Karimipour, Tayefi, Shimia, and Mahmoudi:

Integration of the Neural Stem and Progenitor Cells into Existing Neuronal Circuitry and Adult Neurogenesis in the Dentate Gyrus of the Hippocampus

Mohammad Karimipourac*, Hamid Tayefia, Mohammad Shimiad, Javad Mahmoudib

aDepartment of Anatomical Sciences, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
bNeurosciences Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
cInstitute for Stem Cell Biology and Regenerative Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
dDepartment of Neurosurgery, Tabriz University of Medical Sciences, Tabriz, Iran

Correspondence
Mohammad Karimipour, Department of Anatomical Sciences, Faculty of Medicine, Golgasht St. Tabriz, Iran. Tel/Fax: +98-4133342086, Email: karimipourm@tbzmed.ac.ir

Received: 2016-09-03
Accepted: 2017-01-29

DOI: 10.13183/jecns.v4i1.56

Abstract


Objective: For a long time there was a central dogma that new neurons are not generated in the adult mammalian brain. But nowadays, it is well accepted that new neurons are continuously generated in discrete neurogenic regions of the brain throughout adult life. Neurological disorders are characterized by neuronal cell death, glial scar formation, axonal damage and deterioration of the cerebral cytoarchitecture and neuronal circuitry which result in functional deficits. Since the promising strategy for regenerating the damaged Central Nervous System (CNS) is the activation and recruitment of the endogenous regenerative capacity, therefore we investigated the neurogenesis potential of endogenous neural stem cells in an adult mouse brain.

Materials and Methods: Since the some documented experimental studies have shown that the long-term injection of the BrdU induces cell senescence and astroglial differentiation, in the present study, BrdU intraperitoneal injection was applied in a short period of time. The number of animals sacrificed for evaluation of the proliferation of neural stem and progenitor cells and 4 weeks following the last BrdU injection, the rest of the animals was used for investigation of adult neurogenesis.

Results: Findings showed that the neural stem and progenitor cells produce in the subgranular zone (SGZ) of the dentate gyrus and after 4 weeks differentiate into mature neurons. Also, this experiment showed that the possibility of the evaluation of adult neurogenesis mechanism will be provided with BrdU injection in a short time.

Conclusion: The generation and integration of new neurons is a continuous physiological process that occurs in the brain. Therefore, the use of endogenous neural stem cells is a promising approach for neural tissue regeneration in the neurological conditions.

Keywords: Neural Stem Cells, Proliferation, Differentiation, Neurogenesis, Subgranular Zone

© 2017 Swedish Science Pioneers, All rights reserved.


Introduction

In the past years, there was a fundamental assumption that new neurons are not produced in the adult mammalian brain and it has been thought that neurogenesis, or the production of new neurons, occurs only during development and stops in the adult period [1]. After a long time experimental researches about the Central Nervous System (CNS) regenerative possibility, now, it is well accepted that new neurons are continuously generated in particular neurogenic regions of the brain throughout adult life [2]. Sixty years ago in the early 1965, Altman and Das for the first time discovered the newly generated microneurons in the adult mammalian brain by using thymidine autoradiographic devices [1] and in 1992, Neural stem cells (NSCs) were successfully isolated from the mammalian brain by in-vitro culture assays [3]. After that, Kuhn et al, with the powerful cell proliferation markers such as BrdU detected NSCs in vivo experimental assays [4]. Ultimately NSCs were determined in the developing and adult nervous system neurogenic regions including the subventricular zone (SVZ) of the lateral ventricle, the subgranular zone (SGZ) of the dentate gyrus in the hippocampus, the cortical neuroepithelium, and the spinal cord [3-5]. NSCs are defined as cells that have the ability to self-renew and to give rise to the three major cell types of the CNS: neurons, astrocytes, and oligodendrocytes and these such possibilities, represent them as a promising approach for the treatment of brain neurological disorders [2, 6, 7]. Neurological conditions, such as stroke, Traumatic Brain Injury (TBI) and neurodegenerative diseases (Parkinson, Alzheimer, Huntington and Multiple Sclerosis), are characterized by neuronal cell death, glial scar formation and axonal damage, with subsequent loss of cerebral architecture and neuronal circuitry resulting in functional deficits. Unfortunately there are very limited therapeutic options without effective treatments for these conditions [8, 9]. But in recent decades, the discovery of the NSCs and adult neurogenesis, that could generate neural tissue, has raised new possibilities for repairing the nervous system [7]. Currently, the regenerative approaches for the replacement of lost cells in the CNS and regeneration of the damaged brain and spinal cord could be organized into 2 relevant strategies: The first one, the gold strategy of the regenerative medicine, is the using of the endogenous NSCs. And the second is the transplantation of exogenous NSCs. In the case of first strategy, there is no need to external cell sources and its important advantage is that it hasn’t ethical issues as well as immune and rejection problems [8, 10, 11]. Some methodologies have been presented to detect and characterize neurogenesis phenomenon including Bromodeoxyuridine (BrdU) as a thymidine analog, retroviral vectors and transgenic animals [4, 12-14]. Even though, the BrdU has been widely used to label newly generated cells for birth dating, cell fate studies and specially neurogenesis as well as tracking of cells after transplantation but it has some limitations and pitfalls [15-18]. In the some experimental studies, it has been shown that the administration of the BrdU for a long time induces NSCs senescence, cell death and astroglial differentiation [19-24]. Therefore, the present study aimed to investigate the potential neurogenesis and regenerative effects of the endogenous neural stem and progenitor cells by means of BrdU injection for labeling in a short time.

