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Biologie du développement

仪表近期胎羊多元慢性非麻醉的录音

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

慢性病仪表非麻醉的胎羊模型被用于研究在健康和疾病的人类胎儿发育，因为它允许外科安置和维护导管和电极，反复血液采样，物质喷射，记录的生物电活动的，以及在体内成像 。我们描述了建立这种模式所需的程序。

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

对正常和损害怀孕研究各种动物模型的存在，包括实验室啮齿类，非人类的灵长类动物和家养反刍动物。1,2,3,4,5长期仪器妊娠母羊已被广泛使用50年人类胎儿发育和答复的模型的病理生理刺激如脂多糖（LPS）。6-10以下的LPS暴露病变模仿正是由于这两个物种的一个类似的成熟信息见于早产儿脑室周围白质软化，这是。 11，12

其他妊娠并发症也已经研究的很详细，如发现产前糖皮质激素促进肺的发育13-15和了解胎儿宫内发育迟缓（IUGR）对胎儿16,17的影响。

广泛使用的胎羊模型的是由于uniq的非麻醉胎羊到外科位置和维护导管和电极的UE顺从，允许重复的血液采样，记录的生物电活动的，应用电刺激和体内脑成像。18遥测也是可能的，虽然不经常使用的但由于较高的复杂性来设置，以及初始和维护成本。19

此外，胎羊模型是非常通用的仪器的许多变化是可能根据目标的措施。例如，有可能在数天来记录到周实时多元信号如胎儿呼吸运动，电脑活动，心血管反应，心电图，使用流量探针或微球体等由于局部血流的范围器官这种多功能性，已经进行了包括CARDI的发展广泛的研究ovascular系统20,21，下丘脑-垂体-肾上腺轴（HPA）22，大脑发育23和睡眠状态的发展，特别是24，缺氧的影响/窒息25，低温治疗26，炎症6-11，无论是27的组合，糖皮质激素28,29，抗抑郁药30，支气管肺发育不良（BPD）31,32，胎儿编程33,34,35,36,37,38,39或新颖的胎儿监护模式开发之前和分娩期间的名字，但调查的几个领域。40,41,42,43

提出的方法的总体目标是展示这种多功能车型的基本实现。其允许建立多种研究胎儿生理和病理生理上一体化，器官，细胞和分子水平的急性和慢性实验方案。

Protocol

动物保健随后加拿大议会关于动物饲养和批准蒙特利尔大学理事会动物保健（协议＃10言语报 – 1560年）的指导方针。详细资料，使用的材料和方法是表1中提供。 1.麻醉插入一个单腔导管插入颈静脉。 稳重的使用母羊乙酰丙嗪（Atravet 10毫克/毫升）2毫克静脉内约30分钟前，麻醉诱导，以减少与该过程从而降低皮质醇水平有关应力。 施用西泮（地西泮5毫克…

Representative Results

38怀孕的时间，日期母羊仪器在128±2天的妊娠期（DGA，〜0.88妊娠，期限145 DGA）与动脉，静脉和羊膜导管和心电图（ECG）与全身麻醉下无菌技术（包括母羊和胎儿电极）。如果双胎妊娠的胎儿较大基于触诊和估计跨期直径选择;替代地，胎儿必须被启动，可以随机选择，以避免任何可能的偏差或两者胎儿可能进行检测。该过程的总持续时间为124±27分钟。胎儿上半身必须被启动的部分仍然是子宫外为…

Discussion

麻醉及手术过程中呈现所需要的建立动物模型，为研究胎儿生理和病理生理：长期仪器非麻醉的胎羊。

该协议中的四个关键步骤应该强调。首先，通过母亲侧面传递导管和电极：重要的是，这是在一次以避免任何内部器官损伤。第二，固定之前外缝合胎儿uterotomy运行地点：这是至关重要的，以防止或尽量减少羊水和前子宫闭合羊膜随后缝合的损失。三，动脉插管：胎羊动脉?…

Divulgations

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
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Tags

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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