Generating Virus-Induced Neuroinflammation in a Mouse Model

Protocollo

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

1. Mouse foot pad 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 (PRVBecker) 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.

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Materiali

Name	Company	Catalog Number	Comments
C57BL/6 mice (5-7 weeks)	The Jackson Laboratories
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
Isothesia Isoflurane	Henry Schein	NDC 11695-6776-2
Microcentrifuge tube 2ml	Denville Scientific	1000945
Microtube 1.5ml	SARSTEDT	72692005
Penicillin/Streptomycin	Gibco	154022
Precision Glide needle 18G	BD	305196