• Users Online: 283
  • Print this page
  • Email this page


 
 
Table of Contents
RESEARCH ARTICLE
Year : 2017  |  Volume : 2  |  Issue : 4  |  Page : 1-6

Effects of exercise training on systolic and diastolic function of mice with diabetic cardiomyopathy


1 Department of School of Exercise Science, Shanghai University of Sport, Shanghai 200438, China
2 Department of Cardiology, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

Date of Web Publication12-Mar-2018

Correspondence Address:
Dr. Tingliang Liu
Department of Cardiology, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, No. 1678 Dongfang Road, Shanghai 200127
China
Xin Xu
School of Exercise Science, Shanghai University of Sport, No. 399 Changhai Road, Shanghai 200438
China
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cp.cp_1_18

Get Permissions

  Abstract 

Objective: This study aims to evaluate the effects of exercise training on heart function of mice with diabetic cardiomyopathy.
Materials and Methods: Twenty-four healthy C57 mice were randomly divided into three groups high-fat exercise group (n = 8), high-fat control group (n = 8), and low-fat control group (n = 8). High-fat groups were fed with a high-fat diet for 16 weeks, and the high-fat exercise group was subjected to aerobic treadmill exercise and resistance exercise for 8 weeks. After 24 weeks, the cardiac structure and function of the three groups were detected, and the indexes of the mouse heart were analyzed and compared. Results: The high-fat control group maintained hyperglycemia. The results of echocardiography showed that left ventricular eject fraction in the high-fat exercise group and the low-fat control group was significantly higher than that of the high-fat control group (68.99% ± 2.04% vs. 60.41% ± 2.31%, 66.16% ± 2.12% vs. 60.41% ± 2.31%, P < 0.01). In the diastolic function of the heart, blood flow peak velocities of the early peak at the mitral valve in the high-fat exercise group and the low-fat control group were significantly higher than that in the high-fat control group (709.73 ± 45.48 mm/s vs. 441.51 ± 44.83 mm/s, 632.92 ± 27.22 mm/s vs. 441.51 ± 44.83 mm/s, P < 0.01), the peak velocities of the atrial peak at the mitral valve were significantly lower than that of the high-fat control group (443.77 ± 18.09 mm/s vs. 523.67 ± 57.87 mm/s, 460.36 ± 18.24 mm/s vs. 523.67 ± 57.87 mm/s, P < 0.05), and the ratio of E/A was significantly higher than that of the high-fat control group (1.63 ± 0.06 vs. 0.85 ± 0.045, 1.38 ± 0.072 vs. 0.85 ± 0.045, P < 0.01). Myocardial performance index decreased (0.86 ± 0.095 vs. 0.97 ± 0.091, 0.88 ± 0.073 vs. 0.97 ± 0.091, P > 0.05), but there was no significant difference. Conclusion: The above data indicated that 8 weeks of exercise training can improve the heart function of mice with diabetic cardiomyopathy, especially diastolic heart function. Left ventricular systolic function had some trend to improve, but there is no statistical difference. Exercise intervention may promote the rehabilitation of diabetic cardiomyopathy.

Keywords: Cardiac function, diabetic cardiomyopathy, echocardiography, exercise


How to cite this article:
Lu G, Zhang X, Sun Z, Shi X, Liu T, Xu X. Effects of exercise training on systolic and diastolic function of mice with diabetic cardiomyopathy. Cardiol Plus 2017;2:1-6

How to cite this URL:
Lu G, Zhang X, Sun Z, Shi X, Liu T, Xu X. Effects of exercise training on systolic and diastolic function of mice with diabetic cardiomyopathy. Cardiol Plus [serial online] 2017 [cited 2018 Sep 24];2:1-6. Available from: http://www.cardiologyplus.org/text.asp?2017/2/4/1/227167

Guo Lu and Xu Zhang. These authors have contributed equally to this work.





