Assessing Motor Function in an Experimental Autoimmune Neuritis Mouse Model

Abstract

Source: Gonsalvez, D. G., et al., A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments. 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.

Protocol

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

1. EAN (Experimental Autoimmune Neuritis) Induction

NOTE: EAN can be successfully induced in male C57BL/6 mice aged between 6-8 weeks. The induction protocol takes 9 days in total. Day 0 refers to the day of the first immunization. For this protocol, injections were conducted under anesthesia (see step 1.1.2 below).

1. On Day -1:
   1. Prepare the Pertussis Toxin (see Table of Materials) solution of 1.6 µg/mL using sterile 0.1 M mouse-isotonic phosphate-buffered saline (MT-PBS).
   2. Anesthetization with isoflurane:
      1. Place the mouse (C57BL/6, male, 6-8 weeks) in the anesthetization chamber and adjust the oxygen flow to 1 L/min.
      2. Turn on the isoflurane vaporizer, adjust it to 2.5% for anesthetization, monitor breathing, and wait for 2 min or until primary reflexes (corneal and hind limb) are no longer responsive.
   3. Remove the mouse from the anesthetization chamber and administer 250 µL of the above Pertussis Toxin solution via an intraperitoneal (i.p.) injection using a 0.5 mL syringe with a 30_1/2 G needle.
   4. Prepare the injectable inoculum for immunization:
      1. Prepare Solution A using 2 mg/mL solution of P0180-199 peptide (with > 98% purity, sequence S-S-K-R-G-R- Q-T-P-V-L-Y-A-M-L-D-H-S-R-S) in 0.9% saline.
      2. Prepare Solution B using a 20 mg/mL solution of Mycobacterium tuberculosis (see Table of Materials) in Freund's complete adjuvant (FCA, comprising 15% mannide monoolate + 85% paraffin oil and 0.5 mg/mL of desiccated killed and dried Mycobacterium butyricum).
      3. To make the final inoculum for injecting, combine equal volume parts of Solutions A and B in a bead beater and mix them at a maximum speed for 1 min at room temperature.
         NOTE: Solutions A and B can be kept as stock and stored at 4 °C for a maximum of one month, but the combined final inoculum must be freshly prepared on the day of the mouse injection.
2. On Day 0, administer 50 µL of the inoculum (preparation outlined in step 1.1.4) into a mouse via a subcutaneous injection using a 23 G needle.
   NOTE: The injection can be placed between the shoulder blades or between the hind limb and tail over the caudal dorsum.
3. On Day 1, make the Pertussis Toxin solution of 1.2 µg/mL (in MT-PBS) and administer 250 µL into a mouse via i.p. injection using a 0.5 mL syringe with a 301/2 G needle.
4. On Day 3, repeat step 1.3 above.
   NOTE: Stock solutions A and B can be made up and stored at 4 ˚C for a maximum of one month. However, the final injectable inoculum, produced by combining stock solutions A and B, must be done on the day of the injection.
5. On Day 8, administer 50 µL of the inoculum (preparation outlined in step 1.1.4) into a mouse via subcutaneous injection (see step 1.2 above) at the same injection site as in step 1.2 (for each animal)

2. Motor Function Assessment

NOTE: The motor performance is assessed in parallel with clinical scoring for the same cohort of animals. The motor function assessment apparatus must be connected to a computer that has a gait function imaging system (see Table of Materials) installed. It is also recommended that all mice to be assessed should be habituated to the running task prior to EAN induction. To do this, practice runs (2 test runs per mouse) are performed three days prior to disease induction (Day -3).

1. Turn on the motor function assessment apparatus and switch on the light button.
2. Scruff the mouse firmly and ink its feet by lowering it onto a container filled with red ink while holding its tail.
   NOTE: This step is required for the C57BL/6 strain but can be skipped if a mouse strain with a white coat color is being used.
3. Place the mouse into the walking compartment and set the treadmill speed at 15 cm/s.
4. Turn on the treadmill and click the "record" button to capture the mouse's running motion using the gait function imaging system (see Table of Materials).
5. Use a timer and measure each running for 36 s. After 36 s, stop recording and stop the treadmill.
   NOTE: Mice that cannot successfully complete the running task for this 36 s interval are considered to have failed.
6. Save the video file in the designated folder.
7. Repeat the above steps for each mouse to be assessed. Test the mice with the same running task every 3 days.
8. Analyze the edited video files using software for gait function parameters (see Table of Materials).
   NOTE: The details of how to analyze different motor parameters vary among software, so please refer to the manufacturer's instructions before analysis. Depending on the specific aims of the research, animal tissues can be taken at any stage of post-motor function assessment to gain histological evidence of axonal and myelin damage via either immunohistochemistry or electron microscopy.

