Volume 11, Issue 4 (July & August - Special Issue on Memory, Reward & Stress 2020)
BCN 2020, 11(4): 457-464 |
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Soleimani Asl S, Ghasemi Moravej F, Kowsari G, Farhadi M H, Pourhaydar B, Ghasemi Hamidabadi H et al . The Effects of 3,4-methylenedioxy- methamphetamine on Neurogenesis in the Hippocampus of Male Rats. BCN 2020; 11 (4) :457-464
URL: http://bcn.iums.ac.ir/article-1-1267-en.html
URL: http://bcn.iums.ac.ir/article-1-1267-en.html
Sara Soleimani Asl1 
, Fahimeh Ghasemi Moravej1 , Golshad Kowsari2 , Mohammad Hassan Farhadi3 , Bagher Pourhaydar4 , Hatef Ghasemi Hamidabadi5 , Mehdi Mehdizadeh * 6
, Fahimeh Ghasemi Moravej1 , Golshad Kowsari2 , Mohammad Hassan Farhadi3 , Bagher Pourhaydar4 , Hatef Ghasemi Hamidabadi5 , Mehdi Mehdizadeh * 6
1- Endometrium and Endometriosis Research Centre, Hamadan University of Medical Sciences, Hamadan, Iran.
2- School of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.
3- Researcher of Subsance Abuse and Dependence Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
4- Department of Anatomy, Neurophysiology Research Center, School of Medicine, Urmia University of Medical Sciences, Urmia, Iran.
5- Department of Anatomy and Cell Biology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
6- Department of Anatomy, Cellular and Molecular Research Center, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
2- School of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.
3- Researcher of Subsance Abuse and Dependence Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
4- Department of Anatomy, Neurophysiology Research Center, School of Medicine, Urmia University of Medical Sciences, Urmia, Iran.
5- Department of Anatomy and Cell Biology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
6- Department of Anatomy, Cellular and Molecular Research Center, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
Keywords: N-Methyl-3, 4-methylenedioxyamphetamine, Hippocampus, Nestin, Sox2 Protein, NeuroD Protein
Full-Text [PDF 1189 kb]
| Abstract (HTML)
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The number of expressed nestin-positive cells was significantly lower on days 28 (P<0.01) and 60 (P<0.001) in the control group compared with day 7. We also observed significantly fewer nestin-positive cells in the control group (days 28 (P<0.01) and 60 (P<0.01)) compared with day 14.
The expression level of nestin in the control (A: 7, B:14, C:28, D:60) and 3,4-methylenedioxymethamphetamine (MDMA)-treated groups (E:7, F:14, G:28, H:60). I: Dentate gyrus at low magnification (x10). The arrow shows nestin-positive cells. A: P<0.001 vs. the control group, B: P<0.05 vs. the related controls, C: P< 0.05 vs. the MDMA groups (days 7 and 14), D: P<0.01 vs. the control group (day 7), E: P<0.001 vs. the control group (day 7), F: P<0.01 vs. the control group (day 14). The results showed that Sox2 expression was higher in the control groups.
Further analysis indicated significant differences between the groups (Figure 2). The Sox2 expression level was significantly lower on the days 7 (P<0.01), 14 (P<0.001), 28 (P<0.05), and 60 (P<0.05) in the MDMA groups compared with their related control groups. Sox2 expression significantly decreased on day 60 compared with the days 7 (P<0.001) and 14 (P<0.05) in the MDMA groups.
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There was a difference between the days 7, 14, and 60 in the control groups in the numbers of Sox2-positive cells, and Sox2 expression significantly decreased on day 60 in the control group in comparison with the days 7 and 14 in the control groups (P<0.001).
The expression level of Sox2 in the control (A: 7, B:14, C:28, D:60) and 3,4-methylenedioxymethamphetamine (MDMA)-treated groups (E:7, F:14, G:28, H:60). I: Dentate gyrus at low magnification (x10). The arrow shows Sox2-positive cells. A: P<0.01, B: P<0.001, and C: P<0.05 vs. the related controls; D: P<0.001 vs. the MDMA group (day 7); E: P<0.05 vs. the MDMA group (day 14); F: P<0.001 vs. the control groups (days 7 and 14).
NeuroD expression decreased following MDMA treatment (Figure 3). However, in contrast to nestin and Sox2, no significant differences existed between the control (day 7; 18.74±0.95) and MDMA (day 7) (14.42±1.86) groups; the control (day 14; 17.56±0.41) and MDMA (day 14; 13.4±0.87) groups, the control (day 28; 14.14±0.63) and MDMA (day 28; 11.4±0.91) groups; and the control (day 60; 11.68±0.74) and MDMA (day 60; 8.5±0.36) groups. The NeuroD expression level was significantly higher in the control group (day 7) compared with the days 28 (P<0.05) and 60 (P<0.001). Significant differences were observed between day 14 and day 60 control groups (P<0.001).
.png)
The expression level of NeuroD in the control (A:7, B:14, C:28, D:60) and 3,4-methylenedioxymethamphetamine (MDMA)-treated groups (E:7, F:14, G:28, H:60). I: Dentate gyrus at low magnification (x10). The arrow shows NeuroD-positive cells. A: P<0.05 and B: P<0.001 vs. the control group (day 7), C: P<0.001 vs. the control group (day 14).
4. Discussion
In this study, we found a deficit in neurogenesis following MDMA treatment. We observed that the expression levels of nestin, Sox2, and NeuroD decreased in the hippocampus of MDMA- treated rats compared with the control groups. There was an age-related decline in the neurogenesis of the control and MDMA-treated groups.
Neurogenesis normally occurs in adults in the Subgranular Zone (SGZ) of the DG within the hippocampus and Subventricular Zone (SVZ) of the lateral ventricle, which is influenced by physiological and pathological agents at the levels of survival, proliferation, and differentiation (Zhao, Deng, & Gage, 2008). Lineage tracing studies in rodents have identified two types of neural progenitors in the SGZ that express molecular markers, such as nestin, Sox2, and GFAP (Breunig, Silbereis, Vaccarino, Šestan, & Rakic, 2007). The expressions of these markers are affected by toxic agents in the nervous system (Duman, Malberg, & Nakagawa, 2001; Kang et al., 2017).
