Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Summary

We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans.

Abstract

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed.

Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

Introduction

Neonatal IVH originates from the germinal matrix, a site of rapid cell division that is adjacent to the lateral ventricles of the developing brain. This highly vascular structure is vulnerable to hemodynamic instability related to premature birth. Blood is released into the lateral ventricles in germinal matrix hemorrhage (GMH)-IVH when fragile blood vessels within the germinal matrix rupture. In the case of grade IV IVH, periventricular hemorrhagic infarction may also contribute to the release of blood products within the brain.¹ The combination of GMH-IVH may cause PHH, particularly after high-grade hemorrhage (grades III and IV)

Protocol

NOTE: All animal protocols were approved by the institutions' Animal Care and Use Committee. See the Table of Materials for details about all materials, reagents, equipment, and software used in this protocol.

1. Preparation of hemoglobin and CSF solutions

1. Prepare a sterile artificial CSF (aCSF) solution by adding 500 µL of the aCSF solution to a 1.5 mL microtube and store on ice.
2. Prepare a sterile 150 mg/mL hemoglobin solution by adding 75 mg of hemoglobin to 500 µL of aCSF in a 1.5 mL microtube and store on ice.

2. Preparati....

Results

The success of injection was confirmed by radiologic and immunohistochemical means. Animals that underwent hemoglobin injection developed moderate acute ventriculomegaly when assessed via MRI (Figure 2A), with significantly larger lateral ventricles at 24 h and 72 h post hemoglobin injection compared to aCSF-injected animals (Figure 2B,C). While there was no significant difference in lateral ventricle volume between hemoglobin-injected and aCSF-.......

Discussion

This IVH model utilizing hemoglobin injection allows for the study of the pathology of IVH specifically mediated by hemoglobin. For complementary studies, hemoglobin can also be easily delivered in vitro and does not confound biochemical assays for proteins made by microglia/macrophages that are present in whole blood.

The leading theories of IVH-PHH include the mechanical obstruction of CSF circulation, the disruption of cilia lining the ependymal walls, inflammation, fibrosis, and i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.3 mL insulin syringe	BD Microfine + Insulin Syringe	230-4533	0.3-0.5 mL synringes will work
1.5 mL microtube	USA Scientific	1615-5500	Lot No. K194642H -3 511
4.7T MRI	Agilent/Varian	4.7T/33 cm	Agilent/Varian DirectDrive 4.7-T (200-MHz) MRI system
6-0 monofilament suture	ETHICON	667G
9.4T MRI	Bruker	BioSpec 94/20	Used in this protocol without the cryoprobe
Analytical balance	CCURIS Instruments	W3200-320
Artificial CSF (aCSF)	Tocris Bioscience	3525	Batch No: 72A
Betadine	Purdue Products L.P.	301005-00	NDC 67618-150-09
Carprofen (injectable)	Zoetis Inc.	PI 4019448	Rimadyl
Ethanol	Decon Laboratories	2701
Heating pad	Sunbeam	E12107-819	UL 612A, Z-1228-001
Hemoglobin	MP Biomedicals	100714	LOT NO. SR02321
Isoflurane	Piramal Critical Care	NDC 66794-017-25
Isoflurane vaporizer	VETEQUIP	911103
Light for stereotactic insturment	Dolan-Jenner industries	Fiber-Lite MI-150
Microinjection syringe pump	World Precision Instruments	MICRO21	Serial 184034 T08K
MRI software	Bruker BioSpin	Paravision 360 3.2
Oxygen	Airgas Healthcare	UN1072	LOT NUMBER S1432080XA02
Sprague Dawley rats	Charles River Laboratories	Strain code: 001
Stereotactic instrument	KOPF Instuments	Model 900LS Lazy Susan
Sterile cotton tipped applicator	Fischerbrand	23-400-118
Surgical blade	covetrus	#10
Topical triple antibiotic	Triple Antibiotic Ointment	NDC 51672-2120-1
Ventricle volume quantification software	ITK-SNAP	ITK-SNAP 4.0.0 beta