Name: a simple approach to induce experimental autoimmune neuritis in c57bl/6 mice for functional and neuropathological assessments
Uploaded: 2017-11-09T13:00:00
Description: Watch this Scientific Journal Video about A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments at JoVE.com

DGG is a NHMRC Peter Doherty and Multiple Sclerosis Research Australia (MSRA) Early Career Fellow. JLF is supported by an MSRA Postdoctoral Fellowship. This work was supported by the Australian National Health and Medical Research Council (NHMRC) project grant #APP1058647 to JX.

All procedures described here were approved by the Florey Institute for Neuroscience and Mental Health (Melbourne Brain Centre) Animal Ethics Committee and follow the Australian Code of Practice for the Use of Animals for Scientific Purposes.
1. EAN 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 we...

<ol>
	<li>Newswanger, D. L., Warren, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guillain-Barre+syndrome.">Guillain-Barre syndrome.</a> Am Fam Physician. 69 (10), 2405-2410 (2004).</li><li>Ruiz, E., Ramalle-Gomara, E., Quinones, C., Martinez-Ochoa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+Guillain-Barre+syndrome+mortality+in+Spain+from+1999+to+2013.">Trends in Guillain-Barre syndrome mortality in Spain from 1999 to 2013.</a> Int J Neurosci. 126 (11), 985-988 (2016).</li><li>Walling, A. D., Dickson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guillain-Barre+syndrome.">Guillain-Barre syndrome.</a> Am Fam Physician. 87 (3), 191-197 (2013).</li><li>Gonsalvez, D. G., 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=A+Functional+and+Neuropathological+Testing+Paradigm+Reveals+New+Disability-Based+Parameters+and+Histological+Features+for+P0180-199-Induced+Experimental+Autoimmune+Neuritis+in+C57BL/6+Mice.">A Functional and Neuropathological Testing Paradigm Reveals New Disability-Based Parameters and Histological Features for P0180-199-Induced Experimental Autoimmune Neuritis in C57BL/6 Mice.</a> J Neuropathol Exp Neurol. 76 (2), 89-100 (2017).</li><li>Berg, B., 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=Guillain-Barre+syndrome:+pathogenesis,+diagnosis,+treatment+and+prognosis.">Guillain-Barre syndrome: pathogenesis, diagnosis, treatment and prognosis.</a> Nat Rev Neurol. 10 (8), 469-482 (2014).</li><li>Koller, H., Schroeter, M., Kieseier, B. C., Hartung, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+inflammatory+demyelinating+polyneuropathy--update+on+pathogenesis,+diagnostic+criteria+and+therapy.">Chronic inflammatory demyelinating polyneuropathy--update on pathogenesis, diagnostic criteria and therapy.</a> Curr Opin Neurol. 18 (3), 273-278 (2005).</li><li>Griffin, J. W., 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=Pathology+of+the+motor-sensory+axonal+Guillain-Barre+syndrome.">Pathology of the motor-sensory axonal Guillain-Barre syndrome.</a> Ann Neurol. 39 (1), 17-28 (1996).</li><li>Taylor, W. A., Hughes, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+allergic+neuritis+induced+in+SJL+mice+by+bovine+P2.">Experimental allergic neuritis induced in SJL mice by bovine P2.</a> J Neuroimmunol. 8 (2-3), 153-157 (1985).</li><li>Zou, L. 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=P0+protein+peptide+180-199+together+with+pertussis+toxin+induces+experimental+autoimmune+neuritis+in+resistant+C57BL/6+mice.">P0 protein peptide 180-199 together with pertussis toxin induces experimental autoimmune neuritis in resistant C57BL/6 mice.</a> J Neurosci Res. 62 (5), 717-721 (2000).</li><li>Miletic, H., 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=P0(106-125)+is+a+neuritogenic+epitope+of+the+peripheral+myelin+protein+P0+and+induces+autoimmune+neuritis+in+C57BL/6+mice.">P0(106-125) is a neuritogenic epitope of the peripheral myelin protein P0 and induces autoimmune neuritis in C57BL/6 mice.</a> J Neuropathol Exp Neurol. 64 (1), 66-73 (2005).</li><li>Hafer-Macko, C., 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=Acute+motor+axonal+neuropathy:+an+antibody-mediated+attack+on+axolemma.">Acute motor axonal neuropathy: an antibody-mediated attack on axolemma.</a> Ann Neurol. 40 (4), 635-644 (1996).</li><li>Griffin, J. W., 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=Early+nodal+changes+in+the+acute+motor+axonal+neuropathy+pattern+of+the+Guillain-Barre+syndrome.">Early nodal changes in the acute motor axonal neuropathy pattern of the Guillain-Barre syndrome.</a> J Neurocytol. 25 (1), 33-51 (1996).</li></ol>

