IH induces acute fatigue and greater intrinsic endurance capacity of the diaphragm.

<p>(A) Force-frequency curves and (B) force decrement during repetitive contractions to induce fatigue, in Ctl and IH diaphragms immediately after the last hypoxia session on day 4. (C) Force-frequency curves and (D) force decrement during repetitive contractions of the diaphragm 6 hours after the last hypoxia session on day 4. Data are expressed as means (± SE) of 5–10 mice per group. * p<0.01 compared to Ctl by ANOVA.</p

Indices of mitochondrial function in diaphragm and limb muscle after IH.

<p>Succinate dehydrogenase (SDH) activity in: (A) diaphragm and (B) limb muscle, of Ctl and IH mice. Gene expression levels of oxidative and glycolytic markers in: (C) diaphragm and (D) limb muscle. Quantification of mitochondrial dynamics gene expression in: (E) diaphragm and (F) limb muscle. Data are expressed as means (± SE) of 4–5 mice per group. * p<0.01 compared to Ctl by t-test. Abbreviations used: Mfn = Mitofusin; Drp = Dynamin-related protein; Opa = Optic atrophy; Cox = Cytochrome C oxidase; Sdh = Succinate dehydrogenase; Pgc = PPARgamma coactivator; UCP = Uncoupling protein; HK = Hexokinase.</p

Type I myofibers are increased by IH in diaphragm but not limb muscle.

<p>(A) Low and high magnification images of Ctl and IH diaphragm sections immunostained for type I myosin heavy chain (MyHCI). (B) Proportion of myofibers expressing type I MyHC in the diaphragm. (C) Quantification of the relative area contribution of type I MyHC-expressing fibers to total tissue cross-sectional area of the diaphragm. (D) Proportion of MyHCI-positive myofibers in the limb muscle. (E) Quantification of the relative area contribution of MyHCI-positive fibers to total tissue cross-sectional area of the limb muscle. Data are expressed as means (± SE) of 5 mice per group. * p<0.01 compared to Ctl by t-test. Scale bar = 100μm.</p

Myofiber atrophy in diaphragm but not in limb muscle after IH.

<p>Representative images and quantification of cross-sectional area (CSA) in: (A) diaphragm and (B) tibialis anterior limb muscle, of control (Ctl) and hypoxia-exposed (IH) mice. Frequency distributions of fiber size in: (C) diaphragm and (D) limb muscle. Data are expressed as means (± SE) of 5 mice per group. * p<0.01 compared to Ctl by t-test. Scale bar = 50μm.</p

Influence of IH on proteolysis pathways in diaphragm and limb muscle.

<p>Western blot quantification of cleaved and total calpain proteins in: (A) diaphragm and (B) limb muscle, of Ctl and IH mice. Real-time PCR quantification of E3 ubiquitin ligases MuRF1 and atrogin-1 mRNA in: (C) diaphragm and (D) limb muscle. Western blot analysis and quantification of LC3B-II protein in: (E) diaphragm and (F) limb muscle. Real-time PCR quantification of autophagy-related gene expression in: (G) diaphragm and (H) limb muscle. Data are expressed as means (± SE) of 3–5 mice per group. * p<0.01 compared to Ctl by t-test. Abbreviations used: MuRF = Muscle ring finger; LC3B = Microtubule-associated protein 1 light chain 3; Bnip = Bcl2/adenovirus E1B 19 kDa interacting protein; Gabarapl = GABA(A) receptor-associated protein like; Mul = Mitochondrial ubiquitin ligase.</p

IH promotes lipid droplets and activation of lipid metabolism genes in the diaphragm.

<p>Representative Oil-red O images (left panel) and quantification of lipid droplet staining (right panel) in the diaphragm and limb muscle in (A). Quantification of expression levels for Plin isoforms (B and C) and other lipid metabolism pathway genes (D and E) in: (B and D) diaphragm and (C and E) limb muscle. Data are expressed as means (± SE) of 4–5 mice per group. For (A), * p<0.01 compared to Ctl diaphragm and # p<0.01 compared to IH diaphragm by ANOVA; for (B-E), * p<0.01 compared to Ctl by t-test. Scale bar = 100μm. Abbreviations used: Plin = Perilipin; PDK = Pyruvate dehydrogenase kinase; Fasn = Fatty acid synthase; SCD = Stearoyl-coenzyme A desaturase; SREBF = Sterol regulatory element binding transcription factor; Scap = SREBF cleavage-activating protein.</p

Oxidative stress markers in diaphragm and limb muscle after IH.

