The authors acknowledge Charles River laboratories for their excellent technical support executing the histopathology analyses. This work was funded by National Institute of Neurological Disorders and Stroke (NINDS) (RO1 NS033506 and RO1 NS060699). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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
<ol>
	<li>Anesthetize a C57BL/6 mouse (5&#8211;7 weeks old) with isoflurane gas anesthetic, delivered at a dosage of 3% via a small anesthesia system (chamber).
	<ol>
		<li>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%&#8211;2.0%.</li>
		<li>Closely monitor the mouse for a response to painful stimulus created by forceful pinching of the toe with forceps.</li>
	</ol>
	</li>
	<li>Lightly grasp one hind footpad with flat forceps.
	<ol>
		<li>Gently abrade the glabrous skin of the hind footpad approximately 20x, between the heel and walking pads, with an emery board (100&#8211;180 grit). Do not induce bleeding by abrading too frequently or applying too much pressure.</li>
		<li>Using fine forceps, slowly peel off the stratum corneum detached by abrasion to expose the stratum basale.</li>
	</ol>
	</li>
</ol>
2. PRV footpad inoculation
<ol>
	<li>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).
	<ol>
		<li>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).</li>
		<li>Keep the virus inoculum on ice and gently mix before use.</li>
	</ol>
	</li>
	<li>Add a 20 &#956;L droplet of virus inoculum (8 x 106 PFU) onto the abraded footpad (topical administration). Carry out mock inoculations (medium only) in parallel.</li>
	<li>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.</li>
	<li>Keep the mouse under anesthesia for 30 min until the abraded footpad is dry.</li>
	<li>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.</li>
</ol>
3. Mouse dissection and tissue collection
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Pinch the external layer of fur and skin using forceps and make a small initial incision using fine scissors near the urethral opening.
	<ol>
		<li>From this opening, continue the incision on the mid ventral side up to the chin.</li>
		<li>Extend two lateral incisions towards the extremities of the forelimbs and hind limbs.</li>
		<li>Separate the skin from the underlying muscular layer and pin down to the side.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Collect exposed organs, including heart, lungs, spleen, pancreas, liver, kidneys, and bladder in 1.5 mL microtubes. Keep the tubes on ice.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Place the mouse dorsal side up and wet the fur with 70% ethanol. Cut down the skin layer to expose the vertebral column.
	<ol>
		<li>Make a small incision in the region of the pelvis. Strip the skin from the hind limbs towards the head.</li>
		<li>Remove the legs and arms by cutting with scissors parallel with and close to the spinal column on both sides.</li>
		<li>Cut the head off at the base of the skull (C1&#8211;C2 level).</li>
		<li>Cut open the skull with scissors from the foramen magnum to the frontal bone.</li>
		<li>Pull open the skull in lateral directions using forceps.</li>
		<li>Gently scoop out the brain using forceps and proceed as described in step 3.3.6.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Remove the vertebrae column from ice-cold PBS tube and remove remaining soft tissues, which becomes easier after refrigeration.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Collect both spinal cord halves in 1.5 mL microtubes containing 500 &#956;L of sterile ice-cold PBS. Keep the tubes on ice.</li>
