Author’s Accepted Manuscript Serum levels of nerve growth factor (NGF) in patients with major depression disorder and suicide risk Carolina David Wiener, Sharon de Mello Ferreira, Fernanda Pedrotti Moreira, Guilherme Bittencourt, Jacqueline Flores de Oliveira, Mariane Lopez Molina, Karen Jansen, Luciano Dias de Mattos Souza, Diogo Rizzato Lara, Luiz Valmor Portela, Ricardo Azevedo da Silva, Jean Pierre Oses
PII: DOI: Reference:
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S0165-0327(15)00388-2 http://dx.doi.org/10.1016/j.jad.2015.05.067 JAD7510
To appear in: Journal of Affective Disorders Received date: 26 February 2015 Revised date: 18 May 2015 Accepted date: 27 May 2015 Cite this article as: Carolina David Wiener, Sharon de Mello Ferreira, Fernanda Pedrotti Moreira, Guilherme Bittencourt, Jacqueline Flores de Oliveira, Mariane Lopez Molina, Karen Jansen, Luciano Dias de Mattos Souza, Diogo Rizzato Lara, Luiz Valmor Portela, Ricardo Azevedo da Silva and Jean Pierre Oses, Serum levels of nerve growth factor (NGF) in patients with major depression disorder and suicide risk, Journal of Affective Disorders, http://dx.doi.org/10.1016/j.jad.2015.05.067 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Serum levels of nerve growth factor (NGF) in patients with major depression disorder and suicide risk
Carolina David Wienera, Sharon de Mello Ferreiraa, Fernanda Pedrotti Moreiraa, Guilherme Bittencourta, Jacqueline Flores de Oliveiraª, Mariane Lopez Molinaa, Karen Jansena, Luciano Dias de Mattos Souzaa, Diogo Rizzato Larab, Luiz Valmor Portelac, Ricardo Azevedo da Silvaa, Jean Pierre Osesa*
a
Programa de Pós-Graduação em Saúde e Comportamento, Centro de Ciências da Vida e da Saúde,
Universidade Católica de Pelotas, Av. Gonçalves Chaves 373, 96015-560, Pelotas, RS, Brasil. b
Programa de Pós-Graduação em Biologia Celular e Molecular Pontifícia Universidade do Rio
Grande do Sul, Av. Ipiranga 6681, 90619-900, Porto Alegre, RS, Brasil. c
Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da
Saúde, Universidade Federal do Rio Grande do Sul, Av. Ramiro Barcelos ͒2600, 90035-000, Porto Alegre, RS, Brasil.
*Corresponding author: Jean Pierre Oses, PhD Programa de Pós-Graduação em Saúde e Comportamento, Centro de Ciências da Vida e da Saúde, Universidade Católica de Pelotas, Av. Gonçalves Chaves 373, 96015-560, Pelotas, RS, Brasil. Tel: +55 (53) 2128 8404͒Fax: +55 (53) 2128 8229 Email: [email protected]
Abstract Nerve growth factor (NGF) is an important member of the neurotrophins group and their involvement in the pathophysiology of major depression disorder (MDD) and suicide risk (SR) has been recently suggested. The aim of this study is to evaluate the changes in NGF serum levels in individuals with MDD and with or without risk of suicide, in subjects from a young populationbased sample. This is a paired cross-sectional study nested in a population-based study. Individuals were rated for MDD and SR by a diagnostic interview – Mini International Neuropsychiatric Interview (M.I.N.I). The total population of the sample was comprised of 141 subjects distributed in three groups: 47 healthy controls, 47 subjects with current depressive episode without SR (MDD) and 47 subjects with current depressive episode and with SR (MDD + SR). NGF serum levels were significantly reduced in the MDD and MDD+SR groups when compared with controls (p ≤ 0.001). However, there were no differences in NGF levels between the MDD and MDD+SR groups (p = 1.000). These results suggest that reduced NGF serum levels can be a possible biomarker of MDD.
Keywords: Nerve Growth Factor, major depressive disorder, suicide risk.
Highlights NGF serum levels were significantly reduced in MDD when compared with controls NGF serum levels were significantly reduced in MDD+SR when compared with controls There were no differences in NGF serum levels between the MDD and MDD+SR groups
1.
Introduction Among psychiatric disorders, depression is the most prevalent one, affecting approximately
17% of the population (Schmidt et al., 2011). Individuals with major depressive disorder (MDD) have a high rate of morbidity and mortality, with a suicide risk (SR) rate equivalent to 15% (Mann, 2002). Studies have shown biochemical and morphological abnormalities in certain brain regions in patients with psychiatric disorders and suicide victims (Diniz et al., 2013; Dwivedi et al., 2005; Schmidt et al., 2011). Nerve growth factor (NGF) is an important member of the neurotrophins groups and is produced mainly in the cortex, hippocampus and hypothalamus, but also in the peripheral nervous system and immune system (Martino et al., 2013; Xiong et al., 2011). Recent studies have demonstrated that immune system cells may be an additional source of NGF secretion, and these cells express NGF and p75NTR receptors p140TrkA, promoting both cell survival and proliferation, in addition to inducing proinflammatory cytokines secretion (Takei and Laskey, 2008). Neurotrophins are proteins that play important roles in neuronal survival, differentiation, connectivity and plasticity during development and adulthood (Banerjee et al., 2013; Dwivedi et al., 2005). It has been recently suggested that neurotrophins are involved in the pathophysiology of psychiatric disorders, particularly brain-derived neurotrophic factor (BDNF), which has been found to present reduced serum levels in patients with MDD (Hashimoto, 2010). Clinical studies have detected reduced levels of NGF in patients with MDD when compared with healthy individual controls (Diniz et al., 2013; Xiong et al., 2011). Nevertheless, another study has found no difference in NGF levels in depressed individuals (Ziegenhorn et al., 2007). A study with suicide victims found a decrease in NGF levels in the hippocampus of suicide victims when compared with healthy controls (Banerjee et al., 2013). In addition, treatment with certain antidepressants has increased NGF levels in both clinical and experimental studies (Hassanzadeh and Rahimpour, 2011). Few studies have been performed to examine an association between NGF and suicide risk in a human population. Studies linking the levels of NGF in MDD and SR are
poorly understood. As such, the objective of this study is to evaluate changes in NGF serum levels in patients with MDD and with or without risk of suicide; to investigate the possible involvement of neurotrophins in the pathophysiology of depression and suicide risk and to assist in the early detection and effective treating of this disorder.
