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Abstract

Background: Migraine is a disabling chronic episodic disorder. Attack frequency progressively increases in some patients. Incremental cortical excitability has been implicated as a mechanism underlying progression. Cortical spreading depression (CSD) is the electrophysiological event underlying migraine aura, and a headache trigger. We hypothesized that CSD events during frequent migraine attacks condition the cortex to increase the susceptibility to further attacks.

Methods: A single daily CSD was induced for 1 or 2 weeks in mouse frontal cortex; contralateral hemisphere served as sham control. At the end of CSD conditioning, occipital CSD susceptibility was determined by measuring the frequency of CSDs evoked by topical KCl application.

Results: Sham hemispheres developed 8.4 ± 0.3 CSDs/hour, and did not significantly differ from naïve controls without prior cranial surgery (9.3 ± 0.4 CSDs/hour). After 2 but not 1 week of daily CSD conditioning, CSD frequency (4.9 ± 0.3 CSDs/hour) as well as the duration and propagation speed were reduced significantly in the conditioned hemispheres. Histopathological examination revealed marked reactive astrocytosis without neuronal injury throughout the conditioned cortex after 2 weeks, temporally associated with CSD susceptibility.

Conclusions: These data do not support the hypothesis that frequent migraine attacks predispose the brain to further attacks by enhancing tissue susceptibility to CSD.

Keywords

Spreading depression chronic migraine astrocyte

Introduction

Migraine is a debilitating chronic neurovascular disorder affecting 20% of the general population. In a subset of patients, migraine attack frequency progressively increases over months, transforming into chronic migraine (1). High attack frequency at baseline is among the most important predictors of this progression (2). It has been hypothesized that sensitization in central pain processing pathways or reduced nociceptive thresholds underlie migraine chronification (3). An alternative hypothesis is that frequent migraine attacks augment intrinsic cortical excitability leading to a progressive increase in attack frequency (4–6). In support of this, chronic migraineurs show more severe impairment in cortical processing of sensory stimuli, suggesting higher cortical excitability than episodic migraine (7).

Experimental and clinical evidence suggests that cortical spreading depression (CSD) underlies migraine aura and is implicated in migraine headache pathogenesis (8). CSD is a slowly propagating wave of complete neuronal and glial depolarization triggered when extracellular K⁺ concentration ([K⁺]_e) is raised above a threshold. In experimental models, CSD can be induced chemically by topical application of concentrated KCl, or by direct electrical stimulation. Recent experimental studies showed that genetic, hormonal and pharmacological factors known to modulate migraine susceptibility also modulate CSD susceptibility with good correspondence (9,10).

CSD has profound effects on neuronal and glial structure, function and gene expression (11–13). To test the hypothesis that CSDs occurring during frequent migraine attacks might augment cortical excitability, thereby facilitating the occurrence of further CSDs (6), we induced chronic daily CSDs in mouse cortex, mimicking frequent migraine attacks, and assessed the changes in susceptibility to subsequent CSDs over time.

Methods

Daily CSD conditioning (Figure 1A)

All experimental procedures followed national and institutional guidelines for animal care and use for research purposes, and were approved by an institutional review board. During the conditioning period of 1 or 2 weeks, a single CSD was evoked daily by epidural KCl (1M) application or direct cortical electrical stimulation. We chose to induce a single CSD because there is no evidence to suggest that more than one CSD occurs during a migraine with aura attack. We induced CSDs every day during the 1–2 week conditioning period because the risk of migraine chronification increases exponentially with baseline headache frequency, particularly when patients experience 10–14 days of headache per month (14). To prepare for daily CSD inductions, mice (C57BL6, male, 12–17 weeks old) were anesthetized with isoflurane (2.5% for induction, 1.5% for maintenance in 70% N₂O and 30% O₂) and scalp was reflected laterally via a midline incision. In the KCl stimulation group, one burr hole was drilled under saline cooling over the prefrontal cortex (0.5 mm diameter; 2 mm anterior and 1 mm lateral to bregma) on each hemisphere for epidural KCl or saline application, and the scalp was sutured back. In the electrical stimulation group, two burr holes were drilled on each hemisphere over the prefrontal cortex (0.3 mm diameter; 2 mm anterior and 1 mm lateral to bregma) under saline cooling. Tungsten stimulation electrodes (125 µm diameter) were chronically implanted through each burr hole at a cortical depth of 1 mm, and fixed in place by a plastic ring (diameter 1 cm) attached to the skull surface by translucent epoxy. Adequate pain control was ensured by a small amount of adjunct local anesthesia (1% lidocaine) at the wound edges. One hemisphere of each animal was randomly assigned as sham not subjected to CSD.