Increased environmental complexity enhances neurogenesis by increasing cell survival, proliferation, and differentiation in the hippocampus of adult mice (Brown et al., 2003; Zhao, Deng, & Gage, 2008). In contrast, physiological and psychosocial stressors negatively affect adult neurogenesis (Abbink, Naninck, Lucassen, & Korosi, 2017; Levone, Nolan, Cryan, & O’Leary, 2017). Substance abuse reduces neurogenesis and affects cell proliferation and differentiation (Castilla-Ortega et al., 2017; Venkatesan, Nath, Ming, & Song, 2007; Warner‐Schmidt & Duman, 2006).
Dominguez‐Escriba et al. reported that the chronic administration of cocaine impaired cell proliferation, which supported the results of this study as nestin-positive cells decreased in the DG following MDMA treatment (Domínguez‐Escribà et al., 2006).
Our findings supported the results of Hernandez-Rabaza et al. who have shown that administration of MDMA affects neurogenesis in the DG by impairing neural precursor survival in adult rats (Hernandez-Rabaza et al., 2006). Our results showed that MDMA administration decreased NeuroD expression. NeuroD is expressed in intermediate cells in the initial phases of neurogenesis and serves to expand the population of neuroblasts (Kang et al., 2017).
We found significant differences in the expression of neurogenesis-related proteins between day 7 and day 60 groups of the controls. This finding supported the hypothesis that neurogenesis dramatically decreases during aging.
The current study results supported those by Spalding et al. (2013) who reported a moderate decline in neurogenesis by aging. Although a decrease in synaptic density in the DG has been shown in the aged brain, the mechanism of age-related decline in neurogenesis is unclear and needs to be investigated. Previous results have shown that MDMA caused a neuronal degeneration and reduction in the newborn cells in the hippocampus (Renoir et al., 2008; Soleimani Asl, Saifi, Sakhaie, Zargooshnia, & Mehdizadeh, 2015), but the mechanism, by which MDMA decreases the neurogenesis is not fully understood. Lesions in serotonin neurons in the raphe nuclei led to a decrease in newborn cells. In the adult neurogenesis, serotonin might be considered as a positive regulatory agent (Brezun & Daszuta, 2000). The hippocampus is a target of serotonergic nerve cells. MDMA administration results in serotonin depletion in this area (Gurtman, Morley, Li, Hunt, & McGregor, 2002); therefore, it seems that serotonin alterations in the hippocampus after MDMA administration may play a critical role in the neurogenesis deficits in the hippocampus, which should be investigated further.
Ethical Considerations
Compliance with ethical guidelines
The Ethical Committee of the Iran University of Medical Sciences approved the experiments. (IR.IUMS.REC.1393.24111)
Funding
This study was supported by the Iran University of Medical Sciences (93023024111).
Authors' contributions
Conceptualization: Sara Soleimani Asl, Mehdi Mehdizadeh; Methodology: Sara Soleimani Asl, Mohammad Hassan Farhadi; Investigation: Fahimeh Ghasemi Moravej, Sara Soleimani Asl; Writing – original draft: Sara Soleimani Asl, Bagher Pourhaydar; Writing – review & editing: Hatef Ghasemi Hamidabadi; Funding acquisition: Mehdi Mehdizadeh; Resources, Sara Soleimani Asl, Mehdi Mehdizadeh; Supervision: Mehdi Mehdizadeh.
Conflict of interest
The authors declared no conflict of interest.
Acknowledgments
We thank all colleagues from Anatomy Department of Hamadan University of Medical Sciences.
References
Abbink, M., Naninck, E., Lucassen, P., & Korosi, A. (2017). Early‐life stress diminishes the increase in neurogenesis after exercise in adult female mice. Hippocampus, 27(8), 839-44. [DOI:10.1002/hipo.22745] [PMID]
Alipanahzadeh, H., Soleimani, M., Asl, S. S., Pourheydar, B., Nikkhah, A., & Mehdizadeh, M. (2014). Transforming growth factor-α improves memory impairment and neurogenesis following ischemia reperfusion. Cell Journal (Yakhteh), 16(3), 315–24. https://onlinelibrary.wiley.com/doi/abs/10.1002/hipo.22745
Asi, S., Farhadi, H., Naghdi, N., Choopani, S., Samzadeh-Kermani, A., & Mehdizadeh, M. (2011). Non-acute effects of different doses of 3, 4-methylenedioxymethamphetamine on spatial memory in the Morris water maze in Sprague-Dawley male rats. Neural Regeneration Research, 6(22), 1715-19. [DOI: 10.3969/j.issn.1673-5374.2011.22.006]
Asl, S. S., Saifi, B., Sakhaie, A., Zargooshnia, S., & Mehdizadeh, M. (2015). Attenuation of ecstasy-induced neurotoxicity by N-acetylcysteine. Metabolic Brain Disease, 30(1), 171-81. [DOI:10.1007/s11011-014-9598-0] [PMID]
Barzegar, S., Komaki, A., Shahidi, S., Sarihi, A., Mirazi, N., & Salehi, I. (2015). Effects of cannabinoid and glutamate receptor antagonists and their interactions on learning and memory in male rats. Pharmacology Biochemistry and Behavior, 131, 87-90. [DOI:10.1016/j.pbb.2015.02.005] [PMID]
Breunig, J. J., Silbereis, J., Vaccarino, F. M., Šestan, N., & Rakic, P. (2007). Notch regulates cell fate and dendrite morphology of newborn neurons in the postnatal dentate gyrus. Proceedings of the National Academy of Sciences, 104(51), 20558-63. [DOI:10.1073/pnas.0710156104] [PMID] [PMCID]
Brezun, J. M., & Daszuta, A. (2000). Serotonin may stimulate granule cell proliferation in the adult hippocampus, as observed in rats grafted with foetal raphe neurons. European Journal of Neuroscience, 12(1), 391-96. [DOI:10.1046/j.1460-9568.2000.00932.x] [PMID]
Brown, J., Cooper‐Kuhn, C. M., Kempermann, G., Van Praag, H., Winkler, J., & Gage, F. H., et al. (2003). Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis. European Journal of Neuroscience, 17(10), 2042-6. [DOI:10.1046/j.1460-9568.2003.02647.x] [PMID]
