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 ...

<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&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Hyclone, GE Healthcare life Sciences</td>
			<td>SH30022</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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 ...

This protocol allows the study of the initiation and development of neuroinflammatory responses in the mouse peripheral nervous system during a viral infection. With this model, infections travel a greater distance from the periphery to the peripheral and central nervous system, allowing a maximal assessment of the kinetics of the neuroinflammatory response. After placing the five to seven week old anesthetized C57BL/6 mouse in the supine position on a surgical pad, confirm a lack of response to pedal reflex.And use a 100 to 180 grit emery board, to gently abrade the glabrous skin of one hind footpad between the heel and walking pads with about 20 passes of the board. Then use fine forceps to slowly peel off the stratum corneum detached by the abrasion to expose the stratum basale. After gentle mixing, topically apply 20 microliters of the appropriate titer of virus inoculum onto the abraded footpad.Carry out mock inoculations with medium only in the abraded footpads of control animals in parallel. After each application, gently rub the footpad 10 times with the shaft of a needle to facilitate adsorption of the virus every 10 minutes for 30 minutes. When the abraded footpad is dried, allow the mice to recover with monitoring until sternal recumbency before placing the animals into individual cages for clinical follow-up and sampling.At the appropriate experimental time points, collect the virus exposed organs, including the heart, lungs, spleen, pancreas, liver, kidneys, and bladder into individual 1.5 milliliter microtubes on ice. Then harvest the abraded footpads between the heel and walking pads and place the samples into individual 1.5 milliliter microtubes on ice. Next, place the mouse in the prone position and wet the fur with 70%ethanol.To expose the vertebral column, make a small incision in the region of the pelvis and strip the skin from the hind limbs toward the head. To remove the fore and hind limbs, use scissors to cut parallel with and close to the spinal column on both sides. And remove the head at the base of the skull.Use the scissors to open the skull from the foramen magnum to the frontal bone. And use forceps to pull open the skull in a lateral direction. Then use the forceps to gently scoop out the brain and place the brain into a 1.5 milliliter microtube on ice.Use curved scissors to clean the spinal column of muscle, fat, and soft tissue, and remove the tail. Then place the intact spinal column into a 15 milliliter tube of sterile ice-cold PBS on ice for no more than three hours. After the dissection, transfer the spinal cord into a tissue culture dish to allow removal of any remaining soft tissue.Use a razor blade to make three transverse cuts through the spinal column at the S2, L1, and the T1 vertebrae. Transfer the pieces into individual 15 millimeter dishes containing sterile ice-cold PBS. Keeping track and marking the origins of the segments for later analysis.Next, use forceps to secure one segment dorsal side up and use a razor blade to make a single, longitudinal cut down through the midline to split the column in two equal halves. Place both halves in a new Petri dish containing fresh, sterile ice-cold PBS in the original orientation to be able to distinguish the left and right sides of the spine. Under a dissecting microscope, use fine forceps to gently peel the spinal cord out of the each half of the vertebral column in a rostral to caudal direction.Then place both spinal cord halves in individual 1.5 milliliter microtubes containing 500 microliters of sterile ice-cold PBS on ice. To expose the dorsal root ganglion, gently remove the meninges from one side of the spinal column to the other. And pull the exposed white spinal nerve out of the spinal column.Harvest the round, transparent dorsal root ganglion from the spine segment into a 15 millimeter Petri dish of ice-cold PBS. And collect the ipsilateral and contralateral dorsal root ganglion as just demonstrated. After harvesting the spinal nerve and dorsal root ganglion from the other two spinal column segments as just demonstrated, centrifuge all of the tubes of tissue at high speed.Then aspirate the supernatants and flash-freeze the samples in liquid nitrogen, and store at minus 80 degree Celsius until homogenization. For tissue homogenization, thaw the tubes of frozen tissue on ice before weighing out 100 milligrams of each sample. Transfer into a two milliliter microcentrifuge tube containing sterile beads and 500 microliters of radioimmunoprecipitation assay buffer.Next, disrupt the tissues at room temperature on a homogenizer at 20 cycles per second for two minutes, followed by a one minute waiting period, and 20 cycles per second for an additional two minutes. After the second round of homogenization, centrifuge the tissues at high speed before transferring 500 microliters of each supernatant into a new appropriately labeled tube. Store at minus 20 degree Celsius until downstream analysis.The site of abrasion is visible in the control footpad. And it is typical for a crust to form at the abrasion site as part of the healing process. In contrast, mice inoculated with pseudorabies, demonstrate a severe inflammation of the footpad at the humane endpoint that is characterized by swelling and redness.Histopathological examination of the inoculated footpad and dorsal root ganglion reveals epidermal necrosis and severe dermal inflammation within the pseudorabies infected foot sections. Infiltration of neutrophils is also observed in pseudorabies infected DRG sections. A significant increase in G-CSF protein expression is measured in the footpad and dorsal root ganglion of pseudorabies infected mice compared to controls at both seven and 82-hours post-infection.Significant G-CSF levels are also observed at 82-hours post-infection in the spinal cord, brain, heart, and liver tissues of pseudorabies infected mice compared to controls. Moreover, significant levels of IL-6 are detected in all tested tissues of pseudorabies infected mice starting at 24-hours post-infection. In a moribund state, pseudorabies was detected in the footpad, dorsal root ganglion, spinal cord, and brain.With the high cytoplasmic expression of psuedorabies glycoprotein gB in the neurons of the dorsal root ganglion in pseudorabies infected mice. To ensure a successful infection, it is important that the droplet of virus in a column doesn't fall from the abraded footpad. This animal model may serve platform to test the efficacy of anti-inflammatory and antiviral drugs in order to prevent viral-induced peripheral neuropathies.

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JoVE Journal

Immunology and Infection

10.2K Görüntüleme.  Princeton University. Bu protokol, viral bir enfeksiyon sırasında fare periferik sinir sisteminde nöroinflamatuar yanıtların başlatılması ve geliştirilmesi nin incelenmesine olanak sağlar. Bu model ile, enfeksiyonlar periferik ve merkezi sinir sistemine çevre daha büyük bir mesafe seyahat, nöroinflamatuar yanıt kinetik maksimal bir değerlendirme sağlayan. Beş ila yedi haftalık anestezik C57BL/6 fareyi cerrahi bir ped üzerinde supine pozisyonuna yerleştirdikten sonra, pedal refleksine yanıt ek...