This report outlines a simple method to induce EAN using the P0180-199 peptide in C57BL/6 mice, enabling the quantification of key neuropathological and functional deficits in mice induced with EAN. Distinct to the EAN induction protocol described here is the use of anesthesia while performing the immunization injections. The use of isoflurane anesthesia greatly enhances the ability to ensure that the total volume of inoculum is injected subcutaneously in the desired location with minimal error and stress to t...

Peripheral neuropathies can be either genetic in origin or acquired, with acquired neuropathies having either metabolic, ischaemic, inflammatory, or toxic precipitants. These diseases are also usefully classified as either axonal or demyelinative in origin. The most common acquired demyelinating peripheral neuropathies are Acute Inflammatory Demyelinating Polyneuropathy (AIDP, also known as Guillain-Barr&#233; syndrome, GBS) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP)1,2,3,4; both are pathogenetically characterized by an autoimmune reaction directed against the myelin sheath, causing demyelination of the peripheral nerves. In these diseases, activated T cells cross the blood nerve barrier and generate an immune reaction within the PNS. Activation of macrophages within the nerve then causes demyelination either directly via phagocytic attack or indirectly via secreted inflammatory mediators, resulting in clinical disabilities such as paralysis and sensory dysfunction5. While demyelinated axons retain the ability to be remyelinated following demyelination, remyelination is often delayed or incomplete, resulting in susceptibility of the naked axons to irreversible damage, which is the major cause of permanent clinical disability. Currently, the most effective treatments are immunomodulatory, but despite their efficacy, in many cases the recovery is often slow and ~25% patients will experience residual functional deficits that significantly reduce their quality of life6,7.
EAN is a widely used animal model of demyelinating peripheral neuropathy that has provided valuable insights into pathogenesis and a means to assess novel therapeutic agents4. This model can be induced in different species such as rabbits, rats, mice, and guinea pigs, and is induced by immunization with neurogenic antigens. However, ultimately the successful EAN induction depends on an appropriate immune response for disease to occur. Given the species (and inter-species/strain) variations in immune function, multiple combinations of antigens and adjuvants have been developed to successfully induce EAN. In terms of murine genetic tools, the C57BL/6 is the most widely used; however, the traditional P2 protein peptide 57-81 (P257-81) that results in disease in the susceptible SJL mouse strain8 is unable to illicit pathogenesis leading to functional deficits in the C57BL/6 strain. Fortunately, sensitization paradigms using the P0106-125 or P0180-199 peptides, delivered in adjuvant combined with the injection of pertussis toxin can overcome this barrier, enabling sophisticated genetic tools to be utilized in the murine EAN model.
Here, a simple method for the induction of EAN in the C57BL/6 mice is presented. In addition, a comprehensive detailed approach by which to evaluate the functional and neuropathological deficits associated with the disease is provided. The P0180-199 peptide9 was chosen in preference to the P057-81 alternative10. The P0180-199 model has been described to produce less severe clinical signs compared with the P057-81 alternative10, and is therefore likely to withstand the introduction of potentially deleterious genetic perturbations, recover from surgical procedures (such as osmotic pump implantation), and is amenable to treadmill gait function testing4. However, the treadmill gait function tests and histological protocols described here could easily be applied when studying the disease in a P057-81 induced variant.

