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 hormone1,2. 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 rupture3,4,5. 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 the significantly higher likelihood of brain damage in preterm fetuses4,5.
Currently, no suitable method exists to examine the maturation of the pathways involved in regulating the carotid blood flow with developing fetuses. These studies on carotid blood flow and vasoresponsiveness are crucial from both basic science and clinical perspectives. Currently, to determine the molecular pathways involved in the regulation of arterial contractility, the standard method involves isolating the arterial segments postmortem. Then, the experiments are conducted using wire myography to determine the vasocontractility of different pharmacological molecules that define the regulatory pathways involved in arterial contractility6,7. Of note, the ex vivo findings are not able to fully replicate the in vivo environment because of the blood flow regulation upstream and downstream of the carotid artery. Thus, the present study aimed to develop a technique that can determine the effects of vasoresponsive chemicals or agents on blood flow in an artery in vivo.
The perivascular delivery methodology described in this article provides an in vivo approach to study the effect of the pharmacological or genetic manipulation of signaling pathways on different arterial segments. Using this method, one can manipulate the fetal blood pressure and carotid blood flow. Additionally, experiments with sheep fetuses are demonstrated for studying the effects of signaling molecules in a developing fetus. Hopefully, the detailed methodology provided will lead to new investigations in the field of blood flow studies, especially in relation to fetal physiology and pathology.
Protocol
Representative Results
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Intramural funds from the University of Arizona were used for these studies.
Materials
| 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 |
Riferimenti
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