2.
Methods
2.1.
Subjects This is a paired cross-sectional study nested in a population-based study of people aged 18-
35 years. The sample consisted of 1380 participants living in the city of Pelotas, (Brazil), in the period from June 2011 to October 2012. After the subjects were identified and invited, the volunteers signed a term of free and informed consent and responded to a questionnaire which collected sociodemographic data, drug misuse and The National Economic Indicator – IEN (Barros and Victora, 2005). This study was approved by the university’s Ethics Committee (2010/15). Current MDD and suicide risk diagnosis were both measured by the Mini International Neuropsychiatric Interview (M.I.N.I). This is a short structured interview, lasting around 15-30 minutes, designed to be used in clinical practice and research with the goal of diagnosing the interviewee according to DSM-IV criteria. Suicide risk was dichotomized between absence (low or none) and presence of risk (moderate or high), considering the score of 6 or more for suicide risk presence (Sheehan et al., 1998). Individuals who were taking a psychoactive substance, or in use of psychopharmacological drugs, or who presented any anxiety/psychotic disorder or those who were unable to understand or answer the questions were excluded from the study. After the diagnostic interview, all individuals with current depressive episode and SR without history of (hypo)mania or other disorders were selected for the MDD+SR group. Additionally, two groups of control subjects matched by gender and age were recruited. The active control group (MDD) included individuals with current depression but no past history of
(hypo)mania. We also included a control group of healthy individuals without history of mental illness. 2.2.
Blood sample collection and NGF assessment For the biochemical analyses, 10mL of blood was withdrawn from each subject by
venipuncture, into an anticoagulant-free vacuum tube, after the interview, between the hours of 8:00 AM and 11:00 AM. The blood was immediately centrifuged at 4,000 x g for 10 minutes, and the resulting serum was kept frozen at -80 ºC until analysis. NGF serum levels were measured with a sandwich-ELISA using an available immunoassay kit according to the manufacturer’s instructions (DuoSet ELISA Development, R&D Systems, Inc., USA) and expressed in pg/mL (de Azevedo Cardoso et al., 2014). 2.3.
Statistical analysis Statistical analyses were performed in the Graph Pad Prism 6.0 (GraphPad Software Inc.,
San Diego, USA) and IBM SPSS Statistic 21.0 (IBM Corporation, New York, USA) software for Windows. Sociodemographic and NGF serum levels are shown by absolute and relative frequencies or by means and standard deviations. To verify consistency between groups, the chi-square test and analysis of variance (ANOVA) were used. The comparison between the levels of NGF in the group of individuals with MDD, MDD+SR and healthy controls was performed by ANOVA with posthoc Bonferroni. To control for possible confounding variables, we included in the linear regression adjusted analysis the variables that presented associations with p ≤ 0.20 in the raw analysis. Results with p ≤ 0.05 were considered statistically significant.
3.
Results The total population sample was comprised of 141 individuals: 47 with MDD+SR, 47 with
MDD and 47 healthy individuals as controls. Each group was composed of 35 women and 12 men, with a mean age of 27.32 ± 5.04, 27.34 ± 5.08, and 27.40 ± 5.15 years, respectively. Homogeneous samples showed no differences between diagnostic groups regarding gender (p = 1.000), ethnicity
(p = 0.400) and age (p = 0.996). However, socioeconomic index and years of education were statistically different. The MDD+SR group presented lower economic classes (p = 0.030) and years of education (p = 0.004) (Table 1). NGF serum levels were significantly reduced in the MDD and MDD+SR groups (92.39 ± 41.21 pg/mL and 100.99 ± 35.08 pg/mL, respectively) when compared with the healthy controls group (149.81 ± 65.94 pg/mL) (F = 18:58, p ≤ 0.001). The post-hoc Bonferroni multiple comparison test revealed a significant difference between the healthy controls group and MDD group (p ≤ 0.001) and between the healthy controls group and MDD+SR groups (p ≤ 0.001). Nevertheless, there were no differences in NGF serum levels between the MDD and MDD+SR groups (p = 1.000; Fig. 1). After controlling for years of education and socioeconomic index through linear regression, NGF serum levels between the healthy controls, MDD and MDD+SR groups remained statistically significant (p = 0.004, data not shown).
4.
Discussion In this study, it was possible to observe a significant decrease in NGF serum levels in
individuals with MDD and MDD+SR when compared with healthy controls. Moreover, it was not possible to observe significant differences between individuals with depression and depression plus risk of suicide. It is important to highlight that our sample consisted of young and non-medicated subjects experiencing the early stages of these illnesses and also that the sociodemographic variables analysed here were not associated with changes in NGF levels in our population. The importance of NGF in the behavioral and neuronal responses associated with psychiatric illness, including brain changes, was shown through the decrease of NGF levels in patients with MDD (Diniz et al., 2013; Xiong et al., 2011). Recently, Diniz et al. (2013) found a reduction in the NGF levels of depressed elder patients when compared with a control group. Still, a more detailed analysis showed similarity between past and current depression groups, with no significant differences between them. A significant reduction in NGF levels was found only
between the past depression group and the control group (Diniz et al., 2013). Similarly, in our study, patients with MDD had reduced levels of NGF when compared with healthy controls, demonstrating possible involvement of this neurotrophin in the pathophysiology of depression. Changes in NGF levels after treatment with antidepressants have been described in the literature. In an experimental study, chronic treatment with nortriptyline and citalopram antidepressants increased brain NGF levels (Hassanzadeh and Rahimpour, 2011). On the other hand, Martino et al. (2013) demonstrated that patients with MDD had NGF serum levels reduced when compared with healthy controls, not being reversed by duloxetine antidepressant, possibly reflecting functional impairment in the adaptive neuroplasticity of stress in depressive disorders (Martino et al., 2013). Liu et al. (2014) found a significant improvement in the clinical symptoms of patients with mood disorders after 8 weeks of intensive treatment using antidepressants, but without significant changes in NGF levels (Liu et al., 2014). More importantly, in our study, individuals are at an early stage of the disease and do not make use of psychotherapeutic medication that could alter the measured NGF serum levels. NGF plays an important role in the development of mood disorders and their association with patients under risk of suicide. Dwivedi et al. (2005) analysed NGF levels in suicide victims diagnosed with MDD and other psychiatric disorders. The authors found no differences in the levels of NGF between both groups, but both of them showed significant differences when compared with normal controls, both in the prefrontal cortex and in the hippocampus (Dwivedi et al., 2005). The hippocampus and prefrontal cortex are related to cognition and mood regulation, and these are involved in the pathophysiology of affective disorders and suicide (Dwivedi et al., 2005). In addition, the hippocampus is an area of the brain that is affected by early stress, a major factor in suicidal behavior (Banerjee et al., 2013). Although we observed a reduction in NGF serum levels, there were no significant differences between depressed individuals with and without suicide risk, demonstrating that the presence of risk factors for suicide did not interfere with the levels of NGF.
