Journal of Pharmacy And Bioallied Sciences
Journal of Pharmacy And Bioallied Sciences Login  | Users Online: 627  Print this pageEmail this pageSmall font sizeDefault font sizeIncrease font size 
    Home | About us | Editorial board | Search | Ahead of print | Current Issue | Past Issues | Instructions | Online submission




 
 Table of Contents  
DENTAL SCIENCE - ORIGINAL ARTICLE
Year : 2015  |  Volume : 7  |  Issue : 6  |  Page : 451-456  

A case-control study to detect the extent of DNA damage in oral lichen planus and oral lichenoid reactions using comet assay


1 Department of Oral Medicine, Indira Gandhi Institute of Dental Sciences, Puducherry, India
2 Department of Prosthodontics, Rajah Mutiah Dental College, Chidambaram, Tamil Nadu, India
3 Department of Anatomy, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India

Date of Submission28-Apr-2015
Date of Decision28-Apr-2015
Date of Acceptance22-May-2015
Date of Web Publication1-Sep-2015

Correspondence Address:
Dr. N Madhulika
Department of Oral Medicine, Indira Gandhi Institute of Dental Sciences, Puducherry
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-7406.163499

Rights and Permissions
   Abstract 

Aim: This study aims to quantify the extent of DNA damage in lymphocytes of patients with oral lichen planus (OLP) and oral lichenoid reactions (OLRs) using comet assay. Methodology: Lymphocytes from peripheral blood were subjected to alkaline comet assay. Comet length (CL), head diameter (HD), percentage of DNA in head, tail length (TL), percentage of DNA in tail, tail intensity, tail mean and tail moment were compared between study group (OLP and OLR) and control group using Student's t-test. Pearson's correlation coefficient was used to examine the linear association between the variables. Results: Significantly higher levels of DNA damage was present in study group as reflected by CL, HD and TL, tail intensity and tail moment with P = 0.0001; percentage of DNA in head and tail with P = 0.02 and tail mean with P = 0.012. Conclusion: This study brings out the fact that DNA damage measured by comet assay was greater in the study group when compared to the control group. As a reflection of uniqueness, this study crowns the scenario with respect to early detection and prevention of potentially malignant disorders and the process of malignant transformation.

Keywords: Comet assay, DNA damage, malignant transformation


How to cite this article:
Madhulika N, Rangdhol R V, Sitra G, Ballaiah J, Jaikumar R A, Brooklyin S. A case-control study to detect the extent of DNA damage in oral lichen planus and oral lichenoid reactions using comet assay. J Pharm Bioall Sci 2015;7, Suppl S2:451-6

How to cite this URL:
Madhulika N, Rangdhol R V, Sitra G, Ballaiah J, Jaikumar R A, Brooklyin S. A case-control study to detect the extent of DNA damage in oral lichen planus and oral lichenoid reactions using comet assay. J Pharm Bioall Sci [serial online] 2015 [cited 2019 Aug 19];7, Suppl S2:451-6. Available from: http://www.jpbsonline.org/text.asp?2015/7/6/451/163499

Oral lichen planus (OLP) is a chronic inflammatory disease of unknown etiology while oral lichenoid reaction (OLR) is a condition mimicking OLP. [1] As these conditions are exposed to oxidative stress, they release reactive oxygen species (ROS) which are implicated in the pathogenesis of a plethora of inflammatory conditions to lethal diseases. It has been proved that ROS and reactive nitrogen species (RNS) play an important role in inflammation-mediated carcinogenesis. [2],[3] Malignant transformation of OLP and OLR is 0.4-5.3% and 0.71% respectively. [1],[4] If detected at an early stage these lesions can be prevented and treated successfully. With present advances in genotoxicologic studies, the extent of DNA damage provides a platform to determine the cancer progression. This study is the first of its kind, which was done to detect the extent of DNA damage in OLP and OLR. DNA is constantly subjected to chemical modifications. Several types of DNA damage such as single strand break (SSB), double strand break (DSB), cyclobutane pyrimidine dimers, 6-4 photoproducts and their Dewar valence isomers have been identified that result from alkylating agents, hydrolytic deamination, free radicals and ROS formed by various photochemical processes, including ultraviolet radiation. [5] Oxidative stress, from oxidative and nitrative products, plays an important role in provoking DNA damage resulting in neoplastic changes in potentially malignant disorders. [6] DNA damage can be detected through a number of strategies such as polymerase chain reaction (PCR) and real-time PCR, terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay, fluorescence in situ hybridization, flow cytometry and annexin V labeling. Furthermore, Immunological assays like immunofluorescent and chemiluminescence thymine dimer detection, immunohistochemical assay, enzyme-linked immunosorbent assay and radioimmunoassay, other advanced methods like gas chromatography-mass spectrometry and high performance liquid chromatography-electrospray tandem mass spectrometry and single cell gel electrophoresis or "comet assay," have been used. [5],[7]

