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ORIGINAL ARTICLE
Year : 2019  |  Volume : 22  |  Issue : 12  |  Page : 1680-1684

Investigation the relaxant effects of proton pump ınhibitors and their relaxation mechanism on sheep sphincter of Oddi


1 Department of GeneralSurgery, Cumhuriyet University School of Medicine, Sivas, Turkey
2 Department of Pharmacologyand, Cumhuriyet University School of Medicine, Sivas, Turkey
3 Departments of Gastroenterology, Cumhuriyet University School of Medicine, Sivas, Turkey
4 Department of Biochemistry, Saglik Bilimleri University School of Medicine, Istanbul, Turkey

Date of Submission12-Mar-2018
Date of Acceptance18-Jul-2019
Date of Web Publication3-Dec-2019

Correspondence Address:
Dr. S Soylu
Department of General Surgery, Cumhuriyet University School of Medicine, Sivas
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njcp.njcp_115_18

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   Abstract 


Background: Using a relaxant agent before an endoscopic retrograde cholangiopancreatography (ERCP) might reduce complications. Study Aims: We aimed to investigate the relaxant effects of proton pump inhibitors (PPIs) on sheep sphincter of Oddi (SO) and the mechanisms that might take part in this relaxant effect. Patients and Methods: The sheep SO was mounted in an organ bath filled with Krebs–Ringer bicarbonate solution under 1.5 g tension and the relaxant effects of PPIs were evaluated in the tissues precontracted by carbachol (10−6 mol/l). The relaxant responses to the PPIs were tested in the presence of various blockers to enlighten the underlying mechanism by the PPIs. Results: The PPIs exerted relaxant responses in a concentration-dependent manner in the sheep SO (P < 0.05). Esomeprazole produced the strongest relaxation. The administration of atropine, indomethacin, L-NAME, methylene blue, clotrimazole, glibenclamide, and 4-aminopyridine into the organ baths did not change the relaxations induced by PPIs in vitro (P> 0.05). On the other hand, Ca+2-activated potassium channel blocker tetraethylammonium (TEA) reduced the relaxation responses created by PPIs (P < 0.05). Conclusions: The present study suggests that PPIs create relaxation on SO partially via Ca+2-activated potassium channels. PPIs, especially esomeprazole, may be beneficial during the ERCP procedure. Further clinical studies are needed to confirm our results.

Keywords: Endoscopic retrograde cholangiopancreatography, proton pump inhibitors, sphincter of oddi


How to cite this article:
Soylu S, Sarac B, Kurt A, Altun A, Cakmak E, Pence H H, Kahramanoglu Y E, Bagcivan I. Investigation the relaxant effects of proton pump ınhibitors and their relaxation mechanism on sheep sphincter of Oddi. Niger J Clin Pract 2019;22:1680-4

How to cite this URL:
Soylu S, Sarac B, Kurt A, Altun A, Cakmak E, Pence H H, Kahramanoglu Y E, Bagcivan I. Investigation the relaxant effects of proton pump ınhibitors and their relaxation mechanism on sheep sphincter of Oddi. Niger J Clin Pract [serial online] 2019 [cited 2019 Dec 14];22:1680-4. Available from: http://www.njcponline.com/text.asp?2019/22/12/1680/272194




   Introduction Top


The  Sphincter of Oddi More Details (SO) is an anatomic structure regulating the flow of duodenum, bile, and pancreatic fluids and preventing the reflux of duodenum ingredients into the main bile and pancreatic duct.[1] The SO has a complex formation with smooth muscle fibers and a neural network functioning through hormonal and neural tracts.[2] Anatomical and immunohistochemical analyses have shown that the SO has a rich neural innervation, primarily with cholinergic, adrenergic, nitrergic, and peptidergic neurons.[3] The control mechanisms of SO are not so clear. However, it is believed that excitatory innervation of SO is essentially connected with cholinergic nerves, and the nonadrenergic noncholinergic (NANC) innervation dominates inhibitor innervation.[4]