After surgical preparation, mice were subjected to once-daily CSD inductions under brief isoflurane anesthesia on a stereotactic frame. In 7 mice (n = 2 and 5, for 1-week and 2-week conditioning, respectively), scalp sutures were removed, and CSD was evoked by 1–2 min epidural KCl application using a cotton ball (1 mm) placed onto the test hemisphere, followed by saline wash. A relatively high concentration of KCl (1M) was used to ensure successful CSD induction, which was confirmed by laser speckle flowmetry non-invasively through intact skull as described earlier (15). Sham hemisphere was exposed to isotonic NaCl in a similar fashion. Scalp sutures were replaced after CSD induction. In 8 mice (n = 3 and 5, for 1-week and 2-week conditioning, respectively), CSD was elicited by direct bipolar cathodal stimulation (single square pulses) of the cortex in the test hemisphere only. Stimulation intensity (100–8000 µC) was increased in a stepwise fashion every 5–10 min (100 µA for 1 s; 200 µA for 1 s; 300 µA for 1 s; 400 µA for 1 s; 500 µA for 1 s; 1 mA for 1 s; 1 mA for 2 s; 2 mA for 2 s; 3 mA for 2 s; 4 mA for 2 s) until a CSD was detected non-invasively by laser Doppler flowmetry through the thin epoxy layer covering the skull surface. Each day’s stimulation was started at the intensity that successfully evoked a CSD on the previous day, to minimize the duration of anesthesia and the number of electrical stimulations. Sham hemisphere underwent the same electrode implantation and experimental procedures, except for daily electrical stimulation and CSD induction. CSD conditioning by KCl or electrical stimulation yielded comparable changes in CSD susceptibility and astrocytic activation (not shown); therefore, the data obtained using these complementary techniques were pooled for analysis. Additionally, naïve mice which did not undergo any prior chronic surgical procedure for conditioning were studied to control for the effects of sham surgery (n = 4).

Assessment of CSD susceptibility (Figure 1B)

Twenty-four hours after the last daily CSD induction, mice were anesthetized using isoflurane and N₂O as above, trachea intubated, and femoral artery catheterized for blood pressure and blood gas monitoring (Rapidlab 248 blood gas/pH analyzer, Bayer HealthCare, Walpole, MA, USA). Although isoflurane/N₂O combination mildly suppresses CSD susceptibility (16), it allows a stable and precise anesthetic depth during prolonged experiments with minimal cardiovascular depression and hypotension, and any direct effect on CSD is expected to be comparable between sham and conditioned hemispheres. Mice were then paralyzed (pancuronium, 0.4 mg/kg/hour, intraperitoneal), placed on the stereotactic frame, and connected to the mechanical ventilator (SAR-830, CWE, Ardmore, PA, USA). Rectal temperature was kept at 37°C using a thermostatically controlled heating pad (FHC, Bowdoin, ME, USA). Physiological parameters (mean arterial blood pressure, arterial pH, pO₂ and pCO₂) were within the normal range in all groups (Table 1). After stable mechanical ventilation, the skull surface was washed, and in the daily electrical stimulation group the tungsten electrode assembly was gently removed. Three burr holes were drilled under saline cooling at the following coordinates (mm): (i) anterior 1, lateral 2.5 from lambda (1.5 diameter) for occipital epidural KCl application; (ii) posterior 2, lateral 2 from bregma (0.5 diameter) for parietal recording site 1; (iii) posterior 1, lateral 2 from bregma (0.5 diameter) for parietal recording site 2. Both the occipital and parietal burr holes were located remote from the prefrontal daily CSD induction site, and thus remained naïve until the day of CSD susceptibility testing except for the daily CSDs propagating throughout the ipsilateral cortex. The slow (DC) potential shifts and electrocorticogram were continuously recorded using extracellular glass micropipettes (150 mM NaCl, 300 µm below dura), and a data acquisition system for offline analysis. An Ag/AgCl reference electrode was placed subcutaneously in the neck. Following surgical preparation, the cortex was allowed to rest for more than 10 minutes under saline irrigation. CSD susceptibility was assessed by 30-minute continuous epidural KCl application (300 mM) at the occipital burr hole on conditioned and sham hemispheres in alternating order in different experiments. The frequency, propagation speed and duration of evoked repetitive CSDs were measured as CSD susceptibility endpoints using the characteristic negative DC potential shifts. CSD duration was measured at half-maximal amplitude, whereas the propagation speed was calculated from distance between the two recording sites and the latency between the DC shift onset at each location. In our experience, continuous topical KCl application has been a sensitive and reliable method to assess CSD susceptibility, and correlated well with the electrical stimulation threshold for CSD (9,17,18).

