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

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....

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 (K....

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.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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			5 mg/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