Name: mouse footpad inoculation model to study viral-induced neuroinflammatory responses
Uploaded: 2020-06-14T14:30:00
Description: Watch this Scientific Journal Video about Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses at JoVE.com

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 ...

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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 ...

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