Materials and Methods

Male mice C57BL/6 (12 weeks of age, 25–30 g) were housed 5 per cage and maintained on a 12 h light–dark cycle in an air-conditioned constant temperature (22 ± 1°C) room, with food and water ad libitum. All procedures and animal care were carried out according to the protocols and guidelines of the Tabriz University of Medical Sciences. Initially, 10 animals were randomly chosen for the current study. The protocol of experimental design is summarized in Figure 1.

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Figure 1. Experimental schedule of the research design duringthe course of study.

BrdU labeling as a tracer for proliferation and adult neurogenesis study

BrdU (5-bromo-2-deoxyuridine; Sigma) was dissolved at 10 mg/ml concentration in 0.9% NaCl and sterile-filtered at 0.22 µm. All animals received daily intraperitoneal injection of 50 mg/kg body weight during 3 successive days. One day after the last injection of BrdU, 5 animals for evaluation of proliferation of the adult neural stem and progenitor cells were sacrificed. For investigation of adult neurogenesis, the remaining mice lived for an additional 4 weeks and were killed at this time point.

Tissue processing and stereological analysis for quantification

Five animals were perfused transcardially (under deep anesthesia with chloral hydrate 350 mg/kg/body weight, i.p.) with cold saline (circa 15 minuntil recovery of clear perfusion solution) followed by 4% paraformaldehyde (in 0.1M phosphate buffer, pH 7.4). After complete perfusion, their hippocampus immediately were removed and post-fixed for overnight at 4°C temperature with the same fixative solution. For paraffin embedding, tissues were first dehydrated in series of alcohols, cleared by incubations in xylene and finally embedded in paraffin for 3–6 h and then were blocked. Serial coronal sections with 5 µm thickness and 210 µm intervals were cut throughout the hippocampus using a rotary microtome (Leica, Austria). Then sections were mounted on poly-lysine-coated slides, dried overnight at 4°C temperature. In brief, the sections were first deparaffinized and rehydrate in decreasing ethanol and then washed in tap water and were stored at 4°C until use. For quantification of BrdU-labeled cells, in a 1-in-6 series of sections (5 µm thickness) with 210- µm intervals were used throughout the SGZ. Also for evaluation of phenotype of surviving new born cells, a 1-in-12 series of sections were subjected for double-labeled immunostaining. One hundred BrdU-positive cells per animal were analyzed for co-expression of BrdU and NeuN to assess the neuronal phenotype, and ratio of cellsco-expressing BrdU and NeuN were determined.

