ECED/10-2015-0381-Dia/30.12.2015/MPS
Review
Authors
M. Kanter1, F. Aksu2, M. Takir3, O. Kostek4, B. Kanter5, A. Oymagil6
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ diabates ● ▶ low intensity exercise ● ▶ oxidative stress ● ▶ apoptosis ● ▶ heart tissue ●
Abstract
received 06.10.2015 first decision 09.11.2015 accepted 03.12.2015 Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1569332 Published online: 2016 Exp Clin Endocrinol Diabetes © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence M. Kanter Department of Histology and Embryology Faculty of Medicine Istanbul Medeniyet University Istanbul 34710 Turkey Tel.: + 90/216/2803 333 Fax: + 90/216/6022 805 [email protected]
▼
Background: The aim of this study was to investigate the effects of low intensity exercise on heart of streptozotocine (STZ)-induced diabetic rats. Materials and Methods: The rats were randomly divided into 3 experimental groups: A (control), B (diabetic untreated), and C (diabetic treated with low intensity exercise); each group contains 8 animals. B and C groups received STZ. Diabetes was induced in 2 groups by a single intraperitoneal (i.p) injection of STZ (40 mg/ kg, freshly dissolved in 0,1 M citrate buffer, pH 4.2). 2 days after STZ treatment, diabetes in 2 experimental groups was confirmed by measuring blood glucose levels. Rats with blood glucose levels of 250 mg/dl or higher were considered to be diabetic. Animals in the exercise group were made to run the treadmill once a day for 4 consecutive weeks. Exercise started 3 days prior to STZ administration.
Introduction
▼
Streptozotocin (STZ), an antibiotic produced by Streptomyces achromogenes, possesses pancreatic β-cell cytotoxic effect [1]. Streptozotocin has been widely used for inducing diabetes mellitus in a variety of animals. STZ causes degeneration and necrosis of pancreatic β-cells [2–5]. Although the mechanism of the β-cell cytotoxic action of STZ is not fully understood, experimental evidence has demonstrated that some of its deleterious effects are attributable to induction of metabolic processes, which lead onto an increase in the generation of reactive oxygen species (ROS) [6]. Apart from production of ROS, STZ also inhibits free radical scavenger-enzymes [7]. The superoxide radical has been implicated in lipid peroxidation, DNA damage, and sulfhydryl oxidation [8, 9].
Results: After induction of diabetes, histological abnormalities were observed, including myofibrillar loss, vacuolization of cytoplasm and irregularity of myofibrils. These alterations were attenuated by low intensity exercise. Our data indicates a significant reduction of oxidative stress and apoptosis in cardiomyocytes after exercise. Treatment of diabetic animals with low intensity exercise, decreased the elevated tissue malondialdehyde (MDA) levels and increased the reduced activities of the enzymatic antioxidants superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in cardiac tissue. Conclusion: These findings suggest that low intensity exercise has a therapeutic protective effect in diabetes by decreasing oxidative stress and apoptosis, and by preservation of myocardial integrity.
Diabetes mellitus is an important public health issue due to its high prevalence and increased morbidity and mortality. Cardiovascular disease is a major cause of death in diabetic patients [10]. Cardiac injury is caused by coronary atherosclerosis and diabetes-related cardiomyopathy [11]. As first reported by Rubler et al. [12], diabetic cardiomyopathy is a single form of heart disease characterized by left ventricular systolic and diastolic dysfunction in the absence of underlying coronary artery disease and/or hypertension. Diabetic cardiomyopathy is a common cardiac condition affecting both type 1 and type 2 diabetes patients [12]. The pathophysiology of diabetic cardiomyopathy is not completely understood as several mechanisms can be involved; these include myocyte hypertrophy, myocardial fibrosis, contractile dysfunction, calcium handling and mitochondrial function changes, and nitric
Kanter M et al. Effects of Low Intensity … Exp Clin Endocrinol Diabetes
Downloaded by: University of Connecticut. Copyrighted material.