Table 1.

Systemic physiological parameters during CSD susceptibility testing

		Conditioned
	Naïve (n = 4)	1 week (n = 5)	2 weeks (n = 10)
BP (mmHg)	79 ± 3	82 ± 4	82 ± 4
pH	7.39 ± 0.01	7.39 ± 0.01	7.36 ± 0.01
pCO₂ (mmHg)	33 ± 1	35 ± 2	33 ± 2
pO₂ (mmHg)	141 ± 6	142 ± 3	147 ± 5

Histology and immunohistochemistry

A total of 18 brains (n = 6, 4 and 8, naïve, 1-week and 2-week CSD conditioned brains, respectively) were studied using hematoxylin/eosin staining and glial fibrillary acidic protein (GFAP) immunohistochemistry. Naïve group included four non-conditioned mice that underwent CSD susceptibility testing (30 min topical 300 mM KCl exposure) shortly before sacrifice, and two mice that were not exposed to any CSDs including the susceptibility testing. In addition, four 2-week CSD conditioned brains were studied using propidium iodide and fluoro-Jade staining. Twenty µm thick coronal frozen sections at the level of parietal cortex (0–1.5 mm posterior from bregma; Figure 1B) were studied to avoid the confounding effects of prior direct daily exposure to KCl or electrical stimulation. Adjacent sections were stained with hematoxylin/eosin, propidium iodide and fluoro-Jade, and qualitatively examined for histopathological evidence of neuronal injury (e.g. shrunken eosinophilic cytoplasm and pyknotic nuclei, nuclear fragmentation and debris, positive fluorescent staining with propidium iodide or fluoro-Jade). Propidium iodide (Sigma, St. Louis, MI, USA) was injected 1 hour before sacrifice for visualization of membrane permeable cells. To further investigate the presence of degenerating neurons, brain sections were briefly fixed in 10% buffered formalin for 5 minutes, washed in distilled water, incubated in 0.06% potassium permanganate to suppress background staining, and stained in 0.01% fluoro-Jade B (Millipore, Billerica, MA, USA) with 0.1% acetic acid. Immunostaining with rabbit cy3-labeled anti-GFAP (1:1000; Sigma-Aldrich, Cat# C9205) was done after fixation with 4% paraformaldehyde. Astrocytic activation was assessed by two observers blinded to the treatment groups using one section from each brain at the level of parietal cortex as noted above. In naïve cortex, many astrocytic processes and some cell bodies showed a low but detectable level of GFAP immunoreactivity in high-power images. Therefore, we counted astrocytes that only displayed robust GFAP immunoreactivity throughout the soma and processes that were identifiable at low-power images; as such, higher cell counts after CSD likely reflected increased GFAP immunoreactivity rather than an increase in the number of astrocytes. The number of cells per mm² in the conditioned hemisphere was then divided by the number in the sham hemisphere of the same animal, and expressed as percent change. There was excellent inter-observer agreement of cell counts (r = 0.87; total of 36 hemispheres).

Figure 1.

Experimental set-up and protocol. Experiments were conducted in two phases. A. One hemisphere was subjected to a single CSD evoked in the frontal cortex using topical KCl (1M) application or electrical stimulation (100–8000 µC) every day for 1 or 2 weeks. Successful CSD induction was monitored by laser Doppler (LDF) or laser speckle flowmetry (LSF) through intact skull (thick gray line) as CSD propagated across the parietal cortex (dashed lines). Inset shows a representative cerebral blood flow (CBF) tracing during CSD. Contralateral (sham) hemisphere underwent identical surgical procedures but was not subjected to CSD. B One day after the end of 1- or 2-week CSD conditioning, CSD susceptibility was determined by monitoring the frequency of repetitive CSDs evoked during continuous topical KCl application (300 mM) for 30 minutes on occipital cortex. Evoked CSDs were electrophysiologically detected using two glass micropipettes placed in the parietal cortex (E1 and E2). Inset shows a representative 30-minute electrophysiological recording from a sham hemisphere with repetitive CSDs during topical KCl application (horizontal and vertical bars indicate 5 minute and 10 mV, respectively). At the end of the experiments, parietal cortex was histologically examined at a coronal level that was not exposed to KCl or electrical stimulation either during daily CSD conditioning or CSD susceptibility assessment. Horizontal bar = 1 mm.

