Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

Summary

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

Abstract

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

Introduction

This study describes a footpad inoculation model to investigate the replication and spread of viruses from the PNS to CNS and associated neuroinflammatory responses. The footpad inoculation model has been intensively used to study alphaherpesvirus infection in neurons^1,2,3. The main objective of this model is to allow neurotropic viruses to travel a maximal distance through the PNS before reaching the CNS. Here, this model is used to obtain new insights in the development of a particular neuropathy (neuropathic itch) in mice infected with pseudorabies virus (PRV).

PRV is an alphaherpesvirus related to several well-known pathogens (i.e., herpes simplex type 1 and 2 [HSV1 and HSV2] and varicella-zoster virus [VZV]), which cause cold sores, genital lesions, and chicken pox, respectively⁴. These viruses are all pantropic and able to infect many different cell types without showing affinity for a specific tissue type. However, they all exhibit a characteristic neurotropism by invading the PNS (and occasionally, the CNS) of host species. The natural host is the pig, but PRV can infect most mammals. In these non-natural hosts, PRV infects the PNS and induces a severe pruritus called the “mad itch”, followed by peracute death^5,6. The role of the neuroimmune response in the clinical outcome and pathogenesis of PRV infection has been poorly understood.

The footpad inoculation model allows PRV to initiate infection in the epidermal cells of the footpad. Then, the infection spreads into sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads by virus particles moving via the sciatic nerve to the DRG within approximately 60 h. The infection spreads through the spinal cord, ultimately reaching the hindbrain when animals become moribund (82 h post-infection). During this time window, tissue samples can be collected, processed, and analyzed for virus replication and markers of the immune response. For instance, histological examination and viral load quantification can be performed in different tissues to establish correlations between the initiation and development of clinical, virological, and neuroinflammatory processes in PRV pathogenesis.

Using the footpad inoculation model, the cellular and molecular mechanisms of PRV-induced pruritus in mice can be investigated. Moreover, this model can provide new insight into the initiation and development of virus-induced neuroinflammation during herpesvirus infections. A better understanding of the processes underlying alphaherpesvirus-induced neuropathies may lead to the development of innovative therapeutic strategies. For instance, this model is useful to investigate the mechanisms of neuropathic itch in patients with post-herpetic lesions (e.g., herpes zoster, shingles) and test novel therapeutic targets in mice for the corresponding human diseases.

Protocol

All animal experiments were performed in accordance to a protocol (number 2083-16 and 2083-19) reviewed and approved by the Institution Animal Care and Use Committee (IACUC) of Princeton University. This work was done by strictly following the biosafety level-2 (BSL-2) requirements, to which we have a fully equipped lab approved by the Princeton University biosafety committee. The procedures including mouse footpad abrasion, viral inoculation, mouse dissection, and tissue collection were performed in a biological safety cabinet (BSC) in the Princeton University biocontainment animal facility room. Those performing the procedure wore disposable gowns, head cover, eye protection, sterile gloves, surgical masks, and shoe covers.

1. Mouse footpad abrasion

1. Anesthetize a C57BL/6 mouse (5–7 weeks old) with isoflurane gas anesthetic, delivered at a dosage of 3% via a small anesthesia system (chamber).
   1. Place the mouse in the surgical plane of anesthesia on its back, prior to inoculation, in the hind footpad. Attach a nose cone with a diaphragm (a slit sufficiently sized to fit the muzzle of the animal) to the mouse and deliver isoflurane gas anesthetic at a constant dosage of 1.5%–2.0%.
   2. Closely monitor the mouse for a response to painful stimulus created by forceful pinching of the toe with forceps.
2. Lightly grasp one hind footpad with flat forceps.
   1. Gently abrade the glabrous skin of the hind footpad approximately 20x, between the heel and walking pads, with an emery board (100–180 grit). Do not induce bleeding by abrading too frequently or applying too much pressure.
   2. Using fine forceps, slowly peel off the stratum corneum detached by abrasion to expose the stratum basale.

