and arterial blood gas and pH measurements every 15 to 30mins to maintain arterial pCO2 (paCO2) around 30mmHg (Rapidlab 248 blood gas/pH analyzer, Bayer HealthCare, Walpole, MA, USA). Rectal temperature was kept at 37°C using a thermostatically controlled heating pad (Harvard Apparatus, MA, USA). Rats were placed in a stereotaxic frame (Stoelting Co, IL, USA) and three burr holes were drilled under saline cooling over the right hemisphere at the following coordinates (mm from bregma): (1) posterior 6, lateral 3 (parieto-occipital cortex) for topical KCl application; (2) posterior 2, lateral 3 (fronto-parietal cortex) for SD recording, laser Doppler flowmetry and tissue oxygen monitoring; (3) anterior 2, lateral 3mm (frontal cortex) for SD recording. Dura was gently removed at the KCl and laser Doppler sites, and care was taken to avoid bleeding. After surgical preparation, the cortex was allowed to rest for 30mins under saline irrigation and dura covered with mineral oil to prevent drying.
The extracellular steady (DC) potential and electrocorticogram were recorded with glass micropipettes (150mmol/L NaCl), 300μm below the dural surface (EX1 differential amplifier, Dagan Corporation, MN, USA). Ag/AgCl reference electrode was placed subcutaneously in the neck. The amplitude of DC shift, and its duration at half-maximal amplitude were measured. SD propagation speed was calculated based on its latency and the distance between the two recording electrodes.
Regional cerebral blood flow (CBF) was recorded using laser Doppler flowmetry (ADInstruments, CO, USA). The laser Doppler flowmetry probe (0.48mm tip diameter) was placed in the immediate vicinity of SD recording electrode in contact with the pial surface away from large vessels. The morphology of CBF changes during SD measured with this laser Doppler flowmeter reproduced those observed in our earlier study using a different brand, although the magnitudes of hyperemia during SD and induced hypertension were larger possibly due to overestimation at hyperemic states (Fabricius and Lauritzen, 1996). Baseline CBF at the onset of an experiment (i.e., initial baseline preceding the first SD) was taken as 100%. The CBF changes during an SD were quantified using the following deflection points: baseline immediately before SD, onset of hypoperfusion, trough of hypoperfusion, peak hyperemia, recovery of hyperemia, and plateau 1 to 2min later. The CBF amplitude was measured for each deflection point and averaged within each experimental condition. In addition, the latencies to the trough of hypoperfusion and the peak hyperemia from onset of hypoperfusion were measured to accurately represent the time course of CBF changes in the averaged graphs.
Absolute tissue pO2 (ptO2) was measured (n=12) by phosphorescence lifetime using an intraparenchymal optode (350μm tip diameter; OxyLab, Oxford Optronics, UK) placed 1mm deep in the vicinity of glass micropipette. Measurements were made after a stable baseline was achieved.
As ptO2 measurement is invasive, in a separate group of rats (n=5) we used an alternative method to noninvasively assess the impact of hyperoxia on tissue oxygenation using full-field phosphorescence lifetime imaging through a 4 × 4mm2 closed cranial window on the parietal bone, and the phosphorescence probe Oxyphor R2 (Oxygen Enterprises, Ltd, Philadelphia, PA, USA) injected via the femoral vein, as described in detail recently (Sakadzic et al, 2009). This dye remains intravascular and provides a measure of pO2 within the vasculature (pvO2). The dura was removed and the cranial window was filled with 1.5% agarose and sealed with a microscope coverslip. Briefly, pvO2 was imaged with a thermoelectrically cooled camera (Imager QE, La Vision). The frame rate was synchronized with the triggering rate of the pulsed laser (10Hz) for phosphorescence excitation (Brilliant, Big Sky Laser Technologies, Bozeman, MT, USA; 532nm wavelength). The light from the pulsed laser was coupled into the multimode fiber and typically 5 to 10mJ/cm2 was delivered to the brain tissue at ~60° angle with respect to the cranial window surface. For estimation of phosphorescence lifetimes, 4 × 4 binning of the CCD pixels was used and the CCD exposure time was set to 50μs. A sequence of 25 frames was acquired with variable delay times with respect to the laser Q-switch opening. The first frame (200μs before the laser pulse) was used to subtract the background light from the phosphorescence intensity images. On the basis of 24 consecutive frames of decaying phosphorescence intensity, a two-dimensional map was calculated that represents a 2.4-sec average of the pvO2 values, by fitting the exponential decay lifetime τ of the phosphorescence intensity for each CCD pixel followed by the conversion of phosphorescence lifetimes to the pvO2 values using an empirical Stern–Volmer-like relationship. Single exponential decay was assumed, and a nonlinear least square fitting with statistical weighting was performed (Matlab, MathWorks Inc, Natick, MA, USA). As phosphorescence lifetime is extremely short in arterial compartment, pvO2 values monitored by this technique represent mostly venous compartment.
