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)¹. PHH can be treated with the placement of a ventriculoperitoneal shunt, but shunt placement does not reverse the brain injury that may occur from IVH. Although modern neonatal intensive care has lowered rates of IVH^{2, 3}, there are no specific treatments for the brain injury or hydrocephalus caused by IVH once it has occurred. A significant limitation in developing preventative treatments for IVH-induced brain injury and PHH is the incomplete understanding of IVH pathophysiology.

Recently, early CSF levels of key blood breakdown product hemoglobin have been shown to be associated with the later development of PHH in neonates with high-grade IVH⁴. Furthermore, CSF levels of iron-handling pathway proteins—hemoglobin, ferritin, and bilirubin—are associated with ventricle size in neonatal IVH. This was also shown in a multicenter cohort of infants with preterm PHH, where higher ventricular CSF levels of ferritin were associated with larger ventricle size⁵.

In this study, we developed a clinically relevant model of IVH-induced brain injury and hydrocephalus utilizing hemoglobin injection into the brain ventricles, which allows for quantification of brain injury and PHH and the testing of new therapeutic strategies (Figure 1)^{6, 7}. This IVH model utilizes neonatal rat pups, which are placed under general anesthesia for the duration of the procedure. A midline incision is made on the scalp, and coordinates derived from skull landmarks—the bregma or lambda—are used to target the lateral ventricles for injection. Slow injection using an infusion pump delivers hemoglobin into the ventricle. This protocol is easy to use, versatile, and can model different components of IVH that result in PHH.

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. Preparation of the animal for injection

1. Turn the heating pad to the medium setting to maintain the body temperature of the rat.
2. Anesthetize postnatal day 4 (P4) rats in an induction chamber filled with 3% isoflurane.
   NOTE: Confirm sufficient anesthesia using the toe/tail-pinch response every 15 min. Monitor anesthesia with visual observation of tissue color, body temperature, and respiratory rate.
3. Administer pain relief with a 5 mg/kg subcutaneous carprofen injection to the anesthetized rat.
4. Place the anesthetized rat prone in the stereotactic apparatus with the nose positioned in the anesthesia adaptor with a constant flow of 1.5% isoflurane.
5. Tighten non-rupture ear bars on the external auditory meatus to secure the head.
   NOTE: Apply vet ointment to keep the eyes moisturized if the eyes are open at the age of injection.
6. Clean the head, alternating with sterile cotton-tipped applicators soaked in betadine and 70% ethanol.
   1. Touch the betadine-soaked applicator to the center of the scalp and spread the betadine in circles, moving outward.
   2. Repeat step 2.6.1.1 with the ethanol-soaked applicator.
   3. Repeat step 2.6.1.1 and step 2.6.1.2 3x.
7. Apply a sterile surgical drape to protect the surgical field. 
8. Using a sterile scalpel, make a 0.3 cm incision vertically down the center of the head to expose the bregma of the skull.
   NOTE: If injecting from the lambda, expose the lambda of the skull instead of the bregma.
9. Use a sterile cotton-tipped applicator to dry the area.

3. Setting up the stereotactic injector

1. Draw the hemoglobin solution prepared in step 1.2 into a 0.3 mL sterile syringe with a 30G needle and place the syringe into the stereotactic injector system.
   ​NOTE: If generating control conditions, draw aCSF solution prepared in step 1.1 into a 0.3 mL sterile syringe and proceed with the protocol.
2. Turn on the stereotactic injector interface and click on the Configuration button to input the injection volume and rate settings.
   1. Click on Volume and set the volume at 20,000 nL (20 µL).
   2. Click on Infusion rate and set the rate at 8,000 nL/min (8 µL/min).
3. Exit Configuration by clicking on the Reset Pos button.
4. Flush the needle tip by clicking on the Infuse button until a small bead of hemoglobin solution emerges at the needle tip.
5. Gently wick the hemoglobin solution from the needle tip with a sterile cotton-tipped applicator.