	<li>Gently remove the meninges from one side of the spinal column to the other to expose the DRG.</li>
	<li>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.</li>
	<li>Repeat steps 4.3&#8211;4.7 using the two other spinal column segments within 30&#8211;45 min.</li>
	<li>Centrifuge all tubes at high speed (17,900 x g) for 3 min at 4 &#176;C.</li>
	<li>Aspirate supernatant and flash-freeze tubes in liquid nitrogen. Store tubes at -80 &#176;C for further tissue homogenization.</li>
	<li>Alternatively, fix and section the cleaned DRG and other mouse tissues for histopathological analyses and/or immunofluorescence staining after step 4.9.</li>
</ol>
5. Tissue homogenization
<ol>
	<li>Thaw the tubes containing frozen tissue samples on ice.</li>
	<li>Weigh 100 mg of tissue and place it in a 2 mL microcentrifuge tube containing a sterile steel bead and 500 &#956;L 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.</li>
	<li>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.</li>
	<li>Centrifuge the tube at high speed (17,900 x g) for 10 min at RT.</li>
	<li>Collect supernatant (500 &#956;L) in a new tube and store it at -20 &#176;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).</li>
</ol>
6. Preparation and immunofluorescence staining of frozen DRG sections
<ol>
	<li>Tissue processing
	<ol>
		<li>Fix the dissected and cleaned DRG in 1% paraformaldehyde (PFA) for 2 h at RT.</li>
		<li>Transfer the tissue into a 15 mL tube with 10% sucrose in PBS. Incubate overnight at 4 &#176;C.</li>
		<li>Transfer the tissue into a 15 mL tube with 20% sucrose in PBS. Incubate overnight at 4 &#176;C.</li>
		<li>Transfer the tissue into a 15 mL tube with 30% sucrose in PBS. Incubate overnight at 4 &#176;C.</li>
		<li>Embed tissues in OCT using a cryomold and place blocks in dry ice for rapid freezing.</li>
		<li>Store the samples at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>Cryosectioning preparation
	<ol>
		<li>Remove the frozen block from -80 &#176;C and allow it to equilibrate in the cryostat chamber temperature for 30 min.</li>
		<li>Cryosection the DRG at a thickness of 15 &#181;m and mount the sections onto slides.</li>
		<li>Use a pen to draw a hydrophobic circle around the slide-mounted tissue.</li>
		<li>Keep the slides at 4 &#176;C until staining.</li>
	</ol>
	</li>
	<li>Immunofluorescence staining
	<ol>
		<li>Wash the DRG sections 3x in PBS for 10 min at RT.</li>
		<li>Incubate the sections with 100 &#181;L of desired primary antibody (e.g., mouse antibody against PRV glycoprotein B) for 1 h at 37 &#176;C. Dilute the primary antibody in PBS containing 10% negative goat serum.</li>
		<li>Repeat step 6.3.1.</li>
		<li>Incubate the sections with 100 &#181;L of desired secondary antibody (goat anti-mouse Alexa Fluor 488) for 50 min at 37 &#176;C. Dilute secondary antibody in PBS only.</li>
		<li>Add 100 &#181;L of DAPI (4&#8242;,6-diamidino-2-phenylindole) dye diluted in PBS onto the section. Further incubate samples 10 min at 37 &#176;C.</li>
		<li>Repeat step 6.3.1.</li>
		<li>Mount the samples using a fluorescence mounting medium onto glass slides and secure and cover with coverslip.</li>
	</ol>
	</li>
</ol>
7. Preparation and H&#38;E staining of paraffin-embedded tissue sections
<ol>
	<li>Fix the tissue in 10% formalin for 24 h at 4 &#176;C.</li>
	<li>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.</li>
	<li>Perform standard deparaffinization followed by H&#38;E staining of tissue sections, as previously described9.</li>
</ol>