Effects of Low Intensity Exercise Against Apoptosis and Oxidative Stress in Streptozotocin-induced Diabetic Rat Heart
oxide signaling impairment [12–17]. Hyperglycemia-induced oxidative stress is an important factor involved in diabetic cardiomyopathy [18–21]. Oxidative stress has been shown to be involved in triggering cardiomyocyte apoptosis associated with diabetic cardiomyopathy. Moreover, some recent investigations have been proposed that DM causes an inflammatory response through the oxidative mechanisms, which may play a causative role in development of diabetic cardiomyopathy [22]. At skeletal muscle level, it has been documented that oxidative stress results in oxidative damage of muscle proteins that in turn may result in protein denaturation, aggregation, and loss of essential biological function [23]. Oxidative stress plays a role in the development of diabetic complications [24]. In the diabetic state, lipid peroxidation can be induced by protein glycation and glucose auto-oxidation that can further lead to the formation of free radicals [25]. The main free radicals that occur in this diseased state are superoxide, hydroxyl and peroxyl radicals. These free radicals all might play a role in DNA damage, glycation and protein modification reactions, and in lipid oxidative modification in diabetes [26]. The damage that these radicals inflict on cells might be quantitatively determined by measurement of levels of MDA, a product of lipid peroxidation [27]. Certain enzymes play an important role in antioxidant defense, to maintain viable reproductive ability; a protective mechanism against oxidative stress is of importance [28]. These enzymes include SOD, GSH-Px, glutathione reductase and catalase, which convert free radicals or reactive oxygen intermediates to non-radical products [28]. For decades, the 3 cornerstones of medical care in diabetes have been diet, medication, and exercise. In fact, regular exercise has been shown to improve blood glucose control, reduce cardiovascular risk factors, contribute to weight loss, and improve wellbeing [29]. Moreover, considering the reports about the positive effect of resistance exercise on insulin sensitivity, glycemic control, body composition, muscle mass and quality, and cardiac function nowadays both American diabetes association and American heart association have put resistance type exercise in exercise recommendations for diabetics [29, 30]. The present study was planned to investigate the modulatory role of low intensity exercise on diabetes-induced myocardial injury in rat model of cardiomyopathy and explore its antioxidant and anti-apoptotic potential.
Materials and Methods
▼
Animals
24 healthy male Sprague-Dawley rats, weighing 250–300 g and averaging 12 weeks old were utilized in our study. The animals were purchased from Trakya University Animal Care and Research Unit. Rats were fed on a standard rat chow and tap water ad libitum. In the windowless animal quarter automatic temperature (22 ± 2 °C) and lighting controls (light on at 07 AM and off at 09 PM: 14 h light/10 h dark cycle) was performed. Humidity ranged from 50 to 55 %. All animals received human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health. The study was approved by the Institutional Animal Ethical Committee of Trakya University, Edirne, Turkey.
Kanter M et al. Effects of Low Intensity … Exp Clin Endocrinol Diabetes
ECED/10-2015-0381-Dia/30.12.2015/MPS
Experimental design
The rats were randomly allotted into one of 3 experimental groups: A (control), B (diabetic untreated), and C (diabetic treated with low intensity exercise); each group contain 8 animals. B and C groups received STZ (Sigma diabetes was induced in 2 groups by a single intra-peritoneal (i.p) injection of STZ (40 mg/kg, freshly dissolved in 0,1 M citrate buffer, pH 4.2). 2 days after STZ treatment, development of diabetes in 2 experimental groups was confirmed by measuring blood glucose levels in a tail vein blood samples. Rats with blood glucose levels of 250 mg/dl or higher were considered to be diabetic. Serum glucose levels in control animals remained normal for the duration of the study. Diabetes mellitus was confirmed by Ames One Touch Glucometer (LifeScan, Johnson and Johnson, New Brunswick, NJ, USA). Control rats were injected with the same volume of isotonic NaCl as the diabetic animals that received STZ. Groups C was subjected to treadmill exercise training at zero inclination for 4 weeks. Groups A and B were not exercised. The rats were subjected to treadmill exercise protocol for a total of 4 weeks: 30 min daily for 4 week at a speed of 10 m/min [31]. After every run, the treadmill was cleaned with 70 % ethanol solution, wiped and air dried before the next set of 3 rats were put on the treadmill. The initial and final body weight changes of the various groups were recorded. The initial study consisted of 8 rats in each group. No animals died during the experiment. At the end of the experiment, rats were fasted overnight for 12 h, and sacrificed under chloralhydrate (6 ml of 7 % chloralhydrate kg, Sigma) anaesthesia and the heart tissue was evaluated for lipid peroxidation products, antioxidant enzymes and morphological appearance. Blood samples were collected by cardiac puncture using a heparinized syringe. Serum glucose was determined by the hexokinase method with reagents from Boehringer, Mannheim, Germany [32]. Insulin was determined using a double-antibody radioimmunoassay kit (Amersham Radiochemical Centre, Bucks, UK) [33].
