Inadequate brain glycogen or sleep increases spreading depression susceptibility.

OBJECTIVE: Glycogen in astrocyte processes contributes to maintenance of low extracellular glutamate and K+ concentrations around excitatory synapses. Sleep deprivation (SD), a common migraine trigger, induces transcriptional changes in astrocytes, reducing glycogen breakdown. We hypothesize that when glycogen utilization cannot match synaptic energy demand, extracellular K+ can rise to levels that activate neuronal pannexin-1 channels and downstream inflammatory pathway, which might be one of the mechanisms initiating migraine headaches. METHODS: We suppressed glycogen breakdown by inhibiting glycogen phosphorylation with 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and by SD. RESULTS: DAB caused neuronal pannexin-1 large pore opening and activation of the downstream inflammatory pathway as shown by procaspase-1 cleavage and HMGB1 release from neurons. Six-hour SD induced pannexin-1 mRNA. DAB and SD also lowered the cortical spreading depression (CSD) induction threshold, which was reversed by glucose or lactate supplement, suggesting that glycogen-derived energy substrates are needed to prevent CSD generation. Supporting this, knocking down the neuronal lactate transporter MCT2 with an antisense oligonucleotide or inhibiting glucose transport from vessels to astrocytes with intracerebroventricularly delivered phloretin reduced the CSD threshold. In vivo recordings with a K+ -sensitive/selective fluoroprobe, Asante Potassium Green-4, revealed that DAB treatment or SD caused a significant rise in extracellular K+ during whisker stimulation, illustrating the critical role of glycogen in extracellular K+ clearance. INTERPRETATION: Synaptic metabolic stress caused by insufficient glycogen-derived energy substrate supply can activate neuronal pannexin-1 channels as well as lower the CSD threshold. Therefore, conditions that limit energy supply to synapses (eg, SD) may predispose to migraine attacks, as suggested by genetic studies associating glucose or lactate transporter deficiency with migraine. Ann Neurol 2018;83:61-73.

Effects of base-metal casting alloys on cytoskeletal filaments in cultured human fibroblasts.

PURPOSE: The present study was designed to determine the cytotoxic effects of some widely used dental base-metal casting alloys (Ni-Cr and Co-Cr) on the cytoskeleton in cultured human fibroblasts, and to evaluate whether any structural alteration is associated with the application of these alloys. MATERIALS AND METHODS: Ten specimens from six different alloys were prepared as 5-mm disks. Five of ten samples from each group were polished; the remaining five samples were left sandblasted with 50-microm Al2O3. All samples were directly exposed to human fibroblasts in a 24-well cell culture dish for 120 hours. Then, cells were fixed and stained with antibodies against major cytoskeletal elements--actin, vimentin, and microtubules--by immunofluorescent staining methods. Cells were analyzed in 3-D to document the cytoskeletal alterations using a laser confocal microscope. RESULTS: Disintegration of actin filaments was observed in lamellipodia of fibroblasts by the effect of both polished and sandblasted Ni-Cr and Co-Cr samples, with the exception of the polished Co-Cr alloy (Wirocast). Moreover, intracytoplasmic actin-decorated stress fibers were found bent and occasionally tangled in the sandblasted Ni-Cr (Wiron 99) and Co-Cr alloys (Wirocast and Co-Cr Degussa). Vimentin, a mesenchymal cell intermediate filament protein normally showing an intracellular meshwork pattern, was not affected by any of the polished or sandblasted alloys. Microtubules mainly remained intact in all dental alloy-treated groups. CONCLUSION: Taken together, it is possible to postulate that Ni-Cr and Co-Cr dental alloys, especially sandblasted forms, may have detrimental effects on the actin-based cytoskeleton, at least tested in vitro.