Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Summary

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Abstract

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (>12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

Introduction

Hypoxic ischemic encephalopathy (HIE) is a devastating condition resulting from various factors in newborn infants¹. Perinatal asphyxia and/or the disruption of cerebral blood flow may result in focal or global ischemic changes in the brain². The occurrence rate is approximately 1.6 in 1,000 live births but may be as high as 12.1 in 1,000 live births in developing countries³. This condition results in high mortality (20%-50%), while 25% of those who survive are likely to suffer from a long-term neural disability such as mental retardation, epilepsy, or cerebral palsy⁴. The ....

Protocol

All the experimental procedures were approved by the Oklahoma Medical Research Foundation (OMRF) Institutional Animal Care and Use Committee (protocol IACUC #17-17). Pregnant female Sprague-Dawley rat pups at E14 were used for the present study. The animals were obtained from a commercial source (see Table of Materials).

1. Animal preparation

1. Acclimatize the animals in the animal facility prior to delivery of the litters.
2. Maintain all rat.......

Discussion

A research protocol in newborn rat pups was successfully designed to visualize and analyze early markers of cerebral injury in HIE. To date, there is a lack of objective assessment tools to detect early cerebral injury in the newborn population. After HI injury, there is a phase (1-6 h) in which the impairment of cerebral oxidative metabolism has the potential to partially recover before the failure of mitochondrial function¹⁹, which is irreversible. This latent phase is the therapeutic window for.......

Materials

Name	Company	Catalog Number	Comments
0.9% Normal saline	Fisher Scientific	Z1376
2,3,5-triphenyltetrazolium chloride (TTC)	Millipore Sigma	T8877
Abdominal pneumatic pillow	SA Instruments, Inc., Stony Brook, NY
Absorbent Underpads with Waterproof Moisture Barrier, 58.4 x 91.4 cm, 680 mL	Fisher Scientific	501060566
BD 30 G Needle and syringe	Fisher Scientific	Catalog No.14-826-10
Biospec 7.0 Tesla/30 cm horizontal-bore magnet small animal imaging system	Bruker Biospin, Ettlingen, Germany
Buprenorphine			Provided by veterenary medicine
Compact Thermometer with Probe	Fisher Scientific	S01549
Gas mixture 92% nitrogen 8% oxygen	Airgas
Head surface coil	Bruker BioSpin MRI Gmbh, Ettlingen, Germany
Isoflurane gas			Provided by veterenary medicine
Isotemp Immersion Circulator 2100	Fisher Scientific	Discontinued	Immersed in water bath chamber with continous flowing water via tubing
Lead Ring Flask Weights	VWR	29700-060	Water bath weights to ensure rodent chamber stays submerged in water bath
Mathematica Software	Wolfram Research, Champaign, IL, USA	version 6.0
Pedialyte Electrolyte Solution, Hydration Drink, 1 Liter, Unflavored	Pedialyte		Obtained from CVS
Phosphate-buffered saline (DPBS, 1X), Dulbecco's formula	Millipore Sigma	J67670.AP
Plastic clear bucket			We used an old rodent housing cage- this is a good alternative: Cambro 182615CW135 Camwear Food Storage Box, 18" X 26" X 15", Model #:182615CW135
Plexiglass Rodent Restraint Chamber	Pedialyte/CVS		Vetinary medicine provided a small chamber used to restrain rodents. Approximately 6x4x4 inches
Pregnant Sprague Dawley rats at E14	Charles River	Strain Code 400
Purdue Products Betadine Swabsticks	Fisher Scientific	19-061617
Quadrature volume coil (72-mm inner diameter)	Bruker BioSpin MRI Gmbh, Ettlingen, Germany
Stoelting Silk Suture	Fisher Scientific	Catalog No.10-000-656
Vicryl 5-0 suture	Fisher Scientific	NC1985424