Endoscopic retrograde cholangiopancreatography (ERCP) is an invasive method used in the diagnosis and treatment of bile and pancreatic duct pathologies.[5] Unfortunately, ERCP might lead to some serious complications such as pancreatitis (1.6–15.7%), bleeding (1.3%), and perforation (0.6%).[6],[7],[8],[9] Easy cannulation plays a key role in the prevention of this complication and shortens the duration of the operation. The use of pharmacologic agents for relaxation of the SO might ensure easy cannulation, hence, a safer and shorter ERCP procedure.[6],[10]

Proton pump inhibitors (PPIs) are the group of drugs that remarkably reduce the long-term gastric acid production. They are the types of drugs that inhibit acid secretion in the strongest way.[11] PPIs are among the top-selling drugs in the world, and included in the WHO's List of Essential Medicines by WHO 2013.[12] It was reported that PPIs induce smooth muscle relaxation in different tissues, such as an artery, gallbladder, prostate, cavernous body, and myometrium, but no study is available in the literature on the effect of PPIs on SO.[13]

The objective of the present study was to investigate the relaxant effects of frequently used PPIs, namely, esomeprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole on sheep SO, and if these drugs have any impact on SO, then could there be new relaxants that could be used during ERCP. At the same time, we also aimed to investigate the relaxation mechanisms of these drugs on SO.


   Materials and Methods Top


Experimental design and preparation of tissues

In our study, we used 12 adult Anatolian Akkaraman sheep with a weight range of 38 to 45 kg. The permission of the local ethical committee was obtained for the experimental protocol (Date/no:06,04,2016/ 65202830-050-04.04.50). The sheep were slaughtered in the slaughterhouse. Their abdominal cavity was opened through a midline incision, and the SO tissue was rapidly removed and taken to the laboratory. It was placed into Krebs–Ringer bicarbonate solution (ingredients: NaCl 120 mmol/l, KCl 4.6 mmol/l, CaCl2 2.5 mmol/l, MgCl2 1.2 mmol/l, NaHCO3 22 mmol/l, NaH2 PO4 11.5 mmol/l, and glucose 11.5 mmol/l), which was previously aerated in the laboratory (95% O2 and 5% CO2). The SO tissue was put into the 10 ml organ bath containing Krebs–Ringer bicarbonate solution previously aerated and warmed at 37°C and its upper end were hung on the isometric transducer (Grass FT03, Quincy, Mass., USA). Before the study, the SO was preloaded with 1,5 g weight, and was left to equilibrium period for 60-90 minutes, washing it every 15 minutes.[14],[15],[16]

Isometric measurements

After the equilibrium period, the tissues were contracted by submaximal concentration of carbachol (10-6 mol/l) cumulatively in order to investigate relaxant effects of the PPIs. This concentration of carbachol corresponds to the concentration that induces 70% of its maximal contraction. Esomeprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole (10−7–3.10−4 mol/l) were cumulatively introduced after the contraction by carbachol and relaxant responses were received.

The relaxations caused by PPIs were obtained again in the presence of atropine (10−6 mol/l, parasympathetic blocker); indomethacin (10−5 mol/l, the prostaglandin synthase inhibitor); NG-nitro-L-arginine methyl ester (L-NAME, 3.10−5 mol/l, nonspecific inhibitor of nitric oxide synthase [NOS]); methylene blue (10−5 mol/l, the guanylate cyclase inhibitor); clotrimazole (10−6 mol/l, the cytochrome p450 monooxygenase inhibitor); tetraethyl ammonium (TEA) chloride (3.10−4 mol/l Ca+2-activated potassium channel blocker); glibenclamide (10−5 mol/l ATP-sensitive potassium channel blocker); and 4-aminopyridine (10−5 mol/l voltage-dependent potassium channel blocker), which were added to the organ bath 15 min prior to the precontraction by carbachol.[14],[15],[16]

The concentrations antagonists chosen in the present study are the ones that are proven capable of antagonizing the biological effects of specific agonist agents by our own studies and the literature.[17]

Statistical analysis

The contraction induced by carbachol was counted as a reference for each tissue and calculated as the percentage of carbachol contraction while calculating the relaxation induced by each concentration. pD2(-log10 EC50; negative logarithm of the concentrations inducing half maximal response) and Emax(the maximal response induced by the drug) were separately calculated for each drug.