Immunohistochemistry

Immunohistochemistry for BrdU was performed for evaluation of proliferation ofadult neural stem and progenitor cells. In brief, the sections were washed in Tris-buffered saline (TBS; 0.1 M Tris-HCl, pH 7.4, and 0.9% NaCl). For DNA denaturation, sections were incubated in a 2 N HCl bath for 60 min at 37°C, rinsed 10 min in 0.1 M boric acid, pH 8.5, and washed in TBS. Antigen retrieval was performed with incubation of sections in preheated 10 mM sodium citrate buffer solution for 15 min at 100°C. The sections were washed in Tris-buffered saline (TBS; 0.1 M Tris-HCl, pH 7.4, and 0.9% NaCl). Blocking endogenous peroxidase step was performed by incubation of sectionsin 0.6% H2O2 in TBS for 30 min. After several washes in TBS, sections were incubated in TBS 3% donkey serum 0.3% Triton-X for 1 hour and then were incubatedin primary antibody against BrdU (monoclonal from rat; serotec, 1:250) for 12 h at 4°C. Sections were subjected to a biotinylated donkey anti rat IgG secondary antibody (Jackson Immuno-Research; 1:1000) for 1 h. ABC reagent (Vectastain Elite, Vector Laboratories; 1 µl/ml) was applied for 1 h, then the sections incubated in Diaminobenzedine as a chromogen (Sigma; 0.25 mg/ml in TBS with 0.01% H2O2 and 0.04% nickelchloride) for 7 min. After several washes in TBS, Sections were mounted, air dried and covers lipped.

Double immunofluorescence staining for BrdU/NeuN

Immunofluorescence double labeling of BrdU, NeuN was performed to evaluate adult neurogenesis. Briefly, every 12th section throughout the hippocampus were used for immunofluorescence staining for double-labeling of BrdU, NeuNas described previously [25]. After pretreatment and a blocking step with TBS–tritonplus containing 3% donkey serum, to evaluate double-labeling of (BrdU, NeuN)–positive cells, the sections were incubated in a mixture of primary antibodies including mouse anti NeuN (Millipore, Chandlers Ford, Hants, UK, catalogue No: MAB377) (1:100 in 0.3% Triton in TBS and 3% donkey serum) and rat anti-BrdU (Accurate Chemical &Scientific Corporation, Westbury, USA, catalogue No: OBT0030) (1:200 in 0.3% Triton with 3% donkey serum) overnight at 4°C temperature. The next day after two washes with TBS, the sections were incubated in a mixture of secondary antibodies include Alexa Fluor 448 donkey anti rat [green] (1:1000) and donkey anti mouse Alexa Fluor 568 [red] (1:1000 in 0.3% Triton in TBS with 3% donkey serum) in a humid and dark chamber at room temperature for one hour, washed twice with TBS and slides mounted with glycerol buffer, cover slipped, and then visualized with a fluorescence microscope and digitally photographed (Zeiss, Axiophot, Germany).

Results

Adult neural stem and progenitor cells in the SGZ of the dentate gyrus, generate, proliferate and migrate to the granular cell layer

To assess the proliferation rate of adult neural stem and progenitor cells in the hippocampus of the mice, we injected BrdU (50 mg/kg body weight), the proliferation marker, per day for 3 successive days. 24 h after the last injection, BrdU incorporation was analyzed by using immunohistochemistry technique Figure 2. Our analysis showed that the number of BrdU-positive cells was (2242 ± 2.40). Therefore, these results should that the adult neural stem and progenitor cellsproduce every day continuously in the hippocampus.

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Figure 2. BrdU immunohistochemistry 24 h after the last BrdU injection for evaluation of thegeneration and proliferation of the adult neural stem and progenitor cells in the SGZ of dentate gyrus.

Adult neural stem and progenitor cells differentiate to mature neuronsand integrate intoneuronal circuitry in the hippocampus.

To evaluate the differentiation and maturation of the neural stem and progenitor cells, we examined the differentiate potential of BrdU-positive cells into neurons 4 weeks after the last injection by co- labeling for BrdU and the neuron-specific marker, NeuN Figure 3 (a-f). It was found that (78.80 ± 1.30) % of the surviving BrdU-positive cells expressed NeuN. These results showed that the majority of the generated neural stem and progenitor cells differentiate to mature neurons after 4 weeks and eventually integrate into granular cell layer of the dentate gyrus.