<table>
	<tbody>
		<tr>
			<td>C57BL/6, male, 6-8 weeks old</td>
			<td>Australian Bioresources Cenre, WA, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pertussis toxin</td>
			<td>List Biological Laboratories, Inc., CA, USA</td>
			<td>#181</td>
			<td></td>
		</tr>
		<tr>
			<td>0.1M mouse-isotonic phosphated buffered salined (MT-PBS)</td>
			<td></td>
			<td></td>
			<td>Laboratories will have their own protocol.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pharmachem, QLD, Australia</td>
			<td></td>
			<td>Laboratories will have their own protocol for administration.</td>
		</tr>
		<tr>
			<td>P0180&#8211;199 peptide</td>
			<td>Wuxi Nordisk Biotech Co. Lt. SHG, CHN</td>
			<td></td>
			<td>P0180&#8211;199, sequence S&#8211;S&#8211;K&#8211;R&#8211;G&#8211;R&#8211; Q&#8211;T&#8211;P&#8211;V&#8211;L&#8211;Y&#8211;A&#8211;M&#8211;L&#8211;D&#8211;H&#8211;S&#8211;R&#8211;S</td>
		</tr>
		<tr>
			<td>Heat killed Mycobacterium tuberculosis (strain H37RA)</td>
			<td>Difco, MI, USA</td>
			<td>#231141</td>
			<td></td>
		</tr>
		<tr>
			<td>Freund&#39;s complete adjuvant (FCA)</td>
			<td>Difco, MI, USA</td>
			<td>#263910</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Services</td>
			<td>#15710</td>
			<td>Dilute to 4% PFA day of tissue collection.</td>
		</tr>
		<tr>
			<td>25% glutaraldheyde</td>
			<td>ProSciTech Pty Ltd, QLD, Australia</td>
			<td>#11-30-8</td>
			<td>Dilute to 2.5% glutaraldehyde day of fixation.</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Chem-Supply Pty Ltd, SA, Australia</td>
			<td>SL189</td>
			<td>Create 10% (w/v) stock in 0.1M MT-PBS. Use at 0.03% (v/v).</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Chem-Supply Pty Ltd, SA, Australia</td>
			<td>SA030</td>
			<td>Use at 30% (w/v).</td>
		</tr>
		<tr>
			<td>Optimum cutting temperature (OCT) medium</td>
			<td>Sakura Finetek, CA, USA</td>
			<td>#4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Merck Millipore, MA, USA</td>
			<td>#S30-100</td>
			<td>Use as antibody diluent at 10% (v/v) or other concentration determined by own laboratory.</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma Aldrich, MI, USA</td>
			<td>#90o2-31-1</td>
			<td>Use in antibody diluent at 0.3% (v/v) or other concentration determined by own laboratory.</td>
		</tr>
		<tr>
			<td>rabbit anti-amyloid precursor protein (APP)</td>
			<td>Invitrogen (Life Technologies), CA, USA</td>
			<td>S12700</td>
			<td>Used at 1:400 or titrate in own lab.</td>
		</tr>
		<tr>
			<td>rabbit anti-contactin-associated protein-1 (Caspr)</td>
			<td>Gift from Prof Elior Peles, Wiezmann Institute of Science, Israel</td>
			<td></td>
			<td>Used at 1:500 or titrate in own lab.</td>
		</tr>
		<tr>
			<td>Appropriate Alexa Fluor conjugated secondary antibodies</td>
			<td>Molecular Probes (Life Technologies), OR, USA</td>
			<td>Various</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Aqueous mounting solution</td>
			<td>Dako (Agilent), CA, USA</td>
			<td>#S3023</td>
			<td>Each laboratory will have their own preference.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL syringe with 301/2 g needles</td>
			<td>BD</td>
			<td>#326105</td>
			<td></td>
		</tr>
		<tr>
			<td>23 g needles</td>
			<td>BD</td>
			<td>#305143</td>
			<td></td>
		</tr>
		<tr>
			<td>Red ink pad</td>
			<td></td>
			<td></td>
			<td>Any red ink pad or red food dye could be used to mark the animals&#39; feet.</td>
		</tr>
		<tr>
			<td>DigiGate apparatus (includes treadmill)</td>
			<td>eMouse Specifics Inc. Framingham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DigiGate Imaging System</td>
			<td>eMouse Specifics Inc. Framingham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td></td>
			<td></td>
			<td>Any timer may be used.</td>
		</tr>
		<tr>
			<td>DigiGait 8 Software</td>
			<td>eMouse Specifics Inc. Framingham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Any appropriate dissecting microscope may be used.</td>
		</tr>
		<tr>
			<td>Charged slides</td>
			<td>Superfrost Plus, Lomb Scientific Pty Ltd</td>
			<td>SF41296SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyrostat</td>
			<td>Leica</td>
			<td></td>
			<td>Any suitable cyrostat may be used.</td>
		</tr>
		<tr>
			<td>Perfusion equipment and dissecting instruments</td>
			<td></td>
			<td></td>
			<td>Labs will have their own perfusion protoctols.</td>
		</tr>
		<tr>
			<td>Opaque humified chamber</td>
			<td></td>
			<td></td>
			<td>Labs may produce their own using an opaque plastic container.</td>
		</tr>
		<tr>
			<td>PAP pene</td>
			<td>GeneTex (USA)</td>
			<td></td>
			<td>Wax pencil, or surface tension may also be used to create a well around the tissue section.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeis LSM780</td>
			<td></td>
			<td>Any confocal microscope with appropriate laser lines may be used.</td>
		</tr>
		<tr>
			<td>FIJI/Image J</td>
			<td>National Institues of Health</td>
			<td></td>
			<td>Available from www.fiji.sc</td>
		</tr>
	</tbody>
</table>