Biochemical analysis Preparation of tissue samples
All tissues were weighed and homogenized with 0.15 M KCl solution and 10 % homogenates (w/v) of these tissues were prepared. Tissue homogenates were centrifuged for 10 min at 4 °C cold centrifuge 600 × g. Supernatant was centrifuged for 20 min at 10 000 × g so postmitochondrial fraction was obtained. MDA level of tissues were determined in tissue homogenates; SOD and GSH-Px activities were examined in postmitochondrial fraction of these homogenates. The bicinchoninic acid method was used for determining the amount of protein in samples [34].
Lipid peroxidation determination
MDA, as an endpoint of lipid peroxidation (LPO), was calculated by detecting absorbance of thiobarbituric acid reactive substances at 532 nm [35]. MDA levels were expressed as MDA nmol/mg protein.
Superoxide dismutase determination
SOD activity was measured by principle of increasing the ability of photooxidation rate in o-dianisidin sensitived with riboflavin [36]. Colored product was measured spectrophotometrically at 460 nm, and the results IU/mg protein, as specified.
Downloaded by: University of Connecticut. Copyrighted material.
Review
ECED/10-2015-0381-Dia/30.12.2015/MPS
GSH-Px activity was measured according to the protocol of Lawrence et al. [37]. The results were calculated using NADPH in extinction coefficient and nmol NADPH/mg protein/min were expressed.
Catalase determination
Catalase (CAT) activity was determined according to Aebi’s method [38]. The principle of the method was based on the determination of the rate constant (s, − 1 k) of theH2O2 decomposition rate at 240 nm. Results were expressed as k (rate constant) per mg protein.
Histopathological procedures
Cardiac tissues were harvested from the sacrificed animals, and the tissues were fixed in 10 % neutral formalin and embedded in paraffin blocks. Sections of 5 μm were obtained, deparaffinized and stained with hematoxylin and eosin (H&E). The cardiac tissue was examined, evaluated and photographed in random order under blindfold conditions with standard light microscopy (Nikon Optiphot 2, Tokyo). The severity of changes was quantitated none (0) to severe (3) based on the degree of cytoplasmic vacuolization, myocardial disorganization, and myofibrillar loss. The scoring system was as follows: (0) no damage, (1) mild, (2) moderate, (3) severe.
Immunohistochemical procedures
The harvested pancreatic tissues were fixed in 10 % neutral buffered formalin, embedded in parafin and sectioned at 5 μm thickness. Immunocytochemical reactions were performed according to the ABC technique described by Hsu et al. [39]. The procedure involved the following steps: (1) endogenous peroxidase activity was inhibited by 3 % H2O2 in distilled water for 30 min; (2) the sections were washed in distilled water for 10 min; (3) non-specific binding of antibodies was blocked by incubation with normal goat serum (DAKO X 0907, Carpinteria, CA) with PBS, diluted 1:4; (4) the sections were incubated with specific monoclonal mouse antisera against human insulin protein (18–0066; Zymed, San Francisco, CA), diluted 1:50 for 1 h, and then at room temperature; (5) the sections were washed in PBS 3 × 3 min; (6) the sections were incubated with biotinylated anti-mouse IgG (DAKO LSAB 2 Kit); (7) the sections were washed in PBS 3 × 3 min; (8) the sections were incubated with ABC (DAKO LSAB 2 Kit); (9) the sections were washed in PBS 3 × 3 min; (10) peroxidase was detected with an aminoethylcarbazole substrate kit (AEC kit; Zymed Laboratories); (11) the sections were washed in tap water for 10 min and then dehydrated; (12) the nuclei were stained with hematoxylin; and (13) the sections were mounted in DAKO paramount.
