The authors thank the staff of the Center for Comparative Medicine and Research (CCMR) at Dartmouth for their expert care of the mice used for these studies. The authors also acknowledge Emily Clough for her excellent administrative support.

All animal work utilizes protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Geisel School of Medicine at Dartmouth.
1. The Mouse Model
<ol>
	<li>Induction of TMEV-Induced Demyelinating Disease
	<ol>
		<li>Move the cages containing 4- to 6-week-old female SJL/JHan mice from the rack to a comfortable working space. Mark the mice (e.g., with an ear tag or ear punch) to allow for individual evaluation of clinical and histologic...

<ol>
	<li>Lipton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theiler's+virus+infection+in+mice:+an+unusual+biphasic+disease+process+leading+to+demyelination.">Theiler's virus infection in mice: an unusual biphasic disease process leading to demyelination.</a> Infect Immun. 11, 1147-1155 (1975).</li><li>Pachner, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Primer of Neuroimmunological Disease. , (2012).</li><li>Rustay, N. R., Wahlsten, D., Crabbe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+genetic+susceptibility+to+ethanol+intoxication+in+mice.">Assessment of genetic susceptibility to ethanol intoxication in mice.</a> Proc Natl Acad Sci U S A. 100, 2917-2922 (2003).</li><li>McGavern, D. B., Zoecklein, L., Drescher, K. M., Rodriguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+neurologic+deficits+in+a+chronic+progressive+murine+model+of+CNS+demyelination.">Quantitative assessment of neurologic deficits in a chronic progressive murine model of CNS demyelination.</a> Exp Neurol. 158, 171-181 (1999).</li><li>Zoecklein, L. 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=Direct+comparison+of+demyelinating+disease+induced+by+the+Daniel's+strain+and+BeAn+strain+of+Theiler's+murine+encephalomyelitis+virus.">Direct comparison of demyelinating disease induced by the Daniel's strain and BeAn strain of Theiler's murine encephalomyelitis virus.</a> Brain Pathol. 13, 291-308 (2003).</li><li>Gilli, F., Li, L., Campbell, S. J., Anthony, D. C., Pachner, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+B-cell+depletion+in+the+Theiler's+model+of+multiple+sclerosis.">The effect of B-cell depletion in the Theiler's model of multiple sclerosis.</a> J Neurol Sci. 359, 40-47 (2015).</li><li>Li, L., 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=The+effect+of+FTY720+in+the+Theiler's+virus+model+of+multiple+sclerosis.">The effect of FTY720 in the Theiler's virus model of multiple sclerosis.</a> J Neurol Sci. 308, 41-48 (2011).</li><li>Homanics, G. E., Quinlan, J. J., Firestone, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+and+behavioral+responses+of+inbred+C57BL/6J+and+strain+129/SvJ+mouse+lines.">Pharmacologic and behavioral responses of inbred C57BL/6J and strain 129/SvJ mouse lines.</a> Pharmacol Biochem Be. 63, 21-26 (1999).</li><li>Balkaya, M., Krober, J. M., Rex, A., Endres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+post-stroke+behavior+in+mouse+models+of+focal+ischemia.">Assessing post-stroke behavior in mouse models of focal ischemia.</a> J Cerebr Blood F Met. 33, 330-338 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Columbus Instruments Rotamex-5 Manual. , 1-33 (2005).</li><li>Dunham, N. W., Miya, T. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+nociception,+sex+and+peripheral+nerve+innervation+in+the+TMEV+animal+model+of+multiple+sclerosis.">Analysis of nociception, sex and peripheral nerve innervation in the TMEV animal model of multiple sclerosis.</a> Pain. 136, 293-304 (2008).</li><li>Pirko, I., Johnson, A. J., Lohrey, A. K., Chen, Y., Ying, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+gray+matter+T2+hypointensity+correlates+with+disability+in+a+murine+model+of+MS.">Deep gray matter T2 hypointensity correlates with disability in a murine model of MS.</a> J Neurol Sci. 282, 34-38 (2009).</li><li>Oleszak, E. L., Chang, J. R., Friedman, H., Katsetos, C. D., Platsoucas, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theiler's+virus+infection:+a+model+for+multiple+sclerosis.">Theiler's virus infection: a model for multiple sclerosis.</a> Clin Microbiol Rev. 17, 174-207 (2004).</li><li>McCarthy, D. P., Richards, M. H., Miller, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+multiple+sclerosis:+experimental+autoimmune+encephalomyelitis+and+Theiler's+virus-induced+demyelinating+disease.">Mouse models of multiple sclerosis: experimental autoimmune encephalomyelitis and Theiler's virus-induced demyelinating disease.</a> Methods Mol Biol. 900, 381-401 (2012).</li><li>. International Mouse Phenotyping Resource of Standardised Screens Available from: <a target="_blank" href="https://www.mousephenotype.org/impress/protocol/158/1">https://www.mousephenotype.org/impress/protocol/158/1</a> (2016)</li><li>Bohlen, M., Cameron, A., Metten, P., Crabbe, J. C., Wahlsten, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calibration+of+rotational+acceleration+for+the+rotarod+test+of+rodent+motor+coordination.">Calibration of rotational acceleration for the rotarod test of rodent motor coordination.</a> J Neurosci Methods. 178, 10-14 (2009).</li><li>Hopkins, M. E., Bucci, D. 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The authors have nothing to disclose.