Studies suggest that both symptoms of depression and suicide are related to a reduction in synaptic plasticity in the brain, possibly due to the inability of the brain to respond or adapt to aversive stimulus (Dwivedi et al., 2005; Martino et al., 2013). The reduced expression of neurotrophins in tissues, serum and plasma has been demonstrated through the normalization of these values after clinical treatment (Hashimoto, 2010; Hassanzadeh and Rahimpour, 2011). Regarding the role of NGF, recent studies suggest that this neurotrophin can be a powerful modulator of the immune system by mediating proinflammatory responses in several physiological conditions (Diniz et al., 2013). Furthermore, several evidence point that peripheral CNS inflammatory cytokines influence and are able to induce depressive symptoms (Diniz et al., 2013; Martino et al., 2013; Xiong et al., 2011). Xiong et al. (2011) found decreased serum levels of NGF and IL-2 in subjects with major depression and schizophrenia when compared with healthy controls, suggesting that the interaction between cytokines and neurotrophins is vital in maintaining homeostasis in the brain (Xiong et al., 2011). Also, NGF performs an important role in brain functioning, and alterations in its central levels may influence the development of mental disorders. Studies correlating NGF, major depression and suicide risk are scarce and heterogeneous, especially concerning sample characteristics methodology. Additionally, the participants of these studies among the literature are usually hospitalized patients with longer disease duration and severity. As such, our study is derived from a population-based sample, consisting of young adults experiencing the early stages of the disease, no history of prior hospitalizations and without use of psychiatric medication. Despite these strengths, some limitations should be considered. In the present study, severity of depressive symptoms as well as depression subtypes (such as melancholic and atypical) were not evaluated. A better clinical characterization could clarify the relationship between stressors and peripheral biomarkers in the pathophysiology of MDD. However, the presence of suicide risk did not interfere with NGF serum levels, indicating that this neurotrophin may be related to the diagnosis of MDD and not with symptoms severity.
The understanding of the physiological changes related to mood disorders can provide a better understanding of the genesis and mechanisms involved in the progression of these conditions. The identification of peripheral biomarkers capable of helping in the diagnosis or in monitoring the progression of the condition and treatment remain a goal in the field (Wiener et al., 2013). In this work, we observed reduced serum levels of NGF in MDD and MDD+SR subjects, which suggests that this biomarker can be used as a putative marker for MDD when compared with healthy individuals.
Conflict of interest statement None of the authors of this paper have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
Acknowledgements This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ), and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS ) Brazil.
References
Banerjee, R., Ghosh, A.K., Ghosh, B., Bhattacharyya, S., Mondal, A.C., 2013. Decreased mRNA and Protein Expression of BDNF, NGF, and their Receptors in the Hippocampus from Suicide: An Analysis in Human Postmortem Brain. Clinical medicine insights. Pathology 6, 1-11. Barros, A.J., Victora, C.G., 2005. [A nationwide wealth score based on the 2000 Brazilian demographic census]. Revista de saude publica 39, 523-529. de Azevedo Cardoso, T., Mondin, T.C., Wiener, C.D., Marques, M.B., Fucolo Bde, A., Pinheiro, R.T., de Souza, L.D., da Silva, R.A., Jansen, K., Oses, J.P., 2014. Neurotrophic factors, clinical features and gender differences in depression. Neurochemical research 39, 1571-1578.
Diniz, B.S., Teixeira, A.L., Machado-Vieira, R., Talib, L.L., Gattaz, W.F., Forlenza, O.V., 2013. Reduced serum nerve growth factor in patients with late-life depression. The American journal of geriatric psychiatry : official journal of the American Association for Geriatric b Psychiatry 21, 493-496. Dwivedi, Y., Mondal, A.C., Rizavi, H.S., Conley, R.R., 2005. Suicide brain is associated with decreased expression of neurotrophins. Biological psychiatry 58, 315-324. Hashimoto, K., 2010. Brain-derived neurotrophic factor as a biomarker for mood disorders: an historical overview and future directions. Psychiatry and clinical neurosciences 64, 341-357. Hassanzadeh, P., Rahimpour, S., 2011. The cannabinergic system is implicated in the upregulation of central NGF protein by psychotropic drugs. Psychopharmacology 215, 129-141. Liu, X., Zhang, T., He, S., Hong, B., Chen, Z., Peng, D., Wu, Y., Wen, H., Lin, Z., Fang, Y., Jiang, K., 2014. Elevated serum levels of FGF-2, NGF and IGF-1 in patients with manic episode of bipolar disorder. Psychiatry research 218, 54-60. Mann, J.J., 2002. A current perspective of suicide and attempted suicide. Annals of internal medicine 136, 302-311. Martino, M., Rocchi, G., Escelsior, A., Contini, P., Colicchio, S., de Berardis, D., Amore, M., Fornaro, P., Fornaro, M., 2013. NGF serum levels variations in major depressed patients receiving duloxetine. Psychoneuroendocrinology 38, 1824-1828. Schmidt, H.D., Shelton, R.C., Duman, R.S., 2011. Functional biomarkers of depression: diagnosis, treatment, and pathophysiology. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 36, 2375-2394. Sheehan, D.V., Lecrubier, Y., Sheehan, K.H., Amorim, P., Janavs, J., Weiller, E., Hergueta, T., Baker, R., Dunbar, G.C., 1998. The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD10. The Journal of clinical psychiatry 59 Suppl 20, 22-33.
