Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Summary

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Abstract

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Introduction

With over 12% of infants born in the United States before 37 weeks estimated gestational age¹, perinatal brain injury (PBI) from prematurity is a significant cause of permanent disability. PBI from prematurity, also termed encephalopathy of prematurity (EoP), affects the entire central nervous system (CNS). CNS injury often commences in utero, and is exacerbated by antenatal processes including chorioamnionitis and postnatal complications such as hypoxia and sepsis. PBI from systemic insults alters neurodevelopment and leads to cerebral palsy, epilepsy, cognitive delay and numerous neuropsychiatric disorders affecting emotional regulation, memory a....

Protocol

Institutional Care and Use Committees at both Boston Children’s Hospital and the University of New Mexico Health Sciences Center approved all experimental procedures.

NOTE: Prior to commencing the procedure, seal, sterilize and autoclave all surgical instruments and surgical drapes. Additionally, prepare post-operative medications in sterile vials including 0.125% bipivucaine and 0.1 mg/kg buprenorphine. Also prepare the lipopolysaccharide (LPS) solution sterilely: 0.04 mg/ml LPS (0111:B4) in sterile saline containing dilute Evan’s blue dye.

1. Anesthesia

1. Induce anesthesia in an embryonic ....

Results

Following TSHI+LPS at E18, hematoxylin and eosin staining reveals significant histopathological abnormalities in both the placenta (Figure 1) and in the brain (Figure 2). Placentas examined on E19 and E21 are grossly edematous with micro-hemorrhage, and necrosis throughout the decidua and labyrinth. Significant inflammatory infiltrate and increased vascularity is also observed. Brains examined on P2 reveal ventriculomegaly, as well as white matter and subplate neuron loss compared to sha.......

Discussion

Encephalopathy of prematurity is difficult to model in animals because of the complex interaction of etiologies, neurodevelopmental time course, intricacy of human cerebral network formation, overlapping mechanisms of injury, and the diverse phenotypes of CNS insults manifest in human preterm infants. EoP is associated with specific cell-type vulnerabilities (i.e. immature oligodendrocytes)²¹, as well as diverse developmentally-regulated pathways (i.e. subplate, membrane transporters and rece.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Saline Solution, 0.9%	Sigma	S8776
LPS 011B4	Sigma	L2630
Evan's Blue Dye	Sigma	E2129
Surgical gloves	Biogel	40870
OR Towels	Cardinal Health	287000-008	Sterile
PDI Alcohol Prep Pads	Fisherbrand	06-669-62
Mini Arco Rechargeable Clippers	Kent Scientific Corp.	CL8787
Betadine surgical scrub	Purdue Products L.P.	67618-151-17
Eye Lubricant	Refresh Lacri Lube	00023-0312
Blunt Forceps	Roboz	RS-8100
Scissors	Roboz	RS-6808
Surgical Scissors	Roboz	RS-5880
Surgical Scissors	F.S.T.	14002-16
Syringe	BD	309628	1 ml
Needle	BD	305122	25G 5/8
Needle	BD	305128	30G 1
Cotton-tipped Applicators	Fisherbrand	23-400-114	Small, 6 inch sterile
Cotton Gauze Sponge	Fisherbrand	22-362-178
Needle Holders	Kent Scientific Corp.	INS600109	12.5 CM STR
Vessel Clips	Kent Scientific Corp.	INS600120	30G Pressure
3-0 Perma Hand Silk Sutures	Ethicon	1684G	Black braided, 3-0 (2 metric), 18", non-absorbable,  PS-1 24 mm needle, 3/8 circle
Insulin Syringes	BD	328438	0.3 cc 3 mm 31G
Pentobarbital
Buprenorphine
Bupivacaine
Isoflurane
Lithium Carbonate	Acros Chemicals	554-13-2
Superfrost Plus Microscope Slides	VWR	48311-703
Hematoxylin	Leica	3801521	Surgipath Gill II Hematoxylin
Eosin	Leica	3801601	Surgipath Eosin
Xylenes	Fisherbrand	X3S-4	Histological Grade
Permount	Fisherbrand	SP15-100
Coverglass	Fisherbrand	12-548-5P	Fisher Finest Premium Coverglass