A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Summary

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Abstract

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world’s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials^1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (< 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Introduction

Perinatal asphyxia is an acute and frequently unpredicted condition associated with hypoxic-ischemic encephalopathy (HIE). The overall goal of this protocol is to demonstrate a piglet survival model of perinatal hypoxic-ischemic encephalopathy. This model can be used to investigate the effect of various degrees of hypoxia-ischemia on the neonatal brain and of experimental treatments on neuropathology, magnetic resonance imaging and spectroscopy (MRI and MRS) and biomarkers in body fluids such as blood, cerebrospinal fluid and urine. The model has also proven useful for investigating the cardiovascular system, respiratory system, kidney and liver, all of which are affe....

Protocol

The present protocol was approved by the Danish Animal Experiments Inspectorate. All test animals were anesthetized throughout the procedures. Reproduction of this protocol must be carried out in accordance with national ethics and animal welfare guidelines, and approved by local ethics committees.

1. Animals

1. Danish Landrace piglets < 24 hr old weighing approximately 1,500 - 2,000 g.

2. Anesthesia and Maintenance Fluids

1. Prepare the equipment needed for anesthesia (Figure 1): A mask for sevoflurane administration, alcohol swabs, peripheral intravenous catheter, rubbe....

Results

The effects of hypoxia-ischemia on the brain that occur during the induced insult are documented by recording the aEEG trace. A representative aEEG trace is shown in Figure 6.

Figure 6. Representative aEEG trace.

Discussion

Due to its complexity, the described model can only be implemented in facilities accredited and experienced in animal research. Approval by local ethics committees must be obtained prior to initiation of the experiments, and optimal animal welfare must be ensured at all times. As the model is based on the survival of the test animals, it is important that a sterile environment is maintained during invasive procedures to prevent infections.

The choice of anesthesia is important as most if not a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Warm-touch-pediatric blanket	Covidien	5030840
Adhesive Apertrue Drape	Barrier	915447
Utility Drape (sterile) 75x80 cm	Barrier	800530
Neoflon	BD - Luer	391350
Laryngoscope	Miller	85-0045
Endotracheal tube 2.5 mm	Covidien	111-25
Endotracheal tube 3.0 mm with cuff	Unomedical	MM61110030
Endotracheal tube 3.5 mm with cuff	Unomedical	MM61110035
Anesthesia machine	GE Healthcare	1009-9002-000
EEG - electrodes/disposable subdermal needle electrode	Cephalon	ACCE120550
ECG - electrodes	medtronic	3010107-003
ECG-electrodes for MR	philips	ACCE120550
Arterial blood sampler - aspirator	Radiometer medical ApS	956552
Polyurethane Umbilical vein catheter (5 Fr/Ch)	Covidien	8888160341
Polyurethane Umbilical vein catheter (3,5 Fr/ch)	Covidien	8888160333
Suture set (size 3-0)	Covidien	8886 623341
BD Spinal needle 0.7x38mm	BD needles	405254
Gas with 96% Nitrogen / 4% oxygen	Air Liquide	made on order
NeuroMonitor (CFM) system	Natus Medical Incorporated	OBM70002