Name: sagittal plane kinematic gait analysis in c57bl/6 mice subjected to mog35-55 induced experimental autoimmune encephalomyelitis
Uploaded: 2017-11-04T13:00:00
Description: Watch this Scientific Journal Video about Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis at JoVE.com

We would like to acknowledge Sid Chedrawe for his technical assistance with filming. This work was supported by funding from the MS Society of Canada (EGID 2983).

This protocol is in accordance with the Canadian Council on Animal Care guidelines and was approved by the Dalhousie University Committee on Laboratory Animals.
1. Construct Reflective Markers:
<ol>
	<li>Using a hand-held hole punch, punch the desired number of small circles from a sheet of reflective paper. Each animal requires 5 markers for a single recording; two large and three small markers.</li>
	<li>Using fine scissors, make a straight cut extending from the perimeter to the center of...

<ol>
	<li>Giladi, N., Horak, F. B., Hausdorff, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+gait+disturbances:+distinguishing+between+continuous+and+episodic+changes.">Classification of gait disturbances: distinguishing between continuous and episodic changes.</a> Mov Disord. 28 (11), 1469-1473 (2013).</li><li>Kiehn, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+organization+of+spinal+circuits+that+control+locomotion.">Decoding the organization of spinal circuits that control locomotion.</a> Nat Rev Neurosci. 17 (4), 224-238 (2016).</li><li>Akay, T., Tourtellotte, W. G., Arber, S., Jessell, T. 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M., 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=Basso+Mouse+Scale+for+locomotion+detects+differences+in+recovery+after+spinal+cord+injury+in+five+common+mouse+strains.">Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains.</a> J Neurotrauma. 23 (5), 635-659 (2006).</li><li>Tatem, K. S., 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=Behavioral+and+locomotor+measurements+using+an+open+field+activity+monitoring+system+for+skeletal+muscle+diseases.">Behavioral and locomotor measurements using an open field activity monitoring system for skeletal muscle diseases.</a> J Vis Exp. (91), e51785 (2014).</li><li>Hetze, S., Romer, C., Teufelhart, C., Meisel, A., Engel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+analysis+as+a+method+for+assessing+neurological+outcome+in+a+mouse+model+of+stroke.">Gait analysis as a method for assessing neurological outcome in a mouse model of stroke.</a> J Neurosci Methods. 206 (1), 7-14 (2012).</li><li>Preisig, D. F., 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=High-speed+video+gait+analysis+reveals+early+and+characteristic+locomotor+phenotypes+in+mouse+models+of+neurodegenerative+movement+disorders.">High-speed video gait analysis reveals early and characteristic locomotor phenotypes in mouse models of neurodegenerative movement disorders.</a> Behav Brain Res. 311, 340-353 (2016).</li><li>Leblond, H., L'Esperance, M., Orsal, D., Rossignol, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treadmill+locomotion+in+the+intact+and+spinal+mouse.">Treadmill locomotion in the intact and spinal mouse.</a> J Neurosci. 23 (36), 11411-11419 (2003).</li><li>Ueno, M., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinematic+analyses+reveal+impaired+locomotion+following+injury+of+the+motor+cortex+in+mice.">Kinematic analyses reveal impaired locomotion following injury of the motor cortex in mice.</a> Exp Neurol. 230 (2), 280-290 (2011).</li><li>Zorner, 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=Profiling+locomotor+recovery:+comprehensive+quantification+of+impairments+after+CNS+damage+in+rodents.">Profiling locomotor recovery: comprehensive quantification of impairments after CNS damage in rodents.</a> Nat Methods. 7 (9), 701-708 (2010).</li><li>Pearson, K. G., Acharya, H., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+electrode+configuration+for+recording+electromyographic+activity+in+behaving+mice.">A new electrode configuration for recording electromyographic activity in behaving mice.</a> J Neurosci Methods. 148 (1), 36-42 (2005).