  Introduction Top


Diabetes cardiomyopathy is a complication of diabetes mellitus and is the leading cause of heart failure or death in diabetic patients. The pathogenesis of diabetic cardiomyopathy is multifactorial. Hyperglycemia, hyperinsulinemia, and dyslipidemia may trigger an altered cardiac structure caused by cell signaling.[1] The clinical manifestations are cardiac diastolic and/or systolic dysfunction, left ventricular remodeling, which ultimately leads to heart failure, arrhythmia, and cardiogenic shock, and even sudden death in patients with severe illness.[2]

Exercise plays an important role in the prevention of various chronic diseases including cardiovascular disease. Regular exercise is beneficial for both Type 1 and Type 2 diabetes patients. While it possesses great health benefits for cardiovascular fitness, muscle strength, and insulin sensitivity in Type 1 diabetes patients, regular exercise may prevent the development of Type 2 diabetes.[3] Studies have reported that diabetes is associated with chronic low systemic inflammation. Animal studies have shown that exercise intervention has a positive effect on the prevention of diabetes. Exercise has anti-inflammatory effects, where regular exercise can prevent atherosclerosis and insulin resistance, thereby preventing diabetes.[4] Exercise can also improve blood glucose control in type 2 diabetes, reduce cardiovascular risk factors, contribute to weight loss, and improve overall health.

Although diabetes cardiomyopathy is widely recognized, effective treatments have yet to be discovered. Altered myocardial energy metabolic substrates, which are new therapies for the treatment and/or prevention of diabetic cardiomyopathy, may improve the development of diabetic heart dysfunction by activating pyruvate dehydrogenase to restore diastolic dysfunction and blood glucose level.[5] We demonstrated that treatment of type 2 diabetic rats with Zn reduced plasma glucose levels and prevented diabetic cardiomyopathy.[6]

Studies have found that elevated high-density lipoprotein levels can increase the risk of diabetic cardiomyopathy in patients with type 1 diabetes mellitus.[7] As a result, the study proves that exercise training is effective in the treatment and prevention of obesity. In addition to the beneficial effects of changes in diabetes-/obesity-related systems, obesity may also amend many of the metabolic disturbances characterizing the diabetic myocardium.[8]

In this study, exercise intervention was performed with aerobic treadmill exercise and resistance exercise, based on the model of diabetic cardiomyopathy. The study observed the effects of exercise intervention on cardiac systolic and diastolic function in diabetic cardiomyopathy in mice and provided experimental basis for further study.


  Materials and Methods Top


Experimental animals and grouping

Twenty-four healthy C57BL6 mice were purchased from the Animal Experimental Center of the Second Military Medical University and were housed in the specific-pathogen-free-level barrier of the animal laboratory of the Shanghai University of Sport. High-fat exercise group (F + Ex group, n = 8) and low-fat control group (L + Ctr group, n = 8), high-fat control group (F + Ctr group, n = 8), and high-fat exercise group and high-fat control group were fed a high fat and high sugar diet (60% kcal fat, 20% kcal protein, 20% kcal carbohydrate, based on OpenSource Diets No. D12492) from the first 8 weeks and maintained for 16 weeks to establish diabetic cardiomyopathy mice. The diagnosis of diabetic cardiomyopathy was determined by glucose tolerance test and cardiac ultrasonography. The low-fat control group was fed with a normal diet. All mice were free to eat and drink. The incubation temperature was maintained at 21°C–24°C and the relative humidity was 40%–60%. All operations strictly abide by the Shanghai Institute of Physical Education Ethics Committee requirements.

Exercise program

According to Sigal and Kenny[9] and other clinical research on the treatment of type 2 diabetes, aerobic and antiresistance exercise were used for exercise intervention. The high-fat exercise group was exercised at intervals of one day of aerobic exercise and 1 day of resistance exercise, 6 days a week, for a total of 8 weeks.

Before the start of the experiment, 3–5 days of exercise preconditioning, exercise intensity gradually increased, where the speed does not exceed 20 m/min. Time of exercise gradually increased from 30 to 60 m/min. Antiresistance sports ladder gradient gradually increased from 30° to 90°, from the beginning of the two groups, each group twice, gradually increased to four groups, each group four times. Aerobic exercise consisted of treadmill movement, time 60 ± 5 min, speed and deceleration process, the slope is 0.5 min before the start of the movement, the speed gradually increased to 15 m/min, after the end of the movement, the speed from high to low, slow down until the speed is 0. Resistance movement: Ladder movement, each four groups, each group four times, the slope of 90°, the speed is not limited. Each training from 6 to 7 pm. Sound, light, electricity, and other stimulus means are not used to stress mice.

