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Spreading depression (SD), the likely cause of migraine aura and perhaps migraine, is triggered by widespread and unfettered neuronal hyperexcitability. Migraine and the initiating hyperexcitability of seizure, which involve oxidative stress (OS), are likely interrelated. Environmental enrichment (EE) decreases seizure and can reduce migraine. EE's well-characterized neuroprotective effect involves insulin-like growth factor-1 (IGF-1). Accordingly, we asked if IGF-1 could mitigate the hyperexcitability that initiates SD using rat hippocampal slice cultures. We demonstrate that IGF-1 significantly decreased SD susceptibility and related OS. We mimicked OS of SD and observed that IGF-1 abolished hyperexcitability from OS. Application of an antioxidant significantly decreased SD susceptibility and co-administration of an antioxidant with IGF-1 produced no additive effect, whereas an oxidizer significantly increased SD, and this effect was abrogated by IGF-1. Moreover, IGF-1 significantly decreased baseline OS, despite seemingly paradoxically increasing CA3 bursting. These results suggest that IGF-1 increased endogenous antioxidants to levels sufficient to buffer against the OS of SD. Insulin similarly mitigated SD susceptibility, but required a far greater dose. Since brain IGF-1 increases with EE, and, like insulin, independently functions as an EE mimetic, we suggest that EE mimetics are a novel source of therapeutics for SD, and by extension, migraine.
Migraine and its maladaptive transformation to high frequency and chronic migraine (HFCM) are immense health care burdens affecting 11% of the adult population worldwide, with 3% experiencing chronic daily headache (Rasmussen et al., 1991; Stovner et al., 2007). In the United States, these maladies result in annual medical and lost work-time costs of $30 billion (Goldberg, 2005; Hu et al., 1999), yet existing therapies, largely centering on use of anticonvulsants, are only modestly effective for treatment of HFCM (Mack, 2011).
Spreading depression (SD) is a slowly propagating loss of neuronal activity that is the most likely cause of migraine aura and perhaps migraine pain (Lauritzen and Kraig, 2005; Moskowitz et al., 1993). SD is associated with increased brain hydrogen peroxide concentration, likely due to the increased metabolic demands associated with this phenomenon (Viggiano et al., 2011). SD is preceded (Bureš et al., 1974; Somjen, 2001), and importantly, followed by increased synaptic activity, as recently shown (Grinberg et al., 2011). Synchronous and excessively increased brain excitability in a sufficient brain volume is necessary to trigger SD (Bureš et al., 1974; Somjen, 2001; Kunkler et al., 2005). Furthermore, without sufficient time for compensatory adaptation, recurrent epochs of excessively increased synaptic activity from repeated SDs may lower SD initiation threshold, and therefore be a determinant of HFCM (Kraig et al., 2010). Experiments performed using hippocampal slice cultures support this suggestion (Mitchell et al., 2010a).
In contrast, physiologically increased neuronal activity from environmental enrichment [(EE); i.e., increased physical, intellectual and social volitional opportunities], which necessarily occurs with sufficient time for adaptive change, has the opposite effect. EE is a well-recognized preconditioning stimulus that induces neuroprotection (Will et al., 2004). Notably, EE occurs with physiologically increased neuronal activity and increased hippocampus-based learning and memory (van Praag et al., 2000; Kraig et al., 2010) and EE reduces excessive hyperexcitability from seizures (Young et al., 1999; Kraig et al., 2010). Furthermore, there is evidence that EE reduces SD (Guedes et al., 1996) and improves migraine (Darabaneanu et al., 2011).
Since insulin-like growth factor-1 (IGF-1) is a primary determinant of neuroprotection from EE (Carro et al., 2001), we hypothesized that IGF-1 might act as an EE-mimetic that would reduce SD susceptibility. Our results here show that exposure to IGF-1 triggered a significant reduction in SD susceptibility, a protective effect that involves reduced oxidative stress (OS) and reduced hyperexcitability. Furthermore, insulin, which enhances hippocampus-based memory when delivered to brain via nasal administration (Stockhorst et al., 2004; Craft et al., 2011) had a similar protective effect against SD, but at a dose orders of magnitude greater than required for IGF-1. Moreover, while IGF-1 decreased the hyperexcitability for SD, it also increased spontaneous CA3 area bursting activity, consistent with the electrophysiological changes of hippocampus-based learning (Yanovsky et al., 1995). Our results provide the first evidence that EE-based signaling (i.e., involving IGF-1) can lead to development of novel therapeutics to prevent SD, and by extension, perhaps recurrent and HFCM. This work has appeared in preliminary form (Grinberg and Kraig, 2011).
Experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Chicago Medical Center and followed ARRIVE guidelines.
Slice cultures were prepared (Mitchell et al., 2010b) from nine day old male or female pups of Wistar rats (Charles River, Wilmington, MA, USA) and SD induced as previously described (Grinberg, et al., 2010; Pusic et al., 2011). All electrophysiological procedures were performed in serum-free media, containing (per 100 mL): Neurobosal medium (97 mL; #21103, Invitrogen, Carlsbad, CA, USA); Gem-21, (2.0 mL; #400-160-010; Gemini Bioproducts, Sacramento, CA, USA); Glutamax (1 mM; #35050, Invitrogen); Gentamicin (1 μg/mL; #15710-064, Invitrogen); D-glucose [(45%), 680 μL; #G8769, Sigma, St. Louis, MO, USA]; ascorbic acid (0.5 mM; #A4544, Sigma); Fungizone, (1 mg/mL; #15295, Invitrogen); NaCl (41 mM; #S6546, Sigma); Mg2Cl2 (0.8 mM; #M1028, Sigma); CaCl2 (1.6 – 2.4 mM; #21115, Sigma). The normalcy of slice culture electrophysiological function was verified by recording CA3 area field potentials evoked from bipolar electrical stimuli applied to the dentate gyrus (100 μs pulses, ≤ 0.2 Hz). The recording microelectrode was moved along the long axis of pyramidal neurons at the genu of CA3 until field potential excitatory post-synaptic responses were maximal (Fig. 1). Slices with CA3 field post-synaptic potential responses ≥ 3 mV (with applied currents of 10 – 20 μA) were used for experiments. The Matlab commands ‘filter’ and ‘butter’ (Matlab 7.1, Mathworks, Natick, MA, USA) were used to filter the digitized data with a second order high-pass Butterworth filter with a 1 Hz cutoff frequency for the signals shown in Figure 5.
SD threshold was determined by progressively increasing the amount of current applied [10 pulses, 10 Hz (100 μs/pulse)], starting with the current needed to produce a half-maximal field potential from a single 100 μs pulse (Fig. 1). If a given current intensity did not trigger SD, the amount of current applied was doubled and re-applied 1 – 2 minutes later until SD occurred. Applied currents ranged from 10 to 10,000 nC. Unless otherwise stated, experimental agents were applied ‘acutely’ (i.e. 15 – 30 minutes before electrophysiological procedures). For OS measurements, SD was triggered 6 times over an hour (i.e., ~ every 9 minutes).
While the number of SDs that can occur with migraine and HFCM has not been established, others suggest that chronic migraine may occur with only one SD per day. However, this experimental paradigm results in decreased brain excitability in anesthetized animals (Sukhotinsky et al., 2011), suggesting that the number of SDs and recovery period are critical to establishing a migraine model (Kraig et al., 2010). We chose 6 SDs so as to replicate the hyperexcitable phenotype seen in the brain of migraneurs (Palmer et al., 2000; Brennan, 2011; Mulleners et al., 2001; Welch, 2005) and after recurrent SD in hippocampal slice cultures (Mitchell et al., 2010a). Following SD, slice cultures were returned to normal incubation conditions with fresh media until fixation-harvest 24 hours later.
Slice culture excitability change from hydrogen peroxide exposure was assessed by noting the CA3 area field potential response to a single dentate gyrus 100 μs half-maximal current pulse applied 30 min after exposure.
IGF-1 (40 or 100 ng/mL; #4326-RG, R&D Systems, Minneapolis, MN, USA) or insulin (400 μg/mL; #0355, Invitrogen) was added to media either acutely or 3 days before (and during) electrophysiological studies of SD threshold responses. For experiments involving SD and OS, slice culture IGF-1 was refreshed after SD. Hydrogen peroxide (50 or 200 μM; #H1009, Sigma) or ascorbate (2 mM) were added to serum-free media acutely before (and during) electrophysiological recordings. This hydrogen peroxide concentration was chosen to mimic concentrations produced by SD in vivo (Viggiano et al., 2011) and the ascorbate concentration is four-fold that of the serum-free media, consistent with physiological levels (Rice, 1999). Sham control cultures only experienced normal media.
