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

Summary

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.

Abstract

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.

Introduction

Birth causes stress to the fetus, and there is a considerable increase in the levels of catecholamine, the major stress hormone^1,2. This raises the systemic BP, and if this pressure is transmitted to the fragile brain capillaries via the carotid arteries, this can lead to their rupture^3,4,5. Surges in systemic BP are prevented from reaching the brain by the constriction of the carotid arteries in the full-term fetus. However, this mechanism is not developed in the preterm fetus, and this is responsible for th....

Protocol

For the present study, approval for the animal experiments was obtained from the Animal Care and Use Committee of the University of Arizona. One time-mated, pregnant Columbia-Rambouillet ewes between 2-4 years of age were used for the present study. The animals were obtained from the University of Arizona Sheep Unit.

1. Animal maintenance

1. Obtain animals from any sheep ranch.
2. Transport the ewes to the laboratory at 105 days ± 5 days to 137 days ±.......

Discussion

Currently, no method exists to examine vessel contractility and dilatation in vivo in response to drug compounds and gene manipulation. As a standard in the field, in vivo blood flow is measured by Doppler flow probes, microspheres, and radioactive molecules such as tritiated water. However, to manipulate the receptors' functions or downstream signaling, the animals are sacrificed, and experiments are conducted in vitro in organ baths following the isolation of arterial segments. The current me.......

Materials

Name	Company	Catalog Number	Comments
Aaron Bovie Electrosurgical Cautery	Henry Schein, Inc	5905974
Aaron Bovie Electrosurgical Generator	Henry Schein, Inc	1229913
Alfalfa Pellets	Sacate Pellet Mills, Inc. Maricopa AZ	100-80
Analog to Digital Converter	ADI Instruments	Powerlab
Babcock forceps	Roboz Surgicals	RS8020
Bridge Amplifier	ADI Instruments	Bridge Amplifier
Castroviejo scissors	Roboz Surgicals	RS5650SC
Diazepam	Henry Schein, Inc	1278188
Endotracheal Tube	Henry Schein, Inc	7020408
Flow Probes	Transonic Systems Inc.	MC2PSS-JS-WC100-CRS10-GC, MC3PSS-LS-WC100-CRS10-GC
Heparin	Henry Schein, Inc	1162406
Isoflurane	Henry Schein, Inc	1182097
Ketamine	Henry Schein, Inc	1273383
Ketoprofen	Zoetis Inc., Kalamazoo, MI	Ketofen
Manifold Pump Tubing	Fisher Scientific	14-190-508
Metzenbaum scissors	Roboz Surgicals	RS6010
Narkomed 4 Anesthesia Machine	North American Dräger	Narkomed 4
Normal Saline	Fisher Scientific	Z1376
penicillin G procaine suspension	Henry Schein, Inc	7455874
phenylbutazone	VetOne Boise, ID	510226
Phenylephrine	Sigma Aldrich Inc.	P1240000
Pivodine Scrub	VetOne	510094	Germicidal cleanser
PowerLab	ADInstruments		Data acquisition hardware device
Pulse Oximeter	Amazon Inc.	UT100V
Tygon Tubing	Fisher Scientific	ND-100-80
V-Top Surgical Table	VetLine Veterinary Classic Surgery	TSP-4010
Wound Clips	Fisher Scientific	10-001-024