Glucose tolerance test

The mice were subjected to a glucose tolerance test after 16 weeks of diet intervention. The mice were fasted for 12 h before administering glucose tolerance test. After intraperitoneal injection of glucose (2 g/kg body weight), blood samples were taken from the tail of the mice for 0, 30, 60, 90, and 120 min timepoints for glucose tolerance test. Tolerance assessment was based on the area under the glucose tolerance curve (area under the curve [AUC]).

Cardiac ultrasound

We measured the cardiac structure and function of mice by Visual Sonics Vevo1100 imaging system. Measurements include left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), ejection fraction, stroke volume (SV), mitral valvular diastolic E peak blood flow velocity (E), A peak velocity and ratio (E/A), and myocardial comprehensive index (MPI).

Statistical processing

All data were expressed as mean ± standard deviation. Statistical data were analyzed by the Statistical Package for Social Sciences, version 23.0 software (SPSS, Chicago, IL, USA). Single factor analysis of variance was used to compare the data. P < 0.05 for a significant difference, P < 0.01 was very significant difference.


  Results Top


The results of echocardiography in mice at 24 weeks are listed in [Table 1].
Table 1: The results of echocardiography in mice at 24 weeks

Click here to view


After 30 min of glucose injection, all mice had elevated blood glucose levels. The mice reached their maximum at 30 min and began to drop after 30 min. The blood glucose of the F + Ctr mice was higher than the other two groups and showed significant difference at 0, 60, 90, and 120 min timepoints. Compared with low-fat group and high-fat group, the area under 0, 60, 90, and 120 min timepoints and intraperitoneal glucose tolerance test blood glucose curve (AUC) were significantly different (P< 0.01) [Figure 1].
Figure 1: (a) Mice were injected with glucose, 0, 30, 60, 90, and 120 min of blood glucose. (b) The area under the glucose tolerance curve.###P < 0.01 (L + Ctr vs. F + Ctr); **P < 0.01 (F + Ctr vs. F + Ex)

Click here to view


The results of echocardiography showed that the diastolic function of the heart, blood flow peak velocities of the early peak at the mitral valve in the high-fat exercise group, and the low-fat control group were significantly higher than that in the high-fat control group (709.73 ± 45.48 mm/s vs. 441.51 ± 44.83 mm/s, 632.92 ± 27.22 mm/s vs. 441.51 ± 44.83 mm/s, P < 0.01). The peak velocities of the atrial peak at the mitral valve were significantly lower than the high-fat control group (443.77 ± 18.09 mm/s vs. 523.67 ± 57.87 mm/s, 460.36 ± 18.24 mm/s vs. 523.67 ± 57.87 mm/s, P < 0.05), and the ratio of E/A was significantly higher than that of the high-fat control group (1.63 ± 0.06 vs. 0.85 ± 0.045, 1.38 ± 0.072 vs. 0.85 ± 0.045, P < 0.01) [Figure 2].
Figure 2: Mitral valvular diastolic E peak blood flow velocity (E), A peak velocity and ratio (E/A) in three groups. (a) A peak, (b) E peak, (c) E/A ratio.#P < 0.05,##P < 0.01(L + Ctr vs. F + Ctr). *P < 0.05, **P < 0.01 (F + Ctr vs. F + Ex)

Click here to view


The left ventricular eject fraction (LVEF) in the high-fat exercise group and the low-fat control group was significantly higher than that of the high-fat control group (68.99% ± 2.04% vs. 60.41 ± 2.31%, 66.16% ± 2.12% vs. 60.41 ± 2.31%, P < 0.01). There was no significant difference in LVEDD, LVESD, and SV between the high-fat exercise group and the low-fat control group, when compared with the high-fat control group [Figure 3].
Figure 3: Left ventricular end-diastolic dimension, left ventricular end-systolic dimension, ejection fraction, and stroke volume in three groups. (a) Ejection fraction, (b) cardiac output, (c) left ventricular end-diastolic dimension, and (d) left ventricular end-systolic dimension in all groups after 24 weeks.#P < 0.05,##P < 0.01 (L + Ctr vs. F + Ctr); *P < 0.05, **P < 0.01 (F + Ctr vs. F + Ex)