<ol>
	<li>Field, H. J., Hill, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+pseudorabies+in+mice+following+peripheral+inoculation.">The pathogenesis of pseudorabies in mice following peripheral inoculation.</a> Journal of General Virology. 23 (2), 145-157 (1974).</li><li>Engel, J. P., Madigan, T. C., Peterson, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transneuronal+spread+phenotype+of+herpes+simplex+virus+type+1+infection+of+the+mouse+hind+footpad.">The transneuronal spread phenotype of herpes simplex virus type 1 infection of the mouse hind footpad.</a> Journal of Virology. 71 (3), 2425-2435 (1997).</li><li>Guedon, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+changes+induced+by+Varicella+Zoster+Virus+in+a+rat+model+of+postherpetic+neuralgia.">Neuronal changes induced by Varicella Zoster Virus in a rat model of postherpetic neuralgia.</a> Virology. 482, 167-180 (2015).</li><li>Pomeranz, L. E., Reynolds, A. E., Hengartner, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+pseudorabies+virus:+impact+on+neurovirology+and+veterinary+medicine.">Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine.</a> Microbiology and Molecular Biology Reviews. 69 (3), 462-500 (2005).</li><li>Wittmann, G., Rziha, H. J., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aujeszky's+disease+(pseudorabies)+in+pigs.">Aujeszky's disease (pseudorabies) in pigs.</a> Herpesvirus diseases of cattle, horses and pigs. 9, 230-325 (1989).</li><li>Leman, A. D., Glock, R. D., Mengeling, W. L., Penny, R. H. C., Scholl, E., Straw, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Diseases of swine, 6th ed. , 209-223 (1986).</li><li>Sleigh, J. N., Weir, G. A., Schiavo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+step-by-step+dissection+protocol+for+the+rapid+isolation+of+mouse+dorsal+root+ganglia.">A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia.</a> BMC Research Notes. 9, 82 (2016).</li><li>Sands, S. A., Leung-Toung, R., Wang, Y., Connelly, J., LeVine, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Histochemical+Detection+of+Iron+in+Paraffin+Sections+of+Mouse+Central+Nervous+System+Tissue:+Application+in+the+APP/PS1+Mouse+Model+of+Alzheimer's+Disease.">Enhanced Histochemical Detection of Iron in Paraffin Sections of Mouse Central Nervous System Tissue: Application in the APP/PS1 Mouse Model of Alzheimer's Disease.</a> ASN Neuro. 8 (5), (2016).</li><li>Cardiff, R. D., Miller, C. H., Munn, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+hematoxylin+and+eosin+staining+of+mouse+tissue+sections.">Manual hematoxylin and eosin staining of mouse tissue sections.</a> Cold Spring Harbor Protocols. 2014 (6), 655-658 (2014).</li><li>Koyuncu, O. O., MacGibeny, M. A., Hogue, I. B., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmented+neuronal+cultures+reveal+two+distinct+mechanisms+for+alpha+herpesvirus+escape+from+genome+silencing.">Compartmented neuronal cultures reveal two distinct mechanisms for alpha herpesvirus escape from genome silencing.</a> PLoS pathogens. 13 (10), 1006608 (2017).</li><li>Laval, K., Vernejoul, J. B., Van Cleemput, J., Koyuncu, O. O., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulent+Pseudorabies+Virus+Infection+Induces+a+Specific+and+Lethal+Systemic+Inflammatory+Response+in+Mice.">Virulent Pseudorabies Virus Infection Induces a Specific and Lethal Systemic Inflammatory Response in Mice.</a> Journal of Virology. 92 (24), 01614-01618 (2018).</li><li>Laval, K., Van Cleemput, J., Vernejoul, J. B., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alphaherpesvirus+infection+of+mice+primes+PNS+neurons+to+an+inflammatory+state+regulated+by+TLR2+and+type+I+IFN+signaling.">Alphaherpesvirus infection of mice primes PNS neurons to an inflammatory state regulated by TLR2 and type I IFN signaling.</a> PLoS Pathogens. 15 (11), 1008087 (2019).</li><li>Brittle, E. E., Reynolds, A. E., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+pseudorabies+virus+neuroinvasion+and+lethality+in+mice.">Two modes of pseudorabies virus neuroinvasion and lethality in mice.</a> Journal of Virology. 78 (23), 12951-12963 (2004).</li><li>Mancini, M., Vidal, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+pathogenesis+of+herpes+simplex+encephalitis+from+mouse+models.">Insights into the pathogenesis of herpes simplex encephalitis from mouse models.</a> Mammalian Genome: Official Journal of the International Mammalian Genome Society. 29 (7-8), 425-445 (2018).</li><li>Kopp, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+neurons+and+encephalitis+after+intracranial+inoculation+of+herpes+simplex+virus+requires+the+entry+receptor+nectin-1.">Infection of neurons and encephalitis after intracranial inoculation of herpes simplex virus requires the entry receptor nectin-1.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (42), 17916-17920 (2009).</li><li>Wang, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+specific+innate+immune+responses+in+herpes+simplex+virus+infection+of+the+central+nervous+system.">Role of specific innate immune responses in herpes simplex virus infection of the central nervous system.</a> Journal of Virology. 86 (4), 2273-2281 (2012).</li><li>Haberthur, K., Messaoudi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+varicella+zoster+virus+infection.">Animal models of varicella zoster virus infection.</a> Pathogens. 2 (2), 364-382 (2013).</li><li>Sarova-Pinhas, I., Achiron, A., Gilad, R., Lampl, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+neuropathy+in+multiple+sclerosis:+a+clinical+and+electrophysiologic+study.">Peripheral neuropathy in multiple sclerosis: a clinical and electrophysiologic study.</a> Acta Neurologica Scandinavia. 91 (4), 234-238 (1995).</li><li>MacGibeny, M. A., Koyuncu, O. O., Wirblich, C., Schnell, M. J., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+axonal+transport+of+rabies+virus+is+unaffected+by+interferon+treatment+but+blocked+by+emetine+locally+in+axons.">Retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons.</a> PLoS Pathogens. 14 (7), 1007188 (2018).</li><li>Hunsperger, E. A., Roehrig, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+analyses+of+the+neuropathogenesis+of+a+West+Nile+virus+infection+in+mice.">Temporal analyses of the neuropathogenesis of a West Nile virus infection in mice.</a> Journal of Neurovirology. 12 (2), 129-139 (2006).</li><li>Swartwout, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Persistently+and+Productively+Infects+Primary+Adult+Sensory+Neurons+In+Vitro.">Zika Virus Persistently and Productively Infects Primary Adult Sensory Neurons In Vitro.</a> Pathogens. 6 (4), 49 (2017).</li><li>Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+hundred+years+of+poliovirus+pathogenesis.">One hundred years of poliovirus pathogenesis.</a> Virology. 344 (1), 9-16 (2006).</li></ol>

