authors gratefully acknowledge funding support from the Molly Towell Perinatal Research Foundation, Canadian Institutes of Health Research (CIHR), and Fonds de Recherche du Qu&#233;bec &#8211; Sant&#233; (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.

Animal care followed the guidelines of the Canadian Council on Animal Care and the approval by the Universit&#233; de Montr&#233;al Council on Animal Care (protocol #10-Rech-1560). Detailed information on materials and methods used is provided in the Table 1.
1. Anesthesia
<ol>
	<li>Insert a single-lumen catheter into a jugular vein.</li>
	<li>Sedate the ewe using acepromazine (Atravet 10 mg/mL) 2 mg intravenously approximately 30 min&#160;prior to the induction of anesthesia in order to reduce stress associated with the procedure which in turn reduces levels of cortisol.</li>
	<li>Administer diazepam (Diazepam 5 mg/mL) 20 mg, ketamine (Ketalar 100 mg/mL)&#160;4-5 mg/kg and propofol (Propofol 10 mg/mL) 0.5 to 1 mg/kg intravenously to induce general anesthesia.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Administer trimethoprim-sulfadoxine 5 mg/kg IV to the ewe just prior to the skin incision as antibiotic prophylaxis.</li>
	<li>Use standard aseptic techniques with all surgical manipulations of the ewe and fetus.</li>
	<li>Barrier nurse the ewes at all times. This includes also the non-surgical personnel. This will minimize the enzootic potential for e.g.,&#160;Coxiella burnetii. Use gloves and masks (N-95 type) at all times.</li>
</ol>
2. Overview of the Surgical Procedure
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.
		<ol>
			<li>Exteriorize the right and left thoracic limbs and pull the fetus gently out of the uterus up to the xyphoid process.</li>
		</ol>
		</li>
		<li>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&#8217; end to the sternum of the fetus.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Suture stainless steel electrodes to the manubrium, xiphoid process and to each point of the shoulder to monitor the electrocardiogram (ECG).</li>
		<li>Return the fetus to the uterus. All catheters and electrodes exit via a small stab incision in the left flank.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Detailed description of the surgical procedure
	<ol>
		<li>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.
		<ol>
			<li>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%.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>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.
		<ol>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>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.
		<ol>
			<li>Insert a blunt-ended cannula through the placental membranes to obtain an amniotic fluid sample free from hemorrhage. Incise the placental membranes using scissors.</li>
		</ol>
		</li>
		<li>Exteriorize the cranial half of the fetus through this incision. Place a sterile non latex surgical glove filled with sterile saline at 37 &#176;C over the fetus head to help maintain normothermia.
		<ol>
			<li>During removal of the fetal upper body from the uterus, have the assistant hold the Babcock&#39;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.</li>
			<li>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 &#176;C) sterile cloths, respectively.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>Insert polyvinyl catheters into the right and left brachial arteries and the left brachial vein using a standard cut-down technique.
		<ol>
			<li>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.
			<ol>
				<li>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.</li>
			</ol>
			</li>
			<li>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.</li>
			<li>Close the fetal skin using a USP 2-0 braided synthetic absorbable suture, with a continuous suture pattern.</li>
		</ol>
		</li>
		<li>To the right and left shoulder, manubrium and xyphoid process, fixate an insulated stainless steel electrode to facilitate the monitoring of the fetal ECG.</li>
		<li>Suture the amniotic pressure and sampling catheter to the sternum. This catheter is fenestrated at its extremity.</li>
		<li>Secure all the catheters on the dorsum of the fetus using a USP 2-0 braided synthetic absorbable suture material.</li>
		<li>Prior to the replacement of the fetus back into the uterus, administer clenbuterol 30 &#181;g IV slowly over 15 min&#160;to avoid hypotension and to provide uterine relaxation.</li>
		<li>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.</li>
		<li>Using a purse-string suture pattern, secure all the catheters and ECG cables as they exit the left paracostal incision.</li>
		<li>Using a USP 2&#160;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.</li>
		<li>Administer 250 mg of ampicillin intravenously and again via the amniotic catheter into the amniotic cavity. Replace lost amniotic fluids with warm saline.</li>
		<li>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.</li>
		<li>Stop general anesthesia, and extubate the ewe once laryngeal reflexes have returned to normal.</li>
		<li>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.</li>
		<li>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).</li>
		<li>Evaluate the metabolic status of both ewe and fetus using blood gas analyses.</li>
		<li>Flush all catheters with the minimal volume of heparinized saline possible. Caution &#8211; 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.</li>
	</ol>
	</li>
	<li>Data recording and analysis
	<ol>
		<li>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&#160;maternal and fetal ECG first into 1901 pre-amplifier. Record and display all data in manufacturer&#8217;s software.</li>
		<li>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.</li>
		<li>During postoperative recovery, take a 3 mL fetal blood sample to measure the IL-6 and TNF-&#945; inflammatory profiles. Centrifuge the plasma at 4 &#176;C (4 min, 4,000 x g), decant and store the plasma at -80 &#176;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.</li>
	</ol>
	</li>
	<li>Cytokine analyses (optional step)
	<ol>
		<li>Determine cytokine concentrations (IL-6, TNF-&#945;) in plasma using an ovine-specific sandwich&#160;ELISA. Pre-coat mouse anti-sheep monoclonal antibodies (capture antibody IL-6) or mouse anti-bovine monoclonal antibody (TNF-&#945;) at a concentration 4 &#181;g/ml on ELISA plate at 4 &#176;C for O/N, after 3 times wash with washing buffer (0.05% Tween 20 in PBS, PBST).</li>
		<li>Block the plates for 1 h with 1% BSA in PBST. Wash the plates with washing buffer 3 times.</li>
		<li>Use recombinant sheep proteins (IL-6, TNF-&#945;) as ELISA standard, prepare a serial dilutions range from standard 1 of 2,000 ng/ml to standard 7 of 31.25 pg/ml.</li>
		<li>Load 50 &#181;l 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.</li>
		<li>Apply 50 &#181;l of rabbit anti-sheep polyclonal antibodies (detection antibody IL-6) or rabbit anti-bovine polyclonal antibody (TNF-&#945;) at a dilution of 1:250 in wells and incubate for 30 min at RT. Wash the plates with washing buffer 5 times.</li>
		<li>Add 50 &#181;l of the goat anti-rabbit IgG-HRP conjugated (dilution 1:5,000) for 30 min.</li>
		<li>Incubate with 50 &#181;l of TMB substrate solution per well.</li>
		<li>Stop colour development reaction at desired time with 25 &#181;l of 2 N sulphuric acid.</li>
		<li>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-&#945; ELISA was 13.9 pg/ml, respectively. For all assays, the intra-assay and inter-assay coefficients of variance was &#60;5% and &#60;10%, respectively.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Test normal data distribution using Kolmogorov-Smirnov test followed by parametric or non-parametric tests with adjustment for multiple comparisons, as appropriate.</li>
		<li>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 &#60; 20 mmHg or O2Sat &#60; 55% as fetal hypoxia). 44-48</li>
		<li>Use statistical software such as SPSS for the analyses.</li>
		<li>Present data as Mean&#177;SD with statistical differences at p&#60;0.05 considered significant.</li>
	</ol>
	</li>
</ol>