Click here to view


Myocardial performance index (MPI) decreased (0.86 ± 0.095 vs. 0.97 ± 0.091, 0.88 ± 0.073 vs. 0.97 ± 0.091, P > 0.05), but there was no significant difference [Figure 4].
Figure 4: Myocardial performance index in three groups (F + Ex vs. F + Ctr: 0.86 ± 0.095 vs. 0.97 ± 0.091, P > 0.05); (L + Ctr vs. F + Ctr: 0.88 ± 0.073 vs. 0.97 ± 0.091, P > 0.05)

Click here to view



  Discussion Top


The study found that high-fat and high-sugar diet-induced diabetic cardiomyopathy in mice decreased left ventricular function, especially in the diastolic function. Diastolic function can significantly improve by means of 8 weeks of aerobic and resistive exercise intervention.

Previous animal studies used intraperitoneal injection of streptozotocin to develop a diabetic cardiomyopathy model.[10] Long-term high-fat diet may induce the formation of early diabetic cardiomyopathy.[11] The 16-week high-fat diet could induce insulin resistance and increase the supply of exogenous fat, making the weight of the mice in the high-fat control group significantly higher than the low-fat control group. Similarly, the blood glucose of the high-fat control group maintained a stable high level of state. Fasting blood glucose was significantly higher in the low-fat control group. Echocardiographic results showed that the E peak and E/A ratio in the high-fat control group were significantly lower than in the low-fat control group. A peak was significantly higher than in the low control group, and E, E/A, and A are the indicators of left ventricular diastolic function, suggesting that high-fat quiet mice had left ventricular diastolic dysfunction.

The study of diabetic cardiomyopathy prevention and treatment often involves drug therapy, gene therapy, and exercise as forms of intervention. Although much progress has been made in drug therapy as a form of treatment,[12],[13] a specific and effective drug for diabetic cardiomyopathy has not been found. In addition, the cost of drug therapy is expensive and often has side effects as a result.[14],[15]

While gene therapy is under development and takes time to apply in a clinical setting, exercise intervention is considered a relatively cost-effective treatment and presents significant benefits. The benefits of exercise as treatment of type 2 diabetes have been recognized[16] which includes the improvement of cardiac function. 2007 Neil Smart[17] has demonstrated that exercise can improve diastolic function. In this study selected participants have completed three aerobic exercises a week which intensity at 60% -70% in 16 weeks. Participants have completed three aerobic exercises a week at 60%–70% intensity. After 8 weeks, the participants also did strength training. Results showed that the diastolic function of participants improved.

Mice in the high-fat exercise group had higher E peak and E/A ratio than those in high-fat control group. In respect to the systolic function of the heart, the LVEF in the high-fat exercise group was significantly higher than in the high-fat control group. However, there was no significant difference in LVEDD, LVESD, and SV between the high-fat exercise group and the low-fat control group compared with the high-fat control group.

A large number of reports confirm that exercise may improve insulin sensitivity and reduce intracellular fatty acid content. Exercise can also improve damaged myocardial function, which plays an important role in the treatment of diabetic cardiomyopathy.[18],[19]

However, there are very few clinical studies which show on the impact of exercise on diabetes cardiomyopathy. In 2004, Korte et al.[20] prepared a 20-week porcine model of diabetic dyslipidemia. Some of the pigs were subjected to 14 weeks of exercise training. The results of echocardiography showed that the left ventricular shortening fraction in the exercise group was significantly higher than in the control group. They believed that exercise may improve the heart function of diabetic cardiomyopathy. Wang et al.[21] utilized a long-term exercise intervention in mice and found that exercise has a beneficial effect on the treatment of diabetic cardiomyopathy by reducing myocardial cell apoptosis and fibrosis, improving mitochondrial biogenesis.

