Summary

Аппаратура ближней перспективе плода овец многомерных хронических неинфекционных наркозом Recordings

Published: October 25, 2015
doi:

Summary

Хронически приборами без анестезии плода модель овец используется для изучения развития плода человека в норме и патологии, потому что это позволяет хирургического размещение и обслуживание катетеров и электродов, отбор проб повторяющихся крови, инъекции вещества, записи биоэлектрической активности, и в естественных изображений. Опишем процедуры, необходимые для установления этой модели.

Abstract

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as endotoxins, bacteria, umbilical cord occlusions, hypoxia and various pharmacological treatments. The life-saving clinical practices of glucocorticoid treatment in fetuses at risk of premature birth and the therapeutic hypothermia have been developed in this model. This is due to the unique amenability of the non-anesthetized fetal sheep to the surgical placement and maintenance of catheters and electrodes, allowing repetitive blood sampling, substance injection, recording of bioelectrical activity, application of electric stimulation and in vivo organ imaging. Here we describe the surgical instrumentation procedure required to achieve a stable chronically instrumented non-anesthetized fetal sheep model including characterization of the post-operative recovery from blood gas, metabolic and inflammation standpoints.

Introduction

Разнообразие моделей животных существует для изучения нормальных и скомпрометированных беременности, в том числе лабораторных грызунов, приматов и домашних жвачных. 1,2,3,4,5 Хронически приборами беременной овцы широко используется в течение 50 лет, как модель развития плода и ответов человека на патофизиологических стимулы, такие как липополисахарида (ЛПС). 6-10 Поражения следующие экспозиции LPS имитировать именно то, что видел у недоношенных новорожденных с перивентрикулярного лейкомаляции, что из-за подобной созревания профиль обоих видов. 11, 12

Другие осложнения беременности были также изучены в мельчайших подробностях, таких как открытие, что дородовой глюкокортикоиды содействия развитию легких 13-15 и понимания воздействия внутриутробной задержки роста (ЗВУР) плода на 16,17.

Широкое использование модели плода овец связано с UniqUE аменабельность не-наркозом плода овец в хирургической размещения и обслуживания катетеров и электродов, что позволяет проб повторяющиеся крови, записи биоэлектрической активности, применение электрической стимуляции и в естественных изображений мозга. 18 телеметрии тоже можно, хотя и менее часто используются но из-за более высокой сложности в настроить, а также начальной и поддерживающей стоимости. 19

Кроме того, модель плода овцы очень универсальны, как много вариаций приборов возможны в зависимости от мер, представляющих интерес. Например, можно выполнить запись нескольких дней до недель многомерных сигналов в режиме реального времени, такие как плода дыхательных движений, электрической активности головного мозга, сердечно-сосудистых реакций, ЭКГ, регионарного кровотока к ряду органов с использованием зондов потока или микросфер и т.д. Благодаря это универсальность, широкий диапазон исследований были проведены в том числе развития Кардиovascular система 20,21, гипоталамо-гипофизарно-надпочечниковой (HPA) оси 22, развитие мозга развитие 23 и сна государства, в частности, 24, эффекты гипоксии / асфиксия 25, терапевтической гипотермии 26, воспаление 6-11, комбинация обоих 27, глюкокортикоиды 28,29, антидепрессанты 30, бронхо-легочной дисплазии (БЛД) 31,32 плода программирование 33,34,35,36,37,38,39 или разработка новых методов мониторинга плода до и во время родов, чтобы имя, но несколько областей исследования. 40,41,42,43

Общая цель метода, изложенного, чтобы показать базовую реализацию этой разносторонней модели. Это позволяет устанавливать широкий спектр острых и хронических экспериментальных протоколов, обучающихся плода физиологии и патофизиологии на интегративной, орган, клеточном и молекулярном уровнях.

Protocol

Уход за животными следуют рекомендациям Канадского совета по уходу за животными и утверждения Университета Монреаля совета по уходу за животными (протокол # 10-Речь-1560). Подробная информация о материалах и методах, используемых предоставляется в таблице 1. 1. Анестезия <ol…

Representative Results

38 беременных временем от овец, были оборудованы в 128 ± 2 дней беременности (DGA, ~ 0,88 беременности, срок 145 DGA) с артериальной, венозной и амниотической катетеров и ЭКГ (ЭКГ) электродов с стерильных условиях общей анестезии (как овцы и плода ). В случае двойной беременности чем больше плода был?…

Discussion

Анестезии и хирургических процедур представлены, которые необходимы для создания животной модели для изучения плода физиологии и патофизиологии: хронически инструментальной без наркозом плода овец.

