Determination of reactive oxygen and nitrogen species in rat aorta using the dichlorofluorescein assay

Abstract

A method for the determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in macroscopic sections of vessels has been developed on the basis of the dichlorofluorescein (DCF) assay. DCF was measured by fluorescence in extracts of vessels. The main artifact of the method is the oxidation of dichlorodihydrofluorescein (DCFH(2)) which is released from vessels together with DCF during the extraction procedure. This problem was resolved by decreasing pH during the extraction. The optimal conditions and the time for aorta incubation with DCFH(2)-DA and for the extraction of DCF from aorta have been determined. The ROS/RNS production in different aorta segments and the dependence of ROS/RNS production on rat age have been studied. It was shown that thoracic aorta sections produced the same amounts of ROS/RNS and the intermediate between the thoracic and the abdominal aorta part produced ROS and RNS by 14% more than the thoracic aorta. It was found that ROS/RNS production in aorta increases with rat age: the doubling time of ROS/RNS production rate is 113 days from birth.

Ramipril Protects the Endothelium from High Glucose–Induced Dysfunction through CaMKKβ/AMPK and Heme Oxygenase-1 Activation

“…Cells were washed three times in phosphate-buffered saline, followed by measurement of fluorescence intensity at 485-nm excitation and 538-nm emission spectra. The ROS formation in rat aorta was determined, as described previously (Korystov et al, 2009;Merry et al, 2010). In brief, the aorta was removed and rinsed with cold (4°C) 10 mM Hanks'-HEPES solution (pH 7.4).…”

Tranilast Alleviates Endothelial Dysfunctions and Insulin Resistance via Preserving Glutathione Peroxidase 1 in Rats Fed a High-Fat Emulsion

“…Cells were washed three times in PBS, followed by measurement of fluorescence intensity at 485-nm excitation and 538-nm emission spectra using a microplate reader (Wallac 1420 VICTOR2; PerkinElmer, Waltham, MA, USA). The ROS formation in rat aorta and skeletal muscle (soleus) was determined as described previously (26,27). These procedures were performed in dark conditions to avoid photo-oxidation.…”

Effects of taxifolin on the activity of angiotensin-converting enzyme and reactive oxygen and nitrogen species in the aorta of aging rats and rats treated with the nitric oxide synthase inhibitor and dexamethasone

“…At this rate of water drinking, each rat received daily 100 mg of L-NAME and/or from 10 to 100 μg of taxifolin/kg of body weight. The aorta was prepared according to Korystov et al (2009). At the end of the treatment protocols, animals were anesthetized by ether, and the thorax was dissected before the injection of heparin (500 U) into the heart to prevent blood clotting.…”

“…Measurements of ROS/RNS in the aorta ROS/RNS were determined according to Korystov et al (2009). The aorta from the aortic arch to the point of branching of kidney arteries was cut into seven 5-mm sections.…”

“…In addition, the ACE activity in homogenate can considerably differ from that (determined) in tissue . We have previously developed methods for measuring the ACE activity Korystova et al 2012) and the amount of reactive oxygen species (ROS)/reactive nitrogen species (RNS) in rat aorta sections (Korystov et al 2009). In the present study, we have examined the effect of taxifolin (dihydroquercetin) on the ACE activity in aortas of aging rats and rats treated with the nitric oxide (NO) synthase inhibitor or dexamethasone.…”