Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

Summary

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

Abstract

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 µl of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-µl syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 µl of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Introduction

When studying viral encephalitis, the initial distribution of viral particles is very important to understand disease pathogenesis and to identify viral targets in the brain. Most viruses range in size from 20 to 300 nm, although the Pandoravirus is more than 700 nm in size¹. The distribution of the viral particles in the acute phase of infection may depend on the size of the particles, the distribution of cellular receptors, or the affinity of the cellular receptors for viruses. In animal models, intracerebroventricular (ICV), intraperitoneal, direct placental, and intravenous (IV) infections have been used to study the pathogenesis of viral encephalitis. ....

Protocol

All the experimental protocols were approved by the Animal Care Committee of Hamamatsu University of School of Medicine.

1. Preparation of MCMV (Smith strain) and Recombinant M32-enhanced Green Fluorescent Protein (EGFP)-MCMV

1. Generate recombinant M32-EGFP-MCMV according to the method as follows (1.2 - 1.9) and as previously described⁸.
2. Use recombinant viruses derived from the Smith strain of wild-type MCMV (accession number: U68299). Insert EGFP (4,361 base pairs; bp) between 37,089 and 41,450 bp (M32 - M31 locus) in the MCMV genome by homologous recombination.
3. Amplify by polymerase chain ....

Results

In studies on the pathogenesis of viral encephalitis, the infection method is important. The hematogenous route represents an acute infection of the endothelial cells and pericytes of the brain, while the ICV route represents an acute infection spreading via the CSF through the subarachnoid space, reaching to the meninges and choroid plexus. To analyze the first distribution of particles in acute encephalitis, in situ hybridization detecting the MCMV genomes and direct observatio.......

Discussion

In animal models, ICV, intraperitoneal, direct placental, and IV infections have been used to study the pathogenesis of viral encephalitis. We focused on the ICV and IV injection models of neonatal mice for the simplicity of the procedures and the benefit of direct injection of particles into the target region. Although intraperitoneal infection is an easy method, viral particles spread systemically via an indirect process^5,24. Direct placental infection is a good method to study embryonic systemic infection. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tris; tris(hydroxymethyl)- aminomethane	Sigma-Aldrich	T-6791
HCl	Sigma-Aldrich	H-1758
pEGFP-N1 vector	Clontech	#6085-1
D-sorbitol	Sigma-Aldrich	S-1876
SPHERO TM Fluorescent Polystyrene Nile Red 0.04 - 0.06	Spherotech, Inc.	FP-00556-2
SPHERO TM Fluorescent Polystyrene Nile Red 0.1 - 0.3	Spherotech, Inc.	FP-0256-2
SPHERO TM Fluorescent Polystyrene Nile Red 1.7 - 2.2	Spherotech, inc.	FP-2056-2
10% mouse serum	DAKO	X0910
C57BL/6 mouse	SLC, Inc.
ICR mouse	SLC, Inc.
Modified Microliter Syringes (7000 Series)	Hamilton company
35-gauge needle	Saito Medical
A Wee Sight Transilluminator	Phillips Healthcare	1017920
O.C.T.Compound	Sakura Finetek	4583
RNase A	Sigma-Aldrich	R4642
Nonidet(R) P-40	Nacalai	25223-04
citrate buffer (pH 6) x 10	Sigma-Aldrich	C9999-100ml
pepsin	Sigma-Aldrich	P6887
EDTA	dojindo	N001
Formamide	TCI	F0045
Dextran sulfate sodium salt	Sigma-Aldrich	42867-5G
Denhardt's Solution (50x)	ThermoFishcer sceintific	750018
Yeast tRNA (10 mg/ml)	ThermoFishcer sceintific	AM7119
SSC 20x	Sigma-Aldrich	S6639
DAPI	ThermoFishcer sceintific	D1306
n-Hexane	Sigma-Aldrich	296090
superfrost plus glass	ThermoFishcer sceintific	12-55-18
Cytokeep II	Nippon Shoji Co.
FITC-conjugated Griffonia simplicifolia isolectin B4	Vector laboratories, Inc.	L1104
Anti-Mouse CD31 (PECAM-1) PE	ebioscience	12-0311
ProLong  Gold	ThermoFishcer sceintific	P36934
BIOREVO	KEYENCE	BZ-9000E