At present, the effect of different exercise intensity on the cardiac function of diabetic cardiomyopathy is not clear. Previous studies have used moderately intensive exercise to prevent dystrophic dysfunction in diabetic cardiomyopathy by restoring the mitochondrial function of mice in db/db (leptin receptor gene deficiency-induced obese spontaneous type 2 diabetic mice).[22]

Hafstad et al. utilized differing intensities of exercise interventions on diet-induced obese C57 mice to confirm whether high-intensity intermittent training is superior to moderate-intensity training in counteracting obesity-induced impairment of left ventricular performance and function. They found that both exercise programs could improve cardiac function and structural remodeling caused by obesity.[23] It was also found that low-intensity swimming training can reduce the pathological changes of cardiomyocyte systolic dysfunction in diabetic rats. Diabetic cardiomyopathy is closely related to cardiac remodeling, myocardial dysfunction, and low inflammation in type 2 diabetes mellitus.[24]

At present, exercise intensity and time in patients with diabetic cardiomyopathy still present controversy. In the future, this study will also investigate the effects of different exercise time on diabetes mellitus, which will provide the theoretical basis for patients with clinical diabetic cardiomyopathy to select the best exercise regimen.


  Conclusion Top


The above data indicates that 8 weeks of exercise intervention can improve diastolic heart function of mice with diabetic cardiomyopathy. In addition, improvement in left ventricular systolic function was found, but there is no statistical difference. Overall, the study suggests that exercise intervention has beneficial therapeutic effects on diabetic cardiomyopathy.

Financial support and sponsorship

This work was financially supported by the grant from Science and Technology Development Fund of Science and technology committee of Shanghai Pudong (Grant No.: PKJ2011-Y38).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Spillmann F, Van Linthout S, Tschöpe C. Cardiac effects of HDL and its components on diabetic cardiomyopathy. Endocr Metab Immune Disord Drug Targets 2012;12:132-47.  Back to cited text no. 1
    
2.
Abe T, Ohga Y, Tabayashi N, Kobayashi S, Sakata S, Misawa H, et al. Left ventricular diastolic dysfunction in type 2 diabetes mellitus model rats. Am J Physiol Heart Circ Physiol 2002;282:H138-48.  Back to cited text no. 2
    
3.
Colberg SR, Sigal RJ, Yardley JE, Riddell MC, Dunstan DW, Dempsey PC, et al. Physical activity/Exercise and diabetes: A Position statement of the American diabetes association. Diabetes Care 2016;39:2065-79.  Back to cited text no. 3
    
4.
Pedersen BK. The anti-inflammatory effect of exercise: Its role in diabetes and cardiovascular disease control. Essays Biochem 2006;42:105-17.  Back to cited text no. 4
    
5.
Le Page LM, Rider OJ, Lewis AJ, Ball V, Clarke K, Johansson E, et al. Increasing pyruvate dehydrogenase flux as a treatment for diabetic cardiomyopathy: A Combined 13C hyperpolarized magnetic resonance and echocardiography study. Diabetes 2015;64:2735-43.  Back to cited text no. 5
    
6.
Korkmaz-Icöz S, Al Said S, Radovits T, Li S, Brune M, Hegedűs P, et al. Oral treatment with a zinc complex of acetylsalicylic acid prevents diabetic cardiomyopathy in a rat model of type-2 diabetes: Activation of the Akt pathway. Cardiovasc Diabetol 2016;15:75.  Back to cited text no. 6
    
7.
Drew BG, Duffy SJ, Formosa MF, Natoli AK, Henstridge DC, Penfold SA, et al. High-density lipoprotein modulates glucose metabolism in patients with type 2 diabetes mellitus. Circulation 2009;119:2103-11.  Back to cited text no. 7
    
8.
Hafstad AD, Boardman N, Aasum E. How exercise may amend metabolic disturbances in diabetic cardiomyopathy. Antioxid Redox Signal 2015;22:1587-605.  Back to cited text no. 8
    
9.
Sigal RJ, Kenny GP. Combined aerobic and resistance exercise for patients with type 2 diabetes. JAMA 2010;304:2298-9.  Back to cited text no. 9
    
10.
Zhang CH, Zang WJ, Xu J, Yu Xj, Lv J, Jing AY, et al. A method to produce the animal model of diabetic cardiomyopathy. Journal of hygiene research 2006;35:707-711. [In Chinese].  Back to cited text no. 10
    
11.
Qin F, Siwik DA, Luptak I, Hou X, Wang L, Higuchi A, et al. The polyphenols resveratrol and S17834 prevent the structural and functional sequelae of diet-induced metabolic heart disease in mice. Circulation 2012;125:1757-64, S1-6.  Back to cited text no. 11
    