Следует подчеркнуть, четыре критические шаги в протоколе. Во-первых, …

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

authors gratefully acknowledge funding support from the Molly Towell Perinatal Research Foundation, Canadian Institutes of Health Research (CIHR), and Fonds de Recherche du Québec – Santé (FRQS) (to MGF) and CIHR-Quebec Training Network in Perinatal Research (QTNPR) (to LDD).

The authors wish to thank Esther Simard, Marco Bosa, Carl Bernard and Carmen Movila for technical assistance.

Materials

ACE Light source Schott-Fostec A20500
Dissecting scissors Fine Science Tools 14060 – 11
Angled dissecting scissors Fine Science Tools 15006 – 09
Scalpel handle Fine Science Tools 10003 – 12 alternating dissecting tool
Curved scalpel blades #12 Fine Science Tools 10012 – 00 alternating dissecting tool
Bone scissors Fine Science Tools 16044 – 10
S & T suture tying forceps Fine Science Tools 00272 – 13
Dumont SS forceps – angled Fine Science Tools 11203 – 25 
Braided silk suture size 6-0 Teleflex Medical 07 – 30  – 10
Medical Tape transpore 3M
Ketamine hydrochloride 100 mg/ml Hospira NDC 0409 – 2051 – 05 Final Does is 80 mg/kg
Tranqui Ved Injection (xylazine 100 mg/ml) Vecdo NDC 50989 – 234 – 11 Final Does is 10 mg/kg
Reactive orange 14 Sigma – Aldrich R – 8254
Ringers Solution Components Solution is gas equilibrated with 95% O2 and 5% Co2, final pH 7.4
Sodium chloride Sigma – Aldrich S7653 Final Concentration: 118 mM
Potassium chloride Fisher Scientific P217 – 3 Final Concentration: 4.7 mM
Calcium chloride dihydrate Fisher Scientific C79 – 500 Final Concentration: 2.5 mM
Potassium phosphate monobasic Fisher Scientific P -285 Final Concentration: 1.2 mM
Magnesium sulfate J.T. Baker Jan-00 Final Concentration: 0.57 mM
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) Fisher Scientific BP 310 – 500 Final Concentration: 5.95 g/L
Glucose Sigma – Aldrich G8270 Final Concentration: 5.5 mM
LifeWindow Digicare Biomedical Technology
CED bioamplifier and ADC units Cambridge Electronic Design Limited,
Unit 4, Science Park,
Milton Road,
Cambridge CB4 0FE
ENGLAND.
Bioamp: 1902; ADC: micro1401; Data acquisition software: Spike 2, V7.13
Neurolog analog signal bioamplifier Digitimer Ltd
37 Hydeway
Welwyn Garden City
Hertfordshire, AL7 3BE, England
NL108A
ABL800Flex Radiometer Canada; 200 Aberdeen Dr, London, ON N5V 4N2
Eppendorf 5804R Eppendorf Canada; 2810 Argentia Road, #2
Mississauga, Ontario, L5N 8L2
Arrow Jugular Catheterization Set Arrow International, Inc., 2400 Bernville Road, Reading, PA 19605 USA
Atravet 10 mg/mL
Diazepam 5mg/mL
Ketamine Ketalar 100 mg/mL
Propofol 10 mg/mL
SurgiVeT Endotracheal Tubes; Smiths Medical ASD, Inc. St. Paul, MN 55112, USA
Cook Airway Exchange Catheter with RAPI-FIT Adapters Cook Critical Care 750, Bloomington IN 47402-0489 USA
Dispomed Ventilator Dispomed Ltd., 745 Nazaire-Laurin, Joliette, Quebec J6E 0L6
BD Insyte-W Becton Dickinson, Infusion Therapy Systems Inc., 9450 S State St, Sandy Utah 84070 USA 22 to 20 G; 1 in [0.9 x 25 mm] to 1.16 in [1.1 x 30 mm]
Edwards Lifesciences Ref: PX272 Pressure monitoring kit with TruWave Disposable Pressure
LifeWindow LW6000 Digicare Biomedical Technology 107 Commerce Road, Boynton Beach, FL 33426-9365 USA
Gaymar
Babcock
Polyvinyl catheters SCI (Scientific Commodities Inc.) 2 meters
2-0 Vicryl
Castroviejo scissors
electrocardiogram (ECG) LIFYY, Metrofunk Kabel-Union, Berlin, Germany four copper electrodes in single sheath, 2 meters
2-O Vicryl
3-0 Vicryl
PDS II USP
Trimethoprim sulfadoxine
Ampicillin
Stopcock Argon Medical, Cat 041220001A Double 4-way Stopcock with male luer lock
Needles Tyco Healthcare 8881202389 Monoject aluminum hub blunt needles, 22Gx, 0.7mmx 38.1mm: for fetal arterial and venous catheters
Needles Tyco Healthcare 8881202322 Monoject aluminum hub blunt needles, 16Gx, 1.6mmx38.1mm: for amniotic catheters

