Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 
Print this page Email this page Users Online: 51

  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 13  |  Issue : 2  |  Page : 156-160  

The effect of sitagliptin on hepatic ischemic reperfusion injury in rats


1 Pathophysiology Department, Basic Faculty, Pyongyang Medical College, Kim Il Sung University, Democratic People's Republic of Korea
2 Pathophysiology Department, Graduate School, Pyongyang Medical College, Kim Il Sung University, Pyongyang, Democratic People's Republic of Korea

Date of Submission18-Sep-2018
Date of Decision14-Aug-2019
Date of Acceptance03-Sep-2019
Date of Web Publication28-Feb-2020

Correspondence Address:
Hye-Sun Hong
Graduate School, Pyongyang Medical College, Kim Il Sung University, Pyongyang
Democratic People's Republic of Korea
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mjdrdypu.mjdrdypu_155_18

Rights and Permissions
  Abstract 


Background: Dipeptidyl peptidase-4 (DPP4, DPPIV, CD26, EC 3.4.14.5) was found out more than four decades ago as a serine protease that severs N-terminal dipeptides from peptide substrates. DPP-4 inhibitors have been used in many animal models of lung and heart illness, in which injury was obtained by an ischemic attack followed by the following reperfusion. Here, we present the large body of experimental study that now gives irresistible evidence for the useful impact of DPP-4 targeting in ischemia/reperfusion injury. In this study, we discuss the effect of DPP-4 inhibitor (Sitagliptin) on DPP-4 expression in the rat model. Materials and Methods: We made a rat model of liver ischemia (90 min)-reperfusion (180 min), collected blood and liver samples after reperfusion. The possible inhibitory effect of Sitagliptin on DPP-4 in a rat model of hepatic ischemia-reperfusion (IR) damage was evaluated. Hepatic malondialdehyde (MDA) levels were evaluated spectrophotometrically to know the degree of oxidizing reaction in the liver. We evaluated the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in the model. We used hematoxylin and eosin (H and E) staining to remark the change of liver morphologically. Results: Significantly, the expression of DPP-4 levels was declined after treatment with Sitagliptin in the IR group. MDA, TNF-α, and IL-6 levels were significantly increased in the IR group but decreased in the groups treated with Sitagliptin, 5 mg/kg. H and E staining show exact edema and necrosis were remarked in the IR group, but in the Sitagliptin pretreatment group, they were decreased. Conclusion: The study showed that pretreatment with Sitagliptin might inhibit DPP-4 activation and reduce hepatic IR damage.

Keywords: Dipeptidyl peptidase-4, dipeptidyl peptidase-4 inhibitor, hepatic ischemia-reperfusion injury, sitagliptin


How to cite this article:
Mun SC, Hong HS. The effect of sitagliptin on hepatic ischemic reperfusion injury in rats. Med J DY Patil Vidyapeeth 2020;13:156-60

How to cite this URL:
Mun SC, Hong HS. The effect of sitagliptin on hepatic ischemic reperfusion injury in rats. Med J DY Patil Vidyapeeth [serial online] 2020 [cited 2020 Jul 14];13:156-60. Available from: http://www.mjdrdypv.org/text.asp?2020/13/2/156/279623




  Introduction Top


Dipeptidyl peptidase-4 (DPP-4) is a membrane-associated peptidase and this is known as CD26. DPP-4 is widely spread in organs throughout the body and presents pleiotropic effects by its peptidase activity.[1],[2],[3],[4] It is connected with immune stimulation, combining to and degradation of the extracellular matrix, resistance to anti-cancer agents, and lipid accumulation.[5],[6],[7],[8] In the liver, DPP-4 is presented to a high degree, and recent accumulation shows that DPP-4 is connected with the development of various chronic liver diseases including hepatitis C virus infection, nonalcoholic fatty liver disease,[9],[10] and hepatocellular carcinoma.[11],[12] In addition, DPP-4 is involved in hepatic stem cells and plays an important role in hepatic regeneration.[8]

Liver ischemia-reperfusion injury (IRI) is observed condition, which is caused by restoring blood supply after ischemia in the liver which involved a series of pathophysiological processes, such as radical generation, neutrophil infiltration, and release of inflammatory mediators. Liver surgery often needs clamping of the portal triad, reducing intraoperative blood loss, and is necessary to cause liver IRI which can increase postoperative liver insufficiency and even liver failure.[13],[14]

However, there are no data connected oxidative injury and inflammation reaction with DPP-4 expression and effect of DPP-4 inhibitor Sitagliptin in liver IRI in vivo. In our study, we present the relation of oxidative injury and inflammation reaction with DPP4 expression and the effect of DPP-4 inhibitor (Sitagliptin) on DPP-4 expression in liver IRIin vivo in rat model 227.


