Users Online: 61
Home Print this page Email this page
Home About us Editorial board Search Browse articles Submit article Ahead of Print Instructions Subscribe Contacts Login 


 
Previous article Browse articles Next article 
ORIGINAL ARTICLE
Adv Biomed Res 2013,  2:42

Study of the variations in apoptotic factors in hippocampus of male rats with posttraumatic stress disorder


1 Department of Anatomical Science and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences; Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan; Neuroscience Research Center, Shahid Beheshi University of Medical Sciences, Tehran, Iran
2 Neuroscience Research Center, Shahid Beheshi University of Medical Sciences, Tehran, Iran
3 Research Group on Health Psychology, University of Leuven, Belgium, Iran
4 Department of Physiology, Faculty of Medicine, Kashan University of Medical Sciences; Physiology Research Center, Kashan University of Medical Sciences, Kashan, Iran
5 Neuroscience Research Center, Shahid Beheshi University of Medical Sciences; Behavioural Science Research Center, Department of Psychiatry, Imam Hossein Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Submission28-Apr-2012
Date of Acceptance29-Apr-2012
Date of Web Publication30-Mar-2013

Correspondence Address:
Jamal Shams
Behavioural Science Research Center, Shahid Beheshti University of Medical Sciences, PO Box 19615-1178, Tehran
Iran
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2277-9175.109757

Rights and Permissions
  Abstract 

Background: Post-traumatic stress disorder (PTSD) is a stress-related psychosomatic disorder caused by occurrence of a traumatic event and the hippocampus volume of the patients with Post-traumatic stress disorder decreased. However, the mechanisms that cause such damage are not well-understood. The aim of this study is to detect the expression of apoptosis-related Bax, Bcl-2, Caspase-3 and Insulin-like growth Factor-I proteins in the hippocampus region in the Predatory stress rats.
Materials and Methods: A total of 70 male wistar rats were divided into Predatory stress groups of 1d, 2d, 3d, 7d, 14d, 30d and a normal control group (N = 10). Rats were subjected to 5 min of predatory stress and then exposed to the elevated plus-maze (EPM). Serum corticosterone and Insulin-like growth factor-1 level of Hippocampus were measured by ELISA technique. The expression of Bax, Bcl-2, and Caspase-3 were detected by western blotting.
Results: Rats spent significantly more time in closed arms of the elevated plus maze (EPM) than control group after exposure to stress. Serum levels of corticosterone significantly increased at 2d-3d. The expression of hippocampal IGF-1 was significantly up-regulated at 1d-2d after stress. Both Bax and the ratio of Bax/Bcl-2 significantly peaked at Predatory stress 2d-14d. Caspase3 was significantly active among 2d-30 compared to the normal control.
Conclusion: The activation of caspase-3 in the stress groups indicates that apoptosis may be one of the reasons inducing hippocampus atrophy and play roles in the pathogenesis of PTSD. Increase in hippocampus levels of IGF-1 during early PTSD might be involved in the early molecular inhibitory mechanism of apoptosis in PTSD.

Keywords: Apoptosis, Bax, Bcl2, caspase-3, corticosterone, insulin-like growth factor-1, post-traumatic stress disorder, predator stress


How to cite this article:
Alani B, Maghsoudi N, Khatibi A, Noureddini M, Asefifar F, Shams J. Study of the variations in apoptotic factors in hippocampus of male rats with posttraumatic stress disorder. Adv Biomed Res 2013;2:42

How to cite this URL:
Alani B, Maghsoudi N, Khatibi A, Noureddini M, Asefifar F, Shams J. Study of the variations in apoptotic factors in hippocampus of male rats with posttraumatic stress disorder. Adv Biomed Res [serial online] 2013 [cited 2020 Sep 25];2:42. Available from: http://www.advbiores.net/text.asp?2013/2/1/42/109757