CA3 area OS was determined using CellROX™ Deep Red Reagent (#C10422; Invitrogen), a fixable fluorogenic probe that fluoresces (near infrared) when oxidized. After SD, slice cultures were incubated for 24 hours in normal media supplemented with CellROX™ (5 μM) followed by fixation using 10% phosphate buffered formalin (#SF100-4; Fisher Chemicals, Fair Lawn, NJ, USA) for 24 hours. Then, slice cultures were mounted on gelatin-coated glass slides and coverslipped using Prolong® Gold antifade reagent (#P36930; Invitrogen).
To indirectly assess antioxidant content, slice cultures were exposed to a standard load of OS, namely 50 μmol/L menadione (#47775; Sigma-Aldrich) for 1 hour. Menadione participates in redox cycling reactions at the mitochondrial electron transport chain, leading to production of superoxide anions (Thor et al., 1982). Slices were then incubated in 5 μM CellROX™ for 30 minutes, followed by fixation in 10% phosphate buffered formalin for 24 hours. Hydrogen peroxide could not be used as a mimetic of SD-induced OS here, since we found that it interferes with the CellROX™ reporter molecule (data not shown). Cell death, as measured by Sytox Green (Hulse et al., 2008; Mitchell et al., 2010b), was not observed following hydrogen peroxide or menadione treatment at harvest times described above. There was also no pyramidal neuron death following SD induction, IGF-1 exposure, or any combination thereof (data not shown).
Fluorescence intensity of CellROX™ was measured using a Cool Snap fx CCD camera (Photometrics, Tucson, AZ, USA) on an inverted Leica DM-IRBE microscope (Leica Mikroskopie und Systeme, Wetzlar, Germany) and MetaMorph (v. 7.0.4) software (Molecular Devices, Sunnyvale, CA, USA). Fluorescence intensity (i.e., average fluorescence intensity/area) was registered for a uniform CA3 area of interest (used throughout experiments) at 10× magnification (i.e., 1.70 mm2). Before acquisitions, the imaging system was calibrated to register uniform full image intensity (1500/4096) to a standard (480 nm excitation; 527 nm emission) 125 μmol/L acridine orange (100 mg/L; #A6014; Sigma; St. Louis) solution imaged through a hemacytometer.
Data was analyzed using SigmaStat (V. 3.5) software (Systat Software, Chicago, IL, USA). Control data for each experiment was set to 1.0 with related group data scaled proportionally to allow for inter-experimental comparisons. All experimental group measurements were compared to same-day sham and/or control cultures and reported as mean ± standard error of mean. Specific statistical tests used are indicated in figure legends. CorelDraw (v. X3; Corel, Ottawa, ON, Canada) and Photoshop (v. CS2; Adobe, San Jose, CA, USA) were used to produce figures.
Hippocampal slices were exposed to IGF-1 either acutely (i.e., 15 - 30 minutes), for 3 days, or for 7 days prior to assessing SD threshold. The 7-day IGF-1 exposure was performed phasically to better mimic anticipated effects of EE [i.e., exercise – rest intervals (Will et al., 2004; Kraig et al., 2010)], where slices were exposed to IGF-1-supplemented media in the day and returned to regular media at night. Acute, 3-day, and 7-day exposure to IGF-1 all significantly increased SD threshold compared to control by 24, 75, and 22-fold (Fig. 1). Furthermore, 3-day exposure to insulin [(400 μg/mL); but not lower insulin doses, i.e., 6, 12, and 100 μg/mL (n = 3-9/group)] resulted in a significantly (p = 0.03) higher SD threshold versus control [i.e., 22.60 ± 9.60 (n = 8) and 1.00 ± 0.20 (n = 9), respectively]. However, the insulin dose needed for this protective effect was 15,500-fold higher than IGF-1 (i.e., 70 μM versus 4.5 or 10 nM), suggesting that IGF-1 has greater therapeutic utility against SD. Accordingly, we focused our subsequent work to IGF-1.
Since SD may increase OS (Viggiano et al., 2011), OS can enhance brain excitability (Gulati et al., 2005; Waldbaum & Patel, 2010; Muller et al., 1993), and IGF-1 is involved in antioxidant signaling (see Discussion), we next tested whether IGF-1 treatment altered SD-induced OS. Results show that acute, 3-day and 7-day treatment with IGF-1 significantly reduced OS from SD (Fig. 2). 7-day exposure was again phasic, as described for SD threshold studies above. While acute treatment with IGF-1 led to a 20% decrease in OS from SD, 3-day exposure to IGF-1 afforded an even greater level of protection, with a 30% decrease in OS from SD, and 7-days offered a 73% decrease in OS from SD.