Despite some limitations, the Rotarod performance test represents an important tool for assessing motor function and dysfunction in TMEV-IDD as well as the effect of pharmacological interventions on disability progression in mice.
The Rotarod test was first described in 1957 as a tool for measuring neurological deficits in rodents11. Rodents have to walk on a rotating rod, with increasing rotating speed, and try to avoid falling to the ground. The latency to fall is recorded and use...

Theiler&#39;s Murine Encephalomyelitis Virus (TMEV) is a neurotropic single-stranded RNA virus that persistently infects the murine central nervous system (CNS). In susceptible mice, infection with TMEV causes an immune-mediated, chronic-progressive demyelinating disease, known as TMEV-induced demyelinating disease (TMEV-IDD). Experimental infection of mice takes a disease course resembling that seen in progressive forms of multiple sclerosis (MS). TMEV-IDD is characterized by two distinct phases: the acute phase and the chronic phase. The acute phase is a mild, usually subclinical encephalitis1,2. The second, chronic phase, beginning about a month after infection, consists of a slowly progressing disability characterized by demyelination, inflammation, and axonal damage1,2. The weakness observed in mice is associated with spasticity and, occasionally, severe tonic spasms.
Because there are currently no medications to ameliorate the progressive disability in patients, researchers are particularly attracted by TMEV-IDD, which represents an optimal animal model for monitoring the impact of disease-modifying drugs on disease progression. However, in mice as well as in MS patients, the monitoring of disability progression requires a continuous clinical observation over extended periods of time. In mice, long-term monitoring for disability progression can be accomplished with the Rotarod performance test.
The Rotarod performance test is a standard behavioral test that evaluates motor-associated functions such as coordination, balance, and fatigue in rodents. The mice have to keep their balance on a turning rod, which is rotating under continuous acceleration; the time latency to fall from this rod is recorded. Animals with neurological dysfunction are unable to stay on the rotating rod as long as controls, and they normally drop off when the rotation speed exceeds their motor capacity. The more neurological impairment the animals have, the sooner they fall off the rod, and the shorter the time latency is.
The advantage of the Rotarod test over the traditional visual scoring systems is that it generates an objective, measurable variable-the time latency-which can ultimately be used for statistical analyses to quantify the effects of therapies and experimental procedures3.
In the Laboratory of Neuroimmunology (LONI) at Dartmouth, mice are subjected to an adaptation protocol, where they are tested prior to TMEV infection in order to familiarize them with the machine and to assess their normal &#34;baseline&#34; balance coordination and motor control4,5. Once the baseline is established and the mice are infected with TMEV, they are tested once or twice a week over a period of several months. The actual testing protocol lasts an average of 150 days, thus allowing an assessment of the decline of balance, coordination, and motor control over the entire course of the demyelinating disease.
Several hundred TMEV-IDD and sham-treated mice have been tested so far for neurological dysfunction at Dartmouth. These mice had received various immunomodulatory treatments, but no pharmacological agent has been found to be effective in ameliorating disability progression6,7. The present article and the related protocol describe how to characterize the progressive neurological impairment displayed by TMEV-IDD mice. Particularly, the protocol offers recommendations of specific testing parameters believed to be generally suitable for studying neurological disability in TMEV-IDD mice using the Rotarod test. This procedure provides a baseline against which to assess (1) the relevance of this mouse model to progressive MS and (2) its usefulness for testing therapies aimed at treating progressive neurological conditions such as MS. Clearly, the Rotarod performance test and the current optimized testing parameters and protocol are not only useful at detecting progressive neurological disability in the TMEV-IDD mouse model, but are also useful in uncovering impairments in other virus-induced and/or genetic mouse models of neurodegenerative diseases.

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measuring progressive neurological disability in a mouse model of multiple sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

The aim of this representative experiment was to compare the neurological disability induced by the Daniels (DA) strain and BeAn strain of TMEV. For the purposes of the present study, a group of 32 female SJL mice were infected intracranially with TMEV, either the DA strain (n = 16) or the BeAn strain (n = 16), and their clinical signs were monitored over time. An additional group of 20 mice was sham treated (i.e., saline solution was injected intracranially) and served as health...