2. PRV footpad inoculation

1. Prepare the virus inoculum diluted to the desired titer in DMEM media containing 2% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Table of Materials).
   1. Quantitate the number of plaque-forming units (PFU) in the virus stock on PK15 cells to calculate and standardize the viral dose and dilute the viral inoculum accordingly.
      NOTE: In this study, virulent strain of PRV (PRV-Becker) was used at a dose of 8 x 10⁶ PFU. This dose was optimized in a previous preliminary experiment to ensure that all inoculated animals showed clinical symptoms at 82 h post-inoculation (hpi).
   2. Keep the virus inoculum on ice and gently mix before use.
2. Add a 20 μL droplet of virus inoculum (8 x 10⁶ PFU) onto the abraded footpad (topical administration). Carry out mock inoculations (medium only) in parallel.
3. Gently rub 10x with the shaft of a needle to facilitate adsorption of the virus. Avoid scratching with the needle point. Repeat this step every 10 min.
4. Keep the mouse under anesthesia for 30 min until the abraded footpad is dry.
5. After stopping anesthesia, monitor the mouse until it can maintain sternal recumbency and place it in an individual cage for clinical follow-up and sampling.

3. Mouse dissection and tissue collection

1. At appropriate times after infection, euthanize the mouse by the asphyxiation method (CO₂).
   NOTE: In this study, the humane endpoint (when animals begin to exhibit terminal symptoms) for PRV-Becker infected mice is 82 hpi. Euthanize control animals in parallel.
2. Place the mouse ventral side facing up on a surgical mat using needles/pins. Secure the limbs of the mouse with pins to a surgical foam board. Wet the ventral side of the mouse with 70% ethanol to minimize fur contamination.
3. Pinch the external layer of fur and skin using forceps and make a small initial incision using fine scissors near the urethral opening.
   1. From this opening, continue the incision on the mid ventral side up to the chin.
   2. Extend two lateral incisions towards the extremities of the forelimbs and hind limbs.
   3. Separate the skin from the underlying muscular layer and pin down to the side.
   4. Open the abdominal cavity and incise up to the base of the thorax. Complete the opening of the abdominal cavity by making two transversal incisions.
   5. Open the thoracic cavity by cutting the diaphragm and ribs on both lateral sides.
      NOTE: Cutting the sides of the ribcage prevents the risk of cutting the heart.
   6. Collect exposed organs, including heart, lungs, spleen, pancreas, liver, kidneys, and bladder in 1.5 mL microtubes. Keep the tubes on ice.
   7. Cut the abraded footpad between the heel and walking pads and place the piece of tissue in a tube as described in step 3.3.6.
4. Place the mouse dorsal side up and wet the fur with 70% ethanol. Cut down the skin layer to expose the vertebral column.
   1. Make a small incision in the region of the pelvis. Strip the skin from the hind limbs towards the head.
   2. Remove the legs and arms by cutting with scissors parallel with and close to the spinal column on both sides.
   3. Cut the head off at the base of the skull (C1–C2 level).
   4. Cut open the skull with scissors from the foramen magnum to the frontal bone.
   5. Pull open the skull in lateral directions using forceps.
   6. Gently scoop out the brain using forceps and proceed as described in step 3.3.6.
5. Clean the spinal column by cutting muscle, fat and soft tissues using curved scissors. Cut the tail. Place the intact spinal column in a 15 mL tube containing sterile ice-cold phosphate-buffered saline (PBS). Keep tubes on ice until further dissection of the spinal cord and DRG.

4. Spinal cord and DRG extraction

NOTE: Extract spinal cord and DRG from the vertebrae column directly following mouse dissection. The protocol for spinal cord and DRG extraction has been adapted from a previous publication⁷.