A total of 48 rats were used to study a total of 109 SDs (Figure 1). By independently manipulating MABP and paO2, eight different experimental conditions were created as follows: hypoxic hypotension, hypoxic normotension, and hypoxic hypertension; normoxic hypotension, normoxic normotension and normoxic hypertension; and hyperoxic hypotension and hyperoxic normotension. Normobaric hyperoxia was achieved by inhalation of 100% O2, whereas hypoxia was achieved by decreasing the fraction of O2 in inspired air mixture to ~8% this degree of hypoxia decreases brain ptO2 by ~40% to 50% (Meyer et al, 2000; Weiss et al, 1976). Systemic hypotension was achieved by controlled arterial blood withdrawal, targeting an MABP of 45mmHg, without severely altering arterial pH or causing irreversible cardiovascular collapse; this target MABP was chosen to be slightly lower than the previously reported autoregulatory limit in rats (50 to 65mmHg) and did not reduce resting CBF significantly (Pedersen et al, 2003; Tonnesen et al, 2005). Systemic hypertension was induced by intravenous phenylephrine infusion (2mg/mL, 3mL/h, for ~20min). In addition, intravenous phenylephrine was used to counteract hypoxia-induced mild hypotension when necessary.
Two to three conditions were tested in each rat when feasible. The sequence of conditions was alternated in some experiments to avoid temporal bias (Figure 1, hyperoxia alone and hypertension alone). After establishing the desired stable baseline condition for >15min, SD was evoked by a cotton ball (2mm diameter) soaked with 1M KCl placed on the pial surface; as soon as an SD was detected, KCl was removed and the burr hole was irrigated with saline. In addition, a separate time–control group was studied in which a baseline SD was followed by a second one after 30mins of saline infusion. When repeated SDs were evoked under the same experimental condition in a rat, the data were averaged to obtain a single data point per experimental condition per rat. In experiments where ptO2 was measured, SDs were induced using the same protocols as above to reproduce identical experimental conditions during ptO2 measurements. The effects of interventions on plasma chemistry (electrolytes, glucose) were also studied in a subgroup of rats (n=12). Arterial pH and paCO2 were within normal range, except for mild metabolic acidosis during systemic hypotension and induced hypertension, and mild hypercapnia during induced hypertension (Table 1).
All data were continuously recorded using a data acquisition system for off-line analysis (PowerLab). Statistical comparisons were performed using two-way analysis of variance, followed by Student–Newman–Keuls test for multiple comparisons. In addition, a multiple regression analysis was performed using the pooled data from all rats to independently test the impacts of MABP and paO2 (continuous independent variables) on SD duration (dependent variable). Data are presented as mean±standard deviation, and P<0.05 was considered statistically significant.
The duration, amplitude, and propagation speed of SD were typical for anesthetized rats under normoxic normotensive conditions, (Figures 2 and and3),3), and did not change after saline infusion (not shown). Therefore, data from saline-treated rats were pooled with untreated normoxic normotensive controls for comparisons.
The SD duration was inversely related to MABP. Under normoxic conditions, hypotension (50mmHg) nearly doubled the SD duration whereas induced-hypertension (150mmHg) decreased it by a third compared with normotensive rats (90mmHg) (Figure 3A). Hyperoxia (400mmHg) failed to shorten the SD duration in hypotensive rats, indicating that SD prolongation was not due to tissue hypoxia. In fact, manipulation of paO2 within a range of 40 to 400mmHg had little effect on SD duration (Figure 3B), except when hypoxia was induced in hypotensive rats, which further delayed electrohysiological recovery compared with normoxic hypotensive rats (Figure 3A). Multiple linear regression analysis on pooled, log-transformed data from all eight groups (109 SDs) showed that MABP alone accounted for the variation in SD duration [log10(SD duration)=2.945−0.772 × log10(MABP); adjusted R2=0.7; P<0.001; power=1.0 for α=0.05], whereas paO2 did not (P=0.8).
SD propagation speed was also related to MABP, and was faster in hypertensive rats and slower in hypotensive rats compared with the normotensive group; this relationship did not depend on paO2 (Table 2). Hypoxia also appeared to slow SD propagation in normotensive or hypertensive rats. However, multiple regression analysis once again showed that MABP alone accounted for the variation in SD speed (P<0.01), whereas the effect of paO2 did not reach statistical significance (P=0.07).
Although of less clear significance, SD amplitude was larger in hypotensive rats compared with both normotensive and hypertensive groups, whereas the paO2 did not significantly impact the SD amplitude (Table 2).
