Mechanical performance of the heart
Male rats of the Albino Wistar strain (250-350 g body weight, King Saud University breed), five groups (8 rats each) were used. The experiments were conducted following the ethical guidelines for investigation in laboratory animals, and the College of Medicine, KSU; Institutional Review Board (IRB) approved the protocol of the study. The rats were anesthetized with urethane and the hearts were removed and perfused with Krebs-Henseleit solution (composition in mM: NaCl 118.4, KCl 4.7, MgSO4 1.2, K2HPO4 1.2, NaHCO3 25.0, CaCl2 2.5, glucose 11.5) gassed with 95% O2 ,5% CO2 at a constant flow rate of 10 ml/ min and maintained at 370C with LE Thermostat (Panlab, HARVARD APPARATUS, USA). A saline-filled latex balloon connected via a polyethylene tube to a pressure transducer (MLA844, ADInstruments,Spain) was inserted into the left ventricle, and a baseline enddiastolic pressure was set at 5–10mmHg. The pressure transducer was connected to ADInstrument Powerlab 8/30
via a bridge amplifier (ADInstruments, Sydney Australia). The perfusion pressure was monitored by a second pressure transducer (MLA844, ADInstruments,Spain) connected to ADInstrument Powerlab 8/30 via a bridge amplifier (ADInstruments, Sydney Australia). All hearts were allowed to perfuse for 15 minutes to achieve stabilization. Following the stabilization period, the parameters recorded are Left ventricular pressure (LVP), heart rate (HR), dp/dt (max), dp/dt (min), perfusion pressure, pressure-time index, contractility index and duration of diastole. Results were analyzed using Labchart pro 7 software. Measurements were carried out at one minute before perfusion of cisplatin (Ebewe Pharma, Austria) and 60 minutes after perfusion. In a separate group, rutin trihydrate (Sigma- Aldrich GmbH, Germany) was perfused for 10 minutes before administration of cisplatin and throughout the experiment. A third sham group was perfused with Krebs-Henseleit solution for 60 minutes. Responses obtained 60 minutes after perfusion were expressed as percentages in relation to those obtained at one minute before perfusion of cisplatin.
At the end of the experiment, specimens of the ventricles of control, cisplatin 14mg/l and rutin and cisplatin groups were dissected, divided into two portions, one for detecting the underlying structural changes and the other for endogenous antioxidants analysis. The first specimens were then fixed in 10% buffered formalin and processed with paraffin wax. For histopathological feature examination, 5 µm sections were stained with hematoxylin and eosin for the analysis using a light microscope.
Analysis of endogenous antioxidants
The other heart specimens were washed by PBS then further divided and stored at -80oC till used for quantification of (GSH and MDA) the antioxidant / pro-oxidant variables. On the day of quantification, each specimen was weighed, homogenized in relevant specified buffers, centrifuged, and processed following the methodology of sample preparation for each estimate.
Reduced Glutathione (GSH) Assay
The principle is based on the reaction of reduced GSH with 5.5-dithiobis-(2-nitrobenzoic acid) (DTNB) (E. Merck Ltd., Bombay, India), according to the method of Owens and Belcher . The absorbance was measured by Schimadzu double beam spectrophotometer (UV200S, Japan) at 412 nm. The amount of GSH present was calculated using a standard solution of GSH containing 1 mg of GSH/ml of 3% meta-phosphoric acid. The increase in the extinction at 412 nm was proportional to the amount of GSH present and was expressed as nmol/g wet tissue.
Malondialdehyde (MDA) Assay
The principle is based on the reaction of MDA with thiobarbituric acid (TBA) (E. Merck Ltd., Bombay, India), according to the method described by Buege and Aust . The concentration of the MDA-TBA complex was being quantified spectrophotometrically at 532 nm and expressed as nmol/g wet tissue.
Data reported as mean ± SEM. One-way analysis of variance followed by Tukey’s multiple comparison tests as a post hoc test was used to analyze the data. A p-value of 0.05 or less was taken as a criterion for statistically significant differences. Data were analyzed using Graph Pad Prism 5 software.