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Multiple sclerosis and migraine with aura are clinically correlated and both show imaging changes suggestive of myelin disruption. Furthermore, cortical myelin loss in the cuprizone animal model of multiple sclerosis enhances susceptibility to spreading depression, the likely underlying cause of migraine with aura. Since multiple sclerosis pathology involves inflammatory T cell lymphocyte production of interferon-gamma and a resulting increase in oxidative stress, we tested the hypothesis that spreading depression disrupts myelin through similar signaling pathways. Rat hippocampal slice cultures were initially used to explore myelin loss in spreading depression, since they contain T cells, and allow for controlled tissue microenvironment. These experiments were then translated to the in vivo condition in neocortex. Spreading depression in slice cultures induced significant loss of myelin integrity and myelin basic protein one day later, with gradual recovery by seven days. Myelin basic protein loss was abrogated by T cell depletion, neutralization of interferon-gamma, and pharmacological inhibition of neutral sphingomyelinase-2. Conversely, one day after exposure to interferon-gamma, significant reductions in spreading depression threshold, increases in oxidative stress, and reduced levels of glutathione, an endogenous neutral sphingomyelinase-2 inhibitor, emerged. Similarly, spreading depression triggered significant T cell accumulation, sphingomyelinase activation, increased oxidative stress, and reduction of grey and white matter myelin in vivo. Myelin disruption is involved in spreading depression, thereby providing pathophysiological links between multiple sclerosis and migraine with aura. Myelin disruption may promote spreading depression by enhancing aberrant excitability. Thus, preservation of myelin integrity may provide novel therapeutic targets for migraine with aura.

J. Neurochem. (2011) 117, 187–196. Abstract Cold pre-conditioning reduces subsequent brain injury in small animals but the underlying mechanisms remain undefined. As hypothermia triggers systemic macrophage tumor necrosis factor alpha (TNF-α) production and other neural pre-conditioning stimuli depend on this cytokine, we reasoned that microglia and TNF-α would be similarly involved with cold pre-conditioning neuroprotection. Also, as slice cultures closely approximate their in vivo counterpart and include quiescent microglia, we used rat hippocampal slice cultures to confirm this hypothesis. Furthermore, inflammatory cytokine gene screening with subsequent PCR and immunostaining confirmation of targeted mRNA and related protein changes showed that cold pre-conditioning triggered a significant rise in TNF-α that localized to microglia and a significant rise in interleukin (IL)-11 that localized mainly to hippocampal pyramidal neurons and, more rarely, astrocytes. Importantly, co-stimulation with cold and IL-11, an anti-inflammatory cytokine that inhibits TNF-α expression, abrogated the otherwise evident protection. Instead, cold pre-conditioning coupled with blockade of IL-11 signaling further enhanced neuroprotection from that seen with cold pre-conditioning alone. Thus, physiological activation of brain pro-inflammatory cytokine signaling, and its amplification by inhibition of coincident anti-inflammatory cytokine signaling, may be opportune targets for the development of novel therapeutics that can mimic the protection seen in cold pre-conditioning.

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Environmental enrichment, i.e., increased intellectual, social, and physical activity makes brain more resilient to subsequent neurological disease. The mechanisms for this effect remain incompletely defined, but evidence shows tumor necrosis factor-alpha (TNF-α) is involved. TNF-α, at acutely high levels, possesses the intrinsic capacity to enhance injury associated with neurological disease. Conversely, the effect of TNF-α at low-levels is nutritive over time, consistent with physiological conditioning hormesis. Evidence shows that neural activity triggers low-level pro-inflammatory signaling involving TNF-α. This low-level TNF-α signaling alters gene expression, resulting in an enhanced resilience to disease. Brain-immune signaling may become maladaptive when increased activity is chronic without sufficient periods of reduced activity necessary for nutritive adaptation. Such tonically increased activity may explain, for example, the transformation of episodic to chronic migraine with related increased susceptibility to spreading depression, the most likely underlying cause of this malady. Thus, TNF-α, whose function is to alter gene expression, and its principal cellular source, microglia, seem powerfully positioned to orchestrate hormetic immune signaling that establishes the phenotype of neurological health and disease from brain activity.