12.
Madiraju AK, Erion DM, Rahimi Y, Zhang XM, Braddock DT, Albright RA, et al. Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase. Nature 2014;510:542-6.  Back to cited text no. 12
    
13.
Johannes CB, Koro CE, Quinn SG, Cutone JA, Seeger JD. The risk of coronary heart disease in type 2 diabetic patients exposed to thiazolidinediones compared to metformin and sulfonylurea therapy. Pharmacoepidemiol Drug Saf 2007;16:504-12.  Back to cited text no. 13
    
14.
Zhu J, Ning RB, Lin XY, Chai DJ, Xu CS, Xie H, et al. Retinoid X receptor agonists inhibit hypertension-induced myocardial hypertrophy by modulating LKB1/AMPK/p70S6K signaling pathway. Am J Hypertens 2014;27:1112-24.  Back to cited text no. 14
    
15.
Calligaris SD, Lecanda M, Solis F, Ezquer M, Gutiérrez J, Brandan E, et al. Mice long-term high-fat diet feeding recapitulates human cardiovascular alterations: An animal model to study the early phases of diabetic cardiomyopathy. PLoS One 2013;8:e60931.  Back to cited text no. 15
    
16.
Pieri BL, Souza DR, Luciano TF, Marques SO, Pauli JR, Silva AS, et al. Effects of physical exercise on the P38MAPK/REDD1/14-3-3 pathways in the myocardium of diet-induced obesity rats. Horm Metab Res 2014;46:621-7.  Back to cited text no. 16
    
17.
Smart N, Haluska B, Jeffriess L, Marwick TH. Exercise training in systolic and diastolic dysfunction: Effects on cardiac function, functional capacity, and quality of life. Am Heart J 2007;153:530-6.  Back to cited text no. 17
    
18.
Medeiros C, Frederico MJ, da Luz G, Pauli JR, Silva AS, Pinho RA, et al. Exercise training reduces insulin resistance and upregulates the mTOR/p70S6k pathway in cardiac muscle of diet-induced obesity rats. J Cell Physiol 2011;226:666-74.  Back to cited text no. 18
    
19.
Perseghin G, Lattuada G, De Cobelli F, Ragogna F, Ntali G, Esposito A, et al. Habitual physical activity is associated with intrahepatic fat content in humans. Diabetes Care 2007;30:683-8.  Back to cited text no. 19
    
20.
Korte FS, Mokelke EA, Sturek M, McDonald KS. Exercise improves impaired ventricular function and alterations of cardiac myofibrillar proteins in diabetic dyslipidemic pigs. J Appl Physiol (1985) 2005;98:461-7.  Back to cited text no. 20
    
21.
Wang H, Bei Y, Lu Y, Sun W, Liu Q, Wang Y, et al. Exercise prevents cardiac injury and improves mitochondrial biogenesis in advanced diabetic cardiomyopathy with PGC-1α and Akt activation. Cell Physiol Biochem 2015;35:2159-68.  Back to cited text no. 21
    
22.
Veeranki S, Givvimani S, Kundu S, Metreveli N, Pushpakumar S, Tyagi SC, et al. Moderate intensity exercise prevents diabetic cardiomyopathy associated contractile dysfunction through restoration of mitochondrial function and connexin 43 levels in db/db mice. J Mol Cell Cardiol 2016;92:163-73.  Back to cited text no. 22
    
23.
Hafstad AD, Lund J, Hadler-Olsen E, Höper AC, Larsen TS, Aasum E, et al. High- and moderate-intensity training normalizes ventricular function and mechanoenergetics in mice with diet-induced obesity. Diabetes 2013;62:2287-94.  Back to cited text no. 23
    
24.
da Silva E, Natali AJ, da Silva MF, Gomes Gde J, da Cunha DN, Toledo MM, et al. Swimming training attenuates the morphological reorganization of the myocardium and local inflammation in the left ventricle of growing rats with untreated experimental diabetes. Pathol Res Pract 2016;212:325-34.  Back to cited text no. 24
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed555    
    Printed82    
    Emailed0    
    PDF Downloaded94    
    Comments [Add]    

Recommend this journal