Instrumentación de Ovejas fetal Cerca plazo para multivariados Grabaciones no anestesiados crónicas
Summary
El modelo de oveja fetal no anestesiado instrumentado crónicamente se utiliza para estudiar el desarrollo del feto humano en la salud y la enfermedad, ya que permite la colocación quirúrgica y el mantenimiento de los catéteres y electrodos, muestreo de sangre repetitivo, la inyección de sustancias, registro de la actividad bioeléctrica, y de imágenes in vivo. Se describen los procedimientos necesarios para establecer este modelo.
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
Una variedad de modelos animales existe para el estudio de ambos embarazos normales y comprometidos, incluyendo roedores de laboratorio, primates no humanos y los rumiantes domésticos. 1,2,3,4,5 El ovejas gestantes instrumentado crónicamente ha sido ampliamente utilizado durante 50 años como un modelo de desarrollo fetal humano y las respuestas a los estímulos fisiopatológicos tales como lipopolisacárido (LPS). 10.06 Las lesiones después de la exposición LPS imitan exactamente lo que se ve en los recién nacidos prematuros con leucomalacia periventricular, que se debe a un perfil de maduración similar de ambas especies. 11, 12
Otras complicaciones del embarazo también se han estudiado con gran detalle, como el descubrimiento de que los glucocorticoides prenatales promueven el desarrollo pulmonar 13-15 y comprender el impacto de la restricción del crecimiento intrauterino (RCIU) en el feto 16,17.
El uso extensivo del modelo de oveja fetal se debe a la unique amenability de la oveja fetal no anestesiado a la colocación y el mantenimiento de catéteres y electrodos quirúrgica, lo que permite el muestreo de sangre repetitivo, registro de la actividad bioeléctrica, la aplicación de la estimulación eléctrica y en imágenes del cerebro vivo. 18 de telemetría también es posible, aunque se usa con menos frecuencia sin embargo, debido a la sofisticación más alta para establecer, así como el coste inicial y de mantenimiento. 19
Por otra parte, el modelo de oveja fetal es muy versátil ya que son posibles muchas variaciones de la instrumentación en función de las medidas de interés. Por ejemplo, es posible grabar más de días a semanas señales multivariados en tiempo real tales como los movimientos fetales de respiración, la actividad eléctrica cerebral, respuestas cardiovasculares, electrocardiograma, el flujo sanguíneo regional a una serie de órganos usando sondas de flujo o microesferas, etc. Gracias a esta versatilidad, se han llevado a cabo una amplia gama de estudios que incluye el desarrollo de la cardisistema medades 20,21, (HPA) del eje hipotálamo-pituitario-adrenal 22, el desarrollo del cerebro y el desarrollo de 23 estados del sueño, en particular, de 24 años, los efectos de la hipoxia / asfixia 25, la hipotermia terapéutica 26, inflamación 6-11, combinación de ambos de 27 años, los glucocorticoides 28,29, antidepresivos 30, displasia broncopulmonar (DBP) 31,32, la programación fetal 33,34,35,36,37,38,39 o el desarrollo de modalidades de monitoreo fetal novela antes y durante el parto, por nombrar sólo pocas áreas de investigación. 40,41,42,43
El objetivo general del método presentado es mostrar la aplicación básica de este modelo versátil. Permite establecer una amplia variedad de protocolos experimentales agudos y crónicos que estudian la fisiología y la fisiopatología fetal en el integrador, órgano, los niveles celular y molecular.
Protocol
Representative Results
Discussion
El anestésico y los procedimientos quirúrgicos se presentan que son necesarios para el establecimiento de un modelo animal para el estudio de la fisiología y la fisiopatología fetal: la oveja fetal no anestesiado instrumentado crónicamente.
Cuatro pasos críticos en el protocolo debe hacerse hincapié. En primer lugar, pasar los catéteres y electrodos a través del flanco materna: es importante que esto se hace a la vez para evitar cualquier lesión de órganos internos. En segundo lug…
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
- Barry, J. S., Anthony, R. V. The pregnant sheep as a model for human pregnancy. Theriogenology. 69, 55-67 (2008).