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.

P0180-199 peptide induced EAN in C57BL/6 mice leads to a monophasic disease with clinical score onset from 6 days post first immunization (dpi), and maximal score severity is observed from 25 dpi followed by some clinical score improvement from 40 dpi duration (Figure 1)4,9. In terms of gait function, mice begin to fail at a simple running task as early as 6 dpi, and by 35 dpi mice have lit...

Watch this Scientific Journal Video about A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments at JoVE.com

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

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 ...

The overall goal of this procedure is to successfully induce Experimental Autoimmune Neuritis or EAN in a mouse model for the functional and pathological analysis of the disease process. The EAN model can be used to address key questions relating to peripheral neuropathy. It's also a wonderful model to potentially investigate the efficacy of novel therapeutic agents.The main advantage of this approach is that it provides a simple and objective functional testing paradigm that can be applied when using the EAN model in the mouse. Demonstrating the procedure with me today is Sang Won Yoo, who is an undergraduate student in the neurotrophin and myelin biology lab at the University of Melbourne. Three days before the disease induction, turn on the motor function assessment apparatus and switch on the light button.Pick up the mouse and lower it onto a container of red food dye. Place the mouse into the walking compartment and set the treadmill speed to 15 centimeters per second. Turn on the treadmill and click record to use the gait function imaging system to capture the running motion of the mouse for 36 seconds.After 36 seconds, stop the recording and the treadmill and save the practice run video file of the practice run in the designated folder. One day before the first immunization, use a 0.5 milliliter syringe equipped with a 30-1/2 gauge needle to administer 1.6 micrograms per milliliter of Pertussis toxin in 250 microliters of mouse isotonic PBS IP to anesthetized six to eight-week-old male C57BL/6 mice. Then, score the mice for clinical signs of EAN on a scale from zero to four according to the table and perform a motor function assessment as just demonstrated.The next day, combine equal volumes of freshly prepared solutions of peptide in 0.9%saline and 20 milligrams per milliliter of Mycobacterium tuberculosis in complete Freund's adjuvant in a bead beader and mix the solutions at the maximum speed for one minute at room temperature. Load a syringe equipped with a 23 gauge needle with the inoculum and invert and shake the syringe before evacuating any air within the syringe shaft. Before delivering the inoculum, remove the hair just lateral to the midline between the scapulae and disinfect the exposed skin with 70%ethanol.Then, use a pair of Adson forceps to grasp the skin and deliver 50 microliters of the solution to the Pertussis toxin injected mouse via subcutaneous injection. The next morning, deliver 1.2 micrograms per milliliter of Pertussis toxin in 250 microliters of PBS IP as just demonstrated followed by another 1.2 micrograms per milliliter injection of Pertussis toxin IP 48 hours later. Three days later after the third Pertussis injection, administer another 50 microliters of freshly prepared inoculum at the same injection site as before and monitor the clinical score and motor function of the animals twice a week until the end of the experiment.P0 180-199 peptide-induced EAN in C57BL/6 mice leads to a monophasic disease with a clinical score onset from six days post first immunization and a maximal score severity at 25 days post first immunization, followed by some clinical score improvement at 40 days post first immunization. The mice begin to fail at a simple running task as early as six days post first immunization with little capacity to complete a simple treadmill running task by 35 days post immunization that does not improve in running ability at 55 days post first immunization. The examination of semi-thin sections of the sciatic nerves of