A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings

8.5K views

⸱

DOI:

10.3791/52581

⸱

October 25th, 2015

 ,  ,  ,  ,  ,  ,  , 
1Département de sciences cliniques, CHUV, Université de Montréal, St-Hyacinthe, QC, 2Département d'obstetriques et de gynécologie, CHU Ste-Justine Research Centre, Université de Montréal, 3Département de neurosciences, CHU Ste-Justine Centre de recherche, Université de Montréal, 4Centre de recherche en reproduction animale (CRRA), Université de Montréal, St-Hyacinthe, QC

In This Article

Summary

The chronically instrumented non-anesthetized fetal sheep model is used to study human fetal development in health and disease, because it permits surgical placement and maintenance of catheters and electrodes, repetitive blood sampling, substance injection, recording of bioelectrical activity, and in vivo imaging. We describe the procedures required to establish this model.

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

A variety of animal models exist for the study of both normal and compromised pregnancies, including laboratory rodents, non-human primates and domestic ruminants.1,2,3,4,5 The chronically instrumented pregnant sheep has been used extensively for 50 years as a model of human fetal development and responses to pathophysiologic stimuli such as lipopolysaccharide (LPS).6-10 The lesions following LPS exposure mimic exactly what is seen in preterm infants with periventricular leukomalacia, which is due to a similar maturational profile of both species.11, 12

Other pregnancy complications have also been studied in great detail such as the discovery that antenatal glucocorticoids promote lung development 13-15 and understanding the impact of intrauterine growth restriction (IUGR) on the fetus 16,17.

The extensive use of the fetal sheep model 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, recording of bioelectrical activity, application of electric stimulation and in vivo brain imaging.18 Telemetry is also possible, although less frequently used yet due to the higher sophistication to set up as well as the initial and maintenance cost.19

Moreover, the fetal sheep model is very versatile as many variations of instrumentation are possible depending on the measures of interest. For example, it is possible to record over days to weeks multivariate signals in real time such as fetal breathing movements, electrical brain activity, cardiovascular responses, electrocardiogram, regional blood flow to a range of organs using flow probes or microspheres, etc. Thanks to this versatility, a wide range of studies have been conducted including the development of the cardiovascular system20,21, hypothalamo-pituitary-adrenal (HPA) axis22, brain development23 and sleep states development in particular24, effects of hypoxia/asphyxia25, therapeutic hypothermia 26, inflammation6-11, combination of both27, glucocorticoids28,29, anti-depressants30, broncho-pulmonary dysplasia (BPD) 31,32, fetal programming33,34,35,36,37,38,39 or development of novel fetal monitoring modalities prior and during labor to name but a few areas of investigation. 40,41,42,43

The overall goal of the method presented is to show this versatile model’s basic implementation. It allows establishing a wide variety of acute and chronic experimental protocols studying fetal physiology and pathophysiology on the integrative, organ, cellular and molecular levels.

Access restricted. Please log in or start a trial to view this content.

Protocol

Animal care followed the guidelines of the Canadian Council on Animal Care and the approval by the Université de Montréal Council on Animal Care (protocol #10-Rech-1560). Detailed information on materials and methods used is provided in the Table 1.

1. Anesthesia

  1. Insert a single-lumen catheter into a jugular vein.
  2. Sedate the ewe using acepromazine (Atravet 10 mg/mL) 2 mg intravenously approximately 30 min prior to the induction of anesthesia in order to reduce stress associated with the procedure which in turn reduces levels of cortisol.
  3. Administer diazepam (Diazepam 5 mg/mL) 20 mg, ketamine (Ketalar 100 mg/mL) 4-5 mg/kg and propofol (Propofol 10 mg/mL) 0.5 to 1 mg/kg intravenously to induce general anesthesia.
  4. Insert an airway exchange catheter into the trachea using a laryngoscope with a Wisconsin type blade (Extra-long 350 mm Left-Handed Blade) to aid with the intubation. Slide the silicon endotracheal tube (9 to 12 mm Inner Diameter) off the airway exchange catheter and into the trachea. This technique facilitates the intubation process. Inflate the cuff of the endotracheal tube carefully to avoid pressure-induced ulceration of the trachea and fixate the tube to the head of the ewe.
  5. Connect the endotracheal tube to the respiratory circuit of the anesthetic machine and begin mechanical ventilation immediately. Adjust ventilator settings to maintain a PaCO2 within normal limits of 35 to 45 mmHg.
  6. Insert a catheter into the auricular artery (22 to 20 G; 1 in [0.9 x 25 mm] to 1.16 in [1.1 x 30 mm]) and connect to non-compliant tubing to monitor direct arterial blood pressure.
  7. Use a multi-parameter physiologic monitor to record the electrocardiogram, direct arterial blood pressure, oxygen saturation (SpO2), capnography (PETCO2), and temperature every 5 min. Transfer all physiologic data via a serial cable to a central physiologic data-collecting computer. Maintain normal body temperature using a circulating water blanket.
  8. Administer a balanced poly-ionic solution at 10 mL/kg for the first hour of general anesthesia and then reduce to 5 mL/kg/h.
  9. Administer trimethoprim-sulfadoxine 5 mg/kg IV to the ewe just prior to the skin incision as antibiotic prophylaxis.
  10. Use standard aseptic techniques with all surgical manipulations of the ewe and fetus.
  11. Barrier nurse the ewes at all times. This includes also the non-surgical personnel. This will minimize the enzootic potential for e.g., Coxiella burnetii. Use gloves and masks (N-95 type) at all times.