No disclosures have been made.

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...

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&#8217;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.

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

instrumentation of near-term fetal sheep for multivariate chronic non-anesthetized recordings

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.

38 pregnant time-dated ewes were instrumented at 128&#177;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 tot...

Watch this Scientific Journal Video about Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings at JoVE.com

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

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.

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as ...

instrumentation-near-term-fetal-sheep-for-multivariate-chronic-non

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JoVE Journal

Developmental Biology

9.4K Views.  CHUV, Université de Montréal, St-Hyacinthe, QC.  Chronisch geïnstrumenteerd niet-verdoofde foetale schapen model wordt toegepast om menselijke foetale ontwikkeling in gezondheid en ziekte te bestuderen, omdat het toestaat chirurgische plaatsing en onderhoud van catheters en elektroden herhaalde bloedafname, stof injectie, opname van bio-elektrische activiteit en in vivo imaging. We beschrijven de vereiste om dit model vast te stellen procedures. 

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

Three-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound Receiver

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. When visualization is lost, the medical device can cause trauma to critical tissue structures. Here, a method to track the needle tip during ultrasound image-guided procedures is presented. This method involves the use of a fiber-optic ultrasound receiver that is affixed within the cannula of a medical needle to communicate ultrasonically with the external ultrasound probe. This custom probe comprises a central transducer element array and side element arrays. In addition to conventional two-dimensional (2D) B-mode ultrasound imaging provided by the central array, three-dimensional (3D) needle tip tracking is provided by the side arrays. For B-mode ultrasound imaging, a standard transmit-receive sequence with electronic beamforming is performed. For ultrasonic tracking, Golay-coded ultrasound transmissions from the 4 side arrays are received by the hydrophone sensor, and subsequently the received signals are decoded to identify the needle tip&#39;s spatial location with respect to the ultrasound imaging probe. As a preliminary validation of this method, insertions of the needle/hydrophone pair were performed in clinically realistic contexts. This novel ultrasound imaging/tracking method is compatible with current clinical workflow, and it provides reliable device tracking during in-plane and out-of-plane needle insertions.