Slices were exposed to either ascorbic acid or hydrogen peroxide and SD threshold was assessed. Ascorbate (2 mM) significantly increased SD threshold, while hydrogen peroxide (50 μM) significantly decreased SD threshold (Fig. 3). Co-exposure to IGF-1 and a higher dose of hydrogen peroxide (200 μM) led to a significant decrease in SD threshold when compared to IGF-1 alone. However, 50 μM hydrogen peroxide co-exposed with IGF-1 was an insufficient oxidant stress to overwhelm the protective effect of IGF-1 on SD susceptibility (Fig. 3). Finally co-incubation of slice cultures with ascorbate and IGF-1 (n = 8) did not significantly raise the threshold for SD versus IGF-1 alone (n = 7) [p = 0.28 with relative SD threshold levels of 7.39 ± 6.16 and 1.00 ± 0.31, respectively].
We further assessed the ability of IGF-1 to reduce slice culture excitability by decreasing OS. First, we mimicked OS from SD by application of hydrogen peroxide. This exogenously induced OS significantly increased evoked slice hyperexcitability (Fig. 4), like that seen from SD (Mitchell et al. 2010a). Both 3-day and acute exposure to IGF-1 abrogated this hydrogen peroxide-induced hyperexcitability. Second, we additionally mimicked OS from SD by slice exposure to menadione (Fig. 4). As expected, this treatment triggered a significant increase in slice OS, an effect that was abrogated by acute and 3-day exposure to IGF-1. In fact, 3-day exposure to IGF-1 alone could significantly reduce baseline OS from control levels. Furthermore, 7-day exposure to IGF-1 also significantly reduced baseline OS levels by 26% when compared to controls (p = 0.001; n = 11 and 9 for controls and 7-day IGF-1, respectively). The latter is important because exposure to IGF-1 alone, which led to the significant reductions in baseline OS (Fig. 4), triggered a significant increase in spontaneous CA3 bursting (Fig. 5).
Here we show that IGF-1 mitigated SD susceptibility and decreased its associated OS. Furthermore, we show that OS-induced hyperexcitability and increased SD susceptibility, and that IGF-1 mitigated these effects. Finally, our results revealed that IGF-1 treatment lowered baseline levels of OS and simultaneously increased spontaneous activity of CA3 pyramidal neurons. These are the first results to indicate that a neuroprotective EE mimetic, IGF-1, prevents SD.
Evidence suggests that EE leads to a physiological increase in neuronal excitability that prevents SD. While SD increases aberrant hyperexcitability (Kruger et al., 1996) that makes brain tissue more susceptible to future SD (Mitchell et al., 2010a; Grinberg et al., 2011), EE increases physiological neuronal excitability associated with improved learning and memory (Kumar et al., 2011; Eckert & Abraham, 2010). EE protects from the aberrant hyperexcitability of seizure (Young et al., 1999; Kraig et al., 2010), and has been shown to reduce SD (Guedes et al., 1996). Furthermore, the neuroprotective effects of EE have also recently been shown to include migraine (Darabaneanu et al., 2011). While various molecular mechanisms of how EE produces these neuroprotective effects have been characterized (Gagné et al., 1998; van Praag et al., 2000; Ekstrand et al., 2008; Herring et al., 2010; Kempermann et al., 2010), the role of IGF-1 is particularly noteworthy.
IGF-1 mediates the neuroprotective effects of EE (Carro et al., 2001), and improves learning and memory (Sonntag et al., 2000). With EE, IGF-1 production is increased and its active uptake by the brain increases in an activity-dependent manner (Nishijima et al., 2010). Once in brain, IGF-1 increases spontaneous hippocampal neuronal activity and improves hippocampus-dependent learning and memory test performance (Lupien et al., 2003; Miltiadous et al., 2011; Xing et al., 2007). Mechanisms by which IGF-1 affects neuronal activity may include increasing ionic conductances (Blair & Marshall, 1997; Kanzaki et al., 1999), modulating neurotransmitter receptor activity (Gonzalez de la Vega et al., 2001; Ramsey et al., 2005), and decreasing generation of reactive oxygen species (Csiszar et al., 2008; Pérez et al., 2008). Important to our work here, increased neuronal activity enhances neural antioxidant production (Papadia et al., 2008). Furthermore, IGF-1 has been shown to similarly increase antioxidant production in multiple peripheral tissues (Jallali et al., 2007; Csiszar et al., 2008). Here we show that IGF-1 reduced OS in control hippocampal slices, a preparation which shows spontaneous, physiological neuronal activity. In fact, this spontaneous activity increased after IGF-1 exposure, a phenomenon that should elevate metabolic activity and therefore the generation of reactive oxygen species. Despite this, we found that net OS significantly declined. We speculate that this results from neural activity-dependent signaling involving increased antioxidant production, as first shown by Papadia and coworkers (2008). While beyond the scope of our current report, future studies are designed to directly confirm that IGF-1 can lead to increased antioxidant production in brain.