Referenzen

  1. Barry, J. S., Anthony, R. V. The pregnant sheep as a model for human pregnancy. Theriogenology. 69, 55-67 (2008).
  2. Morrison, J. L. Sheep models of intrauterine growth restriction: fetal adaptations and consequences. Clin Exp Pharmacol Physiol. 35, 730-743 (2008).
  3. Rees, S., Inder, T. Fetal and neonatal origins of altered brain development. Early Hum Dev. 81, 753-761 (2005).
  4. Rees, S., Harding, R., Walker, D. The biological basis of injury and neuroprotection in the fetal and neonatal brain. Int J Dev Neurosci. 29, 551-563 (2011).
  5. Moisiadis, V. G., Matthews, S. G. Glucocorticoids and fetal programming part 1: Outcomes. Nat rev Endocrinol. 10, 391-402 (2014).
  6. Wang, X., Rousset, C. I., Hagberg, H., Mallard, C. Lipopolysaccharide-induced inflammation and perinatal brain injury. Semin Fetal Neonatal Med. 11, 343-353 (2006).
  7. Gotsch, F., et al. The fetal inflammatory response syndrome. Clin Exp Obstet Gynecol. 50, 652-683 (2007).
  8. Svedin, P., Kjellmer, I., Welin, A. K., Blad, S., Mallard, C. Maturational effects of lipopolysaccharide on white-matter injury in fetal sheep. J child neurol. 20, 960-964 (2005).
  9. Nitsos, I., et al. Chronic exposure to intra-amniotic lipopolysaccharide affects the ovine fetal brain. J Soc Gynecol Investig. 13, 239-247 (2006).
  10. Yan, E., Castillo-Melendez, M., Nicholls, T., Hirst, J., Walker, D. Cerebrovascular responses in the fetal sheep brain to low-dose endotoxin. Pedia res. 55, 855-863 (2004).
  11. Dean, J. M., et al. Delayed cortical impairment following lipopolysaccharide exposure in preterm fetal sheep. Ann Neurol. 70, 846-856 (2011).
  12. Dobbing, J., Sands, J. Comparative aspects of the brain growth spurt. Early Hum Dev. 3 (1), 79-83 (1979).
  13. Liggins, G. C. Premature parturition after infusion of corticotrophin or cortisol into foetal lambs. J Endocrinol. 42, 323-329 (1968).
  14. Liggins, G. C. Premature delivery of foetal lambs infused with glucocorticoids. J Endocrinol. 45, 515-523 (1969).
  15. Liggins, G. C., Fairclough, R. J., Grieves, S. A., Kendall, J. Z., Knox, B. S. The mechanism of initiation of parturition in the ewe. Recent Prog Horm Res. 29, 111-159 (1973).
  16. Morrison, J. L. Sheep models of intrauterine growth restriction: fetal adaptations and consequences. Clin Exp Pharmacol Physiol. 35, 730-743 (2008).
  17. Robinson, J. S., Hart, I. C., Kingston, E. J., Jones, C. T., Thorburn, G. D. Studies on the growth of the fetal sheep. The effects of reduction of placental size on hormone concentration in fetal plasma. J Dev Physiol. 2, 239-248 (1980).
  18. Carmel, E., et al. Fetal brain MRI – experiences in the ovine model of cerebral inflammatory response. Repro sci. 19 (3), 347A-348A (2012).
  19. Samson, N., Dumont, S., Specq, M. L., Praud, J. P. Radio telemetry devices to monitor breathing in non-sedated animals. Respir Physiol Neurobiol. 179, 111-118 (2011).
  20. Thakor, A. S., Giussani, D. A. Effects of acute acidemia on the fetal cardiovascular defense to acute hypoxemia. Am J Physiol Regul Integr Comp Physiol. 296, R90-R99 (2009).
  21. Green, L. R., Kawagoe, Y., Homan, J., White, S. E., Richardson, B. S. Adaptation of cardiovascular responses to repetitive umbilical cord occlusion in the late gestation ovine fetus. J Physiol. 535, 879-888 (2001).
  22. Unno, N., et al. Changes in adrenocorticotropin and cortisol responsiveness after repeated partial umbilical cord occlusions in the late gestation ovine fetus. Endocrinology. 138, 259-263 (1997).