Comet assay

Single cell gel electrophoresis or comet assay is one such technique for quantitating DNA damage and repair in vivo and in vitro in any eukaryotic cell and some prokaryotic cells. This technique is rapid, noninvasive, sensitive, visual and inexpensive as compared to the conventional techniques and is a powerful tool to study factors modifying mutagenicity and carcinogenicity. It has rapidly gained importance in the fields of genetic, toxicology and human biomonitoring. It can be used for evaluating lesions with DNA damage caused by DSB, SSB, alkali-labile sites, oxidative base damage, and DNA cross-linking with DNA or protein. [5],[7]

Oxidative stress in oral lichen planus and oral lichenoid reaction

Oxidative stress is defined as a disruption in the balance between oxidants and antioxidants, which leads to the overproduction of free radicals and ROS; the pathology associated with ROS is derived from their ability to modify cellular and extracellular macromolecules, such as proteins, lipids, and DNA to disrupt cellular function. ROS and free radicals have a predominant deleterious role in inducing and promoting carcinogenesis. Based on the literature it is known that oxidative stress plays an important role in the pathogenesis of several inflammatory and autoimmune diseases. Since OLP and OLR are chronic inflammatory diseases, they release large quantities of ROS and RNS, which further causes injury to the surrounding tissues, which has been proved by several studies. [6],[7] Nitric oxide has been used as a salivary biomarker in detecting oxidative stress in patients with OLP and is found to be significantly higher in these patients compared to healthy controls. [8],[9],[10] Role of oxidative stress in OLP and OLR was proved by estimating the levels of salivary melondialdehyde [1],[6],[10],[11],[12],[13],[14] and total antioxidant capacity [1],[6],[10],[12],[13] levels. This leads to the hypothesis that there is an increase in oxidative stress and imbalance in antioxidant defense system in the oral cavity of OLP and OLR patients, which may be involved in the pathogenesis. A further study assessing the DNA damage in peripheral blood lymphocytes using comet assay in oral mucosal lesions like OLP and OLRs is warranted.

Comet assay in detecting DNA damage

Several studies were done on peripheral blood lymphocytes and buccal epithelial cells to detect DNA damage using comet assay in oral mucosal lesions like oral leukoplakia, oral submucous fibrosis (OSMF) and oral cancer. Subjects with deleterious oral habits like smoking, areca nut with lime chewing with or without smoking, areca nut with lime and tobacco chewing with or without smoking were also assessed for DNA damage in different studies. This study was done to detect the extent of DNA damage in OLP and OLRs using comet assay. [15],[16],[17],[18],[19],[20]


   Methodology Top


The study protocol and procedure were approved by the Institutional Internal Review Board and Ethical Committee. Informed consent was obtained from patients after explaining the procedure, aim and objectives of the study. The study comprised of 40 patients including 20 cases (OLP and OLR) and 20 controls. Cases were selected on the basis of modified WHO criteria (2003), [16] following which incisional biopsy was done from the site of lesion under local anesthesia and the soft tissue specimens were sent for histopathological examination. Simultaneously, whole blood was collected from the selected subjects (cases and controls) under aseptic conditions by venipuncture from the antecubital vein and one ml of blood was labeled and subjected to comet assay [Figure 1].
Figure 1: Schematic representation for comet formation