We show that SD induces increased tissue OS, as other work has suggested (Viggiano et al., 2011). OS increases hyperexcitability (Gulati et al., 2005; Waldbaum & Patel, 2010; Muller et al., 1993). We confirm and extend these findings to show that OS-induced CA3 hyperexcitability can lead to SD. Furthermore, we show that IGF-1 mitigated the amount of OS generated by SD, SD susceptibility, OS-induced SD susceptibility, as well as the hyperexcitability of OS. Finally, the impact of IGF-1 protection from SD-induced OS increased with time. Together, these results suggest that IGF-1's effects on OS (and, therefore, SD susceptibility) may involve an adaptive response, consistent with physiological-conditioning hormesis (Radak et al., 2008). These effects may help to entrain brain tissue away from the unfettered hyperexcitability needed for SD and toward physiological excitability and decreased OS.
IGF-1 is highly protective in stroke (Liu et al., 2004; Rizk et al., 2007; Fletcher et al., 2009). SDs occur spontaneously in the penumbra of stroke. The number and cumulative duration of the SDs occurring there are proportional to the growth in infarct volume (Dijkhuizen et al., 1999; Mies et al., 1993; Nakamura et al., 2010). Our results may indicate that the mechanism by which IGF-1 decreases stroke size involves decreasing the spontaneous SDs occurring in the penumbra of stroke.
Although we demonstrate the proof of principle that EE-mimetic IGF-1 decreases OS and SD susceptibility, a complete analysis of the optimal dose, duration, and frequency of treatment requires further study. As a first step, we chose to administer 7-day IGF-1 phasically to better mimic the inherently phasic effects of EE, as well as to avoid potentially harmful effects of prolonged tonic application of agents, such as those seen with corticosterone (de Kloet et al., 1999; Zoladz & Diamond, 2009).
We suspect that the 24-fold reduction in SD susceptibility seen with acute IGF-1 exposure that further increased to 75-fold at three days before settling to 22-fold at seven days reflects an adaptive, damped oscillatory response, commonly seen in biological systems (Stark et al., 2007; Paszek et al., 2010; Wang et al., 2012). In contrast, the progressive reduction of OS from IGF-1 by 20, 30, and 73% at these time points suggests a maximal steady-state has not yet been reached. Thus, whether OS too would show a damped oscillatory or a more simple sigmoid response pattern remains unclear. However, collectively these results suggest that with optimal dosing, the ability of IGF-1 to protect against SD via reduced OS can be expected to be at least 20-fold.
While insulin is already recognized as an agent that increases learning and memory (Stockhorst et al., 2004; Craft et al., 2011), its ability to influence SD has not been previously examined. We show that insulin protects against SD and hypothesize that it does so, like IGF-1, via its actions as an EE mimetic (i.e., by increasing processes associated with improved learning and memory, such as CA3 bursting). However, while insulin mitigated SD susceptibility, we show that IGF-1 has this effect at a 15,500-fold smaller dose, suggesting insulin's effects may occur via cross-reactivity with the IGF-1 receptor. We thus conclude that neuroprotective EE mimetics are promising targets against SD, and by extension migraine and HFCM, with IGF-1 shown here to be a novel and potentially effective therapeutic.
This work was supported by grants from the National Institute of Neurological Disorders and Stroke (NS-19108), the National Institute of Child Health and Human Disorders (5 PO1 HD 09402), ARRA supplement NS-19108-23S1, the Migraine Research Foundation, the White Foundation, and the Dr. Ralph and Marian Falk Medical Research Trust. We thank Heidi M. Mitchell for assistance in the preparation and maintenance of culture systems and comments referable to the early drafts. We thank Aya D. Pusic for reviewing the manuscript and providing advice throughout this work.
The authors have no conflicts or financial interest to disclose.