Results

Table 1: EAN clinical score paradigm.

Score	Criteria	Notes and comments on assessment criteria
0	Normal mouse
1	Less lively OR lathery	This is assessed in response to normal handl...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6, male, 6-8 weeks old	Australian Bioresources Cenre, WA, Australia
Pertussis toxin	List Biological Laboratories, Inc., CA, USA	#181
0.1M mouse-isotonic phosphated buffered salined (MT-PBS)			Laboratories will have their own protocol.
Isoflurane	Pharmachem, QLD, Australia		Laboratories will have their own protocol for administration.
P0180–199 peptide	Wuxi Nordisk Biotech Co. Lt. SHG, CHN		P0180–199, sequence S–S–K–R–G–R– Q–T–P–V–L–Y–A–M–L–D–H–S–R–S
Heat killed Mycobacterium tuberculosis (strain H37RA)	Difco, MI, USA	#231141
Freund's complete adjuvant (FCA)	Difco, MI, USA	#263910
16% Paraformaldehyde (PFA)	Electron Microscopy Services	#15710	Dilute to 4% PFA day of tissue collection.
25% glutaraldheyde	ProSciTech Pty Ltd, QLD, Australia	#11-30-8	Dilute to 2.5% glutaraldehyde day of fixation.
Sodium azide	Chem-Supply Pty Ltd, SA, Australia	SL189	Create 10% (w/v) stock in 0.1M MT-PBS. Use at 0.03% (v/v).
Sucrose	Chem-Supply Pty Ltd, SA, Australia	SA030	Use at 30% (w/v).
Optimum cutting temperature (OCT) medium	Sakura Finetek, CA, USA	#4583
Normal donkey serum	Merck Millipore, MA, USA	#S30-100	Use as antibody diluent at 10% (v/v) or other concentration determined by own laboratory.
Triton-X 100	Sigma Aldrich, MI, USA	#90o2-31-1	Use in antibody diluent at 0.3% (v/v) or other concentration determined by own laboratory.
Rabbit anti-amyloid precursor protein (APP)	Invitrogen (Life Technologies), CA, USA	S12700	Used at 1:400 or titrate in own lab.
Rabbit anti-contactin-associated protein-1 (Caspr)	Gift from Prof Elior Peles, Wiezmann Institute of Science, Israel		Used at 1:500 or titrate in own lab.
Appropriate Alexa Fluor conjugated secondary antibodies	Molecular Probes (Life Technologies), OR, USA	Various	Use at 1:200 or titrate in own lab. Choice of species the antibody was raised in and Alexa Fluor chosen is at the discretion of each laboratory.
Aqueous mounting solution	Dako (Agilent), CA, USA	#S3023	Each laboratory will have their own preference.
Equipment
0.5 mL syringe with 301/2 g needles	BD	#326105
23 g needles	BD	#305143
Red ink pad			Any red ink pad or red food dye could be used to mark the animals' feet.
DigiGate apparatus (includes treadmill)	eMouse Specifics Inc. Framingham, MA
DigiGate Imaging System	eMouse Specifics Inc. Framingham, MA
Stopwatch			Any timer may be used.
DigiGait 8 Software	eMouse Specifics Inc. Framingham, MA
Dissecting microscope	Zeiss		Any appropriate dissecting microscope may be used.
Charged slides	Superfrost Plus, Lomb Scientific Pty Ltd	SF41296SP
Cyrostat	Leica		Any suitable cyrostat may be used.
Perfusion equipment and dissecting instruments			Labs will have their own perfusion protoctols.
Opaque humified chamber			Labs may produce their own using an opaque plastic container.
PAP pene	GeneTex (USA)		Wax pencil, or surface tension may also be used to create a well around the tissue section.
Confocal microscope	Zeis LSM780		Any confocal microscope with appropriate laser lines may be used.
FIJI/Image J	National Institues of Health		Available from www.fiji.sc