Systemic hypotension caused only mild reduction in resting CBF, confirming intact autoregulation, whereas hypertension (MABP 150mmHg) led to significant breakthrough hyperemia (Table 1). Hypoxia or hyperoxia did not significantly impact resting CBF although there was a trend towards mild hyperemia in hypoxic normotensive rats.
Under normoxic normotensive conditions, SD was accompanied by a large monophasic hyperemia occasionally preceded by a brief small hypoperfusion. As reported earlier (Sukhotinsky et al, 2008), hypotension augmented the initial hypoperfusion and greatly diminished the subsequent hyperemic response compared with normotensive rats (Figures 2 and and4).4). Hypertension induced by phenylephrine infusion did not alter the magnitude of the CBF rise during SD; however, peak CBF reached significantly higher levels than other groups because of elevated baseline CBF levels. Manipulation of paO2 did not significantly alter the magnitude of CBF changes during SD.
The DC shift duration was inversely correlated with both CBF trough during the DC shift and CBF peak during the hyperemic phase; the lower the CBF trough and peak, the longer the DC shift (Figure 4B), suggesting a hemodynamic mechanism for modulation of SD recovery by changes in perfusion pressure.
To confirm that hyperoxia restored tissue oxygenation in hypotensive rats, we measured tissue pO2 (ptO2) using an intracortical probe in a separate group of rats (n=12). Under normoxic normotensive conditions, ptO2 was 15±5mmHg, comparable to earlier reports (O'Hara et al, 2005; Rossi et al, 2001). Systemic hypotension decreased the measured ptO2 by ~30% (11±5mmHg), despite normal paO2 (Table 1). This mild reduction in ptO2 was restored to >90% of baseline by normobaric hyperoxia (14±5mmHg). Using a less invasive method, we measured cerebral intravascular pO2 (pvO2) using full-field phosphorescence lifetime optical imaging through a closed cranial window (n=5; see Materials and methods). When the region of interest was placed over a cortical vein, normal resting pvO2 was 62±20mmHg during normoxic normotension (paO2=135±16mmHg; MABP=95±21mmHg), consistent with earlier reports (Estrada et al, 2008; Sakadzic et al, 2009). During systemic hypotension, cortical venous pvO2 was reduced to 37±9mmHg and was partially restored to 48±11mmHg during normobaric hyperoxia; results were similar when the region of interest was placed away from large cortical vessels to assess pvO2 levels in capillary-enriched tissue. These data suggested that normobaric hyperoxia at least partially restored ptO2 in hypotensive rats, as reported earlier (Rossi et al, 2001).
Plasma glucose and electrolytes did not differ among experimental conditions (Table 3). Importantly, animals remained normo- or mildy hyperglycemic during induced hypotension, indicating that hypoglycemia was not the cause of prolonged DC shifts (Gido et al, 1993; Wolf et al, 1997).
We showed that the electrophysiological recovery of SD was significantly delayed when arterial hypotension was induced under tightly controlled physiologic conditions, as reported earlier (Sukhotinsky et al, 2008). The prolongation was not caused by tissue hypoxia because normalizing the ptO2 and pvO2 failed to hasten SD recovery in the presence of hypotension. Indeed, induced hypertension did shorten SD recovery even in hypoxic rats. Therefore, our data strongly suggest that cerebral perfusion pressure and not oxygen delivery is a critical factor that restores the massive transmembrane ionic and water shifts during SD under the stated conditions. Of course, the lack of O2−dependency was found within the tested paO2 ranges of ~40 to 400mmHg; more severe hypoxia compromising baseline energy state would still be expected to delay SD recovery, as was probably the case when hypoxia was combined with hypotension (Figure 3A). The majority of published data also indicate that ptO2 is increased rather than decreased despite a marked increase in the rate of O2 metabolism during SD (Back et al, 1994; Lacombe et al, 1992; Turner et al, 2007; Wolf et al, 1996a; Wolf et al, 1996b), although a biphasic response with increased followed by decreased ptO2 has recently been reported during the DC shift using a microelectrode with good temporal resolution (Piilgaard and Lauritzen, 2009). Mitochondrial enzymes remain oxidized during SD further arguing against O2 shortage (Mayevsky and Chance, 1975; Somjen, 2001). Moreover, SD duration as well as the accompanying CBF changes are not modulated by changes in paO2 within a wide physiologic range (Gido et al, 1994; Hashimoto et al, 2000; Kudo et al, 2008; Schechter et al, 2009). A notable exception is the recent demonstration of a unique pattern of tissue hypoxia during SD in mouse cortex, in vivo, and shortening of SD duration by hyperoxia (Takano et al, 2007); however, the interpretation may have been confounded by preceding severe post-SD oligemia in this species (Ayata et al, 2004). Nevertheless, the techniques we used to measure cortical oxygenation do not have sufficient spatial resolution to assess oxygenation gradients as a function of distance from the capillaries (Takano et al, 2007), or in different cortical layers. Therefore, we cannot rule out the possibility that manipulation of paO2 does not elevate ptO2 within the microregions farthest from the capillaries or at restricted cortical depths.
