In vitro and in vivo comparison of 18F and 123I-labeled ML10 with 68Ga-Cys2-AnxA5 for molecular imaging of apoptosis.

AIM: Recently, 18F-labeled 2-(5-fluoropentyl)-2-methylmalonic acid or ML10 has been proposed as a promising PET tracer for imaging of apoptosis. In this study we compared 18F-ML10, the 123I labeled 5-iodo derivative (123I-ML10) and a 68Ga-labeled Annexin A5 (AnxA5) and evaluated them as apoptosis tracers in several distinct models. METHODS: In vivo stability and biodistribution were studied in healthy mice. Apoptosis imaging was evaluated in anti-Fas treated mice and mice with muscular apoptosis. Furthermore, 18F-ML10 and 68Ga-Cys2-AnxA5 were evaluated in a rat model with reperfused liver infarct and a rat model with cerebral infarct as well as in Daudi tumor bearing mice, before and after treatment with cyclophosphamide and/or radiotherapy. RESULTS: 18F-ML10 and 68Ga-Cys2-AnxA5 were both stable, while 123I-ML10 metabolized very quickly in vivo. All tracers showed a 3-4 times higher uptake in apoptotic muscular tissue in comparison to that in healthy muscular tissue. Animals with anti-Fas induced hepatic apoptosis showed an increased liver uptake which was most pronounced for 18F-ML10. The uptake of both 18F-ML10 and 68Ga-Cys2-AnxA5 increased in the apoptotic region surrounding the cerebral infarction and the reperfused liver infarction. Tumor uptake of 68Ga-Cys2-AnxA5, but not of 18F-ML10, was statistically significantly higher after therapy as measured with PET/MRI. CONCLUSION: All radiotracers were able to detect apoptosis in vitro and in vivo in each of the studied animal models of apoptosis. 68Ga-Cys2-AnxA5, but not 18F-ML10, allowed to visualize the effect of tumor therapy in a statistically significant way.

Metabolic and type 1 cannabinoid receptor imaging of a transgenic rat model in the early phase of Huntington disease.

Several lines of evidence imply early alterations in metabolic and endocannabinoid neurotransmission in Huntington disease (HD). Using [(18)F]MK-9470 and small animal PET, we investigated for the first time cerebral changes in type 1 cannabinoid (CB1) receptor binding in vivo in pre-symptomatic and early symptomatic rats of HD (tgHD), in relation to glucose metabolism, morphology and behavioral testing for motor and cognitive function. Twenty-three Sprague-Dawley rats (14 tgHD and 9 wild-types) were investigated between the age of 2 and 11 months. Relative glucose metabolism and parametric CB1 receptor images were anatomically standardized to Paxinos space and analyzed voxel-wise. Volumetric microMRI imaging was performed to assess HD neuropathology. Within the first 10 months, bilateral volumes of caudate-putamen and lateral ventricles did not significantly differ between genotypes. Longitudinal- and genotype evolution showed that relative [(18)F]MK-9470 binding progressively decreased in the caudate-putamen and lateral globus pallidus of tgHD rats (-8.3%, p≤1.1×10(-5) at 5 months vs. -10.9%, p<1.5×10(-5) at 10 months). In addition, relative glucose metabolism increased in the bilateral sensorimotor cortex of 2-month-old tgHD rats (+8.1%, p≤1.5×10(-5)), where it was positively correlated to motor function at that time point. TgHD rats developed cognitive deficits at 6 and 11 months of age. Our findings point to early regional dysfunctions in endocannabinoid signalling, involving the lateral globus pallidus and caudate-putamen. In vivo CB1 receptor measurements using [(18)F]MK-9470 may thus be a useful early biomarker for HD. Our results also provide evidence of subtle motor and cognitive deficits at earlier stages than previously described.

[About an extreme case of giant lymphoedema of the upper limb: ligasure may be useful and a multidisciplinary approach is mandatory]. / À propos d'un cas de lymphœdème géant du membre supérieur : intérêt du Ligasure(®) et importance du traitement multidisciplinaire.

Treatment of giant lymphoedema remains very difficult. The main problem is not only the obtention of early postoperative favorable results but to keep them long-lasting. The available treatment options are numerous and often combined. The indications have to be adapted to the clinical stage of the lesion. Ligasure(®) give us many advantages in the achievement of the surgical resection due to the quality of the lymphostasis that can be obtained and to the very limited thermic lesions caused to tissues. Long lasting postoperative physiotherapy is of first importance. We here describe a case of a giant lymphoedema of the upper limb after axillary lymph node dissection for breast cancer. The first physiotherapy failed, causing a lymphatic decompensation resulting in a lymphoedematous mass of nearly 7kg. The surgical resection with Ligasure(®) associated to drainage and compressive physiotherapy in the long run allows to obtain good results. The late follow-up after 4 years still shows stable results.

Actions of R- and S-verapamil and nifedipine on rat vascular and intestinal smooth muscle.

