Neuro-Immunotherapies: A 30-year Retrospective of an Overwhelming Success and a Brighter Future

Acute exercise alters skeletal muscle mitochondrial respiration and H2O2 emission in response to hyperinsulinemic-euglycemic clamp in middle-aged obese men

“…The ~10 mg aliquot of muscle was placed in ice-cold preserving solution (BIOPS; in mM: 7.23 K 2 EGTA, 2.77 CaK 2 EGTA, 5.77 Na 2 ATP, 6.56 MgCl 2 -6H 2 O, 20 taurine, 15 phosphocreatine, 20 imidazole, 0.5 dithiothreitol, 50 K + -MES; pH 7.1) until analysis, typically within 3 hours after sampling [ 34 – 36 ]. Using a dissecting microscope, muscle fibers were mechanically separated using fine-point forceps for no more than 3 min while submerged in ice-cold BIOPS.…”

“…Mitochondrial oxygen flux ( J O 2 ) from permeabilized muscle fibers was determined using high resolution respirometry at 37°C, high oxygen concentration (300–450 nmol.ml -1 ; to avoid oxygen diffusion limitation) and continuous stirring (Oxygraph O2k, Oroboros Instruments, Innsbruck, Austria), and mitochondrial hydrogen peroxide emission ( J H 2 O 2 ) was measured simultaneously via fluorimetry (O2k-Fluorescence LED-2 Module; Oroboros Instruments, Innsbruck, Austria) as previously described [ 36 – 39 ]. Briefly, permeabilized fiber bundles were analysed in duplicate, in chambers containing MiR05 respiration buffer (in mM: 0.5 EGTA, 10 KH 2 PO 4 , 3 MgCl 2 -6H 2 O, 60 lactobionic acid, 20 taurine, 20 HEPES, 110 D-sucrose, 1 mg/mL bovine serum albumin; pH 7.1).…”

“…Briefly, permeabilized fiber bundles were analysed in duplicate, in chambers containing MiR05 respiration buffer (in mM: 0.5 EGTA, 10 KH 2 PO 4 , 3 MgCl 2 -6H 2 O, 60 lactobionic acid, 20 taurine, 20 HEPES, 110 D-sucrose, 1 mg/mL bovine serum albumin; pH 7.1). Amplex UltraRed (25 μM; Molecular Probes, Invitrogen), horseradish peroxidase (5 U/mL) and superoxide dismutase (SOD; 5 U/mL) were added for simultaneous fluorimetric measurement of H 2 O 2 at 525/590 nm excitation/emission wavelengths, calibrated with known amounts of H 2 O 2 as described previously [ 36 , 37 , 39 ]. A range of respiratory states were induced using a substrate, uncoupler, and inhibitor titration (SUIT) protocol, added sequentially as follows: malate (2 mM), pyruvate (5 mM) and octanoylcarnitine (0.02 mM) to assess complex I + electron transfer flavoprotein (ETF) supported state-4 leak respiration ( LEAK CI+ETF ); succinate (10 mM) was then added for convergent complex II electron input during leak respiration ( LEAK CI+II+ETF ); ADP (1 & 5 mM) was then added to induce state-3 oxidative phosphorylation ( OXPHOS ); cytochrome-c (10 μM) was added to confirm outer mitochondrial membrane integrity; stepwise 0.05 μM titrations of carbonyl cyanide p -trifloromethoxyphenylhydrazone (FCCP) were then added to uncouple the inner mitochondrial membrane to assess electron transfer system capacity ( ETS CI+II+ETF ).…”

The meningeal and choroidal infiltration routes for leukocytes in stroke

“… 23 Stroke is associated with a leakage of the BBB which was previously associated with propagating the influx of leukocytes into the brain. 24 , 25 However, to date three distinct routes for leukocyte migration from the blood into the brain parenchyma have been described: through the parenchymal vessels of the BBB, via the meningeal blood circulation and across the epithelial cells of the choroid plexus. 26 , 27 …”

“… 14 , 15 Failure to induce consistent neuroprotection by anti-inflammatory therapies in patients with stroke has been discussed elsewhere. 16 , 17 While several aspects of study design, statistical rigor and translational aspects from mouse to men need to be considered in this regard, the inefficient targeting of lymphocytes, at the predominantly investigated transendothelial migration route, might also account for the lack of efficacy of these anti-inflammatory treatments. A better understanding of how cells of the peripheral immune system access the central nervous system (CNS) and interact with the brain microvasculature would likely lead to the development of more efficient immune interventions for individuals with stroke.…”