Takei, Y., Laskey, R., 2008. Interpreting crosstalk between TNF-alpha and NGF: potential implications for disease. Trends in molecular medicine 14, 381-388. Wiener, C.D., Jansen, K., Ghisleni, G., Kaster, M.P., Souza, L.D., Lara, D.R., Portela, L.V., da Silva, R.A., Oses, J.P., 2013. Reduced serum levels of neuron specific enolase (NSE) in drug-naive subjects with major depression and bipolar disorder. Neurochemical research 38, 1394-1398. Xiong, P., Zeng, Y., Wan, J., Xiaohan, D.H., Tan, D., Lu, J., Xu, F., Li, H.Y., Zhu, Z., Ma, M., 2011. The role of NGF and IL-2 serum level in assisting the diagnosis in first episode schizophrenia. Psychiatry research 189, 72-76. Ziegenhorn, A.A., Schulte-Herbruggen, O., Danker-Hopfe, H., Malbranc, M., Hartung, H.D., Anders, D., Lang, U.E., Steinhagen-Thiessen, E., Schaub, R.T., Hellweg, R., 2007. Serum neurotrophins--a study on the time course and influencing factors in a large old age sample. Neurobiology of aging 28, 1436-1445.
Legends Figure 1. NGF serum levels in controls, MDD and MDD+SR individuals. NGF levels were increased in the control group (149.81± 65.94 pg/mL) when compared with the MDD (92.39 ± 41.21 pg/mL) and MDD+SR groups (100.99 ± 35.08pg/mL)(F = 18:58, p ≤ 0.001). The bar represents the median (control: 2.86 ng/mL; MDD: 1.47; BD: 1.52). Control vs MDD (**p ≤ 0.001), control vs MDD+SR (**p ≤ 0.001) MDD vs MDD+SR (p = 1.000) (post-hoc Bonferroni test).
Table 1. Socio-demographic characteristics of the sample according to the diagnostic. Control MDD MDD-SR n (%)/Mean ± S.D. n (%)/Mean ± S.D. n (%)/Mean ± S.D. Gender Female 35 (33.3) 35 (33.3) 35 (33.3) Male 12 (33.3) 12 (33.3) 12 (33.3) Ethnicity Not white White Age (years) Economic Index Class Low Medium High School (years) NGF levels (pg/mL)
p value 1.000
0.400 14 (37.8) 33 (31.7)
9 (24.3) 38 (36.5)
14 (37.8) 33 (31.7)
27.40 ± 5.15
27.34 ± 5.08
27.32 ± 5.04
0.996 0.030
18 (26.9) 20 (41.7) 9 (34.6)
19 (26.9) 16 (33.3) 12 (42.2)
30 (44.8) 12 (25.0) 5 (19.2)
10.72 ± 3.64
11.02 ± 2.84
8.70 ± 4.17
0.004
149.81 ± 65.94
92.39 ± 41.21
100.99 ± 35.08
≤ 0.001
MDD: Major depressive disorder without suicide risk, MDD-RS: Major depressive disorder with suicide risk, S.D.: Standard deviation.
Highlights
NGF serum levels were significantly reduced in MDD compared with controls NGF serum levels were significantly reduced in MDD+SR compared with controls There was no difference in the NGF serum levels between the MDD and MDD+SR groups
Acknowledgements This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ), and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS ) Brazil.
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Role of the funding source The research was funded by a grant from the Brazilian governmental agencies: FAPERGS/PRONEX (10/0055-0), CAPES (23038.001671/2012-16). The agencies had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. Funding and Disclosure FPM received funding from the Brazilian agency for fostering Coordination of Improvement of Higher Education Personnel (CAPES) under the Ministry of Education. CDW received funding from the Brazilian agency Foundation for Research Support of the State of Rio Grande do Sul (FAPERGS) linked to the Secretariat of Science and Technology of the State of Rio Grande do Sul and Coordination of Improvement of Higher Education Personnel (CAPES) under the Ministry of Education.
JFO received funding from the National Counsel of Technological and Scientific
Development (CNPq) under the Ministry of Education. LDMS, RAS, KJ, JPO, LVP and DRL received funding from the National Counsel of Technological and Scientific Development (CNPq) under the Ministry of Education; Brazilian agency promoting National Counsel of Technological and Scientific Development linked to the Ministry of Science andIndividual contributions of each author of the article: Authors:
Wiener CD, conception and design of the study, biochemical analysis, data interpretation, manuscript writing, and approved the version to be published; Ferreira SM, conception and design of the study, drafting the article and approval of the version to be published; Moreira FP, conception and design of the study, drafting the article and approval of the version to be published; Bittencourt G, sample collection, biochemical analysis, drafting the article and approval of the version to be published;
Oliveira JF, sample collection, biochemical analysis, drafting the article and approval of the version to be published; Molina ML, execution of the project and in description of psychological variables; Jansen K, conception and design of the study, analysis and interpretation of data, drafting the article and approval of the version to be published; Souza LDM, conception and design of the study, analysis and interpretation of data, drafting the article and approval of the version to be published; Lara DR, conception and design of the study, drafting the article and approval of the version to be published; Portela LV, conception and design of the study, drafting the article and approval of the version to be published; Silva RA, conception and design of the study, drafting the article and approval of the version to be published. Oses J, conception and design of the study, biochemical analysis, data interpretation, drafting the article and approval of the version to be published;
Technology; Coordination of Improvement of Higher Education Personnel (CAPES) under the Ministry of Education; and Research Foundation of the State of Rio Grande do Sul (FAPERGS) linked to the Secretariat of Science and technology in the State of Rio Grande do Sul. Acknowledgements Support for this study was provided by a grant from Brazilian governmental agencies: FAPERGS/PRONEX (10/0055-0), CAPES (23038.001671/2012-16). Conflict of interest The authors have no conflict of interest to declare.