Accurate and efficient visualization of invasive medical devices is extremely important in many ultrasound-guided minimally invasive procedures. Here, a method for localizing the spatial position of a needle tip relative to the ultrasound imaging probe is presented.

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. ...

Engineering

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Medicine

In Vivo Real-Time Study of Drug Effects on Carotid Blood Flow in the Ovine Fetus

The ability of an organism to maintain a constant blood flow to the brain in response to sudden surges in systemic blood pressure (BP) is known as cerebral autoregulation (CAR), which occurs in the carotid artery. In contrast to full-term neonates, preterm neonates are unable to reduce the cerebral blood flow (CBF) in response to increased systemic BP. In preterm neonates, this exposes the fragile cerebral vessels to high perfusion pressures, leading to their rupture and brain damage. Ex vivo studies using wire myography have demonstrated that carotid arteries from near-term fetuses constrict in response to the activation of adrenergic alpha1 receptors. This response is blunted in the preterm fetus. Thus, to examine the role of alpha1-AR in vivo, presented here is an innovative approach to determine the effects of drugs on a carotid arterial segment in vivo in an ovine fetus during the developmental progression of gestation. The presented data demonstrate the simultaneous measurement of fetal blood flow and blood pressure. The perivascular delivery system can be used to conduct a long-term study over several days. Additional applications for this method could include viral delivery systems to alter the expression of genes in a segment of the carotid artery. These methods could be applied to other blood vessels in the growing organism in utero as well as in adult organisms.

In Vivo Real-Time Study of Drug Effects on Carotid Blood Flow in the Ovine Fetus

The present protocol describes a method to deliver drugs and gene expression-modifying agents perivascularly in an in utero developing fetus. Importantly, the effect of drugs/agents on blood flow can be measured with the progression of pregnancy.

The ability of an organism to maintain a constant blood flow to the brain in response to sudden surges in systemic blood pressure (BP) is known as ...

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...

Neuroscience

The Perinatal Asphyxiated Lamb Model: A Model for Newborn Resuscitation

Birth asphyxia accounts for nearly one million deaths worldwide each year, and is one of the primary causes of early neonatal morbidity and mortality. Many aspects of the current neonatal resuscitation guidelines remain controversial given the difficulties in conducting randomized clinical trials owing to the infrequent and often unpredictable need for extensive resuscitation. Most studies on neonatal resuscitation stem from manikin models that fail to truly reflect physiologic changes or piglet models that have cleared their lung fluid and that have completed the transition from fetal to neonatal circulation. The present protocol provides a detailed step-by-step description on how to create a perinatal asphyxiated fetal lamb model. The proposed model has a transitioning circulation and fluid-filled lungs, which mimics human newborns following delivery, and is, therefore, an excellent animal model to study newborn physiology. An important limitation to lamb experiments is the higher associated cost.

Invasive instrumentation of the fetal lamb provides accurate physiologic measurements of the transitioning circulation in a model that closely mimics the newly born infant.

Birth asphyxia accounts for nearly one million deaths worldwide each year, and is one of the primary causes of early neonatal morbidity and mortality. ...

Subdural Soft Electrocorticography (ECoG) Array Implantation and Long-Term Cortical Recording in Minipigs

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help delineate the epileptic region to resect. In long-term applications such as brain-computer interfaces, these epicortical electrodes are used to record the movement intention of the brain, to control the robotic limbs of paralyzed patients. However, current stiff electrode grids do not answer the need for high-resolution brain recordings and long-term biointegration. Recently, conformable electrode arrays have been proposed to achieve long-term implant stability with high performance. However, preclinical studies for these new implant technologies are needed to validate their long-term functionality and safety profile for their translation to human patients. In this context, porcine models are routinely employed in developing medical devices due to their large organ sizes and easy animal handling. However, only a few brain applications are described in the literature, mostly due to surgery limitations and integration of the implant system on a living animal.
Here, we report the method for long-term implantation (6 months) and evaluation of soft ECoG arrays in the minipig model. The study first presents the implant system, consisting of a soft microfabricated electrode array integrated with a magnetic resonance imaging (MRI)-compatible polymeric transdermal port that houses instrumentation connectors for electrophysiology recordings. Then, the study describes the surgical procedure, from subdural implantation to animal recovery. We focus on the auditory cortex as an example target area where evoked potentials are induced by acoustic stimulation. We finally describe a data acquisition sequence that includes MRI of the whole brain, implant electrochemical characterization, intraoperative and freely moving electrophysiology, and immunohistochemistry staining of the extracted brains.
This model can be used to investigate the safety and function of novel design of cortical prostheses; mandatory preclinical study to envision translation to human patients.