  23. Muller, T., et al. Developmental changes in cerebral autoregulatory capacity in the fetal sheep parietal cortex. J Physiol. 539, 957-967 (2002).
  24. Keen, A. E., Frasch, M. G., Sheehan, M. A., Matushewski, B., Richardson, B. S. Maturational changes and effects of chronic hypoxemia on electrocortical activity in the ovine fetus. Brain Res. 1402, 38-45 (2011).
  25. Ross, M. G., et al. Correlation of arterial fetal base deficit and lactate changes with severity of variable heart rate decelerations in the near-term ovine fetus. Am J Obstet Gynecol. 208, e281-e286 (2013).
  26. Gunn, A. J., Gunn, T. R., de Haan, H. H., Williams, C. E., Gluckman, P. D. Dramatic neuronal rescue with prolonged selective head cooling after ischemia in fetal lambs. J Clin Invest. 99, 248-256 (1997).
  27. Xu, A., Piorkowska, K., Matushewski, B., Hammond, R., Richardson, B. S. Adaptive Brain Shut-Down Counteracts Neuroinflammation in the Near-Term Ovine Fetus. 20 (3), 222A (2013).
  28. Derks, J. B., et al. A comparative study of cardiovascular, endocrine and behavioural effects of betamethasone and dexamethasone administration to fetal sheep. J Physiol Lond. 499, 217-226 (1997).
  29. Lohle, M., et al. Betamethasone effects on fetal sheep cerebral blood flow are not dependent on maturation of cerebrovascular system and pituitary-adrenal axis. J Physiol. 564, 575-588 (2005).
  30. Morrison, J. L., et al. Maternal fluoxetine infusion does not alter fetal endocrine and biophysical circadian rhythms in pregnant sheep. J Soc Gynecol Investig. 12, 356-364 (2005).
  31. Allison, B. J., et al. Ventilation of the very immature lung in utero induces injury and BPD-like changes in lung structure in fetal sheep. Pediatr Res. 64, 387-392 (2008).
  32. Rozance, P. J., et al. Intrauterine growth restriction decreases pulmonary alveolar and vessel growth and causes pulmonary artery endothelial cell dysfunction in vitro in fetal sheep. Am J Physiol Lung Cell Mol Physiol. 301, L860-L871 (2011).
  33. Fowden, A. L., Giussani, D. A., Forhead, A. J. Endocrine and metabolic programming during intrauterine development. Early hum dev. 81, 723-734 (2005).
  34. Nathanielsz, P. W., Hanson, M. A. The fetal dilemma: spare the brain and spoil the liver. J Physiol. 548, 333 (2003).
  35. Manikkam, M., et al. Fetal programming: prenatal testosterone excess leads to fetal growth retardation and postnatal catch-up growth in sheep. Endocrinology. 145, 790-798 (2004).
  36. Savabieasfahani, M., et al. Fetal programming: testosterone exposure of the female sheep during midgestation disrupts the dynamics of its adult gonadotropin secretion during the periovulatory period. Biol Reprod. 72, 221-229 (2005).
  37. Bergen, N. H., et al. Fetal programming alters reactive oxygen species production in sheep cardiac mitochondria. Clin Sci (Lond). 116, 659-668 (2009).
  38. Cox, L. A., et al. A genome resource to address mechanisms of developmental programming: determination of the fetal sheep heart transcriptome. J Physiol. 590, 2873-2884 (2012).
  39. Mahoney, M. M., Padmanabhan, V. Developmental programming: impact of fetal exposure to endocrine-disrupting chemicals on gonadotropin-releasing hormone and estrogen receptor mRNA in sheep hypothalamus. Toxicol Appl Pharmacol. 247, 98-104 (2010).