Figure(s)
PII: DOI: Reference:
www.elsevier.com/locate/jad
S0165-0327(15)00388-2 http://dx.doi.org/10.1016/j.jad.2015.05.067 JAD7510
To appear in: Journal of Affective Disorders Received date: 26 February 2015 Revised date: 18 May 2015 Accepted date: 27 May 2015 Cite this article as: Carolina David Wiener, Sharon de Mello Ferreira, Fernanda Pedrotti Moreira, Guilherme Bittencourt, Jacqueline Flores de Oliveira, Mariane Lopez Molina, Karen Jansen, Luciano Dias de Mattos Souza, Diogo Rizzato Lara, Luiz Valmor Portela, Ricardo Azevedo da Silva and Jean Pierre Oses, Serum levels of nerve growth factor (NGF) in patients with major depression disorder and suicide risk, Journal of Affective Disorders, http://dx.doi.org/10.1016/j.jad.2015.05.067 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Serum levels of nerve growth factor (NGF) in patients with major depression disorder and suicide risk
Carolina David Wienera, Sharon de Mello Ferreiraa, Fernanda Pedrotti Moreiraa, Guilherme Bittencourta, Jacqueline Flores de Oliveiraª, Mariane Lopez Molinaa, Karen Jansena, Luciano Dias de Mattos Souzaa, Diogo Rizzato Larab, Luiz Valmor Portelac, Ricardo Azevedo da Silvaa, Jean Pierre Osesa*
a
Programa de Pós-Graduação em Saúde e Comportamento, Centro de Ciências da Vida e da Saúde,
Universidade Católica de Pelotas, Av. Gonçalves Chaves 373, 96015-560, Pelotas, RS, Brasil. b
Programa de Pós-Graduação em Biologia Celular e Molecular Pontifícia Universidade do Rio
Grande do Sul, Av. Ipiranga 6681, 90619-900, Porto Alegre, RS, Brasil. c
Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da
Saúde, Universidade Federal do Rio Grande do Sul, Av. Ramiro Barcelos ͒2600, 90035-000, Porto Alegre, RS, Brasil.
*Corresponding author: Jean Pierre Oses, PhD Programa de Pós-Graduação em Saúde e Comportamento, Centro de Ciências da Vida e da Saúde, Universidade Católica de Pelotas, Av. Gonçalves Chaves 373, 96015-560, Pelotas, RS, Brasil. Tel: +55 (53) 2128 8404͒Fax: +55 (53) 2128 8229 Email: [email protected]
Abstract Nerve growth factor (NGF) is an important member of the neurotrophins group and their involvement in the pathophysiology of major depression disorder (MDD) and suicide risk (SR) has been recently suggested. The aim of this study is to evaluate the changes in NGF serum levels in individuals with MDD and with or without risk of suicide, in subjects from a young populationbased sample. This is a paired cross-sectional study nested in a population-based study. Individuals were rated for MDD and SR by a diagnostic interview – Mini International Neuropsychiatric Interview (M.I.N.I). The total population of the sample was comprised of 141 subjects distributed in three groups: 47 healthy controls, 47 subjects with current depressive episode without SR (MDD) and 47 subjects with current depressive episode and with SR (MDD + SR). NGF serum levels were significantly reduced in the MDD and MDD+SR groups when compared with controls (p ≤ 0.001). However, there were no differences in NGF levels between the MDD and MDD+SR groups (p = 1.000). These results suggest that reduced NGF serum levels can be a possible biomarker of MDD.
Keywords: Nerve Growth Factor, major depressive disorder, suicide risk.
Highlights NGF serum levels were significantly reduced in MDD when compared with controls NGF serum levels were significantly reduced in MDD+SR when compared with controls There were no differences in NGF serum levels between the MDD and MDD+SR groups
1.
Introduction Among psychiatric disorders, depression is the most prevalent one, affecting approximately
17% of the population (Schmidt et al., 2011). Individuals with major depressive disorder (MDD) have a high rate of morbidity and mortality, with a suicide risk (SR) rate equivalent to 15% (Mann, 2002). Studies have shown biochemical and morphological abnormalities in certain brain regions in patients with psychiatric disorders and suicide victims (Diniz et al., 2013; Dwivedi et al., 2005; Schmidt et al., 2011). Nerve growth factor (NGF) is an important member of the neurotrophins groups and is produced mainly in the cortex, hippocampus and hypothalamus, but also in the peripheral nervous system and immune system (Martino et al., 2013; Xiong et al., 2011). Recent studies have demonstrated that immune system cells may be an additional source of NGF secretion, and these cells express NGF and p75NTR receptors p140TrkA, promoting both cell survival and proliferation, in addition to inducing proinflammatory cytokines secretion (Takei and Laskey, 2008). Neurotrophins are proteins that play important roles in neuronal survival, differentiation, connectivity and plasticity during development and adulthood (Banerjee et al., 2013; Dwivedi et al., 2005). It has been recently suggested that neurotrophins are involved in the pathophysiology of psychiatric disorders, particularly brain-derived neurotrophic factor (BDNF), which has been found to present reduced serum levels in patients with MDD (Hashimoto, 2010). Clinical studies have detected reduced levels of NGF in patients with MDD when compared with healthy individual controls (Diniz et al., 2013; Xiong et al., 2011). Nevertheless, another study has found no difference in NGF levels in depressed individuals (Ziegenhorn et al., 2007). A study with suicide victims found a decrease in NGF levels in the hippocampus of suicide victims when compared with healthy controls (Banerjee et al., 2013). In addition, treatment with certain antidepressants has increased NGF levels in both clinical and experimental studies (Hassanzadeh and Rahimpour, 2011). Few studies have been performed to examine an association between NGF and suicide risk in a human population. Studies linking the levels of NGF in MDD and SR are
poorly understood. As such, the objective of this study is to evaluate changes in NGF serum levels in patients with MDD and with or without risk of suicide; to investigate the possible involvement of neurotrophins in the pathophysiology of depression and suicide risk and to assist in the early detection and effective treating of this disorder.