The data were expressed as the mean ± standard error of the mean (SEM). The analysis of the data was conducted using the Statistical Package for the Social Sciences (SPSS) ver. 14. (Statsoft, Inc., Tusla, Okla.). Repeated measures of analysis of variance (ANOVA) was used during the analysis. Tukey's test was used as a posthoc test. The statistical difference was considered significant when the P value was <0.05.

Drugs

The drugs used in the experiment, namely, carbachol, esomeprazole, lansoprazole, omeprazole, pantoprazole and rabeprazole, atropine, indomethacin, L-NAME, methylene blue, clotrimazole, TEA chloride, glibenclamide, and 4-aminopyridine were supplied from Sigma-Aldrich Corporation (St Louis, MO, USA). All drugs were dissolved in distilled water, and the solutions containing the drugs were prepared fresh daily just before the experiments.


   Results Top


Esomeprazole, lansoprazole, omeprazole, pantoprazole and rabeprazole (10-7-3.10-4 mol/l) induced relaxant responses in the tissues pre-contracted by 10-6 mol/l carbachol (P< 0.05) [Figure 1]. These relaxant responses were observed in the 10−6 mol/l concentration, and they reached a maximum level in the 3.10−4 mol/l concentration. The relaxation caused by esomeprazole was more than the ones caused by other PPIs (P < 0.05) [Figure 1]. Pretreatment with atropine, indomethacin, L-NAME, methylene blue, clotrimazole, glibenclamide, and 4-aminopyridine did not inhibit the relaxation induced by PPIs. The administration of TEA significantly reduced the relaxations by PPIs (P < 0.05) [Figure 2].
Figure 1: Relaxation responses induced by esomeprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole, in the absence of TEA on isolated sheep SO tissues (n = 10). Relaxations are expressed as a percentage of carbachol (10−6 mol/l)-induced precontraction and shown as mean ± SEM. *Statistically different from other PPIs (P < 0.05). TEA: Tetraethylammonium chloride (3.10−4 mol/l Ca+2-activated potassium channel blocker)

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Figure 2: Relaxation responses induced by esomeprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole, in the presence of TEA on isolated sheep SO tissues (n = 10). Relaxations are expressed as a percentage of carbachol (10−6 mol/l)-induced precontraction and shown as mean ± SEM. *Statistically different from other PPIs (P < 0.05). TEA: Tetraethylammonium chloride (3.10−4 mol/l Ca+2-activated potassium channel blocker)

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[Table 1] shows the Emax and pD2 values for the relaxation induced by esomeprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole in the presence and absence of TEA. Emax values are illustrated in a column chart in [Figure 3]. No significant difference was found between the pD2 values of these five drugs in the presence and absence of TEA. (P > 0.05).
Table 1: Emax and pD2 values for the relaxant effects of esomeprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole in the presence and absence of TEA on sheep SO tissues precontracted by carbachol (n=10)

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Figure 3: Comparison of Emax values of the relaxations induced by esomeprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole, in the presence and absence of TEA on isolated sheep SO tissues (n = 10). Relaxations are expressed as a percentage of carbachol (10−6 mol/l)-induced precontraction and shown as mean ± SEM. *Statistically different from other PPIs (P < 0.05). a Statistically different than alone esomeprazole (P < 0.05)

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   Discussion Top


ERCP and endoscopic sphincterotomy, which were introduced in the late 1960s and early 1970s, are two routine procedures used across the world.[18] Today, ERCP is a complex and risky procedure used in the diagnosis and treatment of hepatobiliary and pancreatic diseases (e.g., stones and strictures).[19] During diagnostic or therapeutic ERCP procedure, various short-term complications such as pancreatitis, hemorrhage, and perforation may occur.[19] Complications associated with ERCP can be reduced with the use of local pharmacological agents that induce relaxation in SO to facilitate the cannulation and sphincterotomy.