  40. Blad, S., Welin, A. K., Kjellmer, I., Rosen, K. G., Mallard, C. ECG and Heart Rate Variability Changes in Preterm and Near-Term Fetal Lamb Following LPS Exposure. Reprod Sci. 15, 572-583 (2008).
  41. Frasch, M. G., et al. Heart rate variability analysis allows early asphyxia detection in ovine fetus. Reprod Sci. 16, 509-517 (2009).
  42. Frasch, M. G., Keen, A. E., Gagnon, R., Ross, M. G., Richardson, B. S. Monitoring fetal electrocortical activity during labour for predicting worsening acidemia: a prospective study in the ovine fetus near term. PLoS One. 6, e22100 (2011).
  43. Durosier, L. D., et al. Sampling rate of heart rate variability impacts the ability to detect acidemia in ovine fetuses near-term. Front pedia. 2, 38 (2014).
  44. Danielson, L., McMillen, I. C., Dyer, J. L., Morrison, J. L. Restriction of placental growth results in greater hypotensive response to alpha-adrenergic blockade in fetal sheep during late gestation. J Physiol. 563, 611-620 (2005).
  45. Edwards, L. J., Simonetta, G., Owens, J. A., Robinson, J. S., McMillen, I. C. Restriction of placental and fetal growth in sheep alters fetal blood pressure responses to angiotensin II and captopril. J Physiol. 515 (Pt 3), 897-904 (1999).
  46. Xu, A., et al. Adaptive brain shut-down counteracts neuroinflammation in the near-term ovine fetus. Front neurol. 5, 110 (2014).
  47. Xu, A., et al. The Ovine Fetal and Placental Inflammatory Response to Umbilical Cord Occlusions With Worsening Acidosis. Reprod Sci. 22 (11), (2015).
  48. Wang, X., Durosier, L. D., Ross, M. G., Richardson, B. S., Frasch, M. G. Online detection of fetal acidemia during labour by testing synchronization of EEG and heart rate: a prospective study in fetal sheep. PLoS One. 9, e108119 (2014).
  49. Reid, A., Malone, J. Q fever in Ireland A seroprevalence study of exposure to Coxiella burnettii among Department of Agriculture workers. Occ med. 54, 544-547 (2004).
  50. Roest, H. I., Bossers, A., van Zijderveld, F. G., Rebel, J. M. Clinical microbiology of Coxiella burnetii and relevant aspects for the diagnosis and control of the zoonotic disease Q fever. Vet quart. 33, 148-160 (2013).
  51. Neill, T. J., Sargeant, J. M., Poljak, Z. The effectiveness of Coxiella burnetii vaccines in occupationally exposed populations: a systematic review and meta-analysis. Zoonoses and public health. 61, 81-96 (2014).
  52. Roest, H. I., Bossers, A., Rebel, J. M. Q fever diagnosis and control in domestic ruminants. Dev biol. 135, 183-189 (2013).
  53. Frasch, M. G., et al. Fetal body weight and the development of the control of the cardiovascular system in fetal sheep. J physilo. 579, 893-907 (2007).
  54. Rurak, D., Bessette, N. W. Changes in fetal lamb arterial blood gas and acid-base status with advancing gestation. Am J Physiol Regul Integr Comp Physiol. 304, R908-R916 (2013).
  55. Frasch, M. G., et al. Fetal body weight and the development of the control of the cardiovascular system in fetal sheep. J physiol. 579, 893-907 (2007).
  56. Frasch, M. G., et al. Measures of acidosis with repetitive umbilical cord occlusions leading to fetal asphyxia in the near-term ovine fetus. Am J Obstet Gynecol. 200, 200.e1-207.e1 (2009).
  57. The ESHRE Capri Workshop Group. Multiple gestation pregnancy. Hum reprod. 15, 1856-1864 (2000).
  58. Frasch, M. G. Re The perinatal development of arterial pressure in sheep: effects of low birth weight due to twinning. Reproductive sciences (Thousand Oaks, Calif.). 15, 863-865 (2008).
