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The paper presents clinical diagnostic approaches and therapeutic effects of a specific protocol for the treatment of dogs with cardiovascular dirofilariasis in the Belgrade City (Serbia) territory. The study involved 50 privately owned dogs of different breeds, gender, and age, all showing signs of cardio - respiratory disorders. In addition to a general physical examination, blood tests were done to detect microfilaria and adult forms, and X-ray, ECG, and echocardiography were performed as well. At the first examination, 34 out of 50 examined dogs were positive for microfilaria and adult forms. Because of a lack of drug used as „the golden standard" in dirofilariasis treatment, it involved a combination of doxycycline (10 mg/kg) and ivermectin (6 (µg/kg) supported with Advocate - Bayer spot-on. After six months, the first control was performed while continuing treatment with the aforesaid protocol, and the second control was performed after 12 months. Of the 34 treated dogs, all were negative for microfilaria, as early as after the first six months of the treatment (100%). One dog was positive for adult forms of the parasite after six and 12 months. In echocardiography and X-ray examination after 12 months, six dogs showed evident chronic changes. At controls conducted at sixth month and at one year, the implemented therapy was successful in 97.05% (33/34) of primarily infected dogs.
A sensitive liquid chromatographic method for the determination of doxycycline in animal tissues has been prepared and validated. The extraction of the analyte from biological matrix was performed with the solution of oxalic acid and ethyl acetate. The samples were cleaned up by solid phase extraction (SPE) procedure using a carboxylic acid cartridge. Chromatographic separation was carried out on the C8 analytical column and the mobile phase consisting of acetonitryle-methanol-0.02 M oxalic acid (20:15:65, v/v/v), with the detection by UV detector at λ = 355 nm. This method has been successfully validated and used for the quantitative determination of doxycycline in animal tissues samples. Recoveries from spiked samples were from 65% to 90%. The decision limits (CCα) were 110 µg/kg and 610 µg/kg for muscles and kidneys, respectively.
The experiment was performed on 36 Wistar rats. On the first day of the experiment iodoacetate was administered to the left posterior knee joint of the 18 rats which composed Group I. The second group of 18 rats received additionally doxycycline (doxy) through the gastric tube in doses comparable with those of doxycycline used in humans. The experiment lasted 21 days. The animals were sacrificed after 7, 14 and 21 days in groups of 6 rats each. In sections stained with Safranin 0 semiquantitative histochemical intensity tests were performed on articular cartilage glycosaminoglycans (GAG) using a four-point scale (0–3). In the first group examined destructive lesions in the articular cartilage and weak reactivity on GAG were noted at all stages of the experiment. The intensity of GAG staining was higher in the second group after 14 and especially after 21 days, which may suggest a protective action of doxy on articular cartilage.
An analytical method for the simultaneous determination of tetracycline (TC), oxtetracycline (OTC), chlortetracycline (CTC) with their 4-epimers, and doxycycline (DC) in animal tissues has been developed and validated. The extraction of the analytes from biological matrice was carried out with a 0.02 M oxalic acid (pH=4.0). The samples were cleaned up by using a solid phase extraction procedure with polymeric cartridges. Chromatographic separation was achieved on a C 18 analytical column using a mobile phase consisting of acetonitrile, methanol and 0.02 M oxalic acid in gradient mode. Detection was carried out by UV detector at λ = 355 nm. The method has been validated according to the Commission Decision 2002/657/EC. The recoveries of the analytes from the spiked samples were 50%-80%. The decision limits (CCα) were from 110 to 125 µg/kg and the detection capabilities (CCß) were from 120 to 155 µg/kg, depending on the analytes. The prepared method was successfully verified in the National Residue Control Programme.
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The effect of doxycycline on atherogenesis in apoE-knockout mice

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Doxycycline at subantimicrobial doses inhibits matrix metalloproteinases (MMPs) activity, and is the only MMP inhibitor which is widely available in clinical practice. The aim of the study was to reveal whether non-specific MMPs inhibition by tetracycline could ameliorate development of atherosclerosis in apolipoprotein E (apoE)-knockout mice. Doxycycline (1.5 mg/ kg b.w./day) administered orally attenuated atherogenesis, measured both by "en face" method (10.25±1.7% vs. 15.7±2.0%, p<0.05) and "cross-section" method (66,254±7,468 µm2 vs. 90,687±8,521 µm2, p<0.05). In-situ zymography showed decrease of the extent of non-specific gelatinase activity in doxycycline-treated mice This is the first report to date describing the effect of doxycycline on atherogenesis in apoE-targeted mice.
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