Migraine headache is a disabling neurological disorder affecting 11% of adults worldwide, with 3% experiencing chronic daily headache (Rasmussen et al. 1991; Stovner et al. 2007). However, existing therapies for high frequency and chronic migraine offer only a modest benefit (Mack 2011).
Spreading depression (SD), the most likely cause of migraine aura and perhaps migraine pain (Moskowitz et al. 1993; Lauritzen and Kraig 2005), is a large negative DC shift in the interstitial space that slowly propagates and is associated with transient neuronal silence (Leão 1944). SD is initiated by excessively increased neuronal excitability (i.e., increased excitation, reduced inhibition, or both) synchronously involving a sufficient volume of gray matter (Bureš et al. 1974; Somjen 2001). The increase in neuronal excitability necessary to initiate SD can be elicited by reactive oxygen species (ROS; Grinberg et al. 2012a), and by the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α; Cipolla et al. 2012).
ROS are modulators of neuronal excitability (Kishida and Klann 2007) and appear to be a critical signaling component of SD. Not only do ROS increase SD susceptibility, SD itself also increases ROS and oxidative stress (OS; Viggiano et al. 2011; Grinberg et al. 2012a). OS occurs as a result of an imbalance between pro- and antioxidants. Recent findings suggest that ROS (Grinberg et al. 2012a), like TNF-α (Cipolla et al. 2012), may be the consequence of SD that results in post-SD aberrantly increased neuronal excitability (Beattie et al. 2002) and decreased inhibition (Stellwagen et al. 2005). Determining the cellular sources of ROS generated by SD is thus an important next step in determining the mechanisms underlying this phenomenon.
Here, we induced SD in rat hippocampal slice cultures, then exposed them to a fluorogenic probe for ROS, combined with immunolabeling for specific brain cell types. We found OS generated from SD significantly increased in astrocytes and microglia but not neurons or oligodendrocytes. Since we previously showed that insulin-like growth factor-1 (IGF-1, which functions as a neuroprotective environmental enrichment mimetic in brain; Liu et al. 2001), reduces excitability from ROS and the OS of SD (Grinberg et al. 2012a), we investigated which cell types were involved in this effect. We found that IGF-1 reduced the OS from SD in microglia but not astrocytes. Next, we showed that TNF-α increased OS in microglia (but not astrocytes), and that this effect was abrogated by IGF-1. Since IGF-1 appeared to decrease microglial activation by SD and TNF-α, we next probed for TNF-α involvement in SD susceptibility. We found TNF-α decreased threshold to SD, and that SD itself decreased threshold to subsequent SD, an effect dependent on TNF-α signaling. These findings suggest that controlling microglial activation is an important target for development of novel migraine therapeutics. This work has appeared in preliminary forms (Dibbern 2012; Mitchell et al. 2010b; Grinberg et al. 2012b).
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- Materials and methods
- Supporting Information
Here, we show for the first time that increased OS from SD is specific to astrocytes and microglia in rat hippocampal slice cultures. We also show that IGF-1 mitigation of OS from SD is specific to microglia, and that increased microglial OS from SD is mediated by TNF-α signaling. Lastly, we demonstrate that SD promotes subsequent SD, and does so via TNF-α.
CellROX™ is unique in that it is a fixable and highly photostable ROS marker. Since CellROX™ reacts collisionally with pro-oxidants, thus competing with endogenous antioxidants, it effectively functions as a marker of excessive ROS, and thus OS. We confirmed CellROX™ labeling of OS by also assessing oxidized protein carbonyl content (Fig. 1d), which showed a similar increase in OS from SD using carbonyl levels as we previously observed using CellROX™ (Grinberg et al. 2012a). In addition, while GFAP is a marker of most astrocytes (Walz and Lang 1998), we have no evidence of how OS might change in GFAP- astrocytes. However, both GFAP− and GFAP+ astrocytes have been recently shown to have very similar metabolic gene expression (Lovatt et al. 2007). Therefore, our results are likely to reflect the OS response to SD of astrocytes in general. Finally, it is important to note that we measured cumulative CellROX™ at 24 h following SD, and not simply during the SD repolarization.