<p>Western blot quantification of carbonylated proteins in: (A) diaphragm and (B) limb muscle, of Ctl and IH mice. Real-time PCR quantification of anti-oxidant gene expression in: (C) diaphragm and (D) limb muscle. Data are expressed as means (± SE) of 4–5 mice per group. * p<0.01 compared to Ctl by t-test. Abbreviations used: MnSOD = Manganese superoxide dismutase; Gpx = Glutathione peroxidase; Prx = Peroxiredoxin.</p

Pharmacokinetic Characterization of [¹⁸F]UCB‑H PET Radiopharmaceutical in the Rat Brain

The synaptic vesicle glycoprotein 2A (SV2A), a protein essential to the proper nervous system function, is found in presynaptic vesicles. Thus, SV2A targeting, using dedicated radiotracers combined with positron emission tomography (PET), allows the assessment of synaptic density in the living brain. The first-in-class fluorinated SV2A specific radioligand, [¹⁸F]­UCB-H, is now available at high activity through an efficient radiosynthesis compliant with current good manufacturing practices (cGMP). We report here a noninvasive method to quantify [¹⁸F]­UCB-H binding in rat brain with microPET. Validation study in rats confirmed the need of high enantiomeric purity to target SV2A in vivo. We demonstrated the reliability of a population-based input function to quantify SV2A in preclinical microPET setting. Finally, we investigated the in vivo metabolism of [¹⁸F]­UCB-H and confirmed the negligible amount of radiometabolites in the rat brain. Hence, the in vivo quantification of SV2A using [¹⁸F]­UCB-H microPET seems a promising tool for the assessment of the synaptic density in the rat brain, and opens the way for longitudinal follow-up in neurodegenerative disease rodent models

<i>N</i>¹‑Fluoroalkyltryptophan Analogues: Synthesis and <i>in vitro</i> Study as Potential Substrates for Indoleamine 2,3-Dioxygenase

Indoleamine 2,3-dioxygenase (hIDO) is an enzyme that catalyzes the oxidative cleavage of the indole ring of l-tryptophan through the kynurenine pathway, thereby exerting immunosuppressive properties in inflammatory and tumoral tissues. The syntheses of 1-(2-fluoroethyl)-tryptophan (1-FETrp) and 1-((1-(2-fluoroethyl)-1<i>H</i>-1,2,3-triazol-4-yl)­methyl)-tryptophan, two <i>N</i>¹-fluoroalkylated tryptophan derivatives, are described here. <i>In vitro</i> enzymatic assays with these two new potential substrates of hIDO show that 1-FETrp is a good and specific substrate of hIDO. Therefore, its radioactive isotopomer, 1-[¹⁸F]­FETrp, should be a molecule of choice to visualize tumoral and inflammatory tissues and/or to validate new potential inhibitors

Enabling Efficient Positron Emission Tomography (PET) Imaging of Synaptic Vesicle Glycoprotein 2A (SV2A) with a Robust and One-Step Radiosynthesis of a Highly Potent ¹⁸F‑Labeled Ligand ([¹⁸F]UCB-H)

We herein describe the straightforward synthesis of a stable pyridyl­(4-methoxyphenyl)­iodonium salt and its [¹⁸F] radiolabeling within a one-step, fully automated and cGMP compliant radiosynthesis of [¹⁸F]­UCB-H ([¹⁸F]<b>7</b>), a PET tracer for the imaging of synaptic vesicle glycoprotein 2A (SV2A). Over the course of 1 year, 50 automated productions provided 34 ± 2% of injectable [¹⁸F]<b>7</b> from up to 285 GBq (7.7 Ci) of [¹⁸F]­fluoride in 50 min (uncorrected radiochemical yield, specific activity of 815 ± 185 GBq/μmol). The successful implementation of our synthetic strategy within routine, high-activity, and cGMP productions attests to its practicality and reliability for the production of large doses of [¹⁸F]<b>7</b>. In addition to enabling efficient and cost-effective clinical research on a range of neurological pathologies through the imaging of SV2A, this work further demonstrates the real value of iodonium salts for the cGMP ¹⁸F-PET tracer manufacturing industry, and their ability to fulfill practical and regulatory requirements in that field