  Introduction Top


Post-traumatic stress disorder (PTSD) is continuous psychological disorder, taking place after facing with severe stress. In this disorder, the patient is exposed to a traumatic event, which involves death or a threat of death, and the patient's responses to that event are very intense fear and distress. [1] The patient shows serious disturbed behaviors and clinical disorder in his/her individual, social, and occupational performance, which would not occur before the trauma because the patient repeatedly recalls the traumatic experience. [2] Recent neuro-imaging studies have shown the volume of hippocampus in patients with PTSD decreases. [3],[4] Today, there are lots of evidences on the relationship between hippocampal atrophy, though its mechanism has not been determined properly. [5]

Apoptosis is a genetically-programmed cell process, playing an important role in differentiation and tissue homeostasis. [6] Various pathways for apoptosis have been identified based on different physiological and pathological stimuli among which are the intrinsic mitochondrial and extrinsic death receptor pathways which finally end in caspase-3 and cause protein degradation and cellular integrity. [7],[8] Apoptosis is one of the most important defense mechanisms protecting organisms from the pathogens, although it plays an important role in the pathogenesis of neurodegenerative disease. [9] Activation of intrinsic mitochondrial pathway due to stress and subsequent morphological variations resulting in atrophy in different areas of brain, especially hippocampus has been found in acute and chronic stress. [10],[11],[12]

Insulin-like Growth Factor 1 (IGF-1) is a polypeptide with 70 amino-acids, which chemical structure is similar to that of insulin hormone and is produced in many body tissues, especially in hippocampus. [13],[14] IGF-1 produced in brain cells is obviously effective in growth of primary cells of central nervous system and also maturation of neurons. [15] IGF-1 secretion is influenced by various factors like glucocorticoids. [16] According to researchers, the expression of IGF-1 gene in some parts of adult mice brain, such as hippocampus, remarkably increased number of neurons unlike the failure in expression of the gene or the gene mutation that caused brain development delay both in animals and humans. [17] IGF-1 is known as one of the fewest inhibitors of apoptosis incidence, which prevents the reduction of the anti-apoptotic factor of cell Bcl-2, as, in the absence of IGF-1, the apoptosis was found to increase by 50% in cells. [18],[19],[20] Nowadays, the role of IGF-1 as an effective peptide in neurogenesis processes and also as treatment of some neurodegenerative diseases associated with peripheral nervous system (PNS) and central nervous system (CNS) has been known more than ever, and various applications of this factor and how it acts in many of body vital processes are the concerns of many studies in this regard. [21],[22]

Based on the foregoing and considering that there are no documented results on the correlation between hippocampal atrophy and apoptosis in PSTD and the few reports on the apoptosis incidence are only about single prolong stress in animal model, the present study examined the variations in factors of apoptosis mitochondrial pathway and IGF-1 through an animal model for predator stress of this disorder, during the first procedures of this animal model of PTSD.


  Material and Methods Top


In this experimental study, 70 Wistar male rats with the age of 7 weeks and weight of 160-180 g were kept in animal house of Neuroscience Department of Shahid Beheshti University of Medical Sciences under regulations of protection and research trials with laboratorial animals.

The studied animals were divided into 7 groups [each with 10 rats] called control, after stress 1 st day, 2 nd day, 3 rd day, 7 th day, 14 th day, and 30 th day groups, and the stress was induced using the animal model of predator stress. To do so, each rat was put in a small cage, and this cage was placed in a wooden cage with dimensions of 1 × 1.52 × 1.83 m exposed to a cat. After 5 min, the rat was taken out of the cage and put in a dark place for 5 min. According to the reference, stress induction for all groups was carried out from 8 to 10 AM at the same environmental temperature. [23],[24] In order to identify anxious animals and behavioral changes, the rats were subjected to an elevated plus-maze (EPM) test apparatus for 5 min. Based on the reference, anxious animal's pauses in closed arms would be much longer than their pauses in open arms, and this was the criterion for the animal affected with PTSD in this study. [25],[26]

In order to measure serum corticosterone level, the rats were quickly anesthetized with CO 2 , and blood was taken after beheading the specimens. The blood coming out of rats' neck was collected, and its serum was prepared by centrifuging the blood samples for 10 min at 2000 rpm. The level of serum corticosterone was measured using ELISA test [Sandwich ELISA Kit, DRG Co.].