OS is the result of an imbalance between pro-oxidants and antioxidant systems, and can result in oxidative damage and increased neuronal excitability (Pellmar 1987; Kishida and Klann 2007). Cells generate ROS as byproducts of oxidative metabolism as well as during normal physiological signaling processes (Thannickal and Fanburg 2000; Murphy 2009; Sorce and Krause 2009). To protect against the potentially damaging effects of pro-oxidants, cells produce antioxidant enzymes and utilize small molecule antioxidants such as glutathione and ascorbate (Thannickal and Fanburg 2000; Dringen 2005).
SD results in a rise in brain tissue OS (Grinberg et al. 2012a) and a rise in hydrogen peroxide (Viggiano et al. 2011), which unlike most other ROS can cross cell membranes, travel long distances, and can dismutate into the highly disruptive hydroxyl radical (Cardoso et al. 2012). Since SD results in a 10-fold increase in local brain metabolic rate (Bureš et al. 1974; Dienel and Hertz 2001), and a small fraction of all oxygen utilized by mitochondria become ROS (Murphy 2009), we suggest that in part, the increased OS from SD arises from oxidative metabolism. SD also results in increased production of inflammatory mediators and activation of genes involved in inflammatory pathways such as TNF-α, matrix metalloproteinase-9, interleukin-1β, interleukin-6, cyclooxygenase-2, and nitric oxide synthase (Jander et al. 2001; Kunkler et al. 2004; Thompson and Hakim 2005; Hulse et al. 2008). Along with ROS directly (Zhou et al. 2009), these signaling molecules can activate inflammatory pro-oxidant production pathways (Han et al. 2009).
Surprisingly, SD increased astrocytic but not neuronal OS. Because of the high metabolic load of SD (Bureš et al. 1974; Dienel and Hertz 2001), some OS was expected in neurons, the principal cells traditionally believed to bear the metabolic burden of activity (for review see: Wong-Riley 1989, 2012) and until recently were believed to be the principal cells involved in brain glucose oxidation (Lovatt et al. 2007). Furthermore, astrocytes are widely believed to have a higher antioxidative potential than neurons, and neurons are associated with high vulnerability to OS-induced injury (Wilson 1997; Dringen 2000; Bélanger et al. 2011). However, since we saw increased astrocytic and not neuronal OS, our findings may provide functional evidence to confirm and extend the recent anatomical evidence for the high metabolic capacity and abundant mitochondria of astrocytes (Lovatt et al. 2007; Bélanger et al. 2011).
In addition, astrocytes express NADPH oxidase isoforms, and ROS produced by NADPH oxidase play important roles in astrocytic survival, signaling, and production of inflammatory mediators (Sorce and Krause 2009). Caggiano and Kraig (1998) also observed that astrocytic nitric oxide synthase levels increase following SD in rat brain. CellROX™ is reported to robustly detect a range of pro-oxidants, including superoxide anion, hydroxyl radical, peroxynitrite, and to a lesser extent, nitric oxide (Invitrogen communication). Thus, the above sources of pro-oxidants may all be contributing to the astrocytic CellROX™ signal.
While the literature shows oligodendroglia to be very sensitive to OS (Thorburne and Juurlink 1996; Dewar et al. 2003), their capacity to generate ROS is unclear. Indeed, our results show that these cells do not contain significant levels of OS after SD. However, there is evidence to indicate that oligodendrocytes are affected by SD and its incurred OS (Tamura et al. 2004; Pusic and Kraig 2011). Thus, the lack of CellROX™ labeling of oligodendrocytes suggests that SD-induced demyelination (Pusic and Kraig 2011) may be mediated by other cell types, perhaps microglia (Pusic and Kraig, unpublished observations).