PPIs are drugs that irreversibly block gastric hydrogen (H+)/potassium (K+) ATPase enzyme, alternatively called proton pump, and reduce gastric acid secretion.[20] They block stomach acid secretion in the strongest way, and their effect of acid secretion reduction was stronger than H2-receptor blockers.[20] PPIs are used upon prescription or nonprescription in the long-term treatment of several gastrointestinal (GI) diseases (e.g., peptic ulcer and gastroesophageal reflux).[21] PPIs are currently known to be safe drugs and believed to have no adverse effects.[20],[21] There are various publications indicating that PPIs induce relaxation in the tissues in the GI system (e.g., the lower esophageal tone in rats and human pylorus) and other systems (e.g., artery, gallbladder, prostate, cavernous body, and myometrium) taken from humans or other animals.[22] Similarly, Sossalla et al. demonstrated that various PPIs showed a relaxant effect on the contraction of isolated human myometrium.[23] Rhoden et al. also indicated that some H+/K+ ATPase inhibitors induced relaxation in the airway smooth muscles of guinea pigs and humans.[24] Although studies are available in the literature showing the relaxant effect of PPIs on several tissues, there is no study on the direct or indirect impact of PPIs on SO.

In our study, we tried to find out whether commonly used PPI drugs, namely, esomeprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole induce any relaxation on SO in vitro. In that manner, we applied PPIs to SO tissue precontracted by carbachol. Depending on the concentration, PPIs caused relaxant responses in tissues precontracted by carbachol. The relaxant responses started in 10−6 mol/l concentration and peaked in 3.10−4 mol/l concentration. The relaxant responses induced by esomeprazole were significantly more than the ones induced by the other four drugs. While pretreatment with atropine, indomethacin, L-NAME, methylene blue, clotrimazole, glibenclamide, and 4-aminopyridine did not inhibit the relaxation induced by PPIs, administration of TEA significantly reduced the relaxations by PPIs.

In literature, there are many papers reporting the relaxant effects of PPIs in the smooth muscle,[25],[26],[27] but the findings of the mechanism of action are still unclear. Welsh et al. found that pantoprazole reduced the gastroesophageal muscle tonus in newborn rats via Rho Kinase (ROCK) inhibition.[28] In concordance with this study, Naseri et al. showed that omeprazole and lansoprazole create relaxation in human arteries by adjusting intracellular Ca+2 levels.[29] Furthermore, Kelicen et al. found that omeprazole causes endothelium-dependent relaxations partially in rat aorta.[30]

In the present study, relaxations created by PPIs were not antagonized by atropine, indomethacin, and L-NAME. This finding may show us that relaxations caused by PPIs in sheep SO are independent of the parasympathetic system, synthesis, and/or release of prostaglandins and nitric oxide. Likewise, unchange relaxation responses in the presence of methylene blue and clotrimazole may show that these relaxations are independent of activation of guanylate cyclase and releasing cGMP and system of cytochrome p450 monooxygenases. While potassium channel blocker glibenclamide and 4-aminopyridine did not antagonize the relaxation responses, TEA partially blocked the relaxant responses. This may show us that while relaxations are independent of ATP-sensitive potassium channel and voltage-dependent potassium channel, it may be dependent on Ca+2-activated potassium channel.

Relaxant or inhibitory effects of PPIs at high doses were demonstrated on vascular precontracted smooth muscle, gall bladder, prostate, corpus cavernosum, myometrium, and lower esophageal sphincter.[16] Our result was compliant with the previous ones in the literature showing that the relaxant responses of PPIs in different tissues also appeared in high concentrations. PPIs would perhaps not induce any relaxation in the SO when administered as a drug in therapeutic doses, and their effects would appear in high concentrations.