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Figure 3. (a-f) Double- immunostaining of (BrdU- NeuN) positive cells for study of the adult neurogenesis in the dentate gyrus 4 weeks after the last BrdU injection. Sections were double labeled for NeuN (Green), BrdU (Red). Colocalization was identified using immunofluorescence microscopy. BrdU (a), NeuN (b), DAPI (c), (BrdU/NeuN) (d), (NeuN/DAPI) (e) and (BrdU/NeuN/DAPI) (f).

Discussion

The CNS and PNS when facing the injury show different regenerative strategies since the CNS have limited capacity for regeneration. In the PNS, the functional recovery could be provided because of the presence of Schwann cells, which are able to secrete the growth-promoting molecules, neurotrophic growth factors as well as chemotrophic agents. Also Schwann cells supply the nutrient materials, guidethe axon to the distal segment and remyelinate regenerating axons [26-28]. Meanwhile the regenerative process in the CNSis very difficult because of the multiple factors including inhibitory factors which are produced by neighbor cells and undesirable microenvironment [29]. Therefore restoration of the damaged CNS is a major challenge. The CNS could be damaged under influence conditions including neurodegenerative disorders, stroke, multiple sclerosis, spinal cord and traumatic brain injury. In these cases, neural and glial cells are died and some neuropsychiatric disabilities appear with disease progression [9,30]. The use of endogenous neural stem cell and progenitor cells is a great value to replace the lost neural cells and regeneration of the injured neural tissue [11,31,32]. The application of the endogenous NSCs in regenerative strategies has its own pros and cons. The important advantages of the endogenous NSCs including lack of need to external cell source, devoid of ethical and political considerations, as well as without risk of tumorigenicity and rejection possibilities. Also, access to endogenous NSCs is much easier, simpler and more effective than exogenous stem cells [11,33]. After any kind of injury to the CNS, the endogenous NSCs are recruited to the lesion site, but this natural response is often insufficient and unsatisfactory for recovery of the functional deficits [34-37]. To overcome this important issue, promising strategies have been suggested including the use of growth factors, biomaterials and artificial extracellular matrix for mobilizing, stimulation and recruitment of the endogenous NSCs [38-43]. Although, generally BrdU infusion as an analog for thymidine nucleotide isused for evaluation of neurogenesis, birth dating, proliferation and cell tracking, but the effects of BrdU on the NSCs behavior have not been fully investigated [15-18]. For a long- lasting period it has been known that the long term exposure of the NSCs with BrdU promotes conformational changes in DNA structure, affects the balanced nucleotide pool in NSCs and subsequently alters cell cycle progression in which the majority of cells arrest in the G0/G1- phase of the cell cycle [19, 23, 44]. Also the recent studies have shown that the BrdU alters the DNA structure and stability, raises the risk of sister-chromatid exchange and mutations [18]. Some data from experimental researches clearly have shown that the administration of BrdU for a long time as a chemical substance induces the premature senescence in NSCs and drives the fate of neural progeny into astrocyte and oligodendrocyte lineages [20, 21, 24]. Based on these side effects of the BrdU, we thought that whether we can study the neurogenesis phenomenon using by BrdU injection in a short period or not? Our results showed that the administration of the BrdU for 3 successive days directed us to investigate the proliferation and differentiation of the NSCs. In the present study, it was shown that the NSCs generate daily in SGZ of the dentate gyrus even in adult life period and follow their developmental process, in which they proliferate, migrate and integrate into existing neuronal circuitry in the hippocampus. This biological phenomenon, point out the existing adult neurogenesis as a type of neuroplasticity and neural regenerative potential in the adult brain.

Conclusion

It is concluded that the BrdU injection in a short time could be used for evaluation of NSCs behavior; however, further studies should be performed to further clarify the BrdU application for the investigation of the neurogenesis mechanism in a correct manner. Also, this experiment showed that the continual production of new neurons is a continuous physiological process that occurs in the brain. Thus, regeneration of the damaged CNS is becoming feasible from the clinical aspect and activation of the endogenous NSCs and regenerative capacity are very important strategies for CNS repair.

Acknowledgments

This study was supported by Tabriz University of Medical Sciences (TUOMS).