Castilla-Ortega, E., de Guevara-Miranda, D. L., Serrano, A., Pavón, F. J., Suárez, J., & de Fonseca, F. R., et al. (2017). The impact of cocaine on adult hippocampal neurogenesis: potential neurobiological mechanisms and contributions to maladaptive cognition in cocaine addiction disorder. Biochemical Pharmacology, 141, 100-17. [DOI:10.1016/j.bcp.2017.05.003] [PMID]
Colado, M., & Green, A. (1994). A study of the mechanism of MDMA (‘Ecstasy’)‐induced neurotoxicity of 5‐HT neurones using chlormethiazole, dizocilpine and other protective compounds. British Journal of Pharmacology, 111(1), 131-6. [DOI:10.1111/j.1476-5381.1994.tb14034.x] [PMID] [PMCID]
Costa, G., Morelli, M., & Simola, N. (2017). Progression and persistence of neurotoxicity induced by MDMA in Dopaminergic Regions of the Mouse Brain and Association with Noradrenergic, GABAergic, and serotonergic damage. Neurotoxicity Research, 32(4), 563-74. [DOI:10.1007/s12640-017-9761-6] [PMID]
Domínguez‐Escribà, L., Hernández‐Rabaza, V., Soriano‐Navarro, M., Barcia, J., Romero, F., & García‐Verdugo, J., et al. (2006). Chronic cocaine exposure impairs progenitor proliferation but spares survival and maturation of neural precursors in adult rat dentate gyrus. European Journal of Neuroscience, 24(2), 586-94. [DOI:10.1111/j.1460-9568.2006.04924.x] [PMID]
Duman, R. S., Malberg, J., & Nakagawa, S. (2001). Regulation of adult neurogenesis by psychotropic drugs and stress. Journal of Pharmacology and Experimental Therapeutics, 299(2), 401-7. [DOI: 10.1177/1073858414561303ů7.62]
Euston, D. R., Gruber, A. J., & McNaughton, B. L. (2012). The role of medial prefrontal cortex in memory and decision making. Neuron, 76(6), 1057-70. [DOI:10.1016/j.neuron.2012.12.002] [PMID] [PMCID]
Green, A. R., Mechan, A. O., Elliott, J. M., O’Shea, E., & Colado, M. I. (2003). The pharmacology and clinical pharmacology of 3, 4-methylenedioxymethamphetamine (MDMA,”ecstasy”). Pharmacological Reviews, 55(3), 463-508. [DOI:10.1124/pr.55.3.3] [PMID]
Gurtman, C. G., Morley, K. C., Li, K. M., Hunt, G. E., & McGregor, I. S. (2002). Increased anxiety in rats after 3, 4-methylenedioxymethamphetamine: Association with serotonin depletion. European Journal of Pharmacology, 446(1), 89-96. [DOI:10.1016/S0014-2999(02)01820-4]
Hernandez-Rabaza, V., Dominguez-Escriba, L., Barcia, J., Rosel, J., Romero, F., & Garcia-Verdugo, J., et al. (2006). Binge administration of 3, 4-methylenedioxymethamphetamine (“ecstasy”) impairs the survival of neural precursors in adult rat dentate gyrus. Neuropharmacology, 51(5), 967-73. [DOI:10.1016/j.neuropharm.2006.06.019] [PMID]
Jessberger, S., Clark, R. E., Broadbent, N. J., Clemenson, G. D., Consiglio, A., & Lie, D. C., et al. (2009). Dentate gyrus-specific knockdown of adult neurogenesis impairs spatial and object recognition memory in adult rats. Learning & Memory, 16(2), 147-54. [DOI:10.1101/lm.1172609] [PMID] [PMCID]
Kang, E., Berg, D. A., Furmanski, O., Jackson, W. M., Ryu, Y. K., & Gray, C. D., et al. (2017). Neurogenesis and developmental anesthetic neurotoxicity. Neurotoxicology and Teratology, 60, 33-39. [DOI:10.1016/j.ntt.2016.10.001] [PMID] [PMCID]
Levone, B., Nolan, Y., Cryan, J., & O’Leary, O. (2017). Neural progenitor cells from the ventral hippocampus are more sensitive to long-term exposure to corticosterone. European Neuropsychopharmacology, 27(1), S19-S20. [DOI:10.1016/S0924-977X(17)30087-1]
McDonald, R. J., & White, N. M. (1994). Parallel information processing in the water maze: Evidence for independent memory systems involving dorsal striatum and hippocampus. Behavioral and Neural Biology, 61(3), 260-70. [DOI:10.1016/S0163-1047(05)80009-3]
Ortega-Martínez, S. (2017). Adult Hippocampal Neurogenesis and Memory. International Journal of Advanced Biological and Biomedical Research, 6(1), 360-79. [DOI:10.18869/IJABBR.2017.360]
Paxinos, G., Watson, C., Pennisi, M., & Topple, A. (1985). Bregma, lambda and the interaural midpoint in stereotaxic surgery with rats of different sex, strain and weight. Journal of Neuroscience Methods, 13(2), 139-43. [DOI:10.1016/0165-0270(85)90026-3]
Renoir, T., Païzanis, E., El Yacoubi, M., Saurini, F., Hanoun, N., & Melfort, M., et al. (2008). Differential long-term effects of MDMA on the serotoninergic system and hippocampal cell proliferation in 5-HTT knock-out vs. wild-type mice. International Journal of Neuropsychopharmacology, 11(8), 1149-62. [DOI:10.1017/S1461145708009048] [PMID]
Roberts, C. A., Jones, A., & Montgomery, C. (2016). Meta-analysis of molecular imaging of serotonin transporters in ecstasy/polydrug users. Neuroscience & Biobehavioral Reviews, 63, 158-67. [DOI:10.1016/j.neubiorev.2016.02.003] [PMID]
Schmidt, C. J., Levin, J. A., & Lovenberg, W. (1987). In vitro and in vivo neurochemical effects of methylenedioxymethamphetamine on striatal monoaminergic systems in the rat brain. Biochemical Pharmacology, 36(5), 747-55. [DOI:10.1016/0006-2952(87)90729-5]
Shariati, M. B. H., Sohrabi, M., Shahidi, S., Nikkhah, A., Mirzaei, F., & Medizadeh, M., et al. (2014). Acute effects of ecstasy on memory are more extensive than chronic effects. Basic and Clinical Neuroscience, 5(3), 225-30. https://pubmed.ncbi.nlm.nih.gov/25337384/
Simantov, R. (2004). Multiple molecular and neuropharmacological effects of MDMA (Ecstasy). Life Sciences, 74(7), 803-14. [DOI:10.1016/j.lfs.2003.08.002] [PMID]
Soleimani Asl, S., Saifi, B., Sakhaie, A., Zargooshnia, S., & Mehdizadeh, M. (2015). Attenuation of ecstasy-induced neurotoxicity by N-acetylcysteine. Metabolic Brain Disease, 30(1), 171-81. [DOI:10.1007/s11011-014-9598-0] [PMID]