  59. Hancock, S. N., Oliver, M. H., McLean, C., Jaquiery, A. L., Bloomfield, F. H. Size at birth and adult fat mass in twin sheep are determined in early gestation. J Physiol. 590, 1273-1285 (2012).
  60. Wassink, G., Bennet, L., Davidson, J. O., Westgate, J. A., Gunn, A. J. Pre-existing hypoxia is associated with greater EEG suppression and early onset of evolving seizure activity during brief repeated asphyxia in near-term fetal sheep. PLoS One. 8, e73895 (2013).
  61. Mathai, S., et al. Acute on chronic exposure to endotoxin in preterm fetal sheep. Am J Physiol Regul Integr Comp Physiol. 304, R189-R197 (2013).
  62. Heuij, L. G., et al. Synergistic white matter protection with acute-on-chronic endotoxin and subsequent asphyxia in preterm fetal sheep. J neuroinflam. 11, 89 (2014).
  63. Gagnon, R., Challis, J., Johnston, L., Fraher, L. Fetal endocrine responses to chronic placental embolization in the late-gestation ovine fetus. Am J Obstet Gynecol. 170, 929-938 (1994).
  64. Miller, S. L., Supramaniam, V. G., Jenkin, G., Walker, D. W., Wallace, E. M. Cardiovascular responses to maternal betamethasone administration in the intrauterine growth-restricted ovine fetus. Am J Obstet Gynecol. 201, 613.e1-613.e8 (2009).
  65. Regnault, T. R., et al. The relationship between transplacental O2 diffusion and placental expression of PlGF, VEGF and their receptors in a placental insufficiency model of fetal growth restriction. J Physiol. 550, 641-656 (2003).
  66. Wallace, J. M., Aitken, R. P., Cheyne, M. A. Nutrient partitioning and fetal growth in rapidly growing adolescent ewes. J reprod and fertil. 107, 183-190 (1996).
  67. Rakers, F., et al. Effects of early- and late-gestational maternal stress and synthetic glucocorticoid on development of the fetal hypothalamus-pituitary-adrenal axis in sheep. Stress. 16, 122-129 (2013).
  68. Jiang, Y., et al. The sheep genome illuminates biology of the rumen and lipid metabolism. Science. 344, 1168-1173 (2014).
  69. Begum, G., et al. Epigenetic changes in fetal hypothalamic energy regulating pathways are associated with maternal undernutrition and twinning. FASEB J. 26, 1694-1703 (2012).
  70. Byrne, K., et al. Genomic architecture of histone 3 lysine 27 trimethylation during late ovine skeletal muscle development. Anim Genet. 45, 427-438 (2014).
  71. Lie, S., et al. Impact of embryo number and maternal undernutrition around the time of conception on insulin signaling and gluconeogenic factors and microRNAs in the liver of fetal sheep. Am J physiol Endocrinol. 306, E1013-E1024 (2014).
  72. Nicholas, L. M., et al. Differential effects of maternal obesity and weight loss in the periconceptional period on the epigenetic regulation of hepatic insulin-signaling pathways in the offspring. FASEB J. 27, 3786-3796 (2013).
  73. Wang, K. C., et al. Low birth weight activates the renin-angiotensin system, but limits cardiac angiogenesis in early postnatal life. Physiol rep. 3, (2015).
  74. Zhang, S., et al. Periconceptional undernutrition in normal and overweight ewes leads to increased adrenal growth and epigenetic changes in adrenal IGF2/H19 gene in offspring. FASEB J. 24, 2772-2782 (2010).

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Burns, P., Liu, H. L., Kuthiala, S., Fecteau, G., Desrochers, A., Durosier, L. D., Cao, M., Frasch, M. G. Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings. J. Vis. Exp. (104), e52581, doi:10.3791/52581 (2015).

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