Image analysis
The system used consisted of a PC with hardware and software (Image-Pro Plus 5.0, Media Cybernetics, Silver Spring, MD) for image acquisition and analysis, a Spot Insight QE (Diagnostic Instruments, Silver Spring, MD) camera, and an optical microscope. The method requires preliminary software procedures involving spatial calibration (on a micron scale) and setting of color segmentation for quantitative color analysis. 10 Langerhans islets from each rat (100 islets for each group) were chosen randomly. Then the percentage of the insulin immunoreactive β-cell area in the Langerhans islets (100 islets for each group) was estimated in image analysis system. The percentage of the
insulin immunoreactive β-cells was calculated according to these results. The investigator who obtained these measurements was unaware of the experiment being performed.
Evaluation of cardiomyocytes apoptosis
Cardiomyocytes apoptosis were evaluated by the TUNEL assay. The TUNEL method, which detects fragmentation of DNA in the nucleus during apoptotic cell death in situ, was employed using an apoptosis detection kit (TdT-Fragel™ DNA Fragmentation Detection Kit, cat. no. QIA33, Calbiochem, USA). All reagents listed below are from the kit and were prepared following the manufacturer’s instructions. 5-micrometer-thick cardiac tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series as described previously. The sections were then incubated with 20 mg/ml proteinase K for 20 min and rinsed in PBS. Endogenous peroxidase activity was inhibited by incubation with 3 % hydrogen peroxide. Sections were then incubated with equilibration buffer for 10–30 s and then TdTenzyme, in a humidified atmosphere at 37 °C, for 90 min. Sections were subsequently put into prewarmed working strength stop/ wash buffer at room temperature for 10 min and incubated with blocking buffer for 30 min. Each step was separated by thorough washes in PBS. Labeling was revealed using DAB, counterstaining was performed using methyl green, and sections were dehydrated, cleared, and mounted. TUNEL positive cells were counted, and the TUNEL positive cells/100 cardiomyocyte percentage was used as the index of apoptosis. This percentage represented the apoptotic index of the sample and was compared between groups.
Statistical analysis
The data were expressed as the mean ± standard deviation (SD), and analyzed by repeated measures of variance. A Tukey test was used to test for differences among means when an analysis of variance (ANOVA) indicated a significant (P
Review
Authors
M. Kanter1, F. Aksu2, M. Takir3, O. Kostek4, B. Kanter5, A. Oymagil6
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ diabates ● ▶ low intensity exercise ● ▶ oxidative stress ● ▶ apoptosis ● ▶ heart tissue ●
Abstract
received 06.10.2015 first decision 09.11.2015 accepted 03.12.2015 Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1569332 Published online: 2016 Exp Clin Endocrinol Diabetes © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence M. Kanter Department of Histology and Embryology Faculty of Medicine Istanbul Medeniyet University Istanbul 34710 Turkey Tel.: + 90/216/2803 333 Fax: + 90/216/6022 805 [email protected]
▼
Background: The aim of this study was to investigate the effects of low intensity exercise on heart of streptozotocine (STZ)-induced diabetic rats. Materials and Methods: The rats were randomly divided into 3 experimental groups: A (control), B (diabetic untreated), and C (diabetic treated with low intensity exercise); each group contains 8 animals. B and C groups received STZ. Diabetes was induced in 2 groups by a single intraperitoneal (i.p) injection of STZ (40 mg/ kg, freshly dissolved in 0,1 M citrate buffer, pH 4.2). 2 days after STZ treatment, diabetes in 2 experimental groups was confirmed by measuring blood glucose levels. Rats with blood glucose levels of 250 mg/dl or higher were considered to be diabetic. Animals in the exercise group were made to run the treadmill once a day for 4 consecutive weeks. Exercise started 3 days prior to STZ administration.
Introduction
▼
Streptozotocin (STZ), an antibiotic produced by Streptomyces achromogenes, possesses pancreatic β-cell cytotoxic effect [1]. Streptozotocin has been widely used for inducing diabetes mellitus in a variety of animals. STZ causes degeneration and necrosis of pancreatic β-cells [2–5]. Although the mechanism of the β-cell cytotoxic action of STZ is not fully understood, experimental evidence has demonstrated that some of its deleterious effects are attributable to induction of metabolic processes, which lead onto an increase in the generation of reactive oxygen species (ROS) [6]. Apart from production of ROS, STZ also inhibits free radical scavenger-enzymes [7]. The superoxide radical has been implicated in lipid peroxidation, DNA damage, and sulfhydryl oxidation [8, 9].