peptide-induced EAN mice reveals areas of demyelination compared to sciatic nerves from age-matched healthy controls.High power transmission electron microscopy images allow the identification of specific neuropathological features such as demyelination, damage to the myelin lamella, and signs of axon stress such as mitochondrial swelling. These animals also display acute axonal damage as evidenced by traumatically elevated beta-amyloid precursor protein expression in the sciatic nerves that is not observed in age-matched healthy control tissues. A widening of the nodes and a disruption to the paranodes are also observed.Once mastered, the subcutaneous injections can be performed within about five to 10 minutes per mouse with minimum chance of any injection error. While attempting this procedure, it's really important that you observe the injection site to ensure that all of the inoculum remains within the injection site and that none of it leaks out. Video capture of treadmill gait is great because it enables other parameters to be computed such as swing phase or stride length and these measures may become important when testing the efficacy of novel therapeutics.After watching this video, you should have a really good understanding of how to induce EAN in C57BL/6 male mice. In addition, you should have a really good understanding of how to place subcutaneous injections safely and really accurately ensuring that there is no injecting error. Don't forget that working with needles and inoculum, there is the chance of injury especially when using sharps.So, to avoid any chance of injury, it's a really good idea to always use the forceps and never grasp skin or tissue by hand and also to use single-use syringes, never recapping in the process.

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Neuroscience

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This is mostly due to the difficulty of microdissection of fetal brains and their sectioning for determination of apoptotic cells caused by prenatal exposure to alcohol. The experiments described here provide visualized techniques from mice breeding to the identification of cell death in fetal brain tissue. This study used C57BL/6 mice as the animal model for studying fetal alcohol exposure and the role of trophic peptide against alcohol-induced apoptosis. The breeding consists of a 2-hr matting window to determine the exact stage of embryonic age. An established fetal alcohol exposure model has been used in this study to determine the effects of prenatal alcohol exposure in fetal brains. This involves free access to alcohol or pair-fed liquid diets as the sole source of nutrients for the pregnant mice.
The techniques involving dissection of fetuses and microdissection of fetal brains are described carefully, since the latter can be challenging. Microdissection requires a stereomicroscope and ultra-fine forceps. Step-by-step procedures for dissecting the fetal brains are provided visually. The fetal brains are dissected from the base of the primordium olfactory bulb to the base of the metencephalon.
For investigating apoptosis, fetal brains are first embedded in gelatin using a peel-away mold to facilitate their sectioning with a vibratome apparatus. Fetal brains embedded and fixed in paraformaldehyde are easily sectioned, and the free floating sections can be mounted in superfrost plus slides for determination of apoptosis or cell death.
TUNEL (TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase) assay has been used to identify cell death or apoptotic cells. It is noteworthy that apoptosis and cell-mediated cytotoxicity are characterized by DNA fragmentation. Thus, the visualized TUNEL-positive cells are indicative of cell death or apoptotic cells.
The experimental designs here provide information about the use of an established liquid diet for studying the effects of alcohol and the role of neurotrophic peptides during pregnancy in fetal brains. This involves breeding and feeding pregnant mice, microdissecting fetal brains, and determining apoptosis. Together, these visual and textual techniques might be a source for investigating prenatal exposure of harmful agents in fetal brains.