To examine the variations in proteins associated with apoptosis, the rats' brains were removed out of their skulls, and each hippocampus was isolated on a bar of ice. After homogenization of hippocampus in protein extraction buffer (0.1 M sodium chloride, 10 mM tris-sodium chloride, 0.1 mM EDTA, and protease inhibitor cocktail with pH=8), and centrifuging at 4°C at 1500 rpm for 45 min, the supernatant containing total protein was collected and the concentration of the total protein was determined using Bradford method. [27] IGF-1 level of Hippocampus was measured using ELISA test (Sandwich ELISA Kit, R&D Co.).

To do western blot analysis, at first, 60 μg of the total protein of each sample was added to sodium dodecyl sulphate polyacrylamide gel and was transferred to polyvinylidene difluoride (PVDF) membrane after electrophoresis. Then, the membrane was incubated with each primary monoclonal antibody (Cell Signaling Co.) at dilution of 1:1000 against Bax, Bcl-2, caspase-3, and beta-actin [as a loading control] proteins, and then incubated with secondary antibodies (Cell Signaling Co.) at dilution of 1:10000 and finally, the bonds appeared on photographic film using Ecl-Plus kit (ECL; Amersham Pharmacia Biotech). The films were scanned and quantified by Image J software. Values of each Bax, Bcl-2, and Caspase-3 proteins were divided by the beta-actin value of the same group, and then the proportion of Bax proteins to Bcl-2 proteins was determined.

The means were compared using SPSS software by one-way ANOVA and Tukey tests at different times. Minimum significance level was P < 0.05.


  Results Top


The results of the behavioral experiment for different groups of rats exposed to predator stress are shown in Graph 1. [Additional file 1] In this experiment, behavioral variations, regarding the proportion of time rats spent in the closed arms to the time spent in the open arms of elevated plus-maze, in all groups under predator stress, had a significant upward trend than that in control group (P < 0.05). These variations reached their maximum in proportion to other groups during the second day (P < 0.05), then reduced to a constant level that still differed significantly from that of control group. The level of serum corticosterone significantly increased on the 2 nd day and 3 rd day after stress as compared to that in all groups and the control group (P < 0.05) [Graph 2].[Additional file 2] The amount of IGF-1, on the first and second days after stress, had a significant upward trend than that in control group (P < 0.05). This amount reached its maximum on the second day as compared with that in other groups (P < 0.05) [Graph 3]. [Additional file 3]

In order to study pro-apoptotic and anti-apoptotic variations in the groups under stress and control group, the amount of Bax, Bcl-2, and caspase-3 proteins in rat hippocampus was assessed using western blot analysis. The results showed that the amount of protein on the 2 nd , 3 rd , 7 th , and 14 th days after stress increased significantly as compared to that of the control group (P < 0.05). This amount reached its maximum on the 3 rd day as compared to that in other groups (P < 0.05) [Graph 4] [Additional file 4] and [Figure 1]. Variations in Bax protein on the 1 st and 30 th days were not as significant as those of control group. Variations in Bcl-2 protein, up to the 2 nd day after stress, were not different from those in control group; however, a quite significant reduction was found on the 3 rd and 7 th days (P < 0.05), and then reached its normal level [Graph 5] [Additional file 5] and [Figure 2]. Calculation of ratio Bax/Bcl-2 as an indication of apoptosis showed this ratio in the hippocampus of the groups under stress on the 2 nd , 3 rd , 7 th , and 14 th was significant as compared with that in the control group (P < 0.05). This ratio reached its maximum during the 3 rd day as compared with other groups (P < 0.05) [Graph 6] [Additional file 6].
Figure 1: Immunoblot pattern of Bax and b-actin proteins in hippocampus of rats in groups of 1st, 2nd, 3rd, 7th, 14th, and 30th days after exposed to predator stress and in control group

Click here to view
Figure 2: Immunoblot pattern of Bcl-2 and b-actin proteins in hippocampus of rats in groups of 1st, 2nd, 3rd, 7th, 14th, and 30th days after exposed to predator stress and in control group