Results: After induction of diabetes, histological abnormalities were observed, including myofibrillar loss, vacuolization of cytoplasm and irregularity of myofibrils. These alterations were attenuated by low intensity exercise. Our data indicates a significant reduction of oxidative stress and apoptosis in cardiomyocytes after exercise. Treatment of diabetic animals with low intensity exercise, decreased the elevated tissue malondialdehyde (MDA) levels and increased the reduced activities of the enzymatic antioxidants superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in cardiac tissue. Conclusion: These findings suggest that low intensity exercise has a therapeutic protective effect in diabetes by decreasing oxidative stress and apoptosis, and by preservation of myocardial integrity.
Diabetes mellitus is an important public health issue due to its high prevalence and increased morbidity and mortality. Cardiovascular disease is a major cause of death in diabetic patients [10]. Cardiac injury is caused by coronary atherosclerosis and diabetes-related cardiomyopathy [11]. As first reported by Rubler et al. [12], diabetic cardiomyopathy is a single form of heart disease characterized by left ventricular systolic and diastolic dysfunction in the absence of underlying coronary artery disease and/or hypertension. Diabetic cardiomyopathy is a common cardiac condition affecting both type 1 and type 2 diabetes patients [12]. The pathophysiology of diabetic cardiomyopathy is not completely understood as several mechanisms can be involved; these include myocyte hypertrophy, myocardial fibrosis, contractile dysfunction, calcium handling and mitochondrial function changes, and nitric
Kanter M et al. Effects of Low Intensity … Exp Clin Endocrinol Diabetes
Downloaded by: University of Connecticut. Copyrighted material.
Effects of Low Intensity Exercise Against Apoptosis and Oxidative Stress in Streptozotocin-induced Diabetic Rat Heart
oxide signaling impairment [12–17]. Hyperglycemia-induced oxidative stress is an important factor involved in diabetic cardiomyopathy [18–21]. Oxidative stress has been shown to be involved in triggering cardiomyocyte apoptosis associated with diabetic cardiomyopathy. Moreover, some recent investigations have been proposed that DM causes an inflammatory response through the oxidative mechanisms, which may play a causative role in development of diabetic cardiomyopathy [22]. At skeletal muscle level, it has been documented that oxidative stress results in oxidative damage of muscle proteins that in turn may result in protein denaturation, aggregation, and loss of essential biological function [23]. Oxidative stress plays a role in the development of diabetic complications [24]. In the diabetic state, lipid peroxidation can be induced by protein glycation and glucose auto-oxidation that can further lead to the formation of free radicals [25]. The main free radicals that occur in this diseased state are superoxide, hydroxyl and peroxyl radicals. These free radicals all might play a role in DNA damage, glycation and protein modification reactions, and in lipid oxidative modification in diabetes [26]. The damage that these radicals inflict on cells might be quantitatively determined by measurement of levels of MDA, a product of lipid peroxidation [27]. Certain enzymes play an important role in antioxidant defense, to maintain viable reproductive ability; a protective mechanism against oxidative stress is of importance [28]. These enzymes include SOD, GSH-Px, glutathione reductase and catalase, which convert free radicals or reactive oxygen intermediates to non-radical products [28]. For decades, the 3 cornerstones of medical care in diabetes have been diet, medication, and exercise. In fact, regular exercise has been shown to improve blood glucose control, reduce cardiovascular risk factors, contribute to weight loss, and improve wellbeing [29]. Moreover, considering the reports about the positive effect of resistance exercise on insulin sensitivity, glycemic control, body composition, muscle mass and quality, and cardiac function nowadays both American diabetes association and American heart association have put resistance type exercise in exercise recommendations for diabetics [29, 30]. The present study was planned to investigate the modulatory role of low intensity exercise on diabetes-induced myocardial injury in rat model of cardiomyopathy and explore its antioxidant and anti-apoptotic potential.