  Materials and Methods Top


Animals

Male SD rats (200–250 g) were obtained from the Laboratory Animal Center of Kim Il Sung University Pyongyang Medical College. Animals were fed a standard rodent diet and water, and bred in a controlled environment with 12 h light–dark cycles. All animal procedures were approved by the Institutional Animal Care Committee and conducted in accordance with the Kim Il Sung University Pyongyang Medical College Guidelines for the Care and Use of Laboratory Animals.

Liver ischemia-reperfusion injury model

We used an established rat model of hepatic IRI, as described previously.[13],[14] Briefly, rats were anesthetized with isoflurane and injected with heparin (100 U/kg), and an atraumatic clip was used to interrupt the artery and portal venous blood supply to the left and middle liver lobes. After 90 min of hepatic ischemia, the clamp was removed to generate hepatic reperfusion. Rats were sacrificed 180 min after reperfusion for tissue and plasma collection. To evaluate the role of DPP-4 inhibitor, rats were pretreated with 5 mg/kg of Sitagliptin at 20 min before the ischemia insult. Sham rat underwent the same procedure but without vascular occlusion (n = 10).

Serum levels of alanine aminotransferase and aspartate aminotransferase

The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are one index of hepatocyte injury. A standard automatic analyzer (Hitachi 7600–10, Hitachi High-Technologies Corporation, Japan) was used to determine the serum levels of ALT and AST.

Liver malondialdehyde levels

Hepatic malondialdehyde (MDA) levels were evaluated spectrophotometrically to evaluate the degree of oxidizing reaction in the liver as previously described.[15] The absorbance of the upper layer was read at 532 nm with a spectrophotometer (Kadas 100, Dr. Lange, AG Zurich, Zurich, Switzerland), and the results expressed as nanomoles of MDA per liter of wet liver tissue.

Tumor necrosis factor-α and interleukin-6 in the liver

Tumor necrosis factor-α (TNF-α) concentration in serum and mesenteric lymph was determined by using rat TNF-enzyme-linked immunoabsorbent assay kit (LIFEKEY Biotech, Co., USA) according to the manufacturer's protocol. Interleukin-6 (IL-6) levels were evaluated by IL-enzyme-linked immunosorbent assay according to the manufacturer's protocol (Adlitteram Diagnostic Laboratories). One single treatment was performed on four individual wells.

Histology

Formalin-fixed, paraffin-embedded rat liver specimens were sectioned at 4 μm and stained with hematoxylin and eosin. Liver sections from the left lobe were fixed in 4% formalin. After formalin fixation, the specimens were embedded in paraffin. Sections (4 μm) were cut, air dried and fixed in acetone. Liver sections were stained with hematoxylin (Muto Pure Chemicals, Tokyo, Japan) and eosin (Wako, Osaka, Japan). The sections were used for histopathologic examinations by light microscopy (×100).

Statistical analysis

Statistical analysis was performed using the SPSS software, version 14.0 (SPSS Inc., Chicago, Ill, USA). Results are expressed as means and standard deviations. Parameters were analyzed using Student's t-test. For the above parameters, P < 0.05 was considered to be statistically significant.


