Chemicals
CYP (PubChem CID: 2907) and Q (PubChem CID: 5280343) were purchased from Sigma-Aldrich Scientific International, Inc. CYP and Q were dissolved in saline and corn oil immediately before injection, respectively. All other chemicals that were used were of the highest analytic grade.
Animal housing and experimental design
In this study, 50 male Sprague Dawley rats (250±25 g) were used. The animals were obtained from the Experimental Medicine Research Center in Atatürk University. The experimental studies were conducted according to ethical rules approved by the Local Ethics Committee of Atatürk University for Animal Experiments (Protocol no: 2015/107).
All the animals were housed under standard environment conditions at 21±2 °C and a 12-h light/12-h dark cycle and were allowed access ad libitum to a standard diet and drinking water. The rats were randomly divided into five groups, as described below, with 10 rats in each group:
- The control group was given corn oil via the intragastric (i.g.) route for seven days.
- The CYP group was given corn oil for seven days and injected to a single dose (200 mg/kg) of CYP via the intraperitoneal (i.p.) route on the seventh day.
- The Q50+CYP group was given Q (50 mg/kg) dissolved in corn oil for seven days and injected to a single dose of CYP (200 mg/kg, i.p.) on the seventh day.
- The Q100+CYP group was given Q (100 mg/kg) dissolved in corn oil for seven days and injected with a single dose of CYP (200 mg/kg, i.p.) on the seventh day.
- The Q100 group was given Q (100 mg/kg, i.g.) dissolved in corn oil for seven days.
Twenty-four hours after the CYP treatment according to a previously published protocols (Mansour et al. 2015), the rats were anesthetized with ketamine hydrochloride (ip, 75 mg/kg) (Ketalar, Pfizer, Turkey) and xylazine (15 mg/kg) (Rompun, Bayer, Turkey), and the rats were euthanized by cervical dislocation.
Isolated organ bath analysis
After euthanize, thoracic aortas were quickly removed into 4oC Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM Na2PO4, 0.5 mM MgCl2, 25 mM NaHCO3, 1.12 mM CaCl2, 11 mM glucose-pH 7.4). The connective and fat tissues surrounding the thoracic aorta were dissected and then was cut into approximately 5 mm rings. The endothelium of the aortic rings was kept intact, and the aortic rings were prepared and suspended horizontally in a 10 ml organ bath (EMKA) containing Krebs-Henseleit solution. The aortic rings were mounted on stainless steel triangles and connected to an isometric force transducer. The bath was continuously bubbled with 95% O2 and 5% CO2 at 37 °C. Then, the rings were applied the tension (1g) and equilibration period of 1 h was used. During the equilibration period, the Krebs solution was changed every 15 min. At the end of the equilibration period, phenylephrine (PE) (10-9-10-5 M cumulative) and the submaximal dose (10-6 M) of ACh determined in our previous studies were applied. Dose-response curves to cumulative of PE were obtained. To demonstrate that CYP decreases the relaxation responses by causing to vascular endothelial damage, the relaxation effects of ACh (10-6 M) on PE-induced (10-5 M) contractions were compared among the groups.
Biochemical analysis
Thoracic aortic tissues were disintegrated by using liquid nitrogen. The homogenates from these tissue samples were prepared for analysis of malondialdehyde (MDA) and glutathione (GSH) levels and superoxide dismutase (SOD) activity. MDA and GSH levels and SOD activity were analyzed by using commercial ELISA kits (YL Biotech, Shanghai, China) according to the manufacturer’s instructions. The well plates were read at 450 nm via an ELISA plate reader (BioTekEpoch 2 Microplate Spectrophotometer).
Histopathologic and immunohistochemical analysis
At the end of the study, after the rats had been sacrificed under anesthesia, the thoracic aortas were removed and fixed in 10% buffered neutral formalin solution for 72 h. The tissue samples were embedded to paraffin following exposure to a graded series of xylene and ethanol washes. The paraffin blocks were cut into 5-μm thick sections by using a Leica RM2125RT microtome (Leica Microsystems, Wetzlar, Germany). The thoracic aortic sections were stained with Verhoeff’s elastic tissue stain, and changes in the elastin content based on the structural arrangement of the vessel walls were evaluated. The stained specimens were examined under a light microscope (Nikon eclipse i50, Tokyo, Japan), and photographic images were taken for histopathological evaluation.
For immunohistochemical analysis, from the tissues embedded in paraffin blocks, cross-sections were put on to adhesive-containing slides. The sections were passed through gradients of xylol and alcohol, and deparaffinization and dehydration were performed. The tissues were washed with phosphate-buffered saline (PBS), kept for 10 min in the 3% H2O2 solution. To prevent the antigens in the tissues from being masked, the samples were microwave-treated for 2x5 min with an antigen retrieval solution. After this process, rabbit polyclonal to eNOS primary antibody (catalog no: ab5589, dilution 1:100; Abcam, UK) was added. Afterwards, 3- 3′Diaminobensidine was used as chromogen. The sections that were counterstained by haematoxylin were observed under a light microscope. The pathologists counted the number of positive cells in each high-power field and calculated the average number of positive cells to reflect the intensity of positive expression. The sections were evaluated as none (−), mild (+), moderate (++), and severe (+++) according to their immunopositivity.
Statistical analysis
The statistical evaluation was performed using SPSS, version 20.0 (IBM, Armonk, NY, USA). Tukey’s test and a one-way ANOVA were applied to all the data. The data are expressed as mean±standard deviation (SD). p<0.05 was considered statistically significant.