Modeling Posthemorrhagic Hydrocephalus of Prematurity in Rats

Summary

Posthemorrhagic hydrocephalus of prematurity (PHHP) can be modeled in neonatal rats by combining chorioamnionitis and intraventricular hemorrhage. The combination of these prenatal and postnatal events accurately recapitulates the clinical hallmarks of PHHP, including macrocephaly, ventriculomegaly, and elevated intracranial pressure, through the lifespan.

Abstract

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 weeks gestational age (GA). PHHP is defined by the accumulation of cerebrospinal fluid (CSF) associated with clinical symptoms of elevated intracranial pressure (ICP). Infants with PHHP suffer lifelong shunt dependence, with half requiring repeat surgery in the first year of life and many requiring multiple additional surgeries throughout the lifespan. Prenatal chorioamnionitis predisposes preterm infants to severe IVH and the need for surgical treatment of PHHP trends with neonatal sepsis. These clinical features suggest that systemic inflammation is an integral component of PHHP pathophysiology.

Here, we define an animal model that recapitulates all clinical aspects and essential features of PHHP in rats. The goal of this protocol is to illustrate how in utero chorioamnionitis and postnatal IVH using lysed red blood cells can be combined to yield PHHP. This preclinical approach yields progressive macrocephaly and domed craniums, elevated intracranial pressure, and ventriculomegaly that can be detected via magnetic resonance imaging (MRI) or via microscopy. In addition to sustained disruption in CSF dynamics, rats also have cognitive delay and functional disability into adulthood. Accordingly, this preclinical platform facilitates unique and unparalleled translational studies of PHHP that can incorporate molecular, cellular, biochemical, histologic, imaging, and functional outcome measures. It can also be used for rigorous analysis of the choroid plexus, ependymal motile cilia, and glymphatic system in parallel. Last, it can also be an invaluable preclinical tool for the investigation of novel surgical intervention strategies and non-surgical therapeutic approaches for the treatment of hydrocephalus.

Introduction

Posthemorrhagic hydrocephalus of prematurity (PHHP) remains a substantial public health concern. Defined by symptomatic accumulation of cerebrospinal fluid (CSF) concomitant with elevated intracranial pressure (ICP) secondary to intraventricular hemorrhage (IVH), PHHP is a severe manifestation of encephalopathy of prematurity and a significant contributor to the global burden of prematurity and acquired hydrocephalus^1,2. Globally, approximately 400,000 infants each year are born with or acquire the lifelong burden of hydrocephalus³ and many die due to lack of treatment^....

Protocol

The Animal Care and Use Committee (ACUC) at Johns Hopkins University approved all experimental procedures described herein. This protocol utilizes pregnant Sprague-Dawley rat dams and pups of both sexes.

1. Induction of chorioamnionitis on E18

NOTE: The in utero insult portion of this protocol has been previously published in detail, is summarized above, and is the subject of a separate JOVE protocol and video^{19,37,38,39,40....}

Results

Using this model, hydrocephalus develops in the days and weeks after injection of lysed red blood cells. A representation of a typical experimental design and progression of hydrocephalus is provided in Figure 1. We evaluated 5-6 sham animals and 6-8 PHHP animals per group. As juveniles, rats with PHHP exhibited macrocephaly (Figure 2), elevated intracranial pressure (Figure 3), and ventriculomegaly (Figure 4

Discussion

This protocol for the induction of PHHP allows for rigorous, quantifiable, and clinically translatable outcome measures of brain structure and function concomitant with phenotypic hallmarks of hydrocephalus, including chronic elevation of ICP, ventriculomegaly, and macrocephaly, from birth to adulthood⁴. Biochemical, histological, and functional assays can be used to evaluate the health of the choroid plexus, ependyma, and glymphatic system, as well as gray and white matter¹⁹

Materials

Name	Company	Catalog Number	Comments
70% ethanol	Pharmco	111000200	Diluted to 70%
Betadine surgical scrub	Cardinal Health	NDC-67618-151-17
Blunt Forceps	Roboz	RS-8100
Bravmini Plus Cordless Rechargeable Trimmer	Wahl	41590-0438
Carbon Steel Surgical blades	Bard-Parker	371151-11
centrifuge	Eppendorf	5424R
Cotton Gauze Sponge	Fisherbrand	22-362-178	Small, 6 inch sterile
Cotton-tipped Applicators	Fisherbrand	23-400-114	30 G 1
Eye Lubricant	Refresh Lacri Lube	75929
Far infrared warming pad	Kent scientific	RT-0501
Incubator -  Genie Temp-Shaker 100	Scientific Industries	SI-G100
Insulin Syringes	BD	328438	0.3 cc 3 mm 31 G, ultrafine
Isoflurane	Covetrus	11695067772
Ketamine hydrochloride injection	Dechra	17033-101-10
Kimwipes	Kimtech Science	BXTNI141300
LPS 011B4	Sigma	L2630
microcentrifuge tubes	Thermo Fisher Scientific	3453	2.0 mL
Needle	BD	305122	1 mL
Needle	BD	305128	25 G 5/8
Needle Holders	Kent Scientific Corp.	INS14109	12.5 CM STR
OR Towels	Cardinal Health	287000-008
Paper measuring tape	Cardinal Health	SKU
Saline Solution, 0.9%	Sigma	S8776
Scissors	Roboz	RS-6808
SomnoSuite	Kent Scientific	SS6823B
Sterile Alcohol Prep Pads	Fisherbrand	06-669-62	Sterile
Surgical gloves	Biogel	40870
Surgical Scissors	Roboz	RS-5880
Surgical Scissors	EST	14002-16
Syringe	BD	309628
T/Pump (Heat Therapy Pump)	Stryker Medical	TP700
Vessel Clips	Kent Scientific Corp.	INS14120	30 G Pressure
Xylazine injection	vet one	NDC 13985-704-10