  Results Top


Serum alanine aminotransferase and aspartate aminotransferase levels

The levels of ALT and AST were significantly increased in the IR group (control group) but significantly decreased in groups pretreated with 5 mg/kg Sitagliptin [Figure 1].
Figure 1: The serum level of alanine aminotransferase and alanine asparaginic acid (aspartate aminotransferase) in the sham group, ischemia-reperfusion group (control group), and groups pretreated with 5 mg/kg concentrations of Sitagliptin. The alanine aminotransferase and aspartate aminotransferase levels in the ischemia-reperfusion group were significantly increased, but significantly decreased in groups pretreated with 5 mg/kg Sitagliptin. *P < 0.05

Click here to view


Malondialdehyde, tumor necrosis factor-α, and interleukin-6 levels in the liver

The levels of MDA were significantly increased in the IR group (control group) but significantly decreased in groups pretreated with 5 mg/kg Sitagliptin [Figure 2]. The TNF-α and IL-6 levels in the IR group were significantly increased but significantly decreased in groups pretreated with 5 mg/kg Sitagliptin [Figure 3].
Figure 2: The levels of malondialdehyde in sham group, ischemia-reperfusion group, and 5 mg/kg concentration of Sitagliptin pretreatment groups. **P < 0.01

Click here to view
Figure 3: Serum tumor necrosis factor-α (a) and interleukin-6 (b) in the sham group, ischemia-reperfusion group (control group), and groups pretreated with 5 mg/kg concentrations of Sitagliptin. **P < 0.01, ***P < 0.001

Click here to view


Histological changes

Apparent edema and necrosis were observed in the IR group [Figure 4]b compared to sham group [Figure 4]a. In the Sitagliptin pretreatment group, edema and necrosis in IR modes were reduced. Disrupted lobular architecture and apparent edema were observed in the Sitagliptin group [Figure 4]c.
Figure 4: Representative hematoxylin and eosin staining in the sham group (a), ischemia-reperfusion group (b), and Sitagliptin pretreatment group (c) at 180 min after reperfusion

Click here to view



  Discussion Top


Recently, researchers use partial hepatic ischemia models of rats rather than total hepatic ischemia models and this is because the total ischemia models in liver frequently have hypotension, systemic vascular congestion, and also high mortality.[16] Therefore, in this study, we choose a partial ischemia model to derive hepatic IRI.

It is clear that DPP-4/DPPIV/CD26 cleaves off N-terminal dipeptides from peptides with preferably proline or alanine at the penultimate position.[17] Many DPP-4 inhibitors such as sitagliptin, vildagliptin, saxagliptin, and linagliptin are available for the treatment of Type 2 diabetes. Their pharmacological action is based on the reduced cleavage of incretin hormone glucagon-like peptide-1 by DPP-4, preserving the insulinotropic action of this peptide.[18] Recently, many studies have done regarding DPP-4 inhibitors for their applicability in other conditions pathologically, both in animal studies and in clinical settings.[19]

IRI is characterized by an initial restriction of blood supply to an organ and it is followed by the subsequent reperfusion with concomitant reoxygenation. During ischemia, tissue hypoxia is caused by the severe imbalance of metabolic supply and demand. Restoration of the blood flow and reoxygenation is often accompanied by an exacerbation of tissue damage and profound inflammatory response.[18] IRI is connected with modified local cytokine/chemokine secretion patterns, increased neutrophil recruitment, free-radical accumulation, lipid peroxidation, and impairment of functional and structural integrity of the organ.[13] The study showed that the content of MDA, TNF-α, and IL-6 in the liver tissue are increased in hepatic IRI model than in the normal one and they were decreased by the injection of Sitagliptin, one of the DPP4 inhibitors. The relevance of DPP4 as a target in IRI has been presented in several animal studies, mostly myocardial infarction[13],[15],[20],[21],[22],[23],[24],[25],[26],[27],[28],[29] and experimental lung Tx,[30],[31],[32],[33],[34] either using DPP4 inhibitor treatment or DPP4 knock out animals. Apart from these animal studies, in patients with coronary artery disease,[35] one study in humans showed cardioprotection by sitagliptin. Another research reported a reduction of the infarct size after myocardial IRI on DPP4 inhibitor treatment.[28] The renal IRI studies were either performed in diabetic[36] or nondiabetic animals,[37] both showing a reduction in serum creatinine levels on DPP4 inhibition. Sauvé et al. discovered a decrease of mortality both in DPP-4 and sitagliptin-treated mice.[22] DPP4 inhibitors have capable ability to protect the heart, kidney, and lungs against IRI in preclinical models.

