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Developmental Biology

다변량 만성 비 마취 녹음을위한 단기적 태아 양의 계측

Published: October 25, 2015
doi:
10.3791/52581
, , , , , , ,
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

Summary

이 수술 배치 및 카테터 및 전극, 반복적 인 채혈, 약물 주입, 생체 전기 활동의 기록의 유지 보수 및 생체 내 이미징을 허용하기 때문에 만성적으로 계측 비 마취 태아 양 모델은 건강과 질병에 인간 태아의 발달을 연구하는 데 사용됩니다. 우리는이 모델을 수립하는 데 필요한 절차에 대해 설명합니다.

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

동물 모델의 다양한 실험실 설치류, 비 – 인간 영장류 및 국내 반추 포함한 정상 임신 손상 모두의 연구를 위해 존재한다. 1,2,3,4,5 만성적 계측 양 임신 50 년 등을 위해 광범위하게 사용되어왔다 이러한 리포 폴리 사카 라이드 등의 병태 생리 학적 자극 (LPS)에 인간 태아의 개발 및 응답의 모델. 6-10 LPS 노출을 다음과 같은 병변으로 인해 두 종의 유사한 성숙 프로필입니다 뇌실 주위 백질 연화증,와 미숙아에서 볼을 정확히 모방. 11 12

기타 임신 합병증은 또한 산전 글루코 코르티코이드는 폐 개발 13-15 태아 (16, 17)에 자궁 내 성장 제한 (IUGR)의 영향을 이해를 증진하는 발견으로 아주 자세하게 연구되어왔다.

태아 양 모델의 광범위한 사용은 UNIQ에 기인반복적 채혈, 생체 전기 활동의 기록, 전기 자극 및 생체 뇌 영상에 적용 가능 수술 배치 및 카테터 및 전극의 유지에 비 마취 태아 양 UE 가공성. 자주 사용되지만 (18)의 원격 측정도 가능 아직 더 세련로 인해 초기 및 유지 보수 비용뿐만 아니라 설정합니다. (19)

장비의 많은 변형이 관심에 따라 측정 가능 더욱이, 태아 양 모델은 매우 다재다능하다. 예를 들어, 주 일 동안 기록 할 수있다 이러한 태아 호흡 운동, 전기 뇌 활동, 심혈관 반응, 심전도, 장기의 범위 지역 혈류 흐름 프로브 또는 마이크로 스피어 등에 감사하여 실시간으로 다변량 신호 이 다양성은 연구가 광범위 CARDI의 개발을 포함하여 진행되고있다ovascular 시스템 (20, 21), 시상 하부 – 뇌하수체 – 부신 (HPA) 축 (22), 특히 24 두뇌 발달 (23) 수면 상태 개발, 저산소증의 효과 / 질식 (25), 치료 적 저체온 (26), 염증 6-11, 모두 27의 조합, 글루코 코르티코이드 28, 29, 항우울제 (30), 기관지 폐 이형성증 (BPD) 31, 32, 이전과 노동 동안 이름 만에 태아 프로그래밍 33,34,35,36,37,38,39 또는 소설 태아 모니터링 양식의 개발 조사의 몇 지역. 40,41,42,43

제시된 방법의 전체 목표는이 다용도 모델의 기본 구현을 보여주기위한 것이다. 그것은 셀룰러 통합, 기관, 및 분자 수준에서 태아의 생리학 및 병리학 연구 급성 및 만성 실험 프로토콜의 다양한 설정을 허용한다.

Protocol

동물 케어 동물 관리에 캐나다위원회 및 동물 관리에 몬트리올 대학교 협의회 (프로토콜 # 10 RECH-1560)의 승인의 지침을 따랐다. 사용 된 재료 및 방법에 대한 상세한 정보는 표 1에 제공된다. 1. 마취 경정맥에 단일 루멘 카테터를 삽입합니다. 침착 에웨어 사용 아세 프로 마진 (Atravet 10 ㎎ / ㎖) 2 ㎎을 정맥 내로 약 30 분 차례로 코르티솔의 수준을 감소시키는 절?…

Representative Results

38 임신 한 시간 일자 ​​암양은 임신 128 ± 2 일째 계측 하였다 (DGA, ~ 0.88 임신, 용어 145 DGA) 동맥, 정맥 및 양수 카테터 및 심전도 (ECG) 멸균 전신 마취하에 기술 (에웨어 모두와 태아와 전극 ). 트윈 임신의 경우 태아가 큰과 palpating intertemporal 직경 추정에 기초하여 선택되었다; 대안 적으로, 계측되는 태아 어떤 전위 바이어스를 피하기 위해 무작위로 선택 될 수 있거나 또는 두 태아는 기능화 될 수…

Discussion

만성 계측 비 마취 태아 양 : 마취 및 수술 절차는 태아의 생리 및 병태 생리 연구를위한 동물 모델을 확립 필요가되게됩니다.

프로토콜 내에서 네 가지 중요한 단계가 강조되어야한다. 우선, 모체 플랭크를 통해 카테터 및 전극을 통과 : 이것이 내부 장기 손상을 방지하기 위해 한 번 수행하는 것이 중요하다. 둘째, 태아 exteriorizing 이전 uterotomy 운영 사이트 고정이 방지하거나…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

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

Materials

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

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Fetal SheepChronic InstrumentationMultivariate RecordingsNon-anesthetizedFetal DevelopmentPathophysiologic StimuliGlucocorticoid TreatmentTherapeutic HypothermiaCatheter PlacementBioelectrical ActivityIn Vivo Organ Imaging
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Burns, P., Liu, H. L., Kuthiala, S., Fecteau, G., Desrochers, A., Durosier, L. D., Cao, M., Frasch, M. G. Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings. J. Vis. Exp. (104), e52581, doi:10.3791/52581 (2015).
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