Click here to view


Procedure for assessment of DNA damage

The blood sample collected was centrifuged for 30 min at 1500 rpm, to separate the lymphocytes following which the cells were sandwiched between the layers of agarose gel. These slides were placed in the lysis solution (Nacl) for a minimum of half an hour to a maximum of 24 h followed by electrophoresis for 1 h (without power 30 min and with power 30 min). Later the cells were fixed and stained with silver nitrate; observed under a bright-field light microscope (Olympus BX-51) and images were captured using CCD camera [Figure 2]. Captured images were analyzed for DNA damage using commercially available COMET SCORE (TriTek Corporation) software. The DNA damage was estimated by measuring the length of different parameters using an ocular scale fitted in the eyepiece of the microscope or by visual scoring of the degree of damage from 0 to 4 according to comet appearance. In total, 40-50 randomly selected cells were analyzed per sample. Comets were selected without any bias by eliminating those seen at edges, air bubbles, and overlaps and they represented the whole gel.
Figure 2: Bright field light microscopic pictures (×20): (a) Lymphocytes showing mild DNA damage in cases. (b) Normal lymphocytes in controls (white arrows)

Click here to view


Parameters measured

Tail length (TL), tail moment, comet length (CL), head diameter (HD), the percentage of DNA in the head, the percentage of DNA in tail, tail intensity and tail mean intensity.

Statistical analysis

Statistical analyses were performed with SPSS version 21.0© (IBM Corporation, Armonk, NY, USA). The comparison of cases and control subjects were analyzed by Student's t-test. Pearson correlation coefficient was calculated to estimate the correlation between different comet parameters. Significance was defined at P < 0.05.


   Results Top


Comparison between cases and controls

The mean, standard deviation, P value and level of significance computed for the data collected on CL, HD, percentage of DNA in head, TL, percentage of DNA in tail, tail intensity, tail mean and tail moment (MTM) for group I, and group II consisting of 20 subjects each are presented in [Table 1].
Table 1: Comparison between cases and controls

Click here to view


The results of this study showed statistically significant difference between cases and controls on CL, HD, percentage of DNA in head, TL and percentage of DNA in tail, tail intensity, tail MTM with P values being 0.0001, 0.0001, 0.02, 0.0001, 0.02, 0.0001, 0.012 and 0.0001, respectively. From our study, we could infer, a longer CL, larger HD, lesser percentage of DNA in head, longer TL, higher percentage of DNA in tail; higher tail intensity, tail MTM among the cases compared to controls is suggestive of DNA damage.

Comparison between oral lichen planus cases and controls

The mean, standard deviation, P value and level of significance computed for the data collected on CL, HD, percentage of DNA in head, TL, percentage of DNA in tail, tail intensity, tail MTM of 20 normal control group, and seven OLP cases are presented in [Table 2].
Table 2: Comparison between OLP cases and controls

Click here to view


The results of this study showed statistically significant difference between control and OLP cases on CL, HD, TL and tail intensity with P = 0.0001; whereas percentage of DNA in head, percentage of DNA in tail and tail mean showed no significant difference with P values of 0.06, 0.06 and 0.4, respectively. From our study we could infer, a longer CL, larger HD, longer TL, higher tail intensity and tail mean among OLP cases when compared to controls. But, the percentage of DNA in head, percentage of DNA in tail and tail mean were almost similar to control group.

Comparison between oral lichenoid reaction cases and controls

The mean, standard deviation, P value and level of significance computed for the data collected on CL, HD, percentage of DNA in head, TL, percentage of DNA in tail, tail intensity, tail MTM of 20 normal control group, and 13 OLR cases are presented in [Table 3].
Table 3: Comparison between OLR cases and controls

Click here to view


The results of this study showed statistically significant difference between OLR cases and controls on CL, HD, percentage of DNA in head, TL and percentage DNA of in tail with P values of 0.0001, 0.0001, 0.016, 0.0001 and 0.016, 0.0001, 0.017 and 0.0001 respectively. Based on the study, we could infer, a longer CL, larger HD, lesser percentage DNA in head, longer TL, higher percentage DNA in tail, tail intensity, tail MTM among OLR cases when compared to controls.