Materials and Methods
▼
Animals
24 healthy male Sprague-Dawley rats, weighing 250–300 g and averaging 12 weeks old were utilized in our study. The animals were purchased from Trakya University Animal Care and Research Unit. Rats were fed on a standard rat chow and tap water ad libitum. In the windowless animal quarter automatic temperature (22 ± 2 °C) and lighting controls (light on at 07 AM and off at 09 PM: 14 h light/10 h dark cycle) was performed. Humidity ranged from 50 to 55 %. All animals received human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health. The study was approved by the Institutional Animal Ethical Committee of Trakya University, Edirne, Turkey.
Kanter M et al. Effects of Low Intensity … Exp Clin Endocrinol Diabetes
ECED/10-2015-0381-Dia/30.12.2015/MPS
Experimental design
The rats were randomly allotted into one of 3 experimental groups: A (control), B (diabetic untreated), and C (diabetic treated with low intensity exercise); each group contain 8 animals. B and C groups received STZ (Sigma diabetes was induced in 2 groups by a single intra-peritoneal (i.p) injection of STZ (40 mg/kg, freshly dissolved in 0,1 M citrate buffer, pH 4.2). 2 days after STZ treatment, development of diabetes in 2 experimental groups was confirmed by measuring blood glucose levels in a tail vein blood samples. Rats with blood glucose levels of 250 mg/dl or higher were considered to be diabetic. Serum glucose levels in control animals remained normal for the duration of the study. Diabetes mellitus was confirmed by Ames One Touch Glucometer (LifeScan, Johnson and Johnson, New Brunswick, NJ, USA). Control rats were injected with the same volume of isotonic NaCl as the diabetic animals that received STZ. Groups C was subjected to treadmill exercise training at zero inclination for 4 weeks. Groups A and B were not exercised. The rats were subjected to treadmill exercise protocol for a total of 4 weeks: 30 min daily for 4 week at a speed of 10 m/min [31]. After every run, the treadmill was cleaned with 70 % ethanol solution, wiped and air dried before the next set of 3 rats were put on the treadmill. The initial and final body weight changes of the various groups were recorded. The initial study consisted of 8 rats in each group. No animals died during the experiment. At the end of the experiment, rats were fasted overnight for 12 h, and sacrificed under chloralhydrate (6 ml of 7 % chloralhydrate kg, Sigma) anaesthesia and the heart tissue was evaluated for lipid peroxidation products, antioxidant enzymes and morphological appearance. Blood samples were collected by cardiac puncture using a heparinized syringe. Serum glucose was determined by the hexokinase method with reagents from Boehringer, Mannheim, Germany [32]. Insulin was determined using a double-antibody radioimmunoassay kit (Amersham Radiochemical Centre, Bucks, UK) [33].
Biochemical analysis Preparation of tissue samples
All tissues were weighed and homogenized with 0.15 M KCl solution and 10 % homogenates (w/v) of these tissues were prepared. Tissue homogenates were centrifuged for 10 min at 4 °C cold centrifuge 600 × g. Supernatant was centrifuged for 20 min at 10 000 × g so postmitochondrial fraction was obtained. MDA level of tissues were determined in tissue homogenates; SOD and GSH-Px activities were examined in postmitochondrial fraction of these homogenates. The bicinchoninic acid method was used for determining the amount of protein in samples [34].
Lipid peroxidation determination
MDA, as an endpoint of lipid peroxidation (LPO), was calculated by detecting absorbance of thiobarbituric acid reactive substances at 532 nm [35]. MDA levels were expressed as MDA nmol/mg protein.
Superoxide dismutase determination
SOD activity was measured by principle of increasing the ability of photooxidation rate in o-dianisidin sensitived with riboflavin [36]. Colored product was measured spectrophotometrically at 460 nm, and the results IU/mg protein, as specified.
Downloaded by: University of Connecticut. Copyrighted material.
Review
ECED/10-2015-0381-Dia/30.12.2015/MPS
GSH-Px activity was measured according to the protocol of Lawrence et al. [37]. The results were calculated using NADPH in extinction coefficient and nmol NADPH/mg protein/min were expressed.
Catalase determination
Catalase (CAT) activity was determined according to Aebi’s method [38]. The principle of the method was based on the determination of the rate constant (s, − 1 k) of theH2O2 decomposition rate at 240 nm. Results were expressed as k (rate constant) per mg protein.
