A Novel Model of Intravital Platelet Imaging Using CD41-ZsGreen1 Transgenic Rats

<div><p>Platelets play pivotal roles in both hemostasis and thrombosis. Although models of intravital platelet imaging are available for thrombosis studies in mice, few are available for rat studies. The present effort aimed to generate fluorescent platelets in rats and assess their dynamics in a rat model of arterial injury. We generated CD41-ZsGreen1 transgenic rats, in which green fluorescence protein ZsGreen1 was expressed specifically in megakaryocytes and thus platelets. The transgenic rats exhibited normal hematological and biochemical values with the exception of body weight and erythroid parameters, which were slightly lower than those of wild-type rats. Platelet aggregation, induced by 20 μM ADP and 10 μg/ml collagen, and blood clotting times were not significantly different between transgenic and wild-type rats. Saphenous arteries of transgenic rats were injured with 10% FeCl₃, and the formation of fluorescent thrombi was evaluated using confocal microscopy. FeCl₃ caused time-dependent increases in the mean fluorescence intensity of injured arteries of vehicle-treated rats. Prasugrel (3 mg/kg, p.o.), administered 2 h before FeCl₃, significantly inhibited fluorescence compared with vehicle-treated rats (4.5 ± 0.4 vs. 14.9 ± 2.4 arbitrary fluorescence units at 30 min, respectively, n = 8, <i>P</i> = 0.0037). These data indicate that CD41-ZsGreen1 transgenic rats represent a useful model for intravital imaging of platelet-mediated thrombus formation and the evaluation of antithrombotic agents.</p></div

Representative images of platelet-rich thrombus induced by 10% FeCl₃ in the saphenous artery of CD41-ZsGreen1 transgenic rats 30 min after FeCl₃ application.

<p>Male CD41-ZsGreen1 transgenic rats were anesthetized, the left saphenous artery was exposed, and a filter paper, presoaked with 10% FeCl₃ was attached for 3 min to induce thrombosis 2 h after administration of vehicle or prasugrel. Fluorescence was measured every 1 s for 25 min using a confocal laser microscope.</p

Biochemical parameters of CD41-ZsGreen1 transgenic rats.

<p>Biochemical parameters of CD41-ZsGreen1 transgenic rats.</p

Hematological parameters of CD41-ZsGreen1 transgenic rats.

<p>Hematological parameters of CD41-ZsGreen1 transgenic rats.</p

Representative flow cytometric analysis of CD41-ZsGreen1 transgenic rats.

<p>EDTA-treated blood samples were withdrawn from the abdominal vein for flow cytometric analysis. The blood was mixed with phycoerythrin-labeled hamster anti-mouse CD61 antibody. Flow cytometric analyses were performed using a FACSCalibur. M1: ZsGreen1-positive platelets. WT: wild-type Sprague-Dawley rats, TG: CD41-ZsGreen1 transgenic rats.</p

Representative ADP- and collagen-induced platelet aggregation tracings of CD41-ZsGreen1 transgenic rats.

<p>Citrated blood was collected from the abdominal aorta and centrifuged to obtain platelet-rich plasma. Platelet aggregation induced by ADP (20 μM) and collagen (10 μg/mL) was measured using light transmission aggregometry. WT: wild-type Sprague-Dawley rats, TG: CD41-ZsGreen1 transgenic rats, ADP: adenosine 5’-diphosphate.</p

Specific expression of green fluorescent protein in bone marrow megakaryocytes of CD41-ZsGreen1 transgenic rats.

<p>Bone marrow from the femur was excised, fixed with 10% buffered formalin, and embedded in paraffin. Sections were prepared and stained with DAPI or HE. Arrows in the HE photograph indicate megakaryocytes. WT: wild-type Sprague-Dawley rats, TG: CD41-ZsGreen1 transgenic rats, HE: hematoxylin-eosin, DAPI: 4′,6-diamidino-2-phenylindole.</p

Analysis of the transgene structure in the genome of CD41-ZsGreen1 transgenic rats.

<p>(A) Schematic of transgene constructs and hybridization probe for Southern blot analysis. The start codon of <i>Cd41</i> was replaced by the ZsGreen1 fragment. (B) Southern blot analysis of tail DNA samples of CD41-ZsGreen1 transgenic rats. Genomic DNA isolated from the tail was digested with PstI, electrophoresed through an agarose gel, and transferred to a nylon membrane. The nylon membrane was hybridized to the ZsGreen1 probe to detect the 2.1-kb restriction fragment.</p

Conformationally-Locked <i>N</i>-Glycosides with Selective β-Glucosidase Inhibitory Activity: Identification of a New Non-Iminosugar-Type Pharmacological Chaperone for Gaucher Disease

A series of conformationally locked <i>N</i>-glycosides having a cis-1,2-fused pyranose–1,3-oxazoline-2-thione structure and bearing different substituents at the exocyclic sulfur has been prepared. The polyhydroxylated bicyclic system was built in only three steps by treatment of the corresponding readily available 1,2-anhydrosugar with KSCN using TiO­(TFA)₂ as catalyst, followed by S-alkylation and acetyl deprotection. In vitro screening against several glycosidase enzymes showed highly specific inhibition of mammalian β-glucosidase with a marked dependence of the potency upon the nature of the exocyclic substituent. The most potent representative, bearing an <i>S</i>-(ω-hydroxyhexadecyl) substituent, was further assayed as inhibitor of the human lysosomal β-glucocerebrosidase and as pharmacological chaperone in Gaucher disease fibroblasts. Activity enhancements in N370S/N370S mutants analogous to those achieved with the reference compound ambroxol were attained with a more favorable chaperone/inhibitor balance

Three subtypes of clinical manifestations.

<p>Three subtypes of clinical manifestations.</p