Some microglial ROS production was expected following SD, since SD induces both morphological and functional changes consistent with microglial activation. SD induces microgliosis (Gehrmann et al. 1993; Caggiano and Kraig 1996) and is associated with up-regulation of pro-inflammatory factors (Caggiano and Kraig 1996; Kunkler et al. 2004). Following SD, microglia increase production of TNF-α and other pro-inflammatory cytokines (Jander et al. 2001; Kunkler et al. 2004; Hulse et al. 2008), which can signal to induce ROS production by mitochondria and NADPH oxidase (Shoji et al. 1995; Han et al. 2009; Zhou et al. 2009). We have described microglia to be the predominant cell type producing TNF-α after SD, as well as cold-preconditioning (Hulse et al. 2008; Mitchell et al. 2011) in hippocampal slice cultures. Microglia have also recently been described to move long distances following SD (Grinberg et al. 2011), an effect that likely also includes NADPH oxidase activity at the leading edge of lamellipodia (Ushio-Fukai 2009), which could increase dispersion of ROS and TNF-α to distant regions of brain. Future studies will identify the specific pro-oxidant species involved in SD-induced OS and which species are altered by IGF-1 treatment.
SD-induced microgliosis and astrogliosis have previously been observed to be separable phenomena. Pharmaceutical treatments such as dexamethasone exposure, inactivation of lipoxygenase, or increased nitric oxide signaling inhibits microgliosis but not astrogliosis (Caggiano and Kraig 1996). Here, we extend these observations by showing IGF-1 too can influence OS from SD in microglia but not astrocytes (Fig. 3). Furthermore, we show that TNF-α increased microglial but not astrocytic OS (Fig. 4, S2). While astrocytes possess TNF receptors and have well-described responses to TNF-α (Buffo et al. 2010) their production of pro-oxidants in response to TNF-α, outside of primary culture conditions, has been limited.
We show that IGF-1 decreases microglial OS by altering TNF-α signaling, which is necessary and sufficient for increased microglial but not astrocytic OS (Fig. 4 and Figure S2). IGF-1 receptor activation increases nuclear factor kappa B, promoting the TNF pro-survival complex I formation over that of the TNF pro-apoptotic complex II (Wang et al. 2003; Han et al. 2009). While astrocytes have been well-characterized as capable of producing TNF-α (Buffo et al. 2010), our previous findings show that microglia are the predominant cell type responsible for TNF-α expression after SD (Hulse et al. 2008) and cold-preconditioning (Mitchell et al. 2011), which are non-injurious but pro-inflammatory events in hippocampal slice cultures. The IGF-1 receptor is found on neurons, oligodendroglia, astrocytes, and endothelial cells (Mendez et al. 2006). However, while microglia produce IGF-1 during development and in response to injury (Scheepens et al. 2000), to our knowledge there is no direct evidence that microglia possess the receptor for IGF-1. It is thus possible that IGF-1 alteration of microglial responses to SD involves other brain cell types or is mediated by microglial IGF-II receptor or perhaps insulin receptor cross-reactivity (Sugimoto et al. 2002; Suh et al. 2010).
We have previously shown that two sequelae of SD, TNF-α, and OS can excessively increase neuronal excitability (Cipolla et al. 2012; Grinberg et al. 2012a). SD has previously been shown to decrease neuronal inhibition (Kruger et al. 1996; Grinberg et al. 2012a), and migraine is a disorder characterized by deficient regulation of cortical excitability (Pietrobon and Moskowitz 2012). We thus hypothesized that SD itself may increase susceptibility to subsequent SD. Indeed, here we show that SD decreases electrical threshold to subsequent SD (Fig. 4d). Furthermore, we show that this effect is dependent on TNF-α signaling (Fig. 4c–f).
Though the pathogenesis of migraine remains controversial and incompletely defined, there is substantial evidence for SD involvement in both migraine with and without aura (Moskowitz et al. 1993; Lauritzen and Kraig 2005). Furthermore, SD increases neuronal excitability, consistent with the cortical excitability phenotype seen in migraineurs (for review see Pietrobon and Moskowitz 2012). Our findings support the hypothesis that recurrent SD activates microglia to increase neuronal excitability and prime brain for subsequent SD (Kraig et al. 2010). We have previously shown IGF-1 significantly decreases SD susceptibility (Grinberg et al. 2012a). Here, we show IGF-1 abrogated the microglial responses to SD that would otherwise excessively increase neuronal excitability. This study further supports IGF-1 as a novel therapeutic for mitigation of SD, and thus perhaps migraine. Since IGF-1 is an endogenously produced compound that can easily enter brain (Liu et al. 2001) and is likely to have a high benefit/risk ratio, our findings support further investigation into its efficacy as a potential migraine therapeutic.