Spalding, K. L., Bergmann, O., Alkass, K., Bernard, S., Salehpour, M., & Huttner, H. B., et al. (2013). Dynamics of hippocampal neurogenesis in adult humans. Cell, 153(6), 1219-27. [DOI:10.1016/j.cell.2013.05.002] [PMID] [PMCID]
Venkatesan, A., Nath, A., Ming, G. l., & Song, H. (2007). Adult hippocampal neurogenesis: regulation by HIV and drugs of abuse. Cellular and Molecular Life Sciences, 64(16), 2120-32. [DOI:10.1007/s00018-007-7063-5] [PMID]
Warner‐Schmidt, J. L., & Duman, R. S. (2006). Hippocampal neurogenesis: Opposing effects of stress and antidepressant treatment. Hippocampus, 16(3), 239-49. [DOI:10.1002/hipo.20156] [PMID]
Zhao, C., Deng, W., & Gage, F. H. (2008). Mechanisms and functional implications of adult neurogenesis. Cell, 132(4), 645-60. [DOI:10.1016/j.cell.2008.01.033] [PMID]
Full-Text:
1. Introduction
cstasy or 3, 4-methylenedioxymethamphetamine (MDMA) has been used recreationally since the 1980s. Initially, MDMA was found to cause 5- hydroxytryptamine (5-HT), dopamine (DA), and epinephrine neurotoxicity with increased affinity to 5-HT receptors (Costa, Morelli, & Simola, 2017; Roberts, Jones, & Montgomery, 2016). Pharmacologic studies have shown that MDMA binds to 5-HT transporter and inhibits serotonin absorption and subsequently 5-HT accumulation in the synaptic cleft, which leads to the early release of 5-HT (Simantov, 2004). Following the acute release of 5-HT, serotonin levels are reversibly depleted 3-4 h after MDMA administration. This depletion persists for at least one week (Colado & Green, 1994). It is well established that MDMA-induced serotonin neurotoxicity is area-specific, with the major changes in the prefrontal cortex, hippocampus, and striatum (Green, Mechan, Elliott, Oshea, & Colado, 2003) as important structures involved in spatial memory (Euston, Gruber, & McNaughton, 2012; McDonald & White, 1994). MDMA causes neuronal damage and memory impairment (Asi et al., 2011; Asl, Saifi, Sakhaie, Zargooshnia, & Mehdizadeh, 2015). Given the fundamental role of the hippocampus in learning and memory (Barzegar et al., 2015), it has been speculated that neurogenesis in the hippocampus (newborn granule cells) contribute to memory formation (Ortega-Martínez, 2017).
Jessberger et al. reported that blockage of neurogenesis in the Dentate Gyrus (DG) by a lentivirus-based strategy impaired spatial and objective recognition memory (Jessberger et al., 2009). We previously demonstrated that MDMA treatment could lead to neuronal degeneration and cell death in the hippocampus, with subsequent memory impairment (Shariati et al., 2014).
Although the toxic effects of MDMA on the brain have been widely studied, little is known about the effects of MDMA on different phases of neurogenesis in the hippocampus. MDMA administration leads to memory impairment (Shariati et al., 2014) and researchers have established a correlation between neurogenesis in the hippocampus and memory; therefore, the aim of this study was to investigate the effects of MDMA on neurogenesis in the DG of the rat hippocampus.
2. Materials & Methods
2.1. Chemicals
We obtained MDMA through the Presidency Drug Control Headquarters (Hamadan, Iran). Antibodies (nestin, Sox2, and NeuroD) and other chemicals were purchased from Abcam (Cambridge, UK).
2.2. Experimental design and animal grouping
Male Wistar rats (200-250 g) were housed in the animal house with the standard condition (a 12-h light/12-h dark cycle, the temperature of 22±2°C, and relative humidity of 55±5%) and ad libitum access to food and water.
The Ethical Committee of the Iran University of Medical Sciences approved the experiments (IR.IUMS.REC.1393.24111). According to our previous study and using the formula 1, we obtained the sample size and randomly divided the rats into two groups (n=16 per group): control saline group that received the Intraperitoneal (IP) injections of normal saline (1 ml/kg) and MDMA group that received a single dose of MDMA (IP, 10 mg/kg) (Shariati et al., 2014). Based on Schmidt et al. study, administration of the single dose of MDMA (10 mg/kg) inactivates tryptophan hydroxylase and causes poor expression of the brain 5-HT and serotonin transporter (Schmidt, Levin, & Lovenberg, 1987). Subsequently, we divided the control and MDMA groups into four subgroups (n=4 per group) according to the time that the rats were killed: 7, 14, 28, or 60 days after treatment (Zhao, Deng, & Gage, 2008).
1.
2.3. Tissue preparation and Immunohistochemistry (IHC) protocol
We used our previously reported protocol for the IHC analyses (Alipanahzadeh et al., 2014). In brief, after anesthesia, the rats were transcardially perfused with 4% ParaFormaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). After tissue processing and paraffin embedding and based on the Paxinos and Watson atlas (Paxinos, Watson, Pennisi, & Topple, 1985), 10 µm coronal sections (-3.3 and -3.8 to the Bregma) were prepared from the brain. The slides were deparaffinized, rehydrated in ascending alcohol, and were then retrieved in sodium citrate buffer and incubated in 10% normal serum with 1% BSA in TBS for 2 h. Then, the slides were incubated with antibodies against NeuroD, sSox2, and nestin (1:1000, Abcam, Cambridge, UK ) at 4°C, and also HRP-conjugated anti-rabbit secondary antibody (1:10000, Abcam, Cambridge, UK). All sections were incubated with DAB solution (Abcam, Cambridge, UK) for 20 min and counterstained using hematoxylin. We assessed the DG region of the hippocampus by a light microscope with an attached digital camera.