During ERCP, the development of pancreatitis determines the severity of trauma that occurs during the ERCP procedure. On the other hand, the risks of each patient are different (depending on the age, sex, body mass index (BMI), anatomy, etc.). However, our study evaluated the direct effect of the drug on the tissue (Oddi). All procedures and risks were the same. Therefore, the difference in the results may be related to this condition

In conclusion, PPIs (esomeprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole) induced relaxant responses depending on the concentration of the drug applied on sheep SO. PPIs, especially esomeprazole, may be beneficial during the ERCP procedure. However, further clinical studies are necessary to confirm our results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Bosch A, Peña LR. The sphincter of Oddi. Dig Dis Sci 2007;52:1211-8.  Back to cited text no. 1
    
2.
Talmage EK, Hillsley K, Kennedy, Mave GM. Identification of the cholinergic neurons in guinea pig sphincter of Oddi ganglia. J Auton Nerv Sys 1997;64:12-8.  Back to cited text no. 2
    
3.
Sarac B, Durmus N, Bagcivan I, Altun A, Turan M, Sencan M. Investigation of relaxant effects of new agents affecting nitric oxide/cyclic guanosine monophosphate pathway on sheep oddi sphincter. Pancreas 2010;39:875-8.  Back to cited text no. 3
    
4.
Wells DG, Talmage EK, Mawe GM. Immunohistochemical identification of neurons in ganglia of the guinea pig sphincter of Oddi. J Comp Neurol 1995;352:106-16.  Back to cited text no. 4
    
5.
Sarac B, Durmus N, Altun A, Turan M, Kaya T, Sencan M, et al. Effects of cannabinoid agonists on sheep sphincter of oddi in vitro. Pancreatology 2011;11:428-33.   Back to cited text no. 5
    
6.
Freeman ML. Understanding risk factors and avoiding complications with endoscopic retrograde cholangiopancreatography. Curr Gastroenterol Rep 2003;5:145-53.  Back to cited text no. 6
    
7.
Masci E, Toti G, Mariani A Curioni S, Lomazzi A, Dinelli'yi M, et al. Complications of diagnostic and therapeutic ERCP: a prospective multicenter study. Am J Gastroenterol 2001;96:417-23.  Back to cited text no. 7
    
8.
Andriulli A, Loperfido S, Napolitano G, Niro G, Valvano MR, Spirito F, et al. Incidence rates of post-ERCP complications: A systematic survey of prospective studies. Am J Gastroenterol 2007;102:1781-8.  Back to cited text no. 8
    
9.
Cotton PB, Garrow DA, Gallagher J, Romagnuolo J. Risk factors for complications after ERCP: a multivariate analysis of 11,497 procedures over 12 years. Gastrointest Endosc 2009;70:80-8.  Back to cited text no. 9
    
10.
Barthet M, Lesavre N, Desjeux A, Gasmi, M, Berthezene P, Berdah S, et al. Complications of endoscopic sphincterotomy: results from a single tertiary referral center. Endoscopy 2002;34:991-7.  Back to cited text no. 10
    
11.
Sudhindran S, Bromwich E, Edwards PR. Prospective randomized double-blind placebo-controlled trial of glyceryl trinitrate in endoscopic retrograde cholangiopancreatography induced pancreatitis. Br J Surg 2001;88:1178-82.  Back to cited text no. 11
    
12.
Sachs G, Shin JM, Howden CW. Review article: the clinical pharmacology of proton pump inhibitors. Aliment Pharmacol Ther 2006;23 Suppl 2:2-8.  Back to cited text no. 12
    
13.
World Health Organization. Who Model List of Essential Medicines. 19th ed., 2015a. p. 1-55. Available from: http://www.who.int/medicines/publications/essentialmedicines/en/. [Last accessed on 2015 Apr].  Back to cited text no. 13
    
14.
Yasar NF, Polat E, Duman M, Dagdelen M, Gunal MY, Uzun O, et al. In vitro effects of rabeprazole on human pylorus tone. J Neurogastroenterol Motil. 2015;21:217-21.  Back to cited text no. 14
    
15.
Bagcivan I, Kaya T, Yildirim MK, Turan M. Investigation of the relaxant effects of pinacidil and cromakalim on the sheep sphincter of Oddi. Pancreatology 2006;6:286-90.  Back to cited text no. 15
    
16.
Bagcivan I, Gursoy S, Yildirim MK, Kaya Temiz T, Yildirim S, Yilmaz A, et al. Investigation of relaxant effects of propofol on sheep sphincter of Oddi. Pancreatology 2007;7:174-9.  Back to cited text no. 16
    