2.
Methods
2.1.
Subjects This is a paired cross-sectional study nested in a population-based study of people aged 18-
35 years. The sample consisted of 1380 participants living in the city of Pelotas, (Brazil), in the period from June 2011 to October 2012. After the subjects were identified and invited, the volunteers signed a term of free and informed consent and responded to a questionnaire which collected sociodemographic data, drug misuse and The National Economic Indicator – IEN (Barros and Victora, 2005). This study was approved by the university’s Ethics Committee (2010/15). Current MDD and suicide risk diagnosis were both measured by the Mini International Neuropsychiatric Interview (M.I.N.I). This is a short structured interview, lasting around 15-30 minutes, designed to be used in clinical practice and research with the goal of diagnosing the interviewee according to DSM-IV criteria. Suicide risk was dichotomized between absence (low or none) and presence of risk (moderate or high), considering the score of 6 or more for suicide risk presence (Sheehan et al., 1998). Individuals who were taking a psychoactive substance, or in use of psychopharmacological drugs, or who presented any anxiety/psychotic disorder or those who were unable to understand or answer the questions were excluded from the study. After the diagnostic interview, all individuals with current depressive episode and SR without history of (hypo)mania or other disorders were selected for the MDD+SR group. Additionally, two groups of control subjects matched by gender and age were recruited. The active control group (MDD) included individuals with current depression but no past history of
(hypo)mania. We also included a control group of healthy individuals without history of mental illness. 2.2.
Blood sample collection and NGF assessment For the biochemical analyses, 10mL of blood was withdrawn from each subject by
venipuncture, into an anticoagulant-free vacuum tube, after the interview, between the hours of 8:00 AM and 11:00 AM. The blood was immediately centrifuged at 4,000 x g for 10 minutes, and the resulting serum was kept frozen at -80 ºC until analysis. NGF serum levels were measured with a sandwich-ELISA using an available immunoassay kit according to the manufacturer’s instructions (DuoSet ELISA Development, R&D Systems, Inc., USA) and expressed in pg/mL (de Azevedo Cardoso et al., 2014). 2.3.
Statistical analysis Statistical analyses were performed in the Graph Pad Prism 6.0 (GraphPad Software Inc.,
San Diego, USA) and IBM SPSS Statistic 21.0 (IBM Corporation, New York, USA) software for Windows. Sociodemographic and NGF serum levels are shown by absolute and relative frequencies or by means and standard deviations. To verify consistency between groups, the chi-square test and analysis of variance (ANOVA) were used. The comparison between the levels of NGF in the group of individuals with MDD, MDD+SR and healthy controls was performed by ANOVA with posthoc Bonferroni. To control for possible confounding variables, we included in the linear regression adjusted analysis the variables that presented associations with p ≤ 0.20 in the raw analysis. Results with p ≤ 0.05 were considered statistically significant.
3.
Results The total population sample was comprised of 141 individuals: 47 with MDD+SR, 47 with
MDD and 47 healthy individuals as controls. Each group was composed of 35 women and 12 men, with a mean age of 27.32 ± 5.04, 27.34 ± 5.08, and 27.40 ± 5.15 years, respectively. Homogeneous samples showed no differences between diagnostic groups regarding gender (p = 1.000), ethnicity
(p = 0.400) and age (p = 0.996). However, socioeconomic index and years of education were statistically different. The MDD+SR group presented lower economic classes (p = 0.030) and years of education (p = 0.004) (Table 1). NGF serum levels were significantly reduced in the MDD and MDD+SR groups (92.39 ± 41.21 pg/mL and 100.99 ± 35.08 pg/mL, respectively) when compared with the healthy controls group (149.81 ± 65.94 pg/mL) (F = 18:58, p ≤ 0.001). The post-hoc Bonferroni multiple comparison test revealed a significant difference between the healthy controls group and MDD group (p ≤ 0.001) and between the healthy controls group and MDD+SR groups (p ≤ 0.001). Nevertheless, there were no differences in NGF serum levels between the MDD and MDD+SR groups (p = 1.000; Fig. 1). After controlling for years of education and socioeconomic index through linear regression, NGF serum levels between the healthy controls, MDD and MDD+SR groups remained statistically significant (p = 0.004, data not shown).
4.
Discussion In this study, it was possible to observe a significant decrease in NGF serum levels in
individuals with MDD and MDD+SR when compared with healthy controls. Moreover, it was not possible to observe significant differences between individuals with depression and depression plus risk of suicide. It is important to highlight that our sample consisted of young and non-medicated subjects experiencing the early stages of these illnesses and also that the sociodemographic variables analysed here were not associated with changes in NGF levels in our population. The importance of NGF in the behavioral and neuronal responses associated with psychiatric illness, including brain changes, was shown through the decrease of NGF levels in patients with MDD (Diniz et al., 2013; Xiong et al., 2011). Recently, Diniz et al. (2013) found a reduction in the NGF levels of depressed elder patients when compared with a control group. Still, a more detailed analysis showed similarity between past and current depression groups, with no significant differences between them. A significant reduction in NGF levels was found only
between the past depression group and the control group (Diniz et al., 2013). Similarly, in our study, patients with MDD had reduced levels of NGF when compared with healthy controls, demonstrating possible involvement of this neurotrophin in the pathophysiology of depression. Changes in NGF levels after treatment with antidepressants have been described in the literature. In an experimental study, chronic treatment with nortriptyline and citalopram antidepressants increased brain NGF levels (Hassanzadeh and Rahimpour, 2011). On the other hand, Martino et al. (2013) demonstrated that patients with MDD had NGF serum levels reduced when compared with healthy controls, not being reversed by duloxetine antidepressant, possibly reflecting functional impairment in the adaptive neuroplasticity of stress in depressive disorders (Martino et al., 2013). Liu et al. (2014) found a significant improvement in the clinical symptoms of patients with mood disorders after 8 weeks of intensive treatment using antidepressants, but without significant changes in NGF levels (Liu et al., 2014). More importantly, in our study, individuals are at an early stage of the disease and do not make use of psychotherapeutic medication that could alter the measured NGF serum levels. NGF plays an important role in the development of mood disorders and their association with patients under risk of suicide. Dwivedi et al. (2005) analysed NGF levels in suicide victims diagnosed with MDD and other psychiatric disorders. The authors found no differences in the levels of NGF between both groups, but both of them showed significant differences when compared with normal controls, both in the prefrontal cortex and in the hippocampus (Dwivedi et al., 2005). The hippocampus and prefrontal cortex are related to cognition and mood regulation, and these are involved in the pathophysiology of affective disorders and suicide (Dwivedi et al., 2005). In addition, the hippocampus is an area of the brain that is affected by early stress, a major factor in suicidal behavior (Banerjee et al., 2013). Although we observed a reduction in NGF serum levels, there were no significant differences between depressed individuals with and without suicide risk, demonstrating that the presence of risk factors for suicide did not interfere with the levels of NGF.