Comparison between oral lichen planus and oral lichenoid reaction cases

The mean, standard deviation, P value and level of significance computed for the data collected on CL, HD, percentage of DNA in Head, TL and percentage of DNA in tail of seven OLP cases and 13 OLR cases are presented in [Table 4].
Table 4: Comparison between OLP and OLR cases

Click here to view


The results of this study showed statistically significant difference between OLP and OLR cases on CL, HD and tail intensity with P = 0.0001. But percentage DNA in head, TL, percentage of DNA in tail, tail MTM showed no significant differences with P values of 0.9, 0.8, 0.9, 0.1 and 0.6 respectively.

Pearson's correlation coefficient for comet assay parameters

Correlation coefficients for all the eight comet parameters were calculated along with the P values are presented in [Table 5].
Table 5: Pearson's correlation coefficient for comet assay parameters

Click here to view


Correlation coefficients were calculated with all the eight comet parameters, statistically significant correlation was found with respect to HD and CL; percentage of DNA in head and HD; TL and HD; %percentage of DNA in tail and HD; TL and percentage of DNA in head; % DNA in head and percentage of DNA in tail; percentage of DNA in tail and TL; tail intensity and CL, HD, tail mean with CL, HD, percentage of DNA head, TL, percentage of DNA tail; tail moment with percentage of DNA head, TL, percentage of DNA tail, tail intensity and tail that is, 19 out of 28 variables. Only nine variables did not show statistical significance.


   Discussion Top


This study was done in the Department of Oral Medicine and Radiology, Indira Gandhi Institute of Dental Sciences, Puducherry, India, over a period of 1-year in view of the existing controversies and lack of consensus on published literature on DNA damage in OLP and OLR using comet assay. The principle aim of the study was to quantify the extent of DNA damage in lymphocytes of the patients with OLP and OLR using comet assay. The target population consisted of patients with erosive, atrophic OLP and OLR as confirmed by modified WHO criteria. The study included 40 participants, 20 in the control group and 20 in the study group (OLP-7 and OLR-13).

Detection of DNA damage

In our study, eight parameters such as TL, tail moment, CL, HD, percentage of DNA in head, percentage of DNA in tail, tail intensity and tail mean intensity were selected to assess DNA damage. TL signifies the distance of DNA migration from the body of the nuclear core which contains the damaged DNA, which has moved from cathode to the anode when subjected to electric current. Results showed a significant increase in mean TL in OLP and OLR when compared to controls (P = 0.0001). Potentially malignant disorders like oral leukoplakia [16] and OSMF [16],[17] have shown to have similar increase in the mean TL, indicating the role of oxidative stress leading to DNA damage in these lesions [Table 6].
Table 6: Comparison of TL between different studies

Click here to view


Tail moment is a measure of the DNA damage, which is obtained by combining the amount of DNA in the tail with a distance of migration. The MTM observed in our study, was greater in OLP and OLR when compared to controls which is similar to the higher MTM values observed among smokers in different studies [18],[19],[20] [Table 7].
Table 7: Comparison of tail moment between different studies

Click here to view


The observation on TL and tail Moment in our study shows that the pattern of DNA damage in OLP and OLR was similar to DNA damage seen in leukoplakia, OSMF and tobacco users. Thus, it can be inferred that though the etiopathogenesis of OLP and OLR are different, it is the oxidative stress-induced DNA damage that predisposes these lesions to transform into malignancy. ROS (superoxides, hydroxyl radicals) initiate mutagenic events by causing DNA damage and stimulate malignant transformation and progression. [2],[21] The CL signifies the entire length of the comet, which includes both the intact and damaged DNA. Our study showed a significant difference between cases and controls. HD signifies the diameter of the head which contains only the intact DNA (undamaged DNA is supercoiled and thus, does not migrate very far out of the nucleoid under the influence of electric current). Significant difference was found between the cases and controls. Percentage of DNA in head signifies percentage of intact DNA in the head. Controls had higher percentage of DNA in the head from which we can infer that higher the DNA % in the head, lesser is the damage. Our study showed a significant difference between cases and controls. Percentage of DNA in tail signifies percentage of damaged DNA in the tail. Cases had higher percentage of DNA in the tail, and the obtained values showed a significant difference between cases and controls. Tail intensity signifies the intensity of damaged DNA in the tail which is the sum of all intensity values, and it showed statistically significant difference. Tail mean intensity signifies the average of all intensity values in the comet tail. Both tail intensity and tail mean intensity were higher in cases when compared to controls. Tail moment is a measure of the damage, which is obtained by combining the amount of DNA in the tail with a distance of migration which was observed to be greater in cases than the controls. Since there are no studies in the literature estimating the DNA damage in oral mucosal lesions using the parameters like CL, percentage of DNA head and tail, tail intensity and tail mean intensity in addition to TL and tail moment, our findings are unique and can be used to correlate the DNA damage occurring in the potentially malignant disorders to the process of carcinogenesis.