1. Remove the vertebrae column from ice-cold PBS tube and remove remaining soft tissues, which becomes easier after refrigeration.
2. Make three transverse cuts in the column through the vertebrae (T1, L1, and S2 levels) using a razor blade. Place the three pieces in a 15 mm Petri dish containing sterile ice-cold PBS. Keep track and mark origin of segments for later analysis.
3. Take one segment and secure it between forceps, dorsal side facing up. Make a single, longitudinal cut down through the midline using a razor blade, splitting the column in two equal halves.
4. Place both halves in a new Petri dish containing new sterile ice-cold PBS. Ensure the correct orientation of the segment to be able to distinguish left and right sides.
5. Under a dissecting microscope, gently peel the spinal cord out of the vertebrae column from each half in a rostral to caudal direction using fine forceps.
6. Collect both spinal cord halves in 1.5 mL microtubes containing 500 μL of sterile ice-cold PBS. Keep the tubes on ice.
7. Gently remove the meninges from one side of the spinal column to the other to expose the DRG.
8. Remove each DRG individually by grasping the exposed spinal nerve and pull it gently out of the spinal column. Place the collected DRG in a 15 mm Petri dish containing ice-cold PBS. Harvest all ipsilateral and contralateral DRG separately from the spinal column segment and proceed as described in step 4.5.
   NOTE: The DRG is visible as a round transparent structure along the white spinal nerve.
9. Repeat steps 4.3–4.7 using the two other spinal column segments within 30–45 min.
10. Centrifuge all tubes at high speed (17,900 x g) for 3 min at 4 °C.
11. Aspirate supernatant and flash-freeze tubes in liquid nitrogen. Store tubes at -80 °C for further tissue homogenization.
12. Alternatively, fix and section the cleaned DRG and other mouse tissues for histopathological analyses and/or immunofluorescence staining after step 4.9.

5. Tissue homogenization

1. Thaw the tubes containing frozen tissue samples on ice.
2. Weigh 100 mg of tissue and place it in a 2 mL microcentrifuge tube containing a sterile steel bead and 500 μL of RIPA buffer containing 0.5 M EDTA (pH = 8.0), 1 M Tris-HCl (pH = 8.0), 5 M NaCl, 10% SDS, and protease cocktail inhibitor tablets.
3. Disrupt tissues at room temperature (RT) using a homogenizer at 20 cycles/s for 2 min, followed by a 1 min waiting period, then 20 cycles/s for 2 min.
4. Centrifuge the tube at high speed (17,900 x g) for 10 min at RT.
5. Collect supernatant (500 μL) in a new tube and store it at -20 °C until performing ELISA and qPCR to quantify inflammatory markers and viral loads, respectively.
   NOTE: For qPCR, collect all samples with RNase-free material (i.e., beads, tubes, etc.) and disrupt tissues with RNA lysis buffer containing 1% betamercaptoethanol (Table of Materials).

6. Preparation and immunofluorescence staining of frozen DRG sections

1. Tissue processing
   1. Fix the dissected and cleaned DRG in 1% paraformaldehyde (PFA) for 2 h at RT.
   2. Transfer the tissue into a 15 mL tube with 10% sucrose in PBS. Incubate overnight at 4 °C.
   3. Transfer the tissue into a 15 mL tube with 20% sucrose in PBS. Incubate overnight at 4 °C.
   4. Transfer the tissue into a 15 mL tube with 30% sucrose in PBS. Incubate overnight at 4 °C.
   5. Embed tissues in OCT using a cryomold and place blocks in dry ice for rapid freezing.
   6. Store the samples at -80 °C until use.
2. Cryosectioning preparation
   1. Remove the frozen block from -80 °C and allow it to equilibrate in the cryostat chamber temperature for 30 min.
   2. Cryosection the DRG at a thickness of 15 µm and mount the sections onto slides.
   3. Use a pen to draw a hydrophobic circle around the slide-mounted tissue.
   4. Keep the slides at 4 °C until staining.
3. Immunofluorescence staining
   1. Wash the DRG sections 3x in PBS for 10 min at RT.
   2. Incubate the sections with 100 µL of desired primary antibody (e.g., mouse antibody against PRV glycoprotein B) for 1 h at 37 °C. Dilute the primary antibody in PBS containing 10% negative goat serum.
   3. Repeat step 6.3.1.
   4. Incubate the sections with 100 µL of desired secondary antibody (goat anti-mouse Alexa Fluor 488) for 50 min at 37 °C. Dilute secondary antibody in PBS only.
   5. Add 100 µL of DAPI (4′,6-diamidino-2-phenylindole) dye diluted in PBS onto the section. Further incubate samples 10 min at 37 °C.
   6. Repeat step 6.3.1.
   7. Mount the samples using a fluorescence mounting medium onto glass slides and secure and cover with coverslip.

