A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury

Summary

The method describes inflammation-sensitized hypoxic-ischemic and hyperoxic brain injury in the P17 ferret to model the complex interaction between prolonged inflammation and oxidative brain injury experienced in a number of late preterm infants.

Abstract

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for infants with the neurological sequela of prematurity. Ferrets are ideal candidates for modeling the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32–36 weeks' gestation. We describe an injury model in the P17 ferret, where lipopolysaccharide administration is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This simulates the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of neonates who develop brain injury. Injured animals display a range of gross injury severity, with morphological changes in the brain including narrowing of multiple cortical gyri and associated sulci. Injured animals also show slowed reflex development, slower and more variable speed of locomotion in an automated catwalk, and decreased exploration in an open field. This model provides a platform in which to test putative therapies for infants with neonatal encephalopathy associated with inflammation and HI, study mechanisms of injury that affect cortical development, and investigate pathways that provide resilience in unaffected animals.

Introduction

There is an ongoing need for large animal models that reflect the pathophysiology of prematurity and perinatal hypoxia-ischemia in which therapeutic interventions for infants can be tested. In 2017, 9.93% of the 382,726 infants born in the United States were born preterm, and 84% of these infants were born between 32 and 36 weeks of gestation¹. In premature infants, perinatal exposure to infection or inflammation is common, where maternal immune activation due to viral or bacterial pathogens can initiate preterm labor. Postnatally, preterm infants are at high risk of early or late onset sepsis². Preterm infants also freq....

Protocol

Procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and as part of an approved protocol by the University of Washington Institutional Animal Care and Use Committee.

1. Preparation and LPS Administration

NOTE: Refer to Figure 1 for a timeline of the procedures.

1. Prior to commencing the procedure, seal, sterilize and autoclave all surgical instruments and surgical drapes. Prepare pre-operative medications in sterile vials. Calculate the flow rate required to replace the air in the hypoxia/hyperoxia chamber wi....

Results

Of 34 (n = 18 males, n = 16 females) animals from six litters exposed to the insult, eight animals (24%; n = 4 males, n = 4 females) in the injured group died during the second hypoxia period (n = 5), during temperature management (n = 2), or overnight after the insult (n = 1). In the injured group, nine of 26 survivors (35%) had visible gross injury. Five animals (n = 5 males) had moderate injury, and four animals (n = 2 males, n = 2 females) had severe injury, defined as gross pathology scores of 2–5 and 6–.......

Discussion

Due to the physical and developmental similarities shared between the ferret brain and human brain, the ferret is increasingly being used to model both adult and developmental brain injury.^8,9,10,11,12. However, research to date suggests that the ferret brain is both resistant to initial injury as well as highly-plastic, with behavioral deficits diminishing ov.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
80% Oxygen	Praxair
9% Oxygen	Praxair
Absorbent benchtop protector	Kimtech	7546
Automated catwalk	Noldus
Betadine surgical scrub
Bupivacaine	Patterson Veterinary	07-888-9382
Buprenorphine
Calipers	SRA Measurement Products	ME-CAL-FP-200	200 mm range, 0.01 mm resolution
Cotton Gauze Sponge	Fisher Scientific	22028556
Curved fine hemostat	Roboz	RS-7101
Curved forceps	World Precision Instruments	501215
Curved suture-tying hemostat	Roboz	RS-7111
Ethovision tracking software	Noldus
Eye Lubricant	Rugby	NDC 0536-1970-72
Ferrets (Mustela putorius furo)	Marshall Biosciences		Outbred (no specific strain)
Formalin	Fisher Scientific	SF100-4	10% (Phosphate Buffer/Certified)
Hair Clippers	Conair	GMT175N
Insulin Syringes	BD	329461	0.3 cc 3 mm 31 G
Isoflurane	Piramal	66794-017-25
Lidocaine	Patterson Veterinary	07-808-8202
LPS	List Biological	LPS Ultrapure #423
Oxygen sensor	BW Gas Alert	GAXT-X-DL-2
Pentobarbital
Plastic chamber	Tellfresh	1960	10 L; 373 x 270 x 135 mm3
Saline Solution, 0.9%	Hospira	RL-4492
Scalpel blade	Integra Miltex	297
Scalpel handle	World Precision Instruments	500236	#3, 13 cm
Sterile suture	Fine Science Tools	18020-50	Braided Silk, 5/0
Surgical clip applicator	Fine Science Tools	12020-09
Surgical clip remover	Fine Science Tools	12023-00
Surgical drapes	Medline Unidrape	VET3000
Surgical gloves	Ansell Perry Inc	5785004
Surigical clips	Fine Science Tools	12022-09
Thermometer (rectal)	YSI	Precision 4000A
Thermometer (water)	Fisher Scientific	14-648-26
Umbilical tape	Grafco	3031	Sterile
Water bath	Thermo Scientific	TSCOL19	19 L