2. Overview of the Surgical Procedure

  1. Make a 20 cm midline incision through the lower abdominal wall immediately cranial to the udder through the linea alba to minimise abdominal muscle damage.
    1. Retract the greater omentum cranially and the uterine horns are palpated manually. Palpate each horn from the body of the uterus to the tip of the horn noticing the number of fetus and their size. If there is more than one fetus, choose the larger fetus by evaluating manually the head size and the width between the orbits.
    2. Hold the head of the chosen fetus firmly through the uterus partially exteriorized. Perform a 10 cm hysterotomy on the large curvature with Metzenbaum scissors. Put immediately over the head a non-latex sterile surgical glove filled with sterile saline as if it were a hand. Alternatively, use moistened 4x4s to keep the fetal head moist. Secure the uterus to the abdominal wall.
      1. Exteriorize the right and left thoracic limbs and pull the fetus gently out of the uterus up to the xyphoid process.
    3. Insert polyvinyl catheters into the right and left brachial vein and arteries using a standard cut-down technique. Insert another polyvinyl catheter into the amniotic cavity by fixating its’ end to the sternum of the fetus.
      1. Use sterile catheters only. We recommend gas sterilization in your standard facility. Attach each catheter to a needle and the needle to a double stopcock to allow for later blood sampling or pressure monitoring.
    4. Suture stainless steel electrodes to the manubrium, xiphoid process and to each point of the shoulder to monitor the electrocardiogram (ECG).
    5. Return the fetus to the uterus. All catheters and electrodes exit via a small stab incision in the left flank.
    6. Close the laparotomy incision using a three layer closure. Suture the abdominal wall with a synthetic absorbable monofilament suture USP 2 in a simple continuous fashion. Close the subcutaneous space with a synthetic absorbable braided suture USP 0 in a simple continuous fashion. Use surgical stainless steel staples to close the skin incision.
  2. Detailed description of the surgical procedure
    1. Remove wool by shaving the xyphoid process to the mammary gland and along the fold of the flank on each side with a blade # 40. Clean the ventral abdomen then thoroughly with a 4% chlorhexidine gluconate and a soft brush for 3 min.
      1. Perform a standard sterile scrub with chlorhexidine gluconate 4% starting from the center of the abdomen and progressing in a centrifugal fashion for 3 min. Pour sterile saline on the abdomen to remove the disinfectant soap. For the final step of the surgical preparation, perform three alternate passages of Chlorhexidine gluconate solution 2% and isopropyl alcohol 70%.
    2. Ensure the depth of anesthesia is adequate prior to incision. Make a standard laparotomy incision from the umbilicus to just cranial to the udder through the linea alba to minimise abdominal muscle damage.
    3. Insert a long sponge forceps in the abdomen along the left abdominal wall up to the planned exit site for catheters in the paracostal region.
      1. Push the tips of the forceps against the wall until an assistant can locate it and confirm the appropriate site. Open slightly (1 cm) the jaws of the forceps and let the assistant make a 2 cm full-thickness stab incision.
      2. Exteriorize the tips of the forceps through the incision, open again, and gently grasp the catheters with the forceps that is finally pulled out of the abdomen through the ventral abdominal incision. Of note, some groups implant the catheters and then exteriorize them. This has the drawback that intrasurgical fetal ECG monitoring is not possible.
    4. Palpate the uterus to determine fetal position and numbers. Determine the largest fetus using the inter-aural distance. Incise the uterine wall on the large curvature over the dorsum of the head, avoiding the cotyledons.
      1. Insert a blunt-ended cannula through the placental membranes to obtain an amniotic fluid sample free from hemorrhage. Incise the placental membranes using scissors.
    5. Exteriorize the cranial half of the fetus through this incision. Place a sterile non latex surgical glove filled with sterile saline at 37 °C over the fetus head to help maintain normothermia.
