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. ICV inoculation with virus is often used to establish central nervous system (CNS) infections in mice. Studies using this technique report widespread infection, particularly of cells in the periventricular zones and in regions of the brain in direct contact with the cerebrospinal fluid (CSF), similar to the effects of viral ventriculoencephalitis. The small size of adeno-associated virus (AAV) particles (20 - 25 nm in diameter) facilitates their dissemination throughout the brain in ICV infections^2-4. Intraperitoneal⁵, direct placental⁶, and IV injections⁷ represent hematogenic systemic administration. The penetration of viral particles through the blood-brain barrier (BBB) allows them to reach the parenchyma of the neonatal brain, representing diffuse microglial nodules^8,9.

Cytomegalovirus (CMV) is a common virus that belongs to the herpes virus family. In the United States, 50% - 80% of the people have had CMV infection by age 40. CMV infections are rarely harmful but can cause diseases in immunocompromised patients and fetuses. Of all deliveries, 0.2% - 2% are born with CMV¹⁰, resulting in severe symptoms such as microcephaly, periventricular calcification, cerebellar hypoplasia, microphthalmia, and optic nerve atrophy^11,12. Furthermore, mental retardation, sensorineural hearing loss, visual defects, seizure, and epilepsy occur in about 10% of non-fatally CMV-infected infants^13,14. CNS dysfunction is the most common characteristic symptom of CMV congenital anomaly. More children are permanently disabled each year by congenital CMV than by Down syndrome, fetal alcohol syndrome, or spina bifida¹⁵. There are no vaccinations against CMV available at the present, calling for a need of a safe and effective vaccine. Studying the interaction of CMV particles with their receptors in the earliest phase of infection is important to understand the effect of vaccination.

Ventriculoencephalitis and diffuse microglial nodules are the two main pathological characteristics of CMV encephalitis¹⁶. It has been uncertain how the CMV particles (150 - 300 nm) spread through the brain in the acute phase of infection and how the distribution of cellular receptors and their affinity for viruses contribute to the viral spread. Kawasaki et al. have evaluated ICV and IV infections from the perspective of the distribution of particles and their receptors (β1 integrin) in the earliest phase of infection. We have found that the dissemination of CMV particles and the expression of β1 integrin are well correlated in the earliest phase of infection in both ICV and IV infections⁸. ICV infection is a model of ventriculoencephalitis and IV infection is a model of diffuse microglial nodules. Studying the dynamics of viral or fluorescent particles would give useful information on the effect of particle size, viral interactions with cellular receptors, and the mechanism of BBB penetration in the brain. The following protocol could be used to investigate any viral infection and viral vector in the CNS.

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 reaction the MCMV M32 (41,450 - 39,286 bp, left flanking sequence and M31 (37,089 - 39,246 bp, right flanking sequence gene loci using the following primers: M32, 5′-CTACTAGCTAGCCTTCCGCGAGTCGCTGTATT-3′ (forward primer), 5′-CTACTAGCTAGCCTTCCGCGAGTCGCTGTATT-3′ (reverse primer); M31, 5′-CTAAATTAACTTAAGTCGTCCTCTTCGTCTCACAA-3′ (forward primer), 5′-AACTAGTCTAGATCGCTCCTGGTTGGTTTTTA-3′ (reverse primer).
4. Insert the M32 locus into the EGFP-expressing vector using NheI and BamHI restriction sites. Insert the M31 locus into the M32-EGFP-recombinant plasmid using AflII and XbaI sites. Cleave the M32-EGFP-M31 sequence with NheI and AflII from the M32-EGFP-M31 recombination plasmid and dissolve in H₂O.
5. Transfect the cleaved construct into the nuclei of MCMV (Smith strain)-infected NIH3T3 cells 24 hr post-infection (hpi) using an electroporation system to induce homologous recombination.
6. At 3 days post-infection, co-culture the cells with uninfected mouse embryonic fibroblasts (MEFs) at a ratio of 1/1,000 in six-well plates and screen for EGFP-expressing foci of infected cells.
7. Harvest the virus-containing supernatants from the wells containing green fluorescent foci and dilute tenfold. For purification, choose wells that display single green fluorescent foci.
8. When all foci resulting from the limiting-dilution infection display EGFP expression, the virus preparation is pure, indicating that no wild-type virus remains. Quantify the virus by the plaque-assay method as previously described¹⁷.
9. Treat MCMV in certified biosafety cabinets at biosafety level 2 wearing gloves and mask.
10. Passage MCMV (Smith strain) and recombinant MCMV in MEFs prepared from 12-day-old ICR mouse embryos as previously described¹⁷.
11. Remove cells from the supernatants of infected MEF cultures by centrifugation at 3,000 × g for 20 min at 16 °C.
12. Ultracentrifuge the supernatants for 40 min at 70,000 × g. Resuspend the pellets containing virions in 1 ml of Tris-buffered saline and transfer onto a preformed linear sorbitol gradient (25% - 70%). Ultracentrifuge again at 70,000 x g for 60 min¹⁸.
13. Harvest the virion-containing band with a syringe. Pellet the harvested virions by an additional ultracentrifugation step at 70,000 × g for 40 min.
14. Resuspend the pellet in 1 ml of phosphate-buffered saline (PBS) and store at -80 °C until the infection experiments.
15. Quantify the virus titer by the plaque-assay method as previously described¹⁹.
16. Visualize the EGFP expression of the recombinant MCMV particles (excitation at 489 nm, emission at 508 nm) by fluorescent microscopy (Figure 1A) and the particle structure by transmission electron microscopy (TEM)⁸ (Figure 1B).