Histopathological procedures
Cardiac tissues were harvested from the sacrificed animals, and the tissues were fixed in 10 % neutral formalin and embedded in paraffin blocks. Sections of 5 μm were obtained, deparaffinized and stained with hematoxylin and eosin (H&E). The cardiac tissue was examined, evaluated and photographed in random order under blindfold conditions with standard light microscopy (Nikon Optiphot 2, Tokyo). The severity of changes was quantitated none (0) to severe (3) based on the degree of cytoplasmic vacuolization, myocardial disorganization, and myofibrillar loss. The scoring system was as follows: (0) no damage, (1) mild, (2) moderate, (3) severe.
Immunohistochemical procedures
The harvested pancreatic tissues were fixed in 10 % neutral buffered formalin, embedded in parafin and sectioned at 5 μm thickness. Immunocytochemical reactions were performed according to the ABC technique described by Hsu et al. [39]. The procedure involved the following steps: (1) endogenous peroxidase activity was inhibited by 3 % H2O2 in distilled water for 30 min; (2) the sections were washed in distilled water for 10 min; (3) non-specific binding of antibodies was blocked by incubation with normal goat serum (DAKO X 0907, Carpinteria, CA) with PBS, diluted 1:4; (4) the sections were incubated with specific monoclonal mouse antisera against human insulin protein (18–0066; Zymed, San Francisco, CA), diluted 1:50 for 1 h, and then at room temperature; (5) the sections were washed in PBS 3 × 3 min; (6) the sections were incubated with biotinylated anti-mouse IgG (DAKO LSAB 2 Kit); (7) the sections were washed in PBS 3 × 3 min; (8) the sections were incubated with ABC (DAKO LSAB 2 Kit); (9) the sections were washed in PBS 3 × 3 min; (10) peroxidase was detected with an aminoethylcarbazole substrate kit (AEC kit; Zymed Laboratories); (11) the sections were washed in tap water for 10 min and then dehydrated; (12) the nuclei were stained with hematoxylin; and (13) the sections were mounted in DAKO paramount.
Image analysis
The system used consisted of a PC with hardware and software (Image-Pro Plus 5.0, Media Cybernetics, Silver Spring, MD) for image acquisition and analysis, a Spot Insight QE (Diagnostic Instruments, Silver Spring, MD) camera, and an optical microscope. The method requires preliminary software procedures involving spatial calibration (on a micron scale) and setting of color segmentation for quantitative color analysis. 10 Langerhans islets from each rat (100 islets for each group) were chosen randomly. Then the percentage of the insulin immunoreactive β-cell area in the Langerhans islets (100 islets for each group) was estimated in image analysis system. The percentage of the
insulin immunoreactive β-cells was calculated according to these results. The investigator who obtained these measurements was unaware of the experiment being performed.
Evaluation of cardiomyocytes apoptosis
Cardiomyocytes apoptosis were evaluated by the TUNEL assay. The TUNEL method, which detects fragmentation of DNA in the nucleus during apoptotic cell death in situ, was employed using an apoptosis detection kit (TdT-Fragel™ DNA Fragmentation Detection Kit, cat. no. QIA33, Calbiochem, USA). All reagents listed below are from the kit and were prepared following the manufacturer’s instructions. 5-micrometer-thick cardiac tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series as described previously. The sections were then incubated with 20 mg/ml proteinase K for 20 min and rinsed in PBS. Endogenous peroxidase activity was inhibited by incubation with 3 % hydrogen peroxide. Sections were then incubated with equilibration buffer for 10–30 s and then TdTenzyme, in a humidified atmosphere at 37 °C, for 90 min. Sections were subsequently put into prewarmed working strength stop/ wash buffer at room temperature for 10 min and incubated with blocking buffer for 30 min. Each step was separated by thorough washes in PBS. Labeling was revealed using DAB, counterstaining was performed using methyl green, and sections were dehydrated, cleared, and mounted. TUNEL positive cells were counted, and the TUNEL positive cells/100 cardiomyocyte percentage was used as the index of apoptosis. This percentage represented the apoptotic index of the sample and was compared between groups.
Statistical analysis
The data were expressed as the mean ± standard deviation (SD), and analyzed by repeated measures of variance. A Tukey test was used to test for differences among means when an analysis of variance (ANOVA) indicated a significant (P