References

1. Altman J, Das GD, Post-natal origin of microneurones in the rat brainNature 1965; 207: 5000953-6.

2. Gage FH, Mammalian neural stem cellsScience 2000; 287: 54571433-8.

3. Reynolds BA, Weiss S, Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous systemScience 1992; 255: 50521707-10.

4. Kuhn HG, Dickinson-Anson H, Gage FH, Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferationJ Neurosci 1996; 16: 62027-33.

5. Yao J, Mu Y, Gage FH, Neural stem cells: mechanisms and modelingProtein & cell 2012; 3: 4251-61.

6. Temple S, The development of neural stem cellsNature 2001; 414: 6859112-7.

7. Rossi F, Cattaneo E, Opinion: neural stem cell therapy for neurological diseases: dreams and realityNature reviews Neuroscience 2002; 3: 5401-9.

8. Bjorklund A, Lindvall O, Cell replacement therapies for central nervous system disordersNature neuroscience 2000; 3: 6537-44.

9. Lindvall O, Kokaia Z, Martinez-Serrano A, Stem cell therapy for human neurodegenerative disorders-how to make it workNat Med 2004; 10: SupplS42-50.

10. Lichtenwalner RJ, Parent JM, Adult neurogenesis and the ischemic forebrainJournal of Cerebral Blood Flow & Metabolism 2006; 26: 11-20.

11. Kulbatski I, Mothe A, Nomura H, Tator C, Endogenous and exogenous CNS derived stem/progenitor cell approaches for neurotraumaCurrent drug targets 2005; 6: 1111-26.

12. Kempermann G, Jessberger S, Steiner B, Kronenberg G, Milestones of neuronal development in the adult hippocampusTrends in neurosciences 2004; 27: 8447-52.

13. Lewis PF, Emerman M, Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virusJournal of virology 1994; 68: 1510-6.

14. Hayes N, Nowakowski R, Dynamics of cell proliferation in the adult dentate gyrus of two inbred strains of miceDevelopmental brain research 2002; 134: 177-85.

15. Cooper-Kuhn CM, Kuhn HG, Is it all DNA repair?: methodological considerations for detecting neurogenesis in the adult brainDevelopmental Brain Research 2002; 134: 113-21.

16. Kuhn HG, Cooper-Kuhn CM, Bromodeoxyuridine and the detection of neurogenesisCurrent pharmaceutical biotechnology 2007; 8: 3127-31.

17. Kuhn HG, Eisch AJ, Spalding K, Peterson DA, Detection and Phenotypic Characterization of Adult NeurogenesisCold Spring Harbor perspectives in biology 2016; 8: 3a025981

18. Taupin P, BrdU immunohistochemistry for studying adult neurogenesis: paradigms, pitfalls, limitations, and validationBrain research reviews 2007; 53: 1198-214.

19. Poot M, Hoehn H, Kubbies M, Grossmann A, Chen Y, Rabinovitch PS, Cell-Cycle Analysis Using Continuous Bromodeoxyuridine Labeling and Hoechst 33358—Ethidium Bromide Bivariate Flow CytometryMethods in cell biology 1994; 41: 327-40.

20. Eriko M, Nakabayashi K, Suzuki T, Kaul SC, Ogino H, Fujii M, 5-Bromodeoxyuridine induces senescence-like phenomena in mammalian cells regardless of cell type or speciesJournal of biochemistry 1999; 126: 61052-9.

21. Suzuki T, Minagawa S, Michishita E, Ogino H, Fujii M, Mitsui Y, Induction of senescence-associated genes by 5-bromodeoxyuridine in HeLa cellsExperimental gerontology 2001; 36: 3465-74.

22. Caldwell MA, He X, Svendsen CN, 5-Bromo-2′-deoxyuridine is selectively toxic to neuronal precursors in vitroEuropean Journal of Neuroscience 2005; 22: 112965-70.

23. Lehner B, Sandner B, Marschallinger J, Lehner C, Furtner T, Couillard-Despres S, The dark side of BrdU in neural stem cell biology: detrimental effects on cell cycle, differentiation and survivalCell and tissue research 2011; 345: 3313-28.