2.4. Statistical analysis
Data were presented as Means±SEM. We used SPSS 16 software and One-way Analysis of Variance (ANOVA) and Tukey’s multiple comparison test to analyze the data. P<0.05 was considered statistically significant.
3. Results
We counted five serial sections at 40x magnification to determine the average neuronal counts for each animal. Nestin-positive cells were counted in the hippocampus DG (Figure 1). Significant differences existed between the control and MDMA groups on days 7 (P<0.001), 14 (P<0.001), 28 (P<0.05), and 60 (P<0.05). The MDMA groups from the day7 to 14 had greater nestin expression levels than the day 60 (P<0.05).
cstasy or 3, 4-methylenedioxymethamphetamine (MDMA) has been used recreationally since the 1980s. Initially, MDMA was found to cause 5- hydroxytryptamine (5-HT), dopamine (DA), and epinephrine neurotoxicity with increased affinity to 5-HT receptors (Costa, Morelli, & Simola, 2017; Roberts, Jones, & Montgomery, 2016). Pharmacologic studies have shown that MDMA binds to 5-HT transporter and inhibits serotonin absorption and subsequently 5-HT accumulation in the synaptic cleft, which leads to the early release of 5-HT (Simantov, 2004). Following the acute release of 5-HT, serotonin levels are reversibly depleted 3-4 h after MDMA administration. This depletion persists for at least one week (Colado & Green, 1994). It is well established that MDMA-induced serotonin neurotoxicity is area-specific, with the major changes in the prefrontal cortex, hippocampus, and striatum (Green, Mechan, Elliott, Oshea, & Colado, 2003) as important structures involved in spatial memory (Euston, Gruber, & McNaughton, 2012; McDonald & White, 1994). MDMA causes neuronal damage and memory impairment (Asi et al., 2011; Asl, Saifi, Sakhaie, Zargooshnia, & Mehdizadeh, 2015). Given the fundamental role of the hippocampus in learning and memory (Barzegar et al., 2015), it has been speculated that neurogenesis in the hippocampus (newborn granule cells) contribute to memory formation (Ortega-Martínez, 2017).
Jessberger et al. reported that blockage of neurogenesis in the Dentate Gyrus (DG) by a lentivirus-based strategy impaired spatial and objective recognition memory (Jessberger et al., 2009). We previously demonstrated that MDMA treatment could lead to neuronal degeneration and cell death in the hippocampus, with subsequent memory impairment (Shariati et al., 2014).
Although the toxic effects of MDMA on the brain have been widely studied, little is known about the effects of MDMA on different phases of neurogenesis in the hippocampus. MDMA administration leads to memory impairment (Shariati et al., 2014) and researchers have established a correlation between neurogenesis in the hippocampus and memory; therefore, the aim of this study was to investigate the effects of MDMA on neurogenesis in the DG of the rat hippocampus.
2. Materials & Methods
2.1. Chemicals
We obtained MDMA through the Presidency Drug Control Headquarters (Hamadan, Iran). Antibodies (nestin, Sox2, and NeuroD) and other chemicals were purchased from Abcam (Cambridge, UK).
2.2. Experimental design and animal grouping
Male Wistar rats (200-250 g) were housed in the animal house with the standard condition (a 12-h light/12-h dark cycle, the temperature of 22±2°C, and relative humidity of 55±5%) and ad libitum access to food and water.
The Ethical Committee of the Iran University of Medical Sciences approved the experiments (IR.IUMS.REC.1393.24111). According to our previous study and using the formula 1, we obtained the sample size and randomly divided the rats into two groups (n=16 per group): control saline group that received the Intraperitoneal (IP) injections of normal saline (1 ml/kg) and MDMA group that received a single dose of MDMA (IP, 10 mg/kg) (Shariati et al., 2014). Based on Schmidt et al. study, administration of the single dose of MDMA (10 mg/kg) inactivates tryptophan hydroxylase and causes poor expression of the brain 5-HT and serotonin transporter (Schmidt, Levin, & Lovenberg, 1987). Subsequently, we divided the control and MDMA groups into four subgroups (n=4 per group) according to the time that the rats were killed: 7, 14, 28, or 60 days after treatment (Zhao, Deng, & Gage, 2008).
1.
2.3. Tissue preparation and Immunohistochemistry (IHC) protocol
We used our previously reported protocol for the IHC analyses (Alipanahzadeh et al., 2014). In brief, after anesthesia, the rats were transcardially perfused with 4% ParaFormaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). After tissue processing and paraffin embedding and based on the Paxinos and Watson atlas (Paxinos, Watson, Pennisi, & Topple, 1985), 10 µm coronal sections (-3.3 and -3.8 to the Bregma) were prepared from the brain. The slides were deparaffinized, rehydrated in ascending alcohol, and were then retrieved in sodium citrate buffer and incubated in 10% normal serum with 1% BSA in TBS for 2 h. Then, the slides were incubated with antibodies against NeuroD, sSox2, and nestin (1:1000, Abcam, Cambridge, UK ) at 4°C, and also HRP-conjugated anti-rabbit secondary antibody (1:10000, Abcam, Cambridge, UK). All sections were incubated with DAB solution (Abcam, Cambridge, UK) for 20 min and counterstained using hematoxylin. We assessed the DG region of the hippocampus by a light microscope with an attached digital camera.
2.4. Statistical analysis
Data were presented as Means±SEM. We used SPSS 16 software and One-way Analysis of Variance (ANOVA) and Tukey’s multiple comparison test to analyze the data. P<0.05 was considered statistically significant.
3. Results
We counted five serial sections at 40x magnification to determine the average neuronal counts for each animal. Nestin-positive cells were counted in the hippocampus DG (Figure 1). Significant differences existed between the control and MDMA groups on days 7 (P<0.001), 14 (P<0.001), 28 (P<0.05), and 60 (P<0.05). The MDMA groups from the day7 to 14 had greater nestin expression levels than the day 60 (P<0.05).