Watch this Scientific Journal Video about Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis at JoVE.com

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

The overall goal of this procedure is to assess the progressive neurological impairment in mice with chronic demyelinating diseases. We accomplish this by offering recommendations of testing parameters suitable for studying long term neurological disability in the Theilers virus model of progressive multiple sclerosis, using the Rotarod test. This procedure provides a baseline against which to assess the relevance of mouse model to progressive demyelination and it's usefulness for testing therapies aimed at treating progressive neurological condition such as multiple sclerosis.The advantage of this optimized test over the traditional visual scoring system is that it generates an objective variable to quantify the long term effect of therapies and experimental procedures on motor functions. Demonstrating this procedure will be Darlene Royce, a technician from the laboratory of neuroimmunology at Dartmouth. In order to assess baseline balance coordination and motor control, start the adaption protocol five days prior to TMEV infection by first allowing the mice to acclimate to the testing room for at least 30 minutes.While the mice are acclimating, preset the Rotarod with the minus five DPI training parameters. Then, pickup a mouse by the tail and place it on the rod facing away from the operator. If the mouse falls or jumps, place it back in its lane on the Rotarod until all mice are in position.After loading all four mice, press the enter button to start the experiment. Ensure that the timers start automatically, and observe the rotations per minutes on display for each lane. Then, as each animal falls from the rod, record the speed of the rod at the time of the fall, as well as the duration of time the animal remained on the rod.After all the mice have fallen, use a tissue to remove any fecal boli and urine from the rod as the presence of urine and fecal material may affect the ability of mice to properly grip the rod. After a three minute rest give the mice a second and then a third trial in which the maximum time per single trial is 240 seconds. Administer a total of three trials during each testing day.Finally, return the mice to their home cage. On days negative four, negative three, negative two, and negative one PI, preset the Rotarod with the appropriate training protocol parameters and repeat the Rotarod experiment. Begin by moving cages containing four to six week old female SJL mice from the rack to a comfortable working space.Use ear punches to mark the mice to allow for individual evaluation of clinical and histological disease. Next, draw 30 microliters of TMEV infecting stock in PBS into a 29 gauge insulin syringe needle. Prepare the anesthesia gas machine by ensuring the presence of adequate amounts of oxygen and isoflurane for the duration of the procedure.Place the animal into the induction chamber and seal the top. After a few minutes, remove the animal from the chamber, and test the mouse by pinching the foot pad to ensure adequate anesthesia. Next, clean the injection site with 70%isopropyl alcohol and inject the 30 microliters of TMEV infecting stock into the right cerebral hemisphere by freehand injection.Finally, return the mouse to its holding cage once it is fully alert and mobile. On day seven post infection, preset the Rotarod with the appropriate experimental parameters, and repeat the complete Rotarod training excercise with the appropriate experimental protocol parameters. After the third trial on each day, weigh each mouse and make a note of the body weight on the data sheet.Test the mice twice a week for the following six weeks in the same manner. After six weeks, test the mice once a week with the same experimental protocol for an average of 150 experimental days. Next, analyze the raw data, and express as a neurological functional index, or NFI.To determine the individual NFI value, calculate the baseline performance threshold of each mouse as the mean of all running times from 15 to 45 days post infection. In order to allow for the evaluation of motor performance as a result of progressive demyelination and exclude any contributing deficits due to early encephalitis. Then, calculate the NFI value as the mean of the three most recent average running times divided by the baseline performance threshold of the mouse.Finally, calculate an adjusted NFI by dividing the NFI value by the average NFI value obtained by the sham treated group on that specific day. This figure shows the TMEV infected mice display significantly increased neurological deficits over time when compared with control mice. Chronic infection with two different strains of TMEV negatively affected the running times of the mice.Both groups of TMEV infected mice had significantly lower NFI values than those of sham mice. Furthermore, there was no difference comparing disability progression in mice infected with the BeAn strain, and those infected with the DA strain at all time points. Following this procedure are the traditional visual scoring methods, like the righting reflex test, can be performed in order to rapidly assess general health in mice.After its development, this technique paved the way for researchers in the field of multiple sclerosis and neurodegenerative diseases to test the effectiveness of therapies and other intervention in delaying progressive disability. After watching this video, you should have a good understanding of how to evaluate the long term progressive disability in mice. This procedure is not only useful in detecting the progressive neurological disability in the Theiler virus model, but is also useful in uncovering impairments in other mouse models of neurodegenerative diseases.

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10.9K vistas.  Geisel School of Medicine at Dartmouth. The overall goal of this procedure is to assess the progressive neurological impairment in mice with chronic demyelinating diseases. We accomplish this by offering recommendations of testing parameters suitable for studying long term neurological disability in the Theilers virus model of progressive multiple sclerosis, using the Rotarod test. This procedure provides a baseline against which to assess the relevance of mouse model to progressive demyelination and it's usefulness for testing the...