There are a few data that is related to DPP-4 in the liver model of IRI. The DPP4 expression is increased in the model of liver IRI, resulting in an increase of oxidative procedure and inflammation morphologic change in the liver tissue. These changes were clearly reduced by Sitagliptin, which is known to be one of the DPP-4 inhibitors. These demonstrated that Sitagliptin reduced the content of MDA, TNF-α, and IL-6 and also improved the pathophysiologic findings in liver tissue, inhibiting the expression of DPP4.

In this study, we presented that pretreatment with DPP4 inhibitor Sitagliptin results in reduced MDA, TNF-α, and IL-6 production in hepatic IRI in vivo and this is consistent with previous studies.[38] In addition, we also demonstrated that pretreatment with Sitagliptin results in significantly reduced proinflammatory cytokine production in hepatic IRI modelsin vivo and this is supporting that Sitagliptin might promote anti-inflammatory by inhibiting DPP-4 in vivo.

Our data clearly show that Sitagliptin may inhibit expression of DPP-4 in hepatic IR. In addition, we conclude that targeting DPP-4 represents a useful approach to promoting hepatic IRI. These results give the rationale for promoted approaches to decline hepatic IRI.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Hopsu-Havu VK, Glenner GG. A new dipeptide naphthylamidase hydrolyzing glycyl-prolyl-beta-naphthylamide. Histochemie 1966;7:197-201.  Back to cited text no. 1
    
2.
Misumi Y, Hayashi Y, Arakawa F, Ikehara Y. Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface. Biochim Biophys Acta 1992;1131:333-6.  Back to cited text no. 2
    
3.
Kameoka J, Tanaka T, Nojima Y, Schlossman SF, Morimoto C. Direct association of adenosine deaminase with a T cell activation antigen, CD26. Science 1993;261:466-9.  Back to cited text no. 3
    
4.
Morrison ME, Vijayasaradhi S, Engelstein D, Albino AP, Houghton AN. A marker for neoplastic progression of human melanocytes is a cell surface ectopeptidase. J Exp Med 1993;177:1135-43.  Back to cited text no. 4
    
5.
Heike M, Möbius U, Knuth A, Meuer S, Meyer zum Büschenfelde KH. Tissue distribution of the T cell activation antigen ta1. Serological, immunohistochemical and biochemical investigations. Clin Exp Immunol 1988;74:431-4.  Back to cited text no. 5
    
6.
Gorrell MD, Gysbers V, McCaughan GW. CD26: A multifunctional integral membrane and secreted protein of activated lymphocytes. Scand J Immunol 2001;54:249-64.  Back to cited text no. 6
    
7.
Dinjens WN, ten Kate J, Wijnen JT, van der Linden EP, Beek CJ, Lenders MH, et al. Distribution of adenosine deaminase-complexing protein in murine tissues. J Biol Chem 1989;264:19215-20.  Back to cited text no. 7
    
8.
Mentzel S, Dijkman HB, Van Son JP, Koene RA, Assmann KJ. Organ distribution of aminopeptidase A and dipeptidyl peptidase IV in normal mice. J Histochem Cytochem 1996;44:445-61.  Back to cited text no. 8
    
9.
Miyazaki M, Kato M, Tanaka K, Tanaka M, Kohjima M, Nakamura K, et al. Increased hepatic expression of dipeptidyl peptidase-4 in non-alcoholic fatty liver disease and its association with insulin resistance and glucose metabolism. Mol Med Rep 2012;5:729-33.  Back to cited text no. 9
    
10.
Balaban YH, Korkusuz P, Simsek H, Gokcan H, Gedikoglu G, Pinar A, et al. Dipeptidyl peptidase IV (DDP IV) in NASH patients. Ann Hepatol 2007;6:242-50.  Back to cited text no. 10
    
11.
Itou M, Kawaguchi T, Taniguchi E, Sumie S, Oriishi T, Mitsuyama K, et al. Altered expression of glucagon-like peptide-1 and dipeptidyl peptidase IV in patients with HCV-related glucose intolerance. J Gastroenterol Hepatol 2008;23:244-51.  Back to cited text no. 11
    