The number of expressed nestin-positive cells was significantly lower on days 28 (P<0.01) and 60 (P<0.001) in the control group compared with day 7. We also observed significantly fewer nestin-positive cells in the control group (days 28 (P<0.01) and 60 (P<0.01)) compared with day 14.
The expression level of nestin in the control (A: 7, B:14, C:28, D:60) and 3,4-methylenedioxymethamphetamine (MDMA)-treated groups (E:7, F:14, G:28, H:60). I: Dentate gyrus at low magnification (x10). The arrow shows nestin-positive cells. A: P<0.001 vs. the control group, B: P<0.05 vs. the related controls, C: P< 0.05 vs. the MDMA groups (days 7 and 14), D: P<0.01 vs. the control group (day 7), E: P<0.001 vs. the control group (day 7), F: P<0.01 vs. the control group (day 14). The results showed that Sox2 expression was higher in the control groups.
Further analysis indicated significant differences between the groups (Figure 2). The Sox2 expression level was significantly lower on the days 7 (P<0.01), 14 (P<0.001), 28 (P<0.05), and 60 (P<0.05) in the MDMA groups compared with their related control groups. Sox2 expression significantly decreased on day 60 compared with the days 7 (P<0.001) and 14 (P<0.05) in the MDMA groups.
There was a difference between the days 7, 14, and 60 in the control groups in the numbers of Sox2-positive cells, and Sox2 expression significantly decreased on day 60 in the control group in comparison with the days 7 and 14 in the control groups (P<0.001).
The expression level of Sox2 in the control (A: 7, B:14, C:28, D:60) and 3,4-methylenedioxymethamphetamine (MDMA)-treated groups (E:7, F:14, G:28, H:60). I: Dentate gyrus at low magnification (x10). The arrow shows Sox2-positive cells. A: P<0.01, B: P<0.001, and C: P<0.05 vs. the related controls; D: P<0.001 vs. the MDMA group (day 7); E: P<0.05 vs. the MDMA group (day 14); F: P<0.001 vs. the control groups (days 7 and 14).
NeuroD expression decreased following MDMA treatment (Figure 3). However, in contrast to nestin and Sox2, no significant differences existed between the control (day 7; 18.74±0.95) and MDMA (day 7) (14.42±1.86) groups; the control (day 14; 17.56±0.41) and MDMA (day 14; 13.4±0.87) groups, the control (day 28; 14.14±0.63) and MDMA (day 28; 11.4±0.91) groups; and the control (day 60; 11.68±0.74) and MDMA (day 60; 8.5±0.36) groups. The NeuroD expression level was significantly higher in the control group (day 7) compared with the days 28 (P<0.05) and 60 (P<0.001). Significant differences were observed between day 14 and day 60 control groups (P<0.001).
The expression level of NeuroD in the control (A:7, B:14, C:28, D:60) and 3,4-methylenedioxymethamphetamine (MDMA)-treated groups (E:7, F:14, G:28, H:60). I: Dentate gyrus at low magnification (x10). The arrow shows NeuroD-positive cells. A: P<0.05 and B: P<0.001 vs. the control group (day 7), C: P<0.001 vs. the control group (day 14).
4. Discussion
In this study, we found a deficit in neurogenesis following MDMA treatment. We observed that the expression levels of nestin, Sox2, and NeuroD decreased in the hippocampus of MDMA- treated rats compared with the control groups. There was an age-related decline in the neurogenesis of the control and MDMA-treated groups.
Neurogenesis normally occurs in adults in the Subgranular Zone (SGZ) of the DG within the hippocampus and Subventricular Zone (SVZ) of the lateral ventricle, which is influenced by physiological and pathological agents at the levels of survival, proliferation, and differentiation (Zhao, Deng, & Gage, 2008). Lineage tracing studies in rodents have identified two types of neural progenitors in the SGZ that express molecular markers, such as nestin, Sox2, and GFAP (Breunig, Silbereis, Vaccarino, Šestan, & Rakic, 2007). The expressions of these markers are affected by toxic agents in the nervous system (Duman, Malberg, & Nakagawa, 2001; Kang et al., 2017).
Increased environmental complexity enhances neurogenesis by increasing cell survival, proliferation, and differentiation in the hippocampus of adult mice (Brown et al., 2003; Zhao, Deng, & Gage, 2008). In contrast, physiological and psychosocial stressors negatively affect adult neurogenesis (Abbink, Naninck, Lucassen, & Korosi, 2017; Levone, Nolan, Cryan, & O’Leary, 2017). Substance abuse reduces neurogenesis and affects cell proliferation and differentiation (Castilla-Ortega et al., 2017; Venkatesan, Nath, Ming, & Song, 2007; Warner‐Schmidt & Duman, 2006).
Dominguez‐Escriba et al. reported that the chronic administration of cocaine impaired cell proliferation, which supported the results of this study as nestin-positive cells decreased in the DG following MDMA treatment (Domínguez‐Escribà et al., 2006).
Our findings supported the results of Hernandez-Rabaza et al. who have shown that administration of MDMA affects neurogenesis in the DG by impairing neural precursor survival in adult rats (Hernandez-Rabaza et al., 2006). Our results showed that MDMA administration decreased NeuroD expression. NeuroD is expressed in intermediate cells in the initial phases of neurogenesis and serves to expand the population of neuroblasts (Kang et al., 2017).
We found significant differences in the expression of neurogenesis-related proteins between day 7 and day 60 groups of the controls. This finding supported the hypothesis that neurogenesis dramatically decreases during aging.
The current study results supported those by Spalding et al. (2013) who reported a moderate decline in neurogenesis by aging. Although a decrease in synaptic density in the DG has been shown in the aged brain, the mechanism of age-related decline in neurogenesis is unclear and needs to be investigated. Previous results have shown that MDMA caused a neuronal degeneration and reduction in the newborn cells in the hippocampus (Renoir et al., 2008; Soleimani Asl, Saifi, Sakhaie, Zargooshnia, & Mehdizadeh, 2015), but the mechanism, by which MDMA decreases the neurogenesis is not fully understood. Lesions in serotonin neurons in the raphe nuclei led to a decrease in newborn cells. In the adult neurogenesis, serotonin might be considered as a positive regulatory agent (Brezun & Daszuta, 2000). The hippocampus is a target of serotonergic nerve cells. MDMA administration results in serotonin depletion in this area (Gurtman, Morley, Li, Hunt, & McGregor, 2002); therefore, it seems that serotonin alterations in the hippocampus after MDMA administration may play a critical role in the neurogenesis deficits in the hippocampus, which should be investigated further.