Based on the results obtained we observed that the study group showed significantly higher levels of DNA damage as reflected from the CL, HD and TL, tail intensity and tail moment values.

When the comparison between OLP cases and controls were done, subjects with OLP showed significantly higher levels of DNA damage as reflected by CL, HD and TL and tail intensity. And on further comparison between OLR cases and controls, OLR cases showed significantly higher levels of DNA damage than controls, which can be attributed to the variations reflected from the CL, HD and TL, tail intensity and tail moment. On comparison of OLR and OLP, significantly higher levels of DNA damage was observed in OLP as reflected by the CL and HD and tail intensity, whereas the parameters TL, percentage of DNA in head and tail, tail MTM were not significantly increased. Assimilating the results considering all eight parameters used to assess DNA damage between cases and controls, significant difference was observed with respect to all parameters suggesting the possible role of DNA damage predisposing to these lesions to malignant transformation.

Strengths of the study

  • This study is the first of its kind which was done on OLP and OLR cases for detecting the extent of DNA damage using comet assay
  • All the eight comet parameters in assessing DNA damage showed the statistically significant difference.


Limitations of the study

Smaller sample size of the study group could be attributed to the negative results obtained on a comparison of certain parameters. Further studies with larger sample size could give more conclusive evidence of DNA damage occurring due to oxidative burden in OLP and OLR as a risk factor for malignant transformation.


   Conclusion Top


Early detection and quantification of DNA damage in oral premalignancy or malignancy may help in the management of the disease and improve survival rates. Comet assay has been successfully utilized to detect DNA damage in oral premalignancy or malignancy. This study compares the extent of DNA damage with the comet assay in peripheral blood lymphocytes in OLP and OLR patients with normal controls. The results of the study concluded that the DNA damage was greater in OLP and OLR cases when compared to controls by using comet assay. Demonstrating DNA damage using comet assay may lead to further advancement in understanding the biology of oral cancer and precancer for developing possible worthy early detection tests.

 
   References Top

1.
Georgakopoulou EA, Achtari MD, Achtaris M, Foukas PG, Kotsinas A. Oral lichen planus as a preneoplastic inflammatory model. J Biomed Biotechnol 2012;2012:759626.  Back to cited text no. 1
    
2.
Upadhyay RB, Carnelio S, Shenoy RP, Gyawali P, Mukherjee M. Oxidative stress and antioxidant defense in oral lichen planus and oral lichenoid reaction. Scand J Clin Lab Invest 2010;70:225-8.  Back to cited text no. 2
    
3.
Taghavi Zenouz A, Mehdipour M, Attaran R, Bahramian A, Emamverdi Zadeh P. Squamous cell carcinoma arising from an oral lichenoid lesion: A case report. J Dent Res Dent Clin Dent Prospects 2012;6:29-32.  Back to cited text no. 3
    
4.
Kumari S, Rajesh P. Rastogi, Kanchan L. Singh, Shailendra P. Singh, Rajeshwar P. Sinha. DNA damage: Detection strategies. EXCLI Journal 2008;7:44-62.  Back to cited text no. 4
    
5.
Iannitti T, Rottigni V, Palmieri B. Role of free radicals and antioxidant defences in oral cavity-related pathologies. J Oral Pathol Med 2012;41:649-61.  Back to cited text no. 5
    
6.
Shirzad A, Pouramir M, Seyedmajidi M, Jenabian N, Bijani A, Motallebnejad M. Salivary total antioxidant capacity and lipid peroxidation in patients with erosive oral lichen planus. J Dent Res Dent Clin Dent Prospects 2014;8:35-9.  Back to cited text no. 6
    