2. Preparation of Nile Red Fluorescent Microbeads

1. Purchase carboxyl fluorescent Nile red microbeads and place 500 µl of microbeads into tubes according to diameter (0.04 - 0.06 µm, approximately 3.63 × 10¹² particles; 0.1 - 0.3 µm, approximately 5.75 × 10¹⁰ particles; and 1.7 - 2.2 µm, approximately 6.85 × 10⁷ particles).
2. Treat beads with 500 µl of 0.1 M NaOH for approximately 1 day to remove any endotoxins and resuspend the beads in sterile water at RT.
3. Adsorb the beads O/N with 10% mouse serum obtained from C57BL/6 mice at RT prior to use.
4. To separate aggregates, vortex the beads and sonicate thoroughly before use.

3. ICV Injection of MCMV and Fluorescent Microbeads into Neonatal Mice

1. Maintain normal pregnant ICR mice in a temperature-controlled facility under a 12 hr light/dark cycle. The neonates are designated as P 0.5 on the day of birth.
2. Sterilize a 10-µl syringe and a 27 G needle with 70% alcohol.
3. Load the injection solution (5 µl) containing MCMV or fluorescent microbeads into the needle by carefully pulling the plunger of the syringe.
4. Restrain neonatal mouse (P 0.5) by putting the mouse on ice for 3 - 4 min. Once the animal is under anesthesia, use the toe-pinch response method to determine the depth of anesthesia.
5. Mark the injection site with a non-toxic laboratory pen at a location approximately 0.7 - 1.0 mm lateral to the sagittal suture and 0.7 - 1.0 mm caudal from the neonatal bregma (Figure 2A).
6. Insert the needle 2 mm deep, perpendicular to the skull surface at the marked injection site. For reference, mark 2 mm from the tip of needle with a non-toxic maker.
7. Slowly inject 5 µl of MCMV (approximately 5 × 10⁵ PFU) into the lateral ventricle without opening the scalp (Figure 2B).
8. In another group of mice, inject a 5 µl solution containing fluorescent microbeads (0.1 - 0.3 µm, approximately 5.75 × 10⁸ particles) by the same method.
9. Slowly remove the needle 10 - 20 sec after discontinuing the plunger movement to prevent backflow.
10. To recover, keep the mice for 5 - 10 min in a warm container until movement and general responsiveness are restored.
11. Harvest the brains as described in section 5 at a range of time points (3, 12, 24, 48, and 72 hr) post-injection.

4. IV Injection of MCMV or Fluorescent Microbeads into Neonatal Mice

1. Restrain neonatal mouse (P 0.5) by putting the mouse on ice for 3 - 4 min.
2. Use a 1-ml syringe with a 35 G needle to perform the intravenous injection of MCMV in P 0.5 neonates.
3. Use a transilluminator (vein finder) to visualize the superficial temporal (facial) vein. Before injection, secure the neonate to the transilluminator using surgical tape (Figure 2C).
4. While wearing magnifying glasses (1.5X), slowly infuse 50 µl of MCMV (approximately 5.45 × 10⁹ particles) or fluorescent microbeads (0.04 - 0.06 µm, approximately 3.63 × 10¹¹ particles; 0.1 - 0.3 µm, approximately 5.75 × 10⁹ particles; and 1.7 - 2.2 µm, approximately 6.85 × 10⁶ particles) into the superficial temporal vein (Figure 2D).
5. After removing the needle, use gauze containing 70% alcohol to apply pressure to the injection site until the bleeding ceases.
6. Give the neonate approximately 5 min in a warm container to recover before returning it to the cage.
7. Harvest the brains as described in section 5 at a range of time points (3, 12, 24, and 72 hr) post-injection.