12.
Harada T, Kim DW, Sagawa K, Suzuki T, Takahashi K, Saito I, et al. Characterization of an established human hepatoma cell line constitutively expressing non-structural proteins of hepatitis C virus by transfection of viral cDNA. J Gen Virol 1995;76(Pt 5):1215-21.  Back to cited text no. 12
    
13.
Eltzschig HK, Eckle T. Ischemia and reperfusion – From mechanism to translation. Nat Med 2011;17:1391-401.  Back to cited text no. 13
    
14.
Zwacka RM, Zhou W, Zhang Y, Darby CJ, Dudus L, Halldorson J, et al. Redox gene therapy for ischemia/reperfusion injury of the liver reduces AP1 and NF-kappaB activation. Nat Med 1998;4:698-704.  Back to cited text no. 14
    
15.
Chinda K, Palee S, Surinkaew S, Phornphutkul M, Chattipakorn S, Chattipakorn N, et al. Cardioprotective effect of dipeptidyl peptidase-4 inhibitor during ischemia-reperfusion injury. Int J Cardiol 2013;167:451-7.  Back to cited text no. 15
    
16.
Düşünceli F, Işeri SO, Ercan F, Gedik N, Yeǧen C, Yeǧen BC, et al. Oxytocin alleviates hepatic ischemia-reperfusion injury in rats. Peptides 2008;29:1216-22.  Back to cited text no. 16
    
17.
Lambeir AM, Durinx C, Scharpé S, De Meester I. Dipeptidyl-peptidase IV from bench to bedside: An update on structural properties, functions, and clinical aspects of the enzyme DPP IV. Crit Rev Clin Lab Sci 2003;40:209-94.  Back to cited text no. 17
    
18.
Deacon CF. Dipeptidyl peptidase-4 inhibitors in the treatment of type 2 diabetes: A comparative review. Diabetes Obes Metab 2011;13:7-18.  Back to cited text no. 18
    
19.
Lambeir AM, Scharpé S, De Meester I. DPP4 inhibitors for diabetes – What next? Biochem Pharmacol 2008;76:1637-43.  Back to cited text no. 19
    
20.
Zaruba MM, Theiss HD, Vallaster M, Mehl U, Brunner S, David R, et al. Synergy between CD26/DPP-IV inhibition and G-CSF improves cardiac function after acute myocardial infarction. Cell Stem Cell 2009;4:313-23.  Back to cited text no. 20
    
21.
Post S, Smits AM, van den Broek AJ, Sluijter JP, Hoefer IE, Janssen BJ, et al. Impaired recruitment of HHT-1 mononuclear cells to the ischaemic heart is due to an altered CXCR4/CD26 balance. Cardiovasc Res 2010;85:494-502.  Back to cited text no. 21
    
22.
Sauvé M, Ban K, Momen MA, Zhou YQ, Henkelman RM, Husain M, et al. Genetic deletion or pharmacological inhibition of dipeptidyl peptidase-4 improves cardiovascular outcomes after myocardial infarction in mice. Diabetes 2010;59:1063-73.  Back to cited text no. 22
    
23.
Ye Y, Keyes KT, Zhang C, Perez-Polo JR, Lin Y, Birnbaum Y, et al. The myocardial infarct size-limiting effect of sitagliptin is PKA-dependent, whereas the protective effect of pioglitazone is partially dependent on PKA. Am J Physiol Heart Circ Physiol 2010;298:H1454-65.  Back to cited text no. 23
    
24.
Zhang D, Huang W, Dai B, Zhao T, Ashraf A, Millard RW, et al. Genetically manipulated progenitor cell sheet with diprotin A improves myocardial function and repair of infarcted hearts. Am J Physiol Heart Circ Physiol 2010;299:H1339-47.  Back to cited text no. 24
    
25.
Huisamen B, Genis A, Marais E, Lochner A. Pre-treatment with a DPP-4 inhibitor is infarct sparing in hearts from obese, pre-diabetic rats. Cardiovasc Drugs Ther 2011;25:13-20.  Back to cited text no. 25
    
26.
Ku HC, Chen WP, Su MJ. DPP4 deficiency preserves cardiac function via GLP-1 signaling in rats subjected to myocardial ischemia/reperfusion. Naunyn Schmiedebergs Arch Pharmacol 2011;384:197-207.  Back to cited text no. 26
    