Data analysis and statistics

Data from ipsilateral and contralateral hemispheres in naïve, 1-week and 2-week conditioned groups were analyzed using one-way ANOVA, or two-way ANOVA for paired measures, followed by Bonferroni all pairwise multiple comparisons. Systemic physiological parameters were tested using one-way ANOVA; p < 0.05 was considered significant. Data are presented as mean ± SEM in the text and table, and box and whisker plots in the figures.

Results

Epidural KCl application on the occipital cortex evoked repetitive CSDs in all mice (Figure 2A). Naïve mice without prior cranial surgical intervention or CSD conditioning developed 9.3 ± 0.4 CSDs/hour during KCl application (n = 4 mice, right and left hemispheres combined; Figure 2B). Daily CSD conditioning for 2 weeks significantly reduced the CSD frequency when compared to the sham hemisphere (4.9 ± 0.3 vs. 8.1 ± 0.4 CSDs/hour, respectively; p < 0.001; n = 10). In contrast, 1 week conditioning did not significantly suppress CSD susceptibility (8.4 ± 0.4 vs. 9.0 ± 0.4 CSDs/hour, conditioned vs. sham hemisphere, respectively; p > 0.05, n = 5). CSD propagation speeds and durations were also reduced in the conditioned cortex compared to the sham hemisphere after 2 weeks (Figure 2B).

Figure 2.

CSD conditioning suppresses CSD susceptibility. A. Representative electrophysiological tracings show repetitive CSDs during 30-minute topical KCl application in 2-week CSD conditioned and sham cortices in a representative animal. Fewer CSDs were elicited during topical KCl application in the conditioned cortex compared to sham. Individual CSD durations were also shorter in the conditioned cortex. Vertical and horizontal bars indicate 20 mV and 5 minutes, respectively. B. Bar and whisker plots of CSD frequency, propagation speed and duration in naïve, and 1- or 2-week CSD conditioned and sham cortices. Right and left hemispheres of the naïve group are shown separately. All three parameters showed suppression of CSD susceptibility in conditioned cortex compared to sham at 2 weeks (*p < 0.01 vs. sham). The trend at 1 week did not reach statistical significance. Median (horizontal line), mean (+), 25–75% (box) and 10–90% ranges (whiskers) are shown.

Cortical neuronal injury is one mechanism that can suppress CSD susceptibility (19). To investigate whether chronic daily CSDs cause neuronal injury, we performed post mortem histopathological examination of the cortex using hematoxylin and eosin staining (Figure 3A), as well as propidium iodide and fluoro-Jade stainings (not shown) to detect degenerating neurons. Consistent with previous reports (20), we did not find any evidence of cortical injury in the conditioned or sham hemispheres even after 2 weeks of daily CSDs.

Figure 3.

CSD conditioning induces reactive astrocytosis without histopathological evidence of neuronal injury. A. Representative coronal sections from 2-week CSD conditioned or sham parietal cortices stained with hematoxylin and eosin (scale bar = 140 µm). No histopathological evidence for neuronal injury was noted even after exposure to 2 weeks of once daily CSDs. B. Representative GFAP immunostained coronal sections from 2-week CSD conditioned or sham parietal cortices. Robust reactive astrocytosis is present in conditioned cortex compared to scant staining in the sham cortex (scale same as A). C. Representative high-power views of GFAP immunoreactive astrocytes from a 2-week CSD conditioned or sham hemisphere. Sham hemispheres showed low level of GFAP immunoreactivity in many astrocytic processes and occasional cell bodies mostly detectable at this high-power view. In contrast, CSD-conditioned tissue showed increased staining intensity in both cell bodies and processes, as well as increased arborization and thickness of processes throughout the hemisphere (scale bar = 10 µm). D. Bar and whisker plots show robust reactive astrocytosis compared to sham hemisphere at 2 weeks. Reactive astrocytosis was quantified in a blinded fashion by two investigators by counting cells that showed robust GFAP immunoreactivity in soma and processes in low-power images shown in B (see Methods), and expressed as percent increase in conditioned hemisphere over the sham hemisphere. Median (horizontal line), mean (+), 25–75% (box) and 10–90% ranges (whiskers) are shown. *p < 0.01 vs. the other groups.