The authors have nothing to disclose.

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...

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 neurons1,2,3. 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 &#8220;mad itch&#8221;, followed by peracute death5,6. 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.

<table><tbody><tr><td>Antibody anti-PRV gB</td><td>Made by the lab</td><td></td><td>1/500 dilution</td></tr><tr><td>Aqua-hold2 pap pen red</td><td>Fisher scientific</td><td>2886909</td><td></td></tr><tr><td>Compact emery boards-24 count (100/180 grit nail files)</td><td>Revlon</td><td></td><td></td></tr><tr><td>Complete EDTA-free Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>11836170001</td><td></td></tr><tr><td>C57BL/6 mice (5-7 weeks)</td><td>The Jackson Laboratories</td><td></td><td></td></tr><tr><td>DAPI solution (1mg/ml)</td><td>Fisher scientific</td><td>62248</td><td>1/1000 dilution</td></tr><tr><td>Disposable sterile polystyrene petri dish 100 x 15 mm</td><td>Sigma-Aldrich</td><td>P5731500</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Hyclone, GE Healthcare life Sciences</td><td>SH30022</td><td></td></tr><tr><td>Dulbecco&#039;s Phophate Buffer Saline (PBS) solution</td><td>Hyclone, GE Healthcare life Sciences</td><td>SH30028</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone, GE Healthcare life Sciences</td><td>SH30088</td><td></td></tr><tr><td>Fine curved scissors stainless steel</td><td>FST</td><td>14095-11</td><td></td></tr><tr><td>Fluoromount-G mounting media</td><td>Fisher scientific</td><td>0100-01</td><td></td></tr><tr><td>Formalin solution, neutral buffered 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Isothesia Isoflurane</td><td>Henry Schein</td><td>NDC 11695-6776-2</td><td></td></tr><tr><td>Microcentrifuge tube 2ml</td><td>Denville Scientific</td><td>1000945</td><td></td></tr><tr><td>Microtube 1.5ml</td><td>SARSTEDT</td><td>72692005</td><td></td></tr><tr><td>Negative goat serum</td><td>Vector</td><td>S-1000</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>154022</td><td></td></tr><tr><td>Precision Glide needle 18G</td><td>BD</td><td>305196</td><td></td></tr><tr><td>Razor blades steel back</td><td>Personna</td><td>9412071</td><td></td></tr><tr><td>RNA lysis buffer (RLT)</td><td>Qiagen</td><td>79216</td><td></td></tr><tr><td>Stainless Steel Beads, 5 mm</td><td>Qiagen</td><td>69989</td><td></td></tr><tr><td>Superfrost/plus microscopic slides</td><td>Fisher scientific</td><td>12-550-15</td><td></td></tr><tr><td>Tissue lyser LT</td><td>Qiagen</td><td>69980</td><td></td></tr><tr><td>Tissue-Tek OCT</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>488 (goat anti-mouse)</td><td>Life Technologies</td><td>A11029</td><td>1/2000 dilution</td></tr></tbody></table>

mouse footpad inoculation model to study viral-induced neuroinflammatory responses

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.