- Morrison, J. L. Sheep models of intrauterine growth restriction: fetal adaptations and consequences. Clin Exp Pharmacol Physiol. 35, 730-743 (2008).
- Rees, S., Inder, T. Fetal and neonatal origins of altered brain development. Early Hum Dev. 81, 753-761 (2005).
- 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).
- Moisiadis, V. G., Matthews, S. G. Glucocorticoids and fetal programming part 1: Outcomes. Nat rev Endocrinol. 10, 391-402 (2014).
- Wang, X., Rousset, C. I., Hagberg, H., Mallard, C. Lipopolysaccharide-induced inflammation and perinatal brain injury. Semin Fetal Neonatal Med. 11, 343-353 (2006).
- Gotsch, F., et al. The fetal inflammatory response syndrome. Clin Exp Obstet Gynecol. 50, 652-683 (2007).
- 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).
- Nitsos, I., et al. Chronic exposure to intra-amniotic lipopolysaccharide affects the ovine fetal brain. J Soc Gynecol Investig. 13, 239-247 (2006).
- 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).
- Dean, J. M., et al. Delayed cortical impairment following lipopolysaccharide exposure in preterm fetal sheep. Ann Neurol. 70, 846-856 (2011).
- Dobbing, J., Sands, J. Comparative aspects of the brain growth spurt. Early Hum Dev. 3 (1), 79-83 (1979).
- Liggins, G. C. Premature parturition after infusion of corticotrophin or cortisol into foetal lambs. J Endocrinol. 42, 323-329 (1968).
- Liggins, G. C. Premature delivery of foetal lambs infused with glucocorticoids. J Endocrinol. 45, 515-523 (1969).
- 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).
- Morrison, J. L. Sheep models of intrauterine growth restriction: fetal adaptations and consequences. Clin Exp Pharmacol Physiol. 35, 730-743 (2008).
- 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).
- Carmel, E., et al. Fetal brain MRI – experiences in the ovine model of cerebral inflammatory response. Repro sci. 19 (3), 347A-348A (2012).
- 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).
- 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).
- 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).
- 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).
- Muller, T., et al. Developmental changes in cerebral autoregulatory capacity in the fetal sheep parietal cortex. J Physiol. 539, 957-967 (2002).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- Fowden, A. L., Giussani, D. A., Forhead, A. J. Endocrine and metabolic programming during intrauterine development. Early hum dev. 81, 723-734 (2005).
- Nathanielsz, P. W., Hanson, M. A. The fetal dilemma: spare the brain and spoil the liver. J Physiol. 548, 333 (2003).
- 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).
- 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).
- Bergen, N. H., et al. Fetal programming alters reactive oxygen species production in sheep cardiac mitochondria. Clin Sci (Lond). 116, 659-668 (2009).
- 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).
- 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).
- 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).
- Frasch, M. G., et al. Heart rate variability analysis allows early asphyxia detection in ovine fetus. Reprod Sci. 16, 509-517 (2009).
- 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).
- 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).
- 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).
- 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).
- Xu, A., et al. Adaptive brain shut-down counteracts neuroinflammation in the near-term ovine fetus. Front neurol. 5, 110 (2014).
- Xu, A., et al. The Ovine Fetal and Placental Inflammatory Response to Umbilical Cord Occlusions With Worsening Acidosis. Reprod Sci. 22 (11), (2015).
- 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).
- 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).
- 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).
- 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).
- Roest, H. I., Bossers, A., Rebel, J. M. Q fever diagnosis and control in domestic ruminants. Dev biol. 135, 183-189 (2013).
- 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).
- 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).
- 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).
- 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).
- The ESHRE Capri Workshop Group. Multiple gestation pregnancy. Hum reprod. 15, 1856-1864 (2000).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- Jiang, Y., et al. The sheep genome illuminates biology of the rumen and lipid metabolism. Science. 344, 1168-1173 (2014).
- 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).
- Byrne, K., et al. Genomic architecture of histone 3 lysine 27 trimethylation during late ovine skeletal muscle development. Anim Genet. 45, 427-438 (2014).
- 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).
- 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).
- 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).
- 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).