4. Animal injection

1. Set the bregma as zero on the stereotactic injector system by adjusting the mediolateral and anteroposterior positions of the syringe before lowering the tip of the flushed syringe needle to gently touch the skull at the bregma.
   NOTE: If injecting from the lambda, set lambda as zero.
2. Identify the coordinates of choice.
   1. If injecting from the bregma, in P4 rats described here, use 1.5 mm lateral, 0.4 mm anterior, and 2.0 mm deep from bregma.
   2. If injecting from the lambda, use the following coordinates for P4 rats: 1.1 mm lateral, 4.6 mm anterior, and 3.3 mm deep from lambda.
3. Raise the syringe needle 1 cm above the skull to clear the scalp. When the syringe is raised, proceed to set the mediolateral and anteroposterior coordinates.
4. Lower the syringe needle to gently touch the skull. Check that the needle is touching the skull.
5. Set the dorsoventral coordinate over a 30 s period.
   NOTE: While setting the dorsoventral coordinate, the needle will puncture the skull. Care must be taken to assure that the syringe passes through the skull without deforming the skull. Skull deformation is avoided by slowly withdrawing the needle along the dorsoventral coordinate if deformation occurs, then placing the needle back along the same trajectory. This allows the needle to pass through the hole in the skull with less force and no deformation.
6. On the stereotactic injector interface, click on the Run button to begin injection.
7. After the injection is finished, leave the syringe needle in place for 2 min to minimize backflow of the solution.
8. Withdraw the syringe slowly along the dorsoventral coordinate over 2 min until the needle tip is 2 cm above the scalp.
9. Rotate the stereotactic injector arm away from the operative field.

5. Postoperative care

1. Close the scalp with a 6-0 monofilament suture. Make one simple interrupted suture at the center of the 0.3 cm incision.
2. Remove the pup from anesthesia and place it onto a secure area on the heating pad.
3. Return the rodent to the home cage to recover from the anesthesia under the care of its dam.
   NOTE: Timely return to the care of the dam reduces early postoperative mortality.
4. Monitor the animals for anesthesia by loss of the righting reflex hourly post surgery for 3 h.
5. Monitor the animals daily for 7 days for normal activity, food intake, and weight gain. Monitor the incision site for wound healing, closure, and reappearance of fur at the site of surgery.
   NOTE: In the rare case that neurological changes such as seizures, central depression, or decreased appetite are observed during monitoring, euthanize the animal using intravascular perfusion or cervical dislocation under anesthesia.
6. To prevent infection once the suture is closed and the wound is healing, apply topical triple-antibiotic at the incision site.

6. MRI acquisition and quantification

1. Perform MRI on a 4.7T or 9.4T small-animal scanner.
2. Turn the heating pad to the medium setting to maintain the body temperature of the rat.
3. Induce anesthesia in a chamber using 3% isoflurane.
   NOTE: Confirm sufficient anesthesia using the toe/tail-pinch response every 15 min. Monitor anesthesia with visual observation of tissue color, body temperature, and respiratory rate.
4. Place the anesthetized rat prone in the MRI with the nose positioned in the anesthesia adaptor with a constant flow of 1.5% isoflurane.
5. Perform T2-weighted imaging by selecting a T2-weighted fast spin echo sequence.
   1. If using a 4.7T MRI scanner, enter the following parameters into the MRI software: repetition time = 3,000 ms, echo time = 27.50 ms, number of averages = 3, field of view = 18.0 mm x 18.0 mm, matrix = 128 x 128, number of axial slices = 24, thickness = 0.50 mm.
   2. If using a 9.4T MRI scanner, enter the following parameters into the MRI software: repetition time = 5,000 ms, echo time = 66.00 ms, echo spacing = 16.50 ms, number of averages = 2, repetitions = 1, rare factor = 8, field of view = 16.0 mm x 16.0 mm, matrix = 256 x 256, number of axial slices = 32, thickness = 0.50 mm.
6. Click on the Continue button to start the sequence.

7. Image processing and analysis

1. Use native T2-weighted data to analyze brain volume. Use segmentation software to manually delineate the lateral ventricles⁶. Click on Paintbrush Mode and select square brush style. Adjust the brush size to 1. Click Layout inspector and select axial view. Click zoom to fit. Place the cursor on the image; trace and fill the lateral ventricle space.
2. Click on Segmentation in the toolbar | Volume and Statistics to view the segmented volumes.

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 ...

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

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