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...

Watch this Scientific Journal Video about Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses at JoVE.com

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

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.

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

mouse-footpad-inoculation-model-to-study-viral-induced

Research

JoVE Journal

Immunology and Infection

10.5K Views.  Princeton University. Dit protocol maakt het mogelijk de studie van de initiatie en ontwikkeling van neuro-inflammatoire reacties in de muis perifere zenuwstelsel tijdens een virale infectie. Met dit model, infecties reizen een grotere afstand van de periferie naar het perifere en centrale zenuwstelsel, waardoor een maximale beoordeling van de kinetiek van de neuro-inflammatoire reactie. Na het plaatsen van de vijf tot zeven weken oude verdoofde C57BL/6 muis in de supine positie op een chirurgische pad, bevestig e...

Video: Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

Neuroscience

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice

Receptor-mediated lysophospholipid signaling contributes to the pathophysiology of diverse neurological diseases, especially multiple sclerosis (MS). Lysophosphatidylcholine (LPC) is an endogenous lysophospholipid associated with inflammation, and it could induce rapid damage with toxicity to myelin lipids, leading to focal demyelination. Here, a detailed protocol is presented for stereotactic two-point LPC injection that could directly cause severe demyelination and replicate the experimental demyelination injury quickly and stably in mice by surgical procedure. Thus, this model is highly relevant to demyelination diseases, especially MS, and it can contribute to the related advancing clinically-relevant research. Also, immunofluorescence and Luxol fast blue staining methods were used to depict the time course of demyelination in the corpus callosum of mice injected with LPC. In addition, the behavioral method was used to evaluate the cognitive function of mice after modeling. Overall, the two-point injection of lysophosphatidylcholine via a stereotaxic frame is a stable and reproducible method to generate a demyelination model in mice for further study.

The present protocol describes a two-point injection of lysophosphatidylcholine via a stereotaxic frame to generate a stable and reproducible demyelination model in mice.

Receptor-mediated lysophospholipid signaling contributes to the pathophysiology of diverse neurological diseases, especially multiple sclerosis (MS). ...

Induction of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Source: Schmitz, K., et al. <a href="https://app.jove.com/v/55321">Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice.</a> J. Vis. Exp (2017).
In this video, a mouse model of multiple sclerosis is established using experimental autoimmune encephalomyelitis (EAE). The mouse is injected with an emulsion containing a neuronal peptide and an adjuvant, triggering an immune response that activates autoreactive T cells. This is followed by an injection of pertussis toxin (PTX) to increase blood-brain barrier permeability, allowing the autoreactive T-cells to infiltrate the brain. This infiltration initiates a signaling cascade that ultimately results in myelin damage, mimicking the demyelinating condition of multiple sclerosis.

1. EAE Induction in SJL/J Mice

	Mice
	
		Use 11-week-old female SJL/J mice and allow them to habituate to the experimental room for about 7 days. Use n = ...

JoVE Encyclopedia of Experiments

Neuropathology

Neuromuscular Disorders

Assessing Motor Function in an Experimental Autoimmune Neuritis Mouse Model

Source: Gonsalvez, D. G., et al., <a href="https://app.jove.com/v/56455">A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments.</a> J. Vis. Exp. (2017).
This video demonstrates the process of assessing motor dysfunction in a mouse model through the administration of Pertussis toxin and an inoculum containing myelin-mimicking peptides and adjuvants, followed by treadmill tests to assess motor function and evaluate the resulting nerve damage due to immune activation and demyelination.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. EAN ...

Generation of a Murine Model with a Central Nervous System Infection

Source: Shen, T., et al. <a href="https://app.jove.com/v/59132">A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease.</a> J. Vis. Exp. (2019)
This video demonstrates the generation of a mouse model with a CNS infection. A suckling pup is injected with the virus both intracranially and intraperitoneally. The virus spreads, infecting the CNS causing neuronal cell death and inflammation. The pup is assessed for symptoms of infection as the disease progresses.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video