Subdural Soft Electrocorticography ECoG Array Implantation and Long-Term Cortical Recording in Minipigs

Here, we present a method for the long-term performance and safety assessment of soft subdural electrode arrays in a minipig model, describing surgical method and tools, postoperative magnetic resonance imaging, electrophysiology of the auditory cortex, electrochemical properties of the implant, and postmortem immunochemistry.

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help ...

Bioengineering

Noninvasive Electrocardiography in the Perinatal Mouse

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both human and animal models of disease. Individual heart rate, rhythm, and regularity, combined with quantitative parameters collected from ECG, serve to assess the integrity of the cardiac conduction system as well as the integrated physiology of the cardiac cycle. This article provides a comprehensive description of the methods and techniques used to perform a noninvasive ECG on perinatal and neonatal mouse pups as early as the first postnatal day, without requiring the use of anesthetics. This protocol was designed to directly address a need for a standardized and repeatable method for obtaining ECG in newborn mice. From a translational perspective, this protocol proves to be entirely effective for characterization of congenital cardiopulmonary defects generated using transgenic mouse lines, and particularly for analysis of defects causing lethality at or during the first postnatal days. This protocol also aims to directly address a gap in the scientific literature to characterize and provide normative data associated with maturation of the early postnatal cardiac conduction system. This method is not limited to a specific postnatal timepoint, but rather allows for ECG data collection in neonatal mouse pups from birth to postnatal day 10 (P10), a window that is of critical importance for modeling human diseases in vivo, with particular emphasis on congenital heart disease (CHD).

Here, we present a noninvasive electrocardiography (ECG) protocol, optimized for early postnatal mice, that does not require the use of anesthetics.

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both ...

Wireless Telemetry Device Implantation in a Fontan Ovine Model for Continuous and Long-Term Hemodynamic Monitoring

While the Fontan procedure drastically improves life expectancy for patients with single ventricle, it is well recognized that the resulting circulation causes significant disease burden long term as a consequence of chronically elevated central venous pressures and decreased cardiac output. Chronic Fontan animal models are a valuable asset to studying the late physiological outcomes associated with this operation and a necessary tool in the evaluation of future devices designed to alleviate Fontan failure. However, previous attempts at the creation of chronic Fontan models have been hindered by poor survival rates. Additionally, effective hemodynamic data collection poses a significant challenge in freely moving animals. To this end, the use of wireless implantable telemetry systems provides a novel solution for real-time and long-term monitoring of cardiovascular data. This protocol describes the methodology for surgical implantation of a wireless telemetry device in a Fontan survival ovine model, facilitating the continuous and ongoing recording of several hemodynamic parameters, including heart rate, arterial blood pressure, and localized pressures in the inferior (IVC) and superior vena cava (SVC). Telemetry devices were implanted with cannulation of either the carotid artery and internal jugular vein or femoral artery and vein, for placement of pressure-sensing catheters in the ascending aorta and SVC or abdominal aorta and IVC, respectively. The use of the wireless telemetry systems enabled close postoperative monitoring following a single-stage Fontan operation, which contributed to improved animal welfare and survival.

This protocol describes the surgical methodology for implanting a large animal wireless telemetry device to enable continuous and long-term collection of hemodynamic data, including heart rate, arterial blood pressure, inferior and superior vena cava pressures, and cardiac rhythm.

While the Fontan procedure drastically improves life expectancy for patients with single ventricle, it is well recognized that the resulting circulation ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants with congenital diaphragmatic hernia (CDH) also have lung hypoplasia due to occupation of the chest cavity by the stomach and bowel and, in the most severe cases, the liver. Balloon tracheal occlusion reduces the severity of lung hypoplasia in fetuses with CDH but increases the risk of premature birth. Understanding the optimal occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improving in utero corrective treatments for CDH. The study reports a new method for continuous measurement of the intratracheal and amniotic pressures in an unoccluded and occluded fetal lamb surgical model of CDH. Time-pregnant Merino ewes underwent two recovery hysterotomies: the first at ~80 days of gestation to create the CDH, and the second at ~101 days of gestation to occlude the fetal trachea and implant an intratracheal and amniotic pressure measurement device. Lambs were delivered at ~142 days, and the pressure measurement device was removed and cleaned. The data were downloaded and filtered using a 6 h window. Transrespiratory pressure was calculated.

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

Optimization of occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improve in utero corrective treatments for congenital diaphragmatic hernia (CDH). This study reports a new method for continuous measurement of the tracheal pressure in an occluded and unoccluded fetal lamb surgical model of CDH.

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants ...

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

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