5. Brain Tissue Sample Preparation for Paraffin Sections

1. Place the injected neonates in a small plastic dish on crushed ice.
2. Once the animal is under anesthesia, use the toe-pinch response method to determine the depth of anesthesia.
3. Make one central or two end horizontal end cuts through the rib cage to open up the thoracic cavity.
4. Make a cut in the atrium with sharp scissors. Infuse 4% paraformaldehyde (PFA) solution into the atrium. Stop perfusion when spontaneous movement (PFA dance) and lightened-color of the liver are observed. Do not exsanguinate.
5. Dissect the neonate (as previously described²⁰) by first removing the head using a pair of scissors.
6. Make a midline incision along the integument from the neck to the nose to expose the skull.
7. Place the sharp end of a pair of iris scissors into the foramen magnum on one side, carefully sliding along the inner surface of the skull.
8. Make a cut extending to the distal edge of the posterior skull surface, and make an identical cut on the contralateral side. Clear away the skull around the cerebellum.
9. Carefully peel back the skull on one side to prevent damage to the brain. Repeat this procedure on the other side of the brain.
10. Using a spatula, sever the olfactory bulbs and nervous connections along the ventral surface of the brain.
11. Gently separate the brain from the head, trimming any dura that still connect the brain to the skull using scissors, and remove the brain.
12. Place the brain in a vial of fixative containing 4% PFA at least 10 times the volume of the brain for 24 hr at 4°C.
13. Dehydrate the tissue through a series of graded ethanol baths to displace the water, and then infiltrate with paraffin wax, forming a block. Cut the paraffin block with a microtome into slices 4 µm thick²¹.
14. Deparaffinize and rehydrate the slides of the infected brains²². Proceed to in situ hybridization for paraffin embedded sections.

6. Brain Tissue Sample Preparation for Frozen Sections

1. Remove the brain following the steps outlined in 5.1 - 5.11.
2. To observe the fluorescent microbeads and M32-EGFP-MCMV, place the resected brain on flat bottom cryomolds. Add embedding medium to completely cover the brain sample. Snap freeze the cryomolds in precooled n-hexane (-80 ºC), using forceps to hold the edge of the cryomolds prior to attaching it to the chuck.
3. For sectioning, attach the frozen tissue block on the precooled cryostat chuck. Transfer the frozen tissue with the chuck into a cryostat chamber and lower the temperature to between -10 and -20 °C, and cut the slices approximately 8 - 10 µm thick²³.
4. Fix the sections with cytofixative (mixture of isopropyl alcohol and polyethylene glycol) by spraying. Air dry the sections for 30 min at RT immediately after spraying, and store them at - 80 ºC until further use.
5. Equilibrate the sections back to RT and wash three times with PBS.
6. Stain the sections with fluorescein isothiocyanate (FITC)-conjugated Griffonia simplicifolia isolectin B4 at a concentration of 1:100 for 10 min in PBS at RT (Figure 5) or with PE-conjugated CD31 antibody at a concentration of 1:100 for 30 min in PBS at RT (Figure 6).
7. Wash the sections with PBS three times.
8. After the washing, stain the sections with 4′,6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei. Mount the sections in anti-fade reagent and image DAPI (excitation at 345 nm, emission at 455 nm), EGFP (excitation at 489 nm, emission at 508 nm), and Nile red (excitation at 553 nm, emission at 637 nm) with a fluorescent microscope (Figures 3, 5, 6).

7. Fluorescent in situ Hybridization (FISH) for Paraffin Embedded Sections

1. Prepare the FISH probe labeled directly with fluorophore for DNA in situ hybridization by nick translation using a bacterial artificial chromosome containing the whole MCMV DNA genome (pSM3fr)¹⁹. The concentration of the FISH probe is 0.1 µg/µl.
2. Deparaffinize and rehydrate the slides of the infected brains²².
3. Treat the tissue sections with RNase (100 µg/ml in PBS) to detect viral DNA.
4. Perform antigen retrieval with a 0.05% NP40, 0.01 M citrate buffer (pH 6.0) at 95 - 98°C for 20 min. Cool the slides down to RT for 20 min.
5. In a glass Coplin staining jar, wash the slides in pure water three times for 2 min each time. Perform an additional antigen retrieval step with a 0.06% pepsin, 0.01 N HCl solution for 5 min at 37 °C.
6. Rinse slides three times in pure water for 2 min each time in a glass Coplin staining jar.
7. Dehydrate the tissue sections again by transferring the slides from 70% ethanol, to 85% ethanol, and then to 100% ethanol.
8. For preparing 10 ml of the hybridization buffer, mix 1.25 ml in situ hybridization salts (3 M NaCl, 100 mM Tris-HCl pH 8.0, 100 mM sodium phosphate pH 6.8, 50 mM EDTA) with 5 ml deionized formamide, 2.5 ml 50% Dextran sulfate, 250 µl 50x Denhardt's solution, 125 µl 100 mg/ml tRNA, and 875 µl H₂O.
9. Dilute the DNA probe directly labeled with fluorophores that recognize the whole MCMV genome randomly in the hybridization buffer. The final volume should be 10 µl (7 µl hybridization buffer, 1 µl probe (0.1 µg/µl), and 2 µl distilled water).
10. Add the probe mix (10 µl) to each slide and cover with a coverslip (15 × 15 mm). Seal the coverslip with rubber cement. Denature the probe mix for 5 min at 85 °C, and complete the hybridization step O/N at 42 °C.
11. Wash the slides with 0.3% NP40, 0.4x SSC at 73°C for 2 min; with 0.1% NP40, 0.4x SSC at 73 °C for 1 min; and with 2x SSC twice.
12. Counterstain the nuclei with DAPI (10 ng/ml) and cover the slide with a coverslip.
13. Mount the sections in anti-fade reagent and image DAPI (excitation at 345 nm, emission at 455 nm) and EGFP (excitation at 489 nm, emission at 508 nm) with a fluorescent microscope (Figure 4).

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

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