Studies suggest that both symptoms of depression and suicide are related to a reduction in synaptic plasticity in the brain, possibly due to the inability of the brain to respond or adapt to aversive stimulus (Dwivedi et al., 2005; Martino et al., 2013). The reduced expression of neurotrophins in tissues, serum and plasma has been demonstrated through the normalization of these values after clinical treatment (Hashimoto, 2010; Hassanzadeh and Rahimpour, 2011). Regarding the role of NGF, recent studies suggest that this neurotrophin can be a powerful modulator of the immune system by mediating proinflammatory responses in several physiological conditions (Diniz et al., 2013). Furthermore, several evidence point that peripheral CNS inflammatory cytokines influence and are able to induce depressive symptoms (Diniz et al., 2013; Martino et al., 2013; Xiong et al., 2011). Xiong et al. (2011) found decreased serum levels of NGF and IL-2 in subjects with major depression and schizophrenia when compared with healthy controls, suggesting that the interaction between cytokines and neurotrophins is vital in maintaining homeostasis in the brain (Xiong et al., 2011). Also, NGF performs an important role in brain functioning, and alterations in its central levels may influence the development of mental disorders. Studies correlating NGF, major depression and suicide risk are scarce and heterogeneous, especially concerning sample characteristics methodology. Additionally, the participants of these studies among the literature are usually hospitalized patients with longer disease duration and severity. As such, our study is derived from a population-based sample, consisting of young adults experiencing the early stages of the disease, no history of prior hospitalizations and without use of psychiatric medication. Despite these strengths, some limitations should be considered. In the present study, severity of depressive symptoms as well as depression subtypes (such as melancholic and atypical) were not evaluated. A better clinical characterization could clarify the relationship between stressors and peripheral biomarkers in the pathophysiology of MDD. However, the presence of suicide risk did not interfere with NGF serum levels, indicating that this neurotrophin may be related to the diagnosis of MDD and not with symptoms severity.
The understanding of the physiological changes related to mood disorders can provide a better understanding of the genesis and mechanisms involved in the progression of these conditions. The identification of peripheral biomarkers capable of helping in the diagnosis or in monitoring the progression of the condition and treatment remain a goal in the field (Wiener et al., 2013). In this work, we observed reduced serum levels of NGF in MDD and MDD+SR subjects, which suggests that this biomarker can be used as a putative marker for MDD when compared with healthy individuals.
Conflict of interest statement None of the authors of this paper have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
Acknowledgements This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ), and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS ) Brazil.
References
Banerjee, R., Ghosh, A.K., Ghosh, B., Bhattacharyya, S., Mondal, A.C., 2013. Decreased mRNA and Protein Expression of BDNF, NGF, and their Receptors in the Hippocampus from Suicide: An Analysis in Human Postmortem Brain. Clinical medicine insights. Pathology 6, 1-11. Barros, A.J., Victora, C.G., 2005. [A nationwide wealth score based on the 2000 Brazilian demographic census]. Revista de saude publica 39, 523-529. de Azevedo Cardoso, T., Mondin, T.C., Wiener, C.D., Marques, M.B., Fucolo Bde, A., Pinheiro, R.T., de Souza, L.D., da Silva, R.A., Jansen, K., Oses, J.P., 2014. Neurotrophic factors, clinical features and gender differences in depression. Neurochemical research 39, 1571-1578.
Diniz, B.S., Teixeira, A.L., Machado-Vieira, R., Talib, L.L., Gattaz, W.F., Forlenza, O.V., 2013. Reduced serum nerve growth factor in patients with late-life depression. The American journal of geriatric psychiatry : official journal of the American Association for Geriatric b Psychiatry 21, 493-496. Dwivedi, Y., Mondal, A.C., Rizavi, H.S., Conley, R.R., 2005. Suicide brain is associated with decreased expression of neurotrophins. Biological psychiatry 58, 315-324. Hashimoto, K., 2010. Brain-derived neurotrophic factor as a biomarker for mood disorders: an historical overview and future directions. Psychiatry and clinical neurosciences 64, 341-357. Hassanzadeh, P., Rahimpour, S., 2011. The cannabinergic system is implicated in the upregulation of central NGF protein by psychotropic drugs. Psychopharmacology 215, 129-141. Liu, X., Zhang, T., He, S., Hong, B., Chen, Z., Peng, D., Wu, Y., Wen, H., Lin, Z., Fang, Y., Jiang, K., 2014. Elevated serum levels of FGF-2, NGF and IGF-1 in patients with manic episode of bipolar disorder. Psychiatry research 218, 54-60. Mann, J.J., 2002. A current perspective of suicide and attempted suicide. Annals of internal medicine 136, 302-311. Martino, M., Rocchi, G., Escelsior, A., Contini, P., Colicchio, S., de Berardis, D., Amore, M., Fornaro, P., Fornaro, M., 2013. NGF serum levels variations in major depressed patients receiving duloxetine. Psychoneuroendocrinology 38, 1824-1828. Schmidt, H.D., Shelton, R.C., Duman, R.S., 2011. Functional biomarkers of depression: diagnosis, treatment, and pathophysiology. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 36, 2375-2394. Sheehan, D.V., Lecrubier, Y., Sheehan, K.H., Amorim, P., Janavs, J., Weiller, E., Hergueta, T., Baker, R., Dunbar, G.C., 1998. The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD10. The Journal of clinical psychiatry 59 Suppl 20, 22-33.