Click here to view


The predator stress could significantly increase activation of caspase-3 in the 2 nd to 30 th groups as compared with that in control group (P < 0.05). This activity had a highly significant upward trend on the 3 rd day (P < 0.05), though it followed a downward trend after 72 hours. The caspase-3 activation rate was not significant on the 1 st day [Graph 7] [Additional file 7] and [Figure 3].
Figure 3: Immunoblot pattern of caspase-3 and b-actin proteins in hippocampus of rats in groups of 1st, 2nd, 3rd, 7th, 14th, and 30th days after exposed to predator stress and in control group

Click here to view



  Discussions Top


Studies have shown most of the victims of PTSD express social and behavioral abnormalities due to the hippocampal atrophy in these people. [28] Numerous theories have been published on the causes of atrophy; however, there are not documented evidences on the intervention of apoptosis in hippocampal atrophy. [29]

Today, because of ethical issues with the molecular studies in human models, different animal models including single prolong stress, predator stress, etc. have been suggested for studying the causes of hippocampal atrophy in PTSD, and the behavioral variation in rats on the elevated plus-maze apparatus is considered as a symptom of this disorder. [30] In the animal model of predator stress in Adamec's study and in the animal model of single prolong stress in Shi et al.'s study, behavioral changes in rats in all groups under stress on the elevated plus-maze apparatus were quite significant as compared with those in control group. [31],[32] Shi et al.'s study on the rats' hippocampus, using single prolong stress model, showed the amount of apoptotic Bax, caspase-3 proteins, and the ratio of Bax/Bcl-2 in all stressed groups under 1 month increased significantly as compared with those in the control group, and the amount of anti-apoptotic Bcl-2 protein decreased significantly as compared with that in the control group. [32],[33] The present study also showed significant behavioral variations on the elevated plus-maze apparatus in rats exposed to predator stress than those in the control group. Moreover, the expression rate of Bax protein in hippocampus and the ratio of Bax/Bcl-2 whose variation shows triggering of apoptosis mitochondrial pathway were significant only in the 2 nd to 14 th groups as compared with those in the control group. However, the expression rate of Bcl-2 was significantly reduced only in the 3 rd and 7 th groups as compared with that in the control group. The caspase-3 activity rate in hippocampus in the 2 nd to 30 th groups exposed to predator stress was not significant. Variations in factors related to apoptosis were found in the present study and in Shi et al.'s study; however, one of the reasons for differences in the results of these two studies might be due to the type of stress and subsequently different animal models used in these two studies.

Prevention of cell programmed death may be caused by various molecular mechanisms like neurotrophins, growth factors synthesis, and expression adjustment of apoptotic regulators. [34] IGF-1 produced inside neurons acts as a protective factor, which has been found to be reduced in hippocampus in diseases related to central nervous system. [34],[35] Furthermore, reduction of IGF-1 results in the increase in apoptosis in purkinje cells and anti-apoptotic effects of this factor has been found in dorsal root ganglia and human neuroblastoma cell line. [36],[37] The androgenic IGF-1 exerts its anti-apoptotic effects on Bcl-2 family through preventing the increase in the ratio of intracellular Bax/Bcl-2. [38] Regarding the comparison of intracellular IGF-1 and Bcl-2 on the 1 st and 2 nd days after the stress in the present study, IGF-1 can be considered as a highly preliminary protective factor against apoptotic effects of stress. Therefore, in this study, it seemed that the significant increase in IGF-1 of hippocampus up to the 2 nd day prevented the reduction of Bcl-2, and this inhibited the quick change in ratio of intracellular Bax/Bcl-2 and consequently, the incidence of apoptosis slowed up within 48 hours after stress. However, weak activity of caspase-3 on the 30 th day, despite Bax/Bcl-2 ratio's returning to its normal level, might show the activity of other apoptosis pathways besides mitochondrial pathway due to the stress.


  Acknowledgment Top


This study was conducted by the financial support of Research Deputy of Kashan University of Medical Sciences and Neuroscience Research Center of Shahid Beheshti University of Medical Sciences. The researchers appreciate all the authorities and researchers of these research institutions.