Hypotension markedly diminished the SD-induced hyperemic response, in part due to the emergence of transient hypoperfusion coincident with the DC shift (Sukhotinsky et al, 2008). More severe initial hypoperfusion and smaller hyperemia during the DC shift were tightly correlated with delayed SD recovery, suggesting the importance of tissue perfusion for the restoration of ionic gradients. By contrast, hypoxia alone did not alter the CBF response significantly. In our earlier study, we have found that hypoxia has transformed the CBF response similar to hypotension (Sukhotinsky et al, 2008); however, in light of our current data, this discrepancy is likely because hypoxia has significantly lowered MABP in our earlier study (83±15mmHg), whereas in this study, we tightly maintained MABP within a higher target range (100±11mmHg).
Cerebral glucose delivery may be a rate-limiting step in SD recovery. Although SD recovery seems to be paO2 independent within the studied range (40 to 400mmHg), it is still partially dependent on Na+/K+ ATPase activity and therefore must be energy or ATP utilizing. Resting cerebral metabolism is critically dependent on oxidative phosphorylation as the predominant source for ATP production; however, during acute increases in energy consumption (e.g., functional activation) anaerobic glycolysis is often sufficient to match metabolic demand (Fox et al, 1988; Shinohara et al, 1979). Marked and sustained reductions in tissue levels of creatine phosphate, glucose, glycogen and pH, and increases in lactate occur during SD (Csiba et al, 1985; Gjedde et al, 1981; Krivanek, 1961), suggesting that glucose consumption is rapidly stimulated and exceeds glucose delivery despite elevations in blood flow (Gjedde et al, 1981). Consistent with this notion, hyperglycemia suppresses and hypoglycemia facilitates the ease with which SDs are evoked (Els et al, 1997; Nedergaard and Astrup, 1986), and in the ischemic penumbra even mild reductions in plasma glucose are associated with increased frequency of periinfarct SDs (Strong et al, 2000). In rat cortex, moderate hypoglycemia significantly prolongs the recovery of SD (Gido et al, 1993), and glucose levels but not ptO2 appear critical for SD recovery in an in vitro retina preparation (Vercesi and Martins-Ferreira, 1983). Therefore, it is possible that the attenuated CBF response to SD in hypotensive rats limits glucose delivery, thereby delaying SD recovery.
Reduced vascular clearance of [K+]e provides an alternative explanation for delayed SD recovery in hypotensive animals. For example, artificially increased [K+]e markedly prolongs SD duration in rat brain (Dreier et al, 2001), and intracerebral microdialysis significantly shortens SD duration presumably due to enhanced [K+]e clearance (Obrenovitch et al, 1995). During SD recovery, elevated [K+]e can be restored via Na+/K+ ATPase- or Na+/K+/Cl− cotransporter-dependent reuptake, spatial buffering by glia, and less likely passive diffusion (Gardner-Medwin, 1983a; Gardner-Medwin, 1983b; Kofuji and Newman, 2004). The contributions of each mechanism to [K+]e clearance probably depend on the magnitude, spatial extent, and duration of [K+]e elevations (Somjen, 1979). Although the cerebrovascular endothelium is highly impermeable to K+ (Hansen et al, 1977), active or facilitated transport systems for K+ do exist on the abluminal membranes of the cerebrovascular endothelium (Goldstein, 1979), and may become important for [K+]e clearance into the microvasculature (e.g., capillaries) particularly when [K+]e is diffusely increased to very high levels during SD (Bradbury et al, 1972). If so, hypotension may impede and hypertension facilitate cerebrovascular [K+]e clearance by diminishing and augmenting the CBF response to SD, respectively. Astrocytes may have a function in vascular [K+]e clearance by channeling high [K+]e towards the vasculature as well (Filosa et al, 2006; Newman, 1986).
In conclusion, our data show that hypotension delays the restoration of ionic shifts associated with SD, and SD recovery is independent of tissue oxygenation under the stated conditions. Hence, hypotension may be detrimental to tissue outcome in stroke and subarachnoid hemorrhage, where injury depolarizations have been observed in compromised brain tissue (Dohmen et al, 2008; Dreier et al, 2006), and pharmacologically induced hypertension and plasma volume expansion may be beneficial by hastening the recovery of injury depolarizations (Dreier et al, 2002).
This study was supported by the National Institutes of Health (NS055104).
The authors declare no conflict of interest.