Astrocytes can modulate CSD susceptibility (21–23), and CSD is known to induce reactive astrocytosis (12). We therefore sought a correspondence between CSD susceptibility and the degree of reactive astrocytosis in the conditioned cortex. The number of GFAP-positive astrocytes did not significantly differ between the two hemispheres in naïve brains, or after 1 week of CSD conditioning (Figure 3). In contrast, 2-week CSD conditioned hemisphere showed a more than four-fold increase in the number of astrocytes with robust GFAP immunoreactivity compared to the sham hemisphere.

Discussion

Widely accepted as the electrophysiological event underlying migraine aura and a trigger for trigeminovascular nociception, CSD has transient as well as lasting effects on cerebral perfusion, cell morphology, and gene expression (11,13,24–26). However, the cumulative impact of frequent CSDs occurring in migraineurs with high attack frequency is poorly understood. Clinically, high migraine attack frequency at baseline increases the risk for migraine chronification, which is characterized by a progressive increase in attack frequency in association with cortical hyperexcitability (7,27). In this study we tested whether chronic CSDs facilitate further CSD occurrence as a potential mechanism for migraine progression. We found that two-week exposure to once daily CSDs suppresses CSD susceptibility, as shown by less frequent CSDs during topical KCl application, and reduced CSD propagation speed and duration. Suppression of CSD susceptibility was not associated with neuronal injury. Taken together, our data do not support the hypothesis of facilitative conditioning of CSD susceptibility in migraine chronification, and are consistent with the observations that chronic migraine usually occurs without aura, and often evolves from episodic migraine without aura (4).

Consistent with previous reports, exposure to once daily CSDs for up to 2 weeks did not result in neuronal injury detectable by histological techniques (12,20). Instead, we found marked reactive astrocytosis characterized by astrocyte hypertrophy and hyperplasia that showed good temporal correspondence with the decrease in CSD frequency, propagation speed and duration. Reactive astrocytosis is a response of brain tissue to noxious stimuli, but can be induced by CSD in the absence of injury (12,28). GFAP immunostaining can be upregulated within 2 days when many CSDs are induced within a few hours, and persists for up to a few weeks (12). Moreover, the degree of reactive astrocytosis correlates with the number of induced CSDs (29). In this study we aimed to deliver a more physiological stimulus, and detected only a mild reactive astrocytosis after 1 week of once daily CSDs.

Although our results do not substantiate causality between reactive astrocytosis and suppression of CSD susceptibility after chronic CSD conditioning, published data suggest that astrocytes render the brain less susceptible to CSD. For example, early metabolic challenge to astrocytes can activate adenosine-dependent mechanisms delaying CSD initiation (21), and disruption of astrocytic function augments CSD propagation speed and duration, and predisposes to spontaneous CSD episodes (22,23). Higher astrocyte/neuron ratios (30) have also been proposed as a factor rendering gyrencephalic species less susceptible to CSD. Therefore, astrocytic hypertrophy and hyperplasia may hinder CSD induction in conditioned cortex. Astrocytes are responsible for the uptake and clearance of more than 90% of extracellular glutamate (31,32); however, it is not clear whether reactive astrocytosis augments glutamate uptake (33,34). Another fundamental astrocytic function is to regulate [K⁺]_e within a tight physiological range via spatial buffering both during normal physiological activity and during CSD (35). The massive rise in [K⁺]_e is arguably the most critical determinant of CSD initiation and spread. It is therefore possible that reactive astrocytes buffer the [K⁺]_e surge during CSD more efficiently. Lastly, astrocytes provide major metabolic support to neurons, and may modulate CSD susceptibility in this way. Interestingly, both CSD preconditioning and reactive astrocytosis are protective in focal cerebral ischemia (36,37), and our data suggest that suppression of peri-infarct CSDs may be one mechanism. Of note, CSD also activates microglia (29,38) but it is not known whether microglia modulate CSD.

In summary, we showed that exposure of mouse cortex to once daily CSDs for 2 weeks renders the cortex more resistant to subsequent CSD induction, and reduces both the duration and the propagation speed of induced CSDs. Moreover, CSD suppression shows a temporal association with reactive astrocytosis as a potential mechanism. These data do not support a role for facilitative conditioning of CSD susceptibility to explain migraine chronification.

Footnotes

Acknowledgements

Supported by the National Institutes of Health (NS061505, NS055104).

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Chronic daily cortical spreading depressions suppress spreading depression susceptibility

Abstract

Keywords

Introduction

Methods

Daily CSD conditioning (Figure 1A)

Assessment of CSD susceptibility (Figure 1B)

Histology and immunohistochemistry

Data analysis and statistics

Results

Discussion

Footnotes

Acknowledgements

References