 
  References Top

1.Victor AM, Bernstein GA. Anxiety disorders and posttraumatic stress disorder update. Psychiatr Clin North Am 2009; 32:57-69.  Back to cited text no. 1
    
2.Boscarino JA. Posttraumatic stress disorder and physical illness: Results from clinical and epidemiologic studies. Ann N Y Acad Sci 2004; 1032:141-53.  Back to cited text no. 2
    
3.Bonne O, Brandes D, Gilboa A, Gomori JM, Shenton ME, Pitman RK, et al., Longitudinal MRI study of hippocampal volume in trauma survivors with PTSD. Am J Psychiatry 2001; 158:1248-51.  Back to cited text no. 3
    
4.Sapolsky RM. Atrophy of the hippocampus in posttraumatic stress disorder: How and when?. Hippocampus 2001; 11:90-1.  Back to cited text no. 4
    
5.Kitayama N, Vaccarino V, Kutner M, Weiss P, Bremner JD. Magnetic resonance imaging [MRI] measurement of hippocampal volume in posttraumatic stress disorder: A meta-analysis. J Affect Disord 2005; 88:79-86.  Back to cited text no. 5
    
6.Blagosklonny MV. Apoptosis, proliferation, differentiation: In search of the order. Semin Cancer Biol 2003; 13:97-105.  Back to cited text no. 6
    
7.Thornberry NA, Lazebnik Y. Caspases: Enemies within. Science 1998; 281:1312-6.  Back to cited text no. 7
    
8.Guicciardi M, Gores G. Life and death by death receptors. FASEB J 2009; 23:1625-7.  Back to cited text no. 8
    
9.Waldmeier PC, Tatton WG. Interrupting apoptosis in neurodegenerative disease: Potential for effective therapy? Drug Discov Today 2004;9:210-8.  Back to cited text no. 9
    
10.Shishkina GT, Kalinina TS, Berezova IV, Bulygina VV, Dygalo NN. Resistance to the development of stress-induced behavioral despair in the forced swim test associated with elevated hippocampal Bcl-xl expression. Behav Brain Res 2010; 213:218-24.  Back to cited text no. 10
    
11.Watanabe Y, Gould E, McEwen BS. Stress induces atrophy of pical dendrites of hippocampal CA3 pyramidal neurons. Brain Res 1992; 588:341-5.  Back to cited text no. 11
    
12.Bremner JD. Stress and brain atrophy. CNS Neural Disorder DR 2006; 5:503-12.  Back to cited text no. 12
    
13.Davila D, Piriz J, Trejo JL, Nunez A, Torres-Aleman I. Insulin and insulin-like growth factor I signalling in neurons. Front Biosci 2007; 12:3194-202.  Back to cited text no. 13
    
14.Llorens-Martín M, Torres-Alemán I, Trejo JL. Mechanisms mediating brain plasticity: IGF1 and adult hippocampal neurogenesis. Neuroscientist 2009; 15:134-48.   Back to cited text no. 14
    
15.Aberg D. Role of the growth hormone/insulin-like growth factor 1 axis in neurogenesis. Endocr Dev 2010; 17:63-76.   Back to cited text no. 15
    
16.Bitar MS. Insulin and glucocorticoid-dependent suppression of the IGF-I system in diabetic wounds. Surgery 2000; 127:687-95.  Back to cited text no. 16
    
17.D'Ercole AJ, Ye P, O'Kusky JR. Mutant mouse models of insulin-like growth factor actions in the central nervous system. Neuropeptides 2002; 36:209-20.   Back to cited text no. 17
    
18.Peruzzi F, Prisco M, Dews M, Salomoni P, Grassilli E, Romano G, et al., Multiple signaling pathways of the insulin-like growth factor 1 receptor in protection from apoptosis. Mol Cell Biol 1999; 19:7203-15.  Back to cited text no. 18
    
19.McCarthy NJ, Whyte MK, Gilbert CS, Evan GI. Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J Cell Biol 1997; 136:215-27.  Back to cited text no. 19
    
20.Haunstetter A, Izumo S. Apoptosis: Basic mechanisms and implications for cardiovascular disease. Circ Res 1998; 82:1111-29.  Back to cited text no. 20
    
21.Dietrich MO, Muller A, Bolos M, Carro E, Perry ML, Portela LV. Western style diet impairs entrance of blood-borne insulin-like growth factor-1 into the brain. Neuromol Med 2007; 9:324-30.  Back to cited text no. 21
    