      1. During removal of the fetal upper body from the uterus, have the assistant hold the Babcock's up to prevent the loss of amniotic fluid. Then, again using Babcock forceps, clamp the fetal membranes and uterine wall to the skin to prevent abdominal contamination with amniotic fluid.
      2. Expose only the parts of the fetal body that need to be instrumented and keep the remaining parts inside the uterus or covered by moist and warm (37 °C) sterile cloths, respectively.
    6. Abduct both thoracic limbs to facilitate the exposure to the brachial artery and vein bilaterally. Incise along the medial aspect of both antebrachium and carefully dissect around the brachial artery and vein.
    7. Insert polyvinyl catheters into the right and left brachial arteries and the left brachial vein using a standard cut-down technique.
      1. Free the vessel to be catheterized from adjacent tissue over 1 cm. Ligate the distal portion of the vessel with a braided synthetic absorbable USP 2-0 suture. Preplace a ligature at the proximal aspect of the vessel but keep it untied.
        1. Using Castroviejo scissors, cut the vessel transversally to approximately 30% of its diameter. Stop the blood flow partially by pulling on the proximal suture. Insert the catheter in a proximal direction.
      2. Insert the polyvinyl catheter up to 8 cm proximally or until resistance is detected and then pull back slightly. Temporally secure the proximal aspect of the catheter using a vascular clamp. Place another suture around both the proximal and distal aspect of the catheter. An assistant is continuously aspirating and flushing the catheter to ensure patency of the catheter.
      3. Close the fetal skin using a USP 2-0 braided synthetic absorbable suture, with a continuous suture pattern.
    8. To the right and left shoulder, manubrium and xyphoid process, fixate an insulated stainless steel electrode to facilitate the monitoring of the fetal ECG.
    9. Suture the amniotic pressure and sampling catheter to the sternum. This catheter is fenestrated at its extremity.
    10. Secure all the catheters on the dorsum of the fetus using a USP 2-0 braided synthetic absorbable suture material.
    11. Prior to the replacement of the fetus back into the uterus, administer clenbuterol 30 µg IV slowly over 15 min to avoid hypotension and to provide uterine relaxation.
    12. Suture the fetal membranes using USP 4-0 braided synthetic absorbable suture material with a continuous pattern. Incorporate only one catheter or electrode at a time into the closure to ensure a tight closure. Use a double layer Cushing pattern respecting Halsted principles to close the uterine muscular layer using a USP 0 braided synthetic absorbable suture material. Bury the surgical knots carefully.
    13. Using a purse-string suture pattern, secure all the catheters and ECG cables as they exit the left paracostal incision.
    14. Using a USP 2 monofilament synthetic absorbable suture material secure the linea alba with a continuous pattern. Close the subcutaneous tissues using USP 2-0 braided synthetic absorbable suture material with a continuous pattern. Secure the skin layer with surgical staples.
    15. Administer 250 mg of ampicillin intravenously and again via the amniotic catheter into the amniotic cavity. Replace lost amniotic fluids with warm saline.
    16. Place all the exteriorized catheters and ECG electrodes into a place bag to maintain sterility. Place a stockinette around the torso of the ewe to secure all the catheters and electrodes to the body of the ewe.
    17. Stop general anesthesia, and extubate the ewe once laryngeal reflexes have returned to normal.
    18. Return the ewe to a metabolic cage once she is stable following general anesthesia. The ewe will reside in the metabolic cage for the duration of the experiment. The ewe should be able to stand, lie down and eat ad libitum while monitoring the non-anesthetized fetus without sedating the mother.
    19. For the following three days, administer antibiotics prophylactically to the ewe (Trimethoprim sulfadoxine 5 mg/kg) and fetus (250 mg of ampicillin intravenously and again via the amniotic catheter).