24. Ross HH, Levkoff LH, Marshall GP, Caldeira M, Steindler DA, Reynolds BA, Bromodeoxyuridine induces senescence in neural stem and progenitor cellsStem cells 2008; 26: 123218-27.

25. Kempermann G, Kuhn HG, Gage FH, Genetic influence on neurogenesis in the dentate gyrus of adult miceProc Natl Acad Sci U S A 1997; 94: 1910409-14.

26. Struzyna LA, Katiyar K, Cullen DK, Living scaffolds for neuroregenerationCurrent Opinion in Solid State and Materials Science 2014; 18: 6308-18.

27. Belkas JS, Shoichet MS, Midha R, Peripheral nerve regeneration through guidance tubesNeurological research 2004; 26: 2151-60.

28. Taylor JS, Bampton ET, Factors secreted by Schwann cells stimulate the regeneration of neonatal retinal ganglion cellsJournal of anatomy 2004; 204: 125-31.

29. Tian L, Prabhakaran MP, Ramakrishna S, Strategies for regeneration of components of nervous system: scaffolds, cells and biomoleculesRegenerative Biomaterials 2015; rbu017

30. Bjorklund A, Lindvall O, Cell replacement therapies for central nervous system disordersNature neuroscience 2000; 3: 537-44.

31. Doetsch F, The glial identity of neural stem cellsNature neuroscience 2003; 6: 111127-34.

32. Doetsch F, Caille I, Lim DA, García-Verdugo JM, Alvarez-Buylla A, Subventricular zone astrocytes are neural stem cells in the adult mammalian brainCell 1999; 97: 6703-16.

33. Zhao J, Zhang N, Prestwich GD, Wen X, Recruitment of endogenous stem cells for tissue repairMacromolecular bioscience 2008; 8: 9836-42.

34. Okano H, Sakaguchi M, Ohki K, Suzuki N, Sawamoto K, Regeneration of the central nervous system using endogenous repair mechanismsJournal of neurochemistry 2007; 102: 51459-65.

35. Ke Y, Chi L, Xu R, Luo C, Gozal D, Liu R, Early response of endogenous adult neural progenitor cells to acute spinal cord injury in miceStem cells 2006; 24: 41011-9.

36. Obermair F-J, Schröter A, Thallmair M, Endogenous neural progenitor cells as therapeutic target after spinal cord injuryPhysiology 2008; 23: 5296-304.

37. Picard-Riera N, Nait-Oumesmar B, Evercooren BV, Endogenous adult neural stem cells: limits and potential to repair the injured central nervous systemJournal of neuroscience research 2004; 76: 2223-31.

38. Bauer S, Patterson PH, Leukemia inhibitory factor promotes neural stem cell self-renewal in the adult brainThe Journal of Neuroscience 2006; 26: 4612089-99.

39. Imitola J, Raddassi K, Park KI, Mueller F-J, Nieto M, Teng YD, Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1α/CXC chemokine receptor 4 pathwayProceedings of the National Academy of Sciences 2004; 101: 5218117-22.

40. Cross DP, Wang C, Stromal-derived factor-1 alpha-loaded PLGA microspheres for stem cell recruitmentPharmaceutical research 2011; 28: 102477-89.

41. Kojima A, Tator CH, Epidermal growth factor and fibroblast growth factor 2 cause proliferation of ependymal precursor cells in the adult rat spinal cord in vivoJournal of Neuropathology & Experimental Neurology 2000; 59: 8687-97.

42. Kojima A, Tator CH, Intrathecal administration of epidermal growth factor and fibroblast growth factor 2 promotes ependymal proliferation and functional recovery after spinal cord injury in adult ratsJournal of neurotrauma 2002; 19: 2223-38.

43. Lindberg OR, Brederlau A, Kuhn HG, Epidermal growth factor treatment of the adult brain subventricular zone leads to focal microglia/macrophage accumulation and angiogenesisStem cell reports 2014; 2: 4440-8.

44. Goz B, The effects of incorporation of 5-halogenated deoxyuridines into the DNA of eukaryotic cellsPharmacological reviews 1977; 29: 4249-72.

Notes

Conflict of Interest

The authors have no conflicts of interest to disclose.

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