22.Bateman JM, McNeill H. Insulin/IGF signalling in neurogenesis. Cell Mol Life Sci 2006; 63:1701-5.   Back to cited text no. 22
    
23.Adamec RE, Stark-Adamec C, Livingston KE. The development of predatory aggression and defense in the domestic cat (Felis catus). II. Development of aggression and defense in the first 164 days of life. Behav Neural Biol 1980; 30:410-34.  Back to cited text no. 23
    
24.Adamec RE, Stark-Adamec C, Livingston KE. The development of predatory aggression and defense in the domestic cat (Felis catus). I. Effects of early experience on adult patterns of aggression and defense. Behav Neural Biol 1980; 38:389-409.  Back to cited text no. 24
    
25.Cohen H, Zohar J, Matar MA, Zeev K, Loewenthal U, Richter-Levin G. Setting apart the affected: the use of behavioral criteria in animal models of post traumatic stress disorder. Neuropsychopharmacol 2004; 29:1962-70.  Back to cited text no. 25
    
26.Pellow S, Chopin P, File SE, Briley M. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Meth 1985; 14:149-67.  Back to cited text no. 26
    
27.Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248-54.  Back to cited text no. 27
    
28.Fink G. Stress controversies: Post-traumatic stress disorder, hippocampal volume,gastroduodenal ulceration. J Neuroendocrinol 2011; 23:107-17.  Back to cited text no. 28
    
29.Zhang L, Zhou R, Li X, Ursano RJ, Li H. Stress-induced change of mitochondria membrane potential regulated by genomic and non-genomic GR signaling: A possible mechanism for hippocampus atrophy in PTSD. Med Hypotheses 2006; 66:1205-8.  Back to cited text no. 29
    
30.Cohen H, Kozlovsky N, Alona C, Matar MA, Joseph Z. Animal model for PTSD: From clinical concept to translational research. Neuropharmacol 2012; 62:715-24.   Back to cited text no. 30
    
31.Adamec RE, Burton P, Shallow T, Budgell J. Unilateral block of NMDA receptors in the amygdala prevents predator stress-induced lasting increases in anxiety-like behavior and unconditioned startle--effective hemisphere depends on the behavior. Physiol Behav 1999;65:739-51.  Back to cited text no. 31
    
32.Li X, Han F, Liu D, Shi Y. Changes of Bax, Bcl-2 and apoptosis in hippocampus in the rat model of post-traumatic stress disorder. Neurol Res 2010; 32:579-86.  Back to cited text no. 32
    
33.Li XM, Han F, Liu JD, Shi Y. Single-prolonged stress induced mitochondrial-dependent apoptosis in hippocampus in the rat model of post-traumatic stress disorder. J Chem Neuroanat 2010; 40:248-55.  Back to cited text no. 33
    
34.Lee E, Son H. Adult hippocampal neurogenesis and related neurotrophic factors. BMB Rep 2009; 42:239-44.  Back to cited text no. 34
    
35.Bitar MS. Insulin and glucocorticoid-dependent suppression of the IGF-I system in diabetic wounds. Surgery 2000; 127:687-95.  Back to cited text no. 35
    
36.Zhang W, Ghetti B, Lee WH. Decreased IGF-I gene expression during the apoptosis of Purkinje cells in pcd mice. Brain Res Dev Brain Res 1997; 98:164-76.  Back to cited text no. 36
    
37.Ishii DN. Implication of insulin-like growth factors in the pathogenesis of diabetic neuropathy. Brain Res Rev 1995; 20:47-67.  Back to cited text no. 37
    
38.Anderson MF, Aberg MA, Nilsson M, Eriksson PS. Insulin-like growth factor-I and neurogenesis in the adult mammalian brain. Brain. Res Dev Brain Res 2002; 134:115-22.  Back to cited text no. 38
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

Top
Previous article  Next article
 
  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
Material and Methods
Results
Discussions
Acknowledgment
References
Article Figures

 Article Access Statistics
    Viewed2047    
    Printed77    
    Emailed0    
    PDF Downloaded526    
    Comments [Add]    

Recommend this journal