Takei, Y., Laskey, R., 2008. Interpreting crosstalk between TNF-alpha and NGF: potential implications for disease. Trends in molecular medicine 14, 381-388. Wiener, C.D., Jansen, K., Ghisleni, G., Kaster, M.P., Souza, L.D., Lara, D.R., Portela, L.V., da Silva, R.A., Oses, J.P., 2013. Reduced serum levels of neuron specific enolase (NSE) in drug-naive subjects with major depression and bipolar disorder. Neurochemical research 38, 1394-1398. Xiong, P., Zeng, Y., Wan, J., Xiaohan, D.H., Tan, D., Lu, J., Xu, F., Li, H.Y., Zhu, Z., Ma, M., 2011. The role of NGF and IL-2 serum level in assisting the diagnosis in first episode schizophrenia. Psychiatry research 189, 72-76. Ziegenhorn, A.A., Schulte-Herbruggen, O., Danker-Hopfe, H., Malbranc, M., Hartung, H.D., Anders, D., Lang, U.E., Steinhagen-Thiessen, E., Schaub, R.T., Hellweg, R., 2007. Serum neurotrophins--a study on the time course and influencing factors in a large old age sample. Neurobiology of aging 28, 1436-1445.
Legends Figure 1. NGF serum levels in controls, MDD and MDD+SR individuals. NGF levels were increased in the control group (149.81± 65.94 pg/mL) when compared with the MDD (92.39 ± 41.21 pg/mL) and MDD+SR groups (100.99 ± 35.08pg/mL)(F = 18:58, p ≤ 0.001). The bar represents the median (control: 2.86 ng/mL; MDD: 1.47; BD: 1.52). Control vs MDD (**p ≤ 0.001), control vs MDD+SR (**p ≤ 0.001) MDD vs MDD+SR (p = 1.000) (post-hoc Bonferroni test).
Table 1. Socio-demographic characteristics of the sample according to the diagnostic. Control MDD MDD-SR n (%)/Mean ± S.D. n (%)/Mean ± S.D. n (%)/Mean ± S.D. Gender Female 35 (33.3) 35 (33.3) 35 (33.3) Male 12 (33.3) 12 (33.3) 12 (33.3) Ethnicity Not white White Age (years) Economic Index Class Low Medium High School (years) NGF levels (pg/mL)
p value 1.000
0.400 14 (37.8) 33 (31.7)
9 (24.3) 38 (36.5)
14 (37.8) 33 (31.7)
27.40 ± 5.15
27.34 ± 5.08
27.32 ± 5.04
0.996 0.030
18 (26.9) 20 (41.7) 9 (34.6)
19 (26.9) 16 (33.3) 12 (42.2)
30 (44.8) 12 (25.0) 5 (19.2)
10.72 ± 3.64
11.02 ± 2.84
8.70 ± 4.17
0.004
149.81 ± 65.94
92.39 ± 41.21
100.99 ± 35.08
≤ 0.001
MDD: Major depressive disorder without suicide risk, MDD-RS: Major depressive disorder with suicide risk, S.D.: Standard deviation.
Highlights
NGF serum levels were significantly reduced in MDD compared with controls NGF serum levels were significantly reduced in MDD+SR compared with controls There was no difference in the NGF serum levels between the MDD and MDD+SR groups
Acknowledgements This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ), and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS ) Brazil.
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Role of the funding source The research was funded by a grant from the Brazilian governmental agencies: FAPERGS/PRONEX (10/0055-0), CAPES (23038.001671/2012-16). The agencies had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. Funding and Disclosure FPM received funding from the Brazilian agency for fostering Coordination of Improvement of Higher Education Personnel (CAPES) under the Ministry of Education. CDW received funding from the Brazilian agency Foundation for Research Support of the State of Rio Grande do Sul (FAPERGS) linked to the Secretariat of Science and Technology of the State of Rio Grande do Sul and Coordination of Improvement of Higher Education Personnel (CAPES) under the Ministry of Education.
JFO received funding from the National Counsel of Technological and Scientific
Development (CNPq) under the Ministry of Education. LDMS, RAS, KJ, JPO, LVP and DRL received funding from the National Counsel of Technological and Scientific Development (CNPq) under the Ministry of Education; Brazilian agency promoting National Counsel of Technological and Scientific Development linked to the Ministry of Science andIndividual contributions of each author of the article: Authors:
Wiener CD, conception and design of the study, biochemical analysis, data interpretation, manuscript writing, and approved the version to be published; Ferreira SM, conception and design of the study, drafting the article and approval of the version to be published; Moreira FP, conception and design of the study, drafting the article and approval of the version to be published; Bittencourt G, sample collection, biochemical analysis, drafting the article and approval of the version to be published;
Oliveira JF, sample collection, biochemical analysis, drafting the article and approval of the version to be published; Molina ML, execution of the project and in description of psychological variables; Jansen K, conception and design of the study, analysis and interpretation of data, drafting the article and approval of the version to be published; Souza LDM, conception and design of the study, analysis and interpretation of data, drafting the article and approval of the version to be published; Lara DR, conception and design of the study, drafting the article and approval of the version to be published; Portela LV, conception and design of the study, drafting the article and approval of the version to be published; Silva RA, conception and design of the study, drafting the article and approval of the version to be published. Oses J, conception and design of the study, biochemical analysis, data interpretation, drafting the article and approval of the version to be published;
Technology; Coordination of Improvement of Higher Education Personnel (CAPES) under the Ministry of Education; and Research Foundation of the State of Rio Grande do Sul (FAPERGS) linked to the Secretariat of Science and technology in the State of Rio Grande do Sul. Acknowledgements Support for this study was provided by a grant from Brazilian governmental agencies: FAPERGS/PRONEX (10/0055-0), CAPES (23038.001671/2012-16). Conflict of interest The authors have no conflict of interest to declare.
Figure(s)