    20. Evaluate the metabolic status of both ewe and fetus using blood gas analyses.
    21. Flush all catheters with the minimal volume of heparinized saline possible. Caution – do not exceed the daily dose of heparin and fluids permissible to the fetus. It is possible to fluid overload the fetus. Flush slowly once a day ~5 mL NaCl per line after antibiotic prophylaxis.
  3. Data recording and analysis
    1. During surgery, optionally record the maternal and fetal ECG and heart rates as well as maternal arterial blood pressure and airway pressure (Paw) continuously (Figure 1). Use the Life Window Monitor to acquire all maternal data except ECG. Feed these data into analog-digital converter along with fetal and maternal ECG signals; pass maternal and fetal ECG first into 1901 pre-amplifier. Record and display all data in manufacturer’s software.
    2. Take a 1 mL arterial sample simultaneously from the ewe and fetus for arterial blood gas analysis, lactate, glucose and base excess determination (in plasma) at the begin of the fetal surgery (immediately after inserting the first arterial catheter) and after closing the uterus.
    3. During postoperative recovery, take a 3 mL fetal blood sample to measure the IL-6 and TNF-α inflammatory profiles. Centrifuge the plasma at 4 °C (4 min, 4,000 x g), decant and store the plasma at -80 °C for subsequent ELISA testing.
      Note: For the purpose of the reported representative results, six days after surgery the animals were sacrificed using intravenous injection of 20 mL sodium pentobarbital. Fetal growth was assessed by body, brain, liver and maternal weights. The duration of the experimental period will obviously vary depending on the design chosen for the particular research question and can reach ~6 weeks.
  4. Cytokine analyses (optional step)
    1. Determine cytokine concentrations (IL-6, TNF-α) in plasma using an ovine-specific sandwich ELISA. Pre-coat mouse anti-sheep monoclonal antibodies (capture antibody IL-6) or mouse anti-bovine monoclonal antibody (TNF-α) at a concentration 4 µg/ml on ELISA plate at 4 °C for O/N, after 3 times wash with washing buffer (0.05% Tween 20 in PBS, PBST).
    2. Block the plates for 1 h with 1% BSA in PBST. Wash the plates with washing buffer 3 times.
    3. Use recombinant sheep proteins (IL-6, TNF-α) as ELISA standard, prepare a serial dilutions range from standard 1 of 2,000 ng/ml to standard 7 of 31.25 pg/ml.
    4. Load 50 µl of serial diluted protein standards and samples per well and incubate for 2 hr at RT, wash the plates 3 times. Run all standards and samples in duplicate.
    5. Apply 50 µl of rabbit anti-sheep polyclonal antibodies (detection antibody IL-6) or rabbit anti-bovine polyclonal antibody (TNF-α) at a dilution of 1:250 in wells and incubate for 30 min at RT. Wash the plates with washing buffer 5 times.
    6. Add 50 µl of the goat anti-rabbit IgG-HRP conjugated (dilution 1:5,000) for 30 min.
    7. Incubate with 50 µl of TMB substrate solution per well.
    8. Stop colour development reaction at desired time with 25 µl of 2 N sulphuric acid.
    9. Read the plates on ELISA plate reader at 450 nm, with a 570 nm wavelength correction.
      Note: In our assays, the sensitivity of IL-6 ELISA was 16 pg/ml, the sensitivity of TNF-α ELISA was 13.9 pg/ml, respectively. For all assays, the intra-assay and inter-assay coefficients of variance was <5% and <10%, respectively.
  5. Statistical analyses
    Note: The exact statistical methods will depend on the research question. Here we report the methods used to test for any significant differences in the blood gases reported in Table 2.
    1. Test normal data distribution using Kolmogorov-Smirnov test followed by parametric or non-parametric tests with adjustment for multiple comparisons, as appropriate.
    2. Use K-means cluster analysis to identify the fetuses in the cohort that were spontaneously hypoxic and determine the respective pO2 and O2Sat values (consider pO2 < 20 mmHg or O2Sat < 55% as fetal hypoxia). 44-48
    3. Use statistical software such as SPSS for the analyses.
    4. Present data as Mean±SD with statistical differences at p<0.05 considered significant.