Ethical Considerations
Compliance with ethical guidelines
The Ethical Committee of the Iran University of Medical Sciences approved the experiments. (IR.IUMS.REC.1393.24111)
Funding
This study was supported by the Iran University of Medical Sciences (93023024111).
Authors' contributions
Conceptualization: Sara Soleimani Asl, Mehdi Mehdizadeh; Methodology: Sara Soleimani Asl, Mohammad Hassan Farhadi; Investigation: Fahimeh Ghasemi Moravej, Sara Soleimani Asl; Writing – original draft: Sara Soleimani Asl, Bagher Pourhaydar; Writing – review & editing: Hatef Ghasemi Hamidabadi; Funding acquisition: Mehdi Mehdizadeh; Resources, Sara Soleimani Asl, Mehdi Mehdizadeh; Supervision: Mehdi Mehdizadeh.
Conflict of interest
The authors declared no conflict of interest.
Acknowledgments
We thank all colleagues from Anatomy Department of Hamadan University of Medical Sciences.
References
Abbink, M., Naninck, E., Lucassen, P., & Korosi, A. (2017). Early‐life stress diminishes the increase in neurogenesis after exercise in adult female mice. Hippocampus, 27(8), 839-44. [DOI:10.1002/hipo.22745] [PMID]
Alipanahzadeh, H., Soleimani, M., Asl, S. S., Pourheydar, B., Nikkhah, A., & Mehdizadeh, M. (2014). Transforming growth factor-α improves memory impairment and neurogenesis following ischemia reperfusion. Cell Journal (Yakhteh), 16(3), 315–24. https://onlinelibrary.wiley.com/doi/abs/10.1002/hipo.22745
Asi, S., Farhadi, H., Naghdi, N., Choopani, S., Samzadeh-Kermani, A., & Mehdizadeh, M. (2011). Non-acute effects of different doses of 3, 4-methylenedioxymethamphetamine on spatial memory in the Morris water maze in Sprague-Dawley male rats. Neural Regeneration Research, 6(22), 1715-19. [DOI: 10.3969/j.issn.1673-5374.2011.22.006]
Asl, S. S., Saifi, B., Sakhaie, A., Zargooshnia, S., & Mehdizadeh, M. (2015). Attenuation of ecstasy-induced neurotoxicity by N-acetylcysteine. Metabolic Brain Disease, 30(1), 171-81. [DOI:10.1007/s11011-014-9598-0] [PMID]
Barzegar, S., Komaki, A., Shahidi, S., Sarihi, A., Mirazi, N., & Salehi, I. (2015). Effects of cannabinoid and glutamate receptor antagonists and their interactions on learning and memory in male rats. Pharmacology Biochemistry and Behavior, 131, 87-90. [DOI:10.1016/j.pbb.2015.02.005] [PMID]
Breunig, J. J., Silbereis, J., Vaccarino, F. M., Šestan, N., & Rakic, P. (2007). Notch regulates cell fate and dendrite morphology of newborn neurons in the postnatal dentate gyrus. Proceedings of the National Academy of Sciences, 104(51), 20558-63. [DOI:10.1073/pnas.0710156104] [PMID] [PMCID]
Brezun, J. M., & Daszuta, A. (2000). Serotonin may stimulate granule cell proliferation in the adult hippocampus, as observed in rats grafted with foetal raphe neurons. European Journal of Neuroscience, 12(1), 391-96. [DOI:10.1046/j.1460-9568.2000.00932.x] [PMID]
Brown, J., Cooper‐Kuhn, C. M., Kempermann, G., Van Praag, H., Winkler, J., & Gage, F. H., et al. (2003). Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis. European Journal of Neuroscience, 17(10), 2042-6. [DOI:10.1046/j.1460-9568.2003.02647.x] [PMID]
Castilla-Ortega, E., de Guevara-Miranda, D. L., Serrano, A., Pavón, F. J., Suárez, J., & de Fonseca, F. R., et al. (2017). The impact of cocaine on adult hippocampal neurogenesis: potential neurobiological mechanisms and contributions to maladaptive cognition in cocaine addiction disorder. Biochemical Pharmacology, 141, 100-17. [DOI:10.1016/j.bcp.2017.05.003] [PMID]
Colado, M., & Green, A. (1994). A study of the mechanism of MDMA (‘Ecstasy’)‐induced neurotoxicity of 5‐HT neurones using chlormethiazole, dizocilpine and other protective compounds. British Journal of Pharmacology, 111(1), 131-6. [DOI:10.1111/j.1476-5381.1994.tb14034.x] [PMID] [PMCID]
Costa, G., Morelli, M., & Simola, N. (2017). Progression and persistence of neurotoxicity induced by MDMA in Dopaminergic Regions of the Mouse Brain and Association with Noradrenergic, GABAergic, and serotonergic damage. Neurotoxicity Research, 32(4), 563-74. [DOI:10.1007/s12640-017-9761-6] [PMID]
Domínguez‐Escribà, L., Hernández‐Rabaza, V., Soriano‐Navarro, M., Barcia, J., Romero, F., & García‐Verdugo, J., et al. (2006). Chronic cocaine exposure impairs progenitor proliferation but spares survival and maturation of neural precursors in adult rat dentate gyrus. European Journal of Neuroscience, 24(2), 586-94. [DOI:10.1111/j.1460-9568.2006.04924.x] [PMID]
Duman, R. S., Malberg, J., & Nakagawa, S. (2001). Regulation of adult neurogenesis by psychotropic drugs and stress. Journal of Pharmacology and Experimental Therapeutics, 299(2), 401-7. [DOI: 10.1177/1073858414561303ů7.62]
Euston, D. R., Gruber, A. J., & McNaughton, B. L. (2012). The role of medial prefrontal cortex in memory and decision making. Neuron, 76(6), 1057-70. [DOI:10.1016/j.neuron.2012.12.002] [PMID] [PMCID]