The experimental designs proposed here focus on studying the effects of alcohol exposure in apoptosis and the application of neurotrophic peptide during pregnancy in fetal brain. A detailed description from the breeding to the collection of fetal brains is described. Techniques for determination of apoptosis are also described in detail.

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) remain unclear. In order to adequately study the attributes of endolymphatic hydrops, such as the origins of low-frequency hearing loss, a reliable model is needed. The guinea pig is a&#160;good model because it hears in the low-frequency regions that are putatively affected by endolymphatic hydrops. Previous research has demonstrated that endolymphatic hydrops can be induced surgically via intradural or extradural approaches that involve drilling on the endolymphatic duct and sac. However, whether it was possible to create an endolymphatic hydrops model using an extradural approach that avoided dangerous drilling on the endolymphatic duct and sac was unknown. The objective of this study was to demonstrate a revised extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The sample size consisted of seven guinea pigs. Functional measurements of hearing were made and temporal bones were subsequently harvested for histologic analysis. The approach had a success rate of 86% in achieving endolymphatic hydrops. The risk of cerebrospinal fluid leak was minimal. No perioperative deaths or injuries to the posterior semicircular canal occurred in the sample. The presented method demonstrates a safe and reliable way to induce endolymphatic hydrops at a relatively quick time point of 30 days. The clinical implications are that the presented method provides a reliable model to further explore the origins of low-frequency hearing loss that can be associated endolymphatic hydrops.

This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely afflicting a fairly young population. Several animal models of SAH have been developed to investigate the pathophysiological mechanisms behind SAH and to test pharmacological interventions. The pre-chiasmatic, single injection model in the rat presented in this article is an experimental model of SAH with a predetermined blood volume. Briefly, the animal is anesthetized, intubated, and kept under mechanical ventilation. Temperature is regulated with a heating pad. A catheter is placed in the tail artery, enabling continuous blood pressure measurement as well as blood sampling. The atlantooccipital membrane is incised and a catheter for pressure recording is placed in the cisterna magna to enable intracerebral pressure measurement. This catheter can also be used for intrathecal therapeutic interventions. The rat is placed in a stereotaxic frame, a burr hole is drilled anteriorly to the bregma, and a catheter is inserted through the burr hole and placed just anterior to the optic chiasm. Autologous blood (0.3 mL) is withdrawn from the tail catheter and manually injected. This results in a rise of intracerebral pressure and a decrease of cerebral blood flow. The animal is kept sedated for 30 min and given subcutaneous saline and analgesics. The animal is extubated and returned to its cage. The pre-chiasmatic model has a high reproducibility rate and limited variation between animals due to the pre-determined blood volume. It mimics SAH in humans making it a relevant model for SAH research.

Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented.

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely ...

Induction and Evaluation of Experimental Autoimmune Encephalomyelitis in Mouse

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

Author Spotlight: Creating a Versatile Experimental Autoimmune Encephalomyelitis Model Relevant for Both Male and Female Mice 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.