27.
Theiss HD, Vallaster M, Rischpler C, Krieg L, Zaruba MM, Brunner S, et al. Dual stem cell therapy after myocardial infarction acts specifically by enhanced homing via the SDF-1/CXCR4 axis. Stem Cell Res 2011;7:244-55.  Back to cited text no. 27
    
28.
Yin M, Silljé HH, Meissner M, van Gilst WH, de Boer RA. Early and late effects of the DPP-4 inhibitor vildagliptin in a rat model of post-myocardial infarction heart failure. Cardiovasc Diabetol 2011;10:85.  Back to cited text no. 28
    
29.
Hocher B, Sharkovska Y, Mark M, Klein T, Pfab T. The novel DPP-4 inhibitors linagliptin and BI 14361 reduce infarct size after myocardial ischemia/reperfusion in rats. Int J Cardiol 2013;167:87-93.  Back to cited text no. 29
    
30.
Zhai W, Cardell M, De Meester I, Augustyns K, Hillinger S, Inci I, et al. Ischemia/reperfusion injury: The role of CD26/dipeptidyl-peptidase-IV-inhibition in lung transplantation. Transplant Proc 2006;38:3369-71.  Back to cited text no. 30
    
31.
Zhai W, Cardell M, De Meester I, Augustyns K, Hillinger S, Inci I, et al. Intragraft DPP IV inhibition attenuates post-transplant pulmonary ischemia/reperfusion injury after extended ischemia. J Heart Lung Transplant 2007;26:174-80.  Back to cited text no. 31
    
32.
Zhai W, Jungraithmayr W, De Meester I, Inci I, Augustyns K, Arni S, et al. Primary graft dysfunction in lung transplantation: The role of CD26/dipeptidylpeptidase IV and vasoactive intestinal peptide. Transplantation 2009;87:1140-6.  Back to cited text no. 32
    
33.
Jungraithmayr W, De Meester I, Matheeussen V, Baerts L, Arni S, Weder W, et al. CD26/DPP-4 inhibition recruits regenerative stem cells via stromal cell-derived factor-1 and beneficially influences ischaemia-reperfusion injury in mouse lung transplantation. Eur J Cardiothorac Surg 2012;41:1166-73.  Back to cited text no. 33
    
34.
Jungraithmayr W, De Meester I, Matheeussen V, Inci I, Augustyns K, Scharpé S, et al. Inhibition of CD26/DPP IV attenuates ischemia/reperfusion injury in orthotopic mouse lung transplants: The pivotal role of vasoactive intestinal peptide. Peptides 2010;31:585-91.  Back to cited text no. 34
    
35.
Read PA, Khan FZ, Heck PM, Hoole SP, Dutka DP. DPP-4 inhibition by sitagliptin improves the myocardial response to dobutamine stress and mitigates stunning in a pilot study of patients with coronary artery disease. Circ Cardiovasc Imaging 2010;3:195-201.  Back to cited text no. 35
    
36.
Vaghasiya J, Sheth N, Bhalodia Y, Manek R. Sitagliptin protects renal ischemia reperfusion induced renal damage in diabetes. Regul Pept 2011;166:48-54.  Back to cited text no. 36
    
37.
Glorie LL, Verhulst A, Matheeussen V, Baerts L, Magielse J, Hermans N, et al. DPP4 inhibition improves functional outcome after renal ischemia-reperfusion injury. Am J Physiol Renal Physiol 2012;303:F681-8.  Back to cited text no. 37
    
38.
Natsume H, Tokuda H, Mizutani J, Adachi S, Matsushima-Nishiwaki R, Minamitani C, et al. Synergistic effect of vasoactive intestinal peptides on TNF-alpha-induced IL-6 synthesis in osteoblasts: Amplification of p44/p42 MAP kinase activation. Int J Mol Med 2010;25:813-7.  Back to cited text no. 38
    


    Figures

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



 

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
   References
   Article Figures

 Article Access Statistics
    Viewed267    
    Printed19    
    Emailed0    
    PDF Downloaded42    
    Comments [Add]    

Recommend this journal