17.
Çakmak E, Yönem O, Saraç B, Parlak M, Çelik C, Ataseven H, et al. Comparative Relaxant Effects of Ataciguat and Zaprinast on Sheep Sphincter of Oddi. Balkan Med J 2016;33:453-7.  Back to cited text no. 17
    
18.
Gokce G, Bagcivan I, Kilicarslan H, Yildirim S, Gultekin YE, Sarioglu Y. Relaxation effects of adrenomedullin in isolated rabbit corpus cavernosum smooth muscle. BJU Int 2004;93:859-62.  Back to cited text no. 18
    
19.
Classen M, Demling L. Endoscopic sphincterotomy of the papilla of Vater and extraction of stones from the choledochal duct. Dtsch Med Wochenschr 1974;99:496-7.  Back to cited text no. 19
    
20.
Huang LY, Liu YX, Wu CR. Application ofendoscopic retrograde cholangiopancreatography in biliarypancreatic diseases. Chinese Med J 2009;122:2967-72.  Back to cited text no. 20
    
21.
CADTH Rapid Response Reports. Pre-Endoscopic Intravenous Proton Pump Inhibitors for Emergency Department Patients with Upper Gastrointestinal Bleeds: A Review of Clinical Effectiveness, Cost-Effectiveness and Guidelines. Ottawa (ON): Canadian Agency for Drugs and Technologies in Health. 2016. Available from: https://www.cadth.ca/pre-endoscopic-intravenous-proton-pump-inhibitors-emergency-department-patients-upper-0. [Last accessed on 2016 Feb 05].  Back to cited text no. 21
    
22.
Duman M, Ozer M, Reyhan E, Demirci Y, Atici AE, Dalgıç T, et al. In vitro effect of pantoprazole on lower esophageal sphincter tone in rats. World J Gastroenterol 2011;17:5105-9.  Back to cited text no. 22
    
23.
Sossalla S, Schotola H, Schmitto J, Toischer K, Sohns C, Schwörer H, et al. Effects of different proton pump inhibitors on cardiac contractility in isolated human failing myocardium. J Cardiovasc Surg (Torino) 2011;52:437-44.  Back to cited text no. 23
    
24.
Rhoden KJ, Tallini G, Douglas JS. H+-K+ ATPase inhibitors cause relaxation of guinea pig and human airway smooth muscle in vitro. J Pharmacol Exp Ther 1996;276:897-903.  Back to cited text no. 24
    
25.
Kurt A, Altun A, Bagcivan I, Koyuncu A, Topcu O, Aydın C, et al. Effects of Proton Pump Inhibitors and H2 Receptor Antagonists on the Ileum Motility. Gastroenterology Research and Practice. 2011;1:218342.  Back to cited text no. 25
    
26.
Parkman HP, Urbain JL, Knight LC, Brown KL, Trate DM, Miller MA, et al. Effect of gastric acid suppressants on human gastric motility. Gut 1998;42:243-50.  Back to cited text no. 26
    
27.
Cahan MA, Balduf L, Colton K, Palacioz B, McCartney W, Farrell TM. “Proton pump inhibitors reduce gallbladderfunction,” Surgical Endoscopy and Other Interventional Techniques 2006;20:1364-7.  Back to cited text no. 27
    
28.
Welsh C, Kasirer MY, Pan J, Shifrin Y, Belik J. Pantoprazole decreases gastroesophageal muscle tone in newborn rats via rho-kinase inhibition. Am J Physiol Gastrointest Liver Physiol 2014;307:390-6.  Back to cited text no. 28
    
29.
Naseri E, Yenisehirli A. Proton pump inhibitors omeprazole and lansoprazole induce relaxation of isolated human arteries. Eur J Pharmacol 2006;531:226-31.  Back to cited text no. 29
    
30.
Kelicen P, Pekiner C, Sarioǧlu Y, Uma S. Omeprazole-induced relaxation in rat aorta is partly dependent on endothelium. Pharmacol Res 2002;46:321-3.  Back to cited text no. 30
    


    Figures

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