1 We have investigated the actions of the calcium entry blockers nifedipine, R-verapamil and S-verapamil in rat aorta, colon and vas deferens. 2 In aorta and colon, these agents produced concentration-dependent relaxations of KCl (80 mM)-induced contractions. In both tissues, the order of potency was nifedipine > S-verapamil > R-verapamil. However, nifedipine showed selectivity for aorta (potency ratio, colon/aorta: 4.36), S-verapamil showed no selectivity (0.62), but R-verapamil showed selectivity for colon (0.19). 3 In prostatic portions of rat vas deferens, nifedipine (10 microM) abolished the contraction to a single electrical stimulus, but R- and S-verapamil were without effect. In epididymal portions of rat vas deferens, R- and S-verapamil inhibited alpha1-adrenoceptor-mediated contractions to a single electrical stimulus at concentrations of 10 microM and above. 4 In conclusion, R-verapamil may prove useful as an intestinal selective calcium entry blocker in the treatment of intestinal disease with a hypermotility component, e.g. irritable bowel syndrome.

Lack of involvement of pertussis toxin-sensitive G-proteins in norepinephrine-induced vasoconstriction of rat aorta smooth muscle.

Several studies have shown that stimulation of pertussis toxin (PTX)-sensitive G-proteins amplified alpha-adrenoceptor (alpha-AR) agonist-induced vasoconstriction in small muscular and resistance arteries. The aim of this study was to assess the potential involvement of PTX-sensitive G-proteins in norepinephrine (NE)-induced constriction in a large diameter artery, the rat aorta. PTX (1 microg/mL, 2 hr; 3 microg/mL, 4 hr) did not modify concentration-response curves to NE in endothelium-denuded aortic rings. However, several lines of evidence suggested that aortic smooth muscle cells (SMC) had a PTX-sensitive G-protein pathway. [alpha-(32)P]ADP-ribosylation of G(i/o)-proteins by PTX (3 microg/mL, 4 hr) was demonstrated in situ in the intact aorta without endothelium. alpha(i/o) subunits were identified in vitro by both immunoblotting and ADP-ribosylation experiments in rat aorta SMC membranes. The measurement of G(i/o)-specific GTPase activity evidenced an effective coupling between NE receptors and G(i/o)-proteins, as NE induced an increase in basal G(i/o)-specific GTPase activity (20.7 +/- 2.8 vs 7.2 +/- 2.2 pmol P(i)/mg protein at 5 min; P < 0.05 vs basal). Co-immunoprecipitation revealed the in vitro coupling between alpha(1D)-ARs and G(i)-protein in rat aorta SMC membranes. In conclusion, we identified a PTX-sensitive G(i/o)-protein pathway in rat endothelium-denuded aorta. We showed an effective coupling between NE receptors and G(i)-proteins via alpha(1D)-ARs. Since PTX has no effect on NE-induced vasoconstriction, the PTX-sensitive G(i)-protein pathway does not play a predominant role in NE-induced responses in rat aorta SMC in contrast to small diameter muscular and resistance arteries.

Melatonin potentiates NE-induced vasoconstriction without augmenting cytosolic calcium concentration.

Because little is known of the intracellular mechanisms involved in the vasoconstrictor effect of melatonin (Mel), we examined the in vitro effects of Mel by using perfused cylindrical segments of the rat tail artery loaded with the intracellular Ca(2+) concentration ([Ca(2+)](i))-sensitive fluorescent dye, fura 2. Mel (10(-14) to 10(-4) M) had no effect on baseline perfusion pressure or [Ca(2+)](i) but increased, at submicromolar concentrations, the vasoconstrictor effect of norepinephrine (NE) (P = 0.0029). Mel did not modify NE-induced [Ca(2+)](i) mobilization, and thus the [Ca(2+)](i) sensitivity of NE-induced contraction increased in the presence of Mel. Mel consistently increased KCl-induced vasoconstriction and [Ca(2+)](i) sensitivity of contraction, but differences were not statistically significant. In conclusion, Mel increases the [Ca(2+)](i) sensitivity of vasoconstriction evoked by NE suggesting that Mel may amplify endogenous vasoconstrictor responses to sympathetic outflow.

A microtiter plate assay for total glutathione and glutathione disulfide contents in cultured/isolated cells: performance study of a new miniaturized protocol.

The microtiter plate technique reported by Baker and colleagues for the glutathione reductase-DTNB recycling assay of total glutathione (GSx) and glutathione disulfide (GSSG) has been modified according to Anderson's recommendations, in order to improve the reliability and accuracy of this miniaturized method for the measurement of glutathione status in cultured/isolated cells. Dilute HCl (10 mmol/L) has been used to lyse cells, before protein removal by centrifugation in the presence of 1.3% sulfosalicylic acid. The final DTNB, GSSG-reductase and NADPH concentrations in the reaction mixture have been increased to 0.7 mmol/L, 1.2 IU/ml and 0.24 mmol/L, respectively. The procedure specificity has been tested by spiking and dilution assays, showing that about 90% of the expected GSx amounts could actually be recovered, while no changes of GSSG concentrations were caused in the cells. Accuracy has been assessed by analysis of within-series precision as well as of intra- and interassay reproducibility, showing coefficient variation of < 10%. Glutathione changes measured either in control rat hepatocytes or in primary cultures treated with paracetamol or menadione were in good agreement with well-known literature data. These data suggest that the experimental conditions reported in this paper are suitable for the analysis of total glutathione and glutathione disulfide concentrations in cultured/isolated cells.