Access restricted. Please log in or start a trial to view this content.

Results

38 pregnant time-dated ewes were instrumented at 128±2 days of gestation (dGA, ~0.88 gestation, term 145 dGA) with arterial, venous and amniotic catheters and electrocardiogram (ECG) electrodes with sterile technique under general anesthesia (both ewe and fetus). In case of twin pregnancy the larger fetus was chosen based on palpating and estimating the intertemporal diameter; alternatively, the fetus to be instrumented may be selected randomly to avoid any potential bias or both fetuses may be instrumented. The total du...

Access restricted. Please log in or start a trial to view this content.

Discussion

The anesthetic and surgical procedures are presented that are required for establishing an animal model for studying fetal physiology and pathophysiology: the chronically instrumented non-anesthetized fetal sheep.

Four critical steps within the protocol should be emphasized. First, passing the catheters and electrodes through the maternal flank: it is important that this is done at once to avoid any internal organ injuries. Second, securing the uterotomy operating site prior to exteriorizing t...

Access restricted. Please log in or start a trial to view this content.

Disclosures

No disclosures have been made.

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.

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
ACE Light sourceSchott-FostecA20500
Dissecting scissorsFine Science Tools14060 - 11
Angled dissecting scissorsFine Science Tools15006 - 09
Scalpel handleFine Science Tools10003 - 12alternating dissecting tool
Curved scalpel blades #12Fine Science Tools10012 - 00alternating dissecting tool
Bone scissorsFine Science Tools16044 - 10
S & T suture tying forcepsFine Science Tools00272 - 13
Dumont SS forceps - angledFine Science Tools11203 - 25 
Braided silk suture size 6-0Teleflex Medical07 - 30  - 10
Medical Tapetranspore3M
Ketamine hydrochloride 100 mg/mlHospiraNDC 0409 - 2051 - 05Final Does is 80 mg/kg
Tranqui Ved Injection (xylazine 100 mg/ml)VecdoNDC 50989 - 234 - 11Final Does is 10 mg/kg
Reactive orange 14Sigma - AldrichR - 8254
Ringers Solution ComponentsSolution is gas equilibrated with 95% O2 and 5% Co2, final pH 7.4
Sodium chlorideSigma - AldrichS7653Final Concentration: 118 mM
Potassium chlorideFisher ScientificP217 - 3Final Concentration: 4.7 mM
Calcium chloride dihydrateFisher ScientificC79 - 500Final Concentration: 2.5 mM
Potassium phosphate monobasicFisher ScientificP -285Final Concentration: 1.2 mM
Magnesium sulfateJ.T. BakerJan-00Final Concentration: 0.57 mM
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)Fisher ScientificBP 310 - 500Final Concentration: 5.95 g/L
GlucoseSigma - AldrichG8270Final Concentration: 5.5 mM
LifeWindowDigicare Biomedical Technology
CED bioamplifier and ADC unitsCambridge 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 bioamplifierDigitimer Ltd
37 Hydeway
Welwyn Garden City
Hertfordshire, AL7 3BE, England		NL108A
ABL800FlexRadiometer Canada; 200 Aberdeen Dr, London, ON N5V 4N2
Eppendorf 5804REppendorf Canada; 2810 Argentia Road, #2
Mississauga, Ontario, L5N 8L2
Arrow Jugular Catheterization SetArrow International, Inc., 2400 Bernville Road, Reading, PA 19605 USA
Atravet10 mg/ml
Diazepam5 mg/ml
KetamineKetalar100 mg/ml
Propofol10 mg/ml
SurgiVeTEndotracheal Tubes; Smiths Medical ASD, Inc. St. Paul, MN 55112, USA
Cook Airway Exchange Catheter with RAPI-FIT AdaptersCook Critical Care 750, Bloomington IN 47402-0489 USA
Dispomed VentilatorDispomed Ltd., 745 Nazaire-Laurin, Joliette, Quebec J6E 0L6
BD Insyte-WBecton Dickinson, Infusion Therapy Systems Inc., 9450 S State St, Sandy Utah 84070 USA22 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 LW6000Digicare Biomedical Technology 107 Commerce Road, Boynton Beach, FL 33426-9365 USA
Gaymar
Babcock
Polyvinyl cathetersSCI (Scientific Commodities Inc.)2 meters
2-0 Vicryl
Castroviejo scissors
electrocardiogram (ECG)LIFYY, Metrofunk Kabel-Union, Berlin, Germanyfour copper electrodes in single sheath, 2 meters
2-O Vicryl
3-0 Vicryl
PDS II USP
Trimethoprim sulfadoxine
Ampicillin
StopcockArgon Medical, Cat 041220001ADouble 4-way Stopcock with male luer lock
NeedlesTyco Healthcare 8881202389Monoject aluminum hub blunt needles, 22Gx, 0.7mmx 38.1mm: for fetal arterial and venous catheters
NeedlesTyco Healthcare 8881202322Monoject aluminum hub blunt needles, 16Gx, 1.6mmx38.1mm: for amniotic catheters

References

  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. 60th Meeting of the Society for Gynecologic Investigation, 20 (3), Reproductive Sci. 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).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Tags

Fetal Sheep InstrumentationVascular Catheter PlacementElectrocardiographic Cable ImplantationAmniotic Fluid SamplingMaternal Fetal MonitoringSurgical Uterine ClosurePostoperative Recovery AssessmentPhysiologic Perturbation ResponseBlood Gas Analysis
Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings
Contact Us
Recommend to Library

Privacy

Terms of Use

Policies

Copyright © 2026 MyJoVE Corporation. All rights reserved.