Name: measuring progressive neurological disability in a mouse model of multiple sclerosis
Uploaded: 2016-11-14T13:00:00
Description: Watch this Scientific Journal Video about Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis at JoVE.com

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. 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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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+note+on+a+simple+apparatus+for+detecting+neurological+deficit+in+rats+and+mice.">A note on a simple apparatus for detecting neurological deficit in rats and mice.</a> J Am Pharm Ass. 46, 208-209 (1957).</li><li>Ulrich, R., Kalkuhl, A., Deschl, U., Baumgartner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine+learning+approach+identifies+new+pathways+associated+with+demyelination+in+a+viral+model+of+multiple+sclerosis.">Machine learning approach identifies new pathways associated with demyelination in a viral model of multiple sclerosis.</a> J Cell Mol Med. 14, 434-448 (2010).</li><li>Lynch, J. L., Gallus, N. J., Ericson, M. E., Beitz, A. 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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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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Neuroscience

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Lipidomics and Transcriptomics in Neurological Diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson&#039;s Disease

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, leading to motor dysfunctions, psychical disability, and cognitive impairment during MS progression. Positron emission tomography (PET) is an imaging technique able to quantify in vivo cellular and molecular alterations.
Radiotracers with affinity to intact myelin can be used for in vivo imaging of myelin content changes over time. It is possible to detect either an increase or decrease in myelin content, what means this imaging technique can detect demyelination and remyelination processes of the central nervous system. In this protocol we demonstrate how to use PET imaging to detect myelin changes in the lysolecithin rat model, which is a model of focal demyelination lesion (induced by stereotactic injection) (i.e., a model of multiple sclerosis disease). 11C-PIB PET imaging was performed at baseline, and 1 week and 4 weeks after stereotaxic injection of lysolecithin 1% in the right striatum (4 &#181;L) and corpus callosum (3 &#181;L) of the rat brain, allowing quantification of focal demyelination (injection site after 1 week) and the remyelination process (injection site at 4 weeks).
Myelin PET imaging is an interesting tool for monitoring in vivo changes in myelin content which could be useful for monitoring demyelinating disease progression and therapeutic response.

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...