</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 Cereb Blood Flow Metab. 33 (3), 330-338 (2013).</li><li>Akay, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+measurement+of+muscle+denervation+and+locomotor+behavior+in+individual+wild-type+and+ALS+model+mice.">Long-term measurement of muscle denervation and locomotor behavior in individual wild-type and ALS model mice.</a> J Neurophysiol. 111 (3), 694-703 (2014).</li><li>Taylor, T. N., Greene, J. G., Miller, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+of+mouse+models+of+Parkinson's+disease.">Behavioral phenotyping of mouse models of Parkinson's disease.</a> Behav Brain Res. 211 (1), 1-10 (2010).</li><li>Chen, K., 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=Differential+Histopathological+and+Behavioral+Outcomes+Eight+Weeks+after+Rat+Spinal+Cord+Injury+by+Contusion,+Dislocation,+and+Distraction+Mechanisms.">Differential Histopathological and Behavioral Outcomes Eight Weeks after Rat Spinal Cord Injury by Contusion, Dislocation, and Distraction Mechanisms.</a> J Neurotrauma. 33 (18), 1667-1684 (2016).</li><li>de Bruin, N. M., 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=Multiple+rodent+models+and+behavioral+measures+reveal+unexpected+responses+to+FTY720+and+DMF+in+experimental+autoimmune+encephalomyelitis.">Multiple rodent models and behavioral measures reveal unexpected responses to FTY720 and DMF in experimental autoimmune encephalomyelitis.</a> Behav Brain Res. 300, 160-174 (2016).</li><li>Steinman, L., Zamvil, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+successfully+apply+animal+studies+in+experimental+allergic+encephalomyelitis+to+research+on+multiple+sclerosis.">How to successfully apply animal studies in experimental allergic encephalomyelitis to research on multiple sclerosis.</a> Ann Neurol. 60 (1), 12-21 (2006).</li><li>Emerson, M. R., Gallagher, R. J., Marquis, J. G., LeVine, S. 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(86), (2014).</li><li>Beeton, C., Garcia, A., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+clinical+scoring+of+chronic-relapsing+experimental+autoimmune+encephalomyelitis.">Induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis.</a> J Vis Exp. (5), e224 (2007).</li><li>Barthelmes, 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=Induction+of+Experimental+Autoimmune+Encephalomyelitis+in+Mice+and+Evaluation+of+the+Disease-dependent+Distribution+of+Immune+Cells+in+Various+Tissues.">Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues.</a> J Vis Exp. (111), (2016).</li><li>Shaw, M. K., Zhao, X. Q., Tse, H. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+induction+of+experimental+allergic+encephalomyelitis.">Active induction of experimental allergic encephalomyelitis.</a> Nat Protoc. 1 (4), 1810-1819 (2006).</li><li>Fiander, M. D., Stifani, N., Nichols, M., Akay, T., Robertson, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinematic+gait+parameters+are+highly+sensitive+measures+of+motor+deficits+and+spinal+cord+injury+in+mice+subjected+to+experimental+autoimmune+encephalomyelitis.">Kinematic gait parameters are highly sensitive measures of motor deficits and spinal cord injury in mice subjected to experimental autoimmune encephalomyelitis.</a> Behav Brain Res. 317, 95-108 (2017).</li><li>Jones, M. V., 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=Behavioral+and+pathological+outcomes+in+MOG+35-55+experimental+autoimmune+encephalomyelitis.">Behavioral and pathological outcomes in MOG 35-55 experimental autoimmune encephalomyelitis.</a> J Neuroimmunol. 199 (1-2), 83-93 (2008).</li><li>van den Berg, R., Laman, J. D., van Meurs, M., Hintzen, R. Q., Hoogenraad, C. 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In mice with EAE, the two most common methods of measuring motor deficits are clinical scoring and fall latency from a rotarod27,28. These techniques have several limitations. Although convenient and widely used, clinical scoring is limited by yielding only ordinal level data, meaning that the magnitude of the differences between clinical scores are not known. Clinical scoring also suffers from being unable to provide precise information about the nature of the m...