7.
Nandhakumar S, Parasuraman S, Shanmugam MM, Rao KR, Chand P, Bhat BV. Evaluation of DNA damage using single-cell gel electrophoresis (Comet Assay). J Pharmacol Pharmacother 2011;2:107-11.  Back to cited text no. 7
[PUBMED]  Medknow Journal  
8.
Sunitha M, Shanmugam S. Evaluation of salivary nitric oxide levels in oral mucosal diseases: A controlled clinical trial. Indian J Dent Res 2006;17:117-20.  Back to cited text no. 8
[PUBMED]  Medknow Journal  
9.
Ohashi M, Iwase M, Nagumo M. Elevated production of salivary nitric oxide in oral mucosal diseases. J Oral Pathol Med 1999;28:355-9.  Back to cited text no. 9
    
10.
Agha-Hosseini F, Mirzaii-Dizgah I, Mikaili S, Abdollahi M. Increased salivary lipid peroxidation in human subjects with oral lichen planus. Int J Dent Hyg 2009;7:246-50.  Back to cited text no. 10
    
11.
Rai B, Kaur J, Jacobs R, Singh J. Possible action mechanism for curcumin in pre-cancerous lesions based on serum and salivary markers of oxidative stress. J Oral Sci 2010;52:251-6.  Back to cited text no. 11
    
12.
Ergun S, Trosala SC, Warnakulasuriya S, Özel S, Önal AE, Ofluoglu D, et al. Evaluation of oxidative stress and antioxidant profile in patients with oral lichen planus. J Oral Pathol Med 2011;40:286-93.  Back to cited text no. 12
    
13.
Panjwani S, Bagewadi A, Keluskar V, Malik R, Rai S, Misra D. Estimation and comparison of levels of salivary nitric oxide in patients with oral lichen planus and controls. Int J Prev Med 2013;4:710-4.  Back to cited text no. 13
[PUBMED]  Medknow Journal  
14.
Mehdipour M, Taghavi Zenouz A, Bahramian A, Gholizadeh N, Boorghani M. Evaluation of serum nitric oxide level in patients with oral lichen planus. J Dent (Shiraz) 2014;15:48-51.  Back to cited text no. 14
    
15.
Mukherjee S, Ray JG, Chaudhuri K. Evaluation of DNA damage in oral precancerous and squamous cell carcinoma patients by single cell gel electrophoresis. Indian J Dent Res 2011;22:735-6.  Back to cited text no. 15
[PUBMED]  Medknow Journal  
16.
Smita Jyoti, Saif Khan, Falaq Naz, Rahul. M, Fahad Ali, Yasir Hasan Siddique. Assessment of DNA damage by panmasala, gutkha chewing and smoking in buccal epithelial cells using alkaline single cell gel electrophoresis (SCGE). The Egyptian Journal of Medical Human Genetics 2013;14:391-94.  Back to cited text no. 16
    
17.
Udupa R, Hallikeri. K, Dhiraj J. Trivedi. The comet assay a method to measure DNA damage in oral submucous fibrosis patients: A case-control study. Clin Cancer Invest J 2014;3:299-304.  Back to cited text no. 17
    
18.
Manuel F. Preliminary evaluation of DNA damage related with the smoking habit measured by the comet assay in whole blood cells. Cancer Epidemiol Biomarkers Prev 2004;13:1223-9.  Back to cited text no. 18
    
19.
Manikantan. P, Balachandar. V, Sasikala. K, Mohanadevi. S. Lymphocyte DNA Damage in Chewing Tobacco Users of Coimbatore, Tamilnadu, by Using Comet Assay. J Hum Ecol 2010;31:53-8.  Back to cited text no. 19
    
20.
Soylemez. E, Kayaalti. Z, Aliyev. V, Soylemezoglu. T. Effect of cigarette smoking on DNA damage according to nine comet assay parameters in female and male groups. Ankara University Journal 2012;65:1- 8.  Back to cited text no. 20
    
21.
Metgud R, Bajaj S. Evaluation of salivary and serum lipid peroxidation, and glutathione in oral leukoplakia and oral squamous cell carcinoma. J Oral Sci 2014;56:135-42.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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
   Methodology
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed1081    
    Printed17    
    Emailed0    
    PDF Downloaded73    
    Comments [Add]    

Recommend this journal