Green, A. R., Mechan, A. O., Elliott, J. M., O’Shea, E., & Colado, M. I. (2003). The pharmacology and clinical pharmacology of 3, 4-methylenedioxymethamphetamine (MDMA,”ecstasy”). Pharmacological Reviews, 55(3), 463-508. [DOI:10.1124/pr.55.3.3] [PMID]
Gurtman, C. G., Morley, K. C., Li, K. M., Hunt, G. E., & McGregor, I. S. (2002). Increased anxiety in rats after 3, 4-methylenedioxymethamphetamine: Association with serotonin depletion. European Journal of Pharmacology, 446(1), 89-96. [DOI:10.1016/S0014-2999(02)01820-4]
Hernandez-Rabaza, V., Dominguez-Escriba, L., Barcia, J., Rosel, J., Romero, F., & Garcia-Verdugo, J., et al. (2006). Binge administration of 3, 4-methylenedioxymethamphetamine (“ecstasy”) impairs the survival of neural precursors in adult rat dentate gyrus. Neuropharmacology, 51(5), 967-73. [DOI:10.1016/j.neuropharm.2006.06.019] [PMID]
Jessberger, S., Clark, R. E., Broadbent, N. J., Clemenson, G. D., Consiglio, A., & Lie, D. C., et al. (2009). Dentate gyrus-specific knockdown of adult neurogenesis impairs spatial and object recognition memory in adult rats. Learning & Memory, 16(2), 147-54. [DOI:10.1101/lm.1172609] [PMID] [PMCID]
Kang, E., Berg, D. A., Furmanski, O., Jackson, W. M., Ryu, Y. K., & Gray, C. D., et al. (2017). Neurogenesis and developmental anesthetic neurotoxicity. Neurotoxicology and Teratology, 60, 33-39. [DOI:10.1016/j.ntt.2016.10.001] [PMID] [PMCID]
Levone, B., Nolan, Y., Cryan, J., & O’Leary, O. (2017). Neural progenitor cells from the ventral hippocampus are more sensitive to long-term exposure to corticosterone. European Neuropsychopharmacology, 27(1), S19-S20. [DOI:10.1016/S0924-977X(17)30087-1]
McDonald, R. J., & White, N. M. (1994). Parallel information processing in the water maze: Evidence for independent memory systems involving dorsal striatum and hippocampus. Behavioral and Neural Biology, 61(3), 260-70. [DOI:10.1016/S0163-1047(05)80009-3]
Ortega-Martínez, S. (2017). Adult Hippocampal Neurogenesis and Memory. International Journal of Advanced Biological and Biomedical Research, 6(1), 360-79. [DOI:10.18869/IJABBR.2017.360]
Paxinos, G., Watson, C., Pennisi, M., & Topple, A. (1985). Bregma, lambda and the interaural midpoint in stereotaxic surgery with rats of different sex, strain and weight. Journal of Neuroscience Methods, 13(2), 139-43. [DOI:10.1016/0165-0270(85)90026-3]
Renoir, T., Païzanis, E., El Yacoubi, M., Saurini, F., Hanoun, N., & Melfort, M., et al. (2008). Differential long-term effects of MDMA on the serotoninergic system and hippocampal cell proliferation in 5-HTT knock-out vs. wild-type mice. International Journal of Neuropsychopharmacology, 11(8), 1149-62. [DOI:10.1017/S1461145708009048] [PMID]
Roberts, C. A., Jones, A., & Montgomery, C. (2016). Meta-analysis of molecular imaging of serotonin transporters in ecstasy/polydrug users. Neuroscience & Biobehavioral Reviews, 63, 158-67. [DOI:10.1016/j.neubiorev.2016.02.003] [PMID]
Schmidt, C. J., Levin, J. A., & Lovenberg, W. (1987). In vitro and in vivo neurochemical effects of methylenedioxymethamphetamine on striatal monoaminergic systems in the rat brain. Biochemical Pharmacology, 36(5), 747-55. [DOI:10.1016/0006-2952(87)90729-5]
Shariati, M. B. H., Sohrabi, M., Shahidi, S., Nikkhah, A., Mirzaei, F., & Medizadeh, M., et al. (2014). Acute effects of ecstasy on memory are more extensive than chronic effects. Basic and Clinical Neuroscience, 5(3), 225-30. https://pubmed.ncbi.nlm.nih.gov/25337384/
Simantov, R. (2004). Multiple molecular and neuropharmacological effects of MDMA (Ecstasy). Life Sciences, 74(7), 803-14. [DOI:10.1016/j.lfs.2003.08.002] [PMID]
Soleimani Asl, S., Saifi, B., Sakhaie, A., Zargooshnia, S., & Mehdizadeh, M. (2015). Attenuation of ecstasy-induced neurotoxicity by N-acetylcysteine. Metabolic Brain Disease, 30(1), 171-81. [DOI:10.1007/s11011-014-9598-0] [PMID]
Spalding, K. L., Bergmann, O., Alkass, K., Bernard, S., Salehpour, M., & Huttner, H. B., et al. (2013). Dynamics of hippocampal neurogenesis in adult humans. Cell, 153(6), 1219-27. [DOI:10.1016/j.cell.2013.05.002] [PMID] [PMCID]
Venkatesan, A., Nath, A., Ming, G. l., & Song, H. (2007). Adult hippocampal neurogenesis: regulation by HIV and drugs of abuse. Cellular and Molecular Life Sciences, 64(16), 2120-32. [DOI:10.1007/s00018-007-7063-5] [PMID]
Warner‐Schmidt, J. L., & Duman, R. S. (2006). Hippocampal neurogenesis: Opposing effects of stress and antidepressant treatment. Hippocampus, 16(3), 239-49. [DOI:10.1002/hipo.20156] [PMID]
Zhao, C., Deng, W., & Gage, F. H. (2008). Mechanisms and functional implications of adult neurogenesis. Cell, 132(4), 645-60. [DOI:10.1016/j.cell.2008.01.033] [PMID]
Type of Study: Original |
Subject:
Behavioral Neuroscience
Received: 2018/06/27 | Accepted: 2019/02/19 | Published: 2020/07/1
Received: 2018/06/27 | Accepted: 2019/02/19 | Published: 2020/07/1
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