7. Preparation and H&E staining of paraffin-embedded tissue sections

1. Fix the tissue in 10% formalin for 24 h at 4 °C.
2. Perform standard processing schedule for paraffin embedding and sectioning of fixed tissues. Transfer fixed tissues to 70% ethanol and process through upgraded alcohols to xylene followed by paraffin, as previously described⁸.
   NOTE: Use polyester sponges to keep small tissue samples such as DRG inside cassettes.
3. Perform standard deparaffinization followed by H&E staining of tissue sections, as previously described⁹.

Results

The mouse footpad inoculation model allows for characterization of the immunopathogenesis of alphaherpesvirus infection in vivo, including replication and spread of the infection from the inoculated footpad to the nervous system and the induction of specific neuroinflammatory responses.

In this study, we first abraded the mouse hind footpad and either mock-inoculated or inoculated the abraded region with a virulent strain of PRV (PRV-Becker). The site of abrasion was visible in the control foo...

Discussion

The footpad inoculation model described here is useful to investigate the initiation and development of neuroinflammatory responses during alphaherpesvirus infection. Moreover, this in vivo model is used to establish the kinetics of replication and spread of alphaherpesvirus from the PNS to CNS. This is an alternate to other inoculation models, such as the flank skin inoculation model, which relies on deep dermal scratching¹³, or the intracranial route, which directly introduces the virus into the...

Materials

Name	Company	Catalog Number	Comments
Antibody anti-PRV gB	Made by the lab		1/500 dilution
Aqua-hold2 pap pen red	Fisher scientific	2886909
Compact emery boards-24 count (100/180 grit nail files)	Revlon
Complete EDTA-free Protease Inhibitor Cocktail	Sigma-Aldrich	11836170001
C57BL/6 mice (5-7 weeks)	The Jackson Laboratories
DAPI solution (1mg/ml)	Fisher scientific	62248	1/1000 dilution
Disposable sterile polystyrene petri dish 100 x 15 mm	Sigma-Aldrich	P5731500
Dulbecco's Modified Eagle Medium (DMEM)	Hyclone, GE Healthcare life Sciences	SH30022
Dulbecco's Phophate Buffer Saline (PBS) solution	Hyclone, GE Healthcare life Sciences	SH30028
Fetal bovine serum (FBS)	Hyclone, GE Healthcare life Sciences	SH30088
Fine curved scissors stainless steel	FST	14095-11
Fluoromount-G mounting media	Fisher scientific	0100-01
Formalin solution, neutral buffered 10%	Sigma-Aldrich	HT501128
Isothesia Isoflurane	Henry Schein	NDC 11695-6776-2
Microcentrifuge tube 2ml	Denville Scientific	1000945
Microtube 1.5ml	SARSTEDT	72692005
Negative goat serum	Vector	S-1000
Penicillin/Streptomycin	Gibco	154022
Precision Glide needle 18G	BD	305196
Razor blades steel back	Personna	9412071
RNA lysis buffer (RLT)	Qiagen	79216
Stainless Steel Beads, 5 mm	Qiagen	69989
Superfrost/plus microscopic slides	Fisher scientific	12-550-15
Tissue lyser LT	Qiagen	69980
Tissue-Tek OCT	Sakura	4583
488 (goat anti-mouse)	Life Technologies	A11029	1/2000 dilution