Gait is a series of repetitive movements of the limbs used to achieve locomotion. Gait is comprised of step cycles, which are divided into two phases: the stance phase, which is when the foot is moving backwards on the ground to propel the body forwards; and the swing phase, where the foot is off the ground and moving forwards. Disturbances of gait are hallmark features of many neurodegenerative disorders, such as spinal cord injury (SCI), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Parkinson&#39;s disease (PD), and stroke; preclinical rodent models of these disorders often recapitulate their respective gait impairments1. The basic control mechanisms of locomotion in mice have been intensively studied2,3. Additionally, there are mouse models of many human neurological disorders4. Gait analysis in mice is therefore an attractive approach to measure multiple aspects of motor deficits that have known anatomical correlates. The study of gait in mouse models may provide insights into the neuropathological bases of locomotor deficits in neurodegenerative disorders, and enable the identification of potential treatments.
Some techniques that have been used to measure gait in rodents include visual inspection (e.g., the Basso mouse scale5 and open field test6) and analysis of gait from the ventral plane7. More recently, methods to measure sagittal plane kinematics of hindlimb movements have gained popularity because they provide more information about the execution of movement and consequently are more sensitive to subtle changes in gait8,9,10,11. Kinematic techniques developed to study hindlimb movement in sagittal plane while walking on a treadmill9,12 have been extensively studied in the context of SCI, ALS, traumatic cortical injuries, stroke, and Huntington&#39;s disease8,9,10,11,13,14,15,16. In contrast, these techniques have seen limited use in the study of locomotor deficits for mouse models of multiple sclerosis17.
Experimental autoimmune encephalomyelitis (EAE) is the most commonly used mouse model of MS18. The two main methods of inducing EAE is via active or passive inoculation. In active EAE, mice are immunized with myelin antigens, causing autoreactive T cell-mediated neuroinflammation and demyelination in the spinal cord and cerebellum. Passive EAE, on the other hand, is induced by transferring autoreactive T cells from a mouse with active EAE to a na&#239;ve mouse19. As described elsewhere, the disease course and neuropathology are influenced by the central nervous system (CNS) antigen and mouse strain20,21,22,23,24,25. In EAE experiments, control mice are injected with complete Freund&#39;s adjuvant (CFA) without the myelin antigen. EAE is characterized by ascending paralysis that begins with tail weakness and can potentially involve the forelimbs, resulting in ataxia and paralysis20. We have recently characterized gait changes in C57Bl/6 mice subjected to myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE. These studies have shown gait analysis to be superior than classical behavioral analysis because deviations from normal ankle movement are highly correlated with the degree of white matter loss in the lumbar spinal cord of EAE mice26. By contrast, the strength of the correlation between white matter loss and two other traditional behavioral measures (clinical scoring and rotarod) was much weaker26.
We describe here the use of kinematic gait analysis to detect movement deficits in the sagittal plane of EAE mice walking on a treadmill. Five reflective markers were placed on a hindlimb to identify movement of the hip, knee, and ankle joints in high-speed video recordings. Motion analysis software was used to extract kinematic data about joint excursions. The utility of these techniques to quantify movement deficits for the MOG35-55 model of EAE are discussed. These techniques are also applicable to the study of gait deficits in other mouse models of neurodegenerative disorders.

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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Camera</td>
			<td style="width:167px;">Nikon</td>
			<td style="width:344px;">Nikon D750</td>
			<td style="width:167px;">Used to film the video</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Reflective tape</td>
			<td>B&#38;L Engineering</td>
			<td>MKR-Tape-2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fine scissors</td>
			<td>Fine Science Tools</td>
			<td>15023-10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glue gun</td>
			<td>Craftsmart</td>
			<td>E231647</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">scalpel handle #4</td>
			<td>Roboz</td>
			<td>R5-9884</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Scalpel Blade No.10</td>
			<td>Feather</td>
			<td>2020-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">C57BL/6 mice</td>
			<td>Charles River Laboratories</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anesthetic machine</td>
			<td>EZ Anesthesia</td>
			<td>EZ-AF9000 Auto Flow System</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recirculating water heating blanket</td>
			<td>Androit</td>
			<td>HTP-1500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">topical eye lubricant</td>
			<td>Refresh</td>
			<td>DIN00210889</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Shaver</td>
			<td>Oster</td>
			<td>78997-010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">High speed camera</td>
			<td>Fastec</td>
			<td>Fastec IL3-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">High power light</td>
			<td>Smith Victor Corporation</td>
			<td>Model 700 SG (600 Watt quartz light, 120 Volts)</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Light Stand</td>
			<td>Promaster</td>
			<td>LS1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Treadmill</td>
			<td colspan="2">Custom built at the Zoological Institute, University of Cologne</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microsoft Excel 2016</td>
			<td>Microsoft</td>
			<td>Version 2016</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KinemaJ</td>
			<td>Nicolas Stifani</td>
			<td></td>
			<td>This is a script generated for use with ImageJ</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KinemaR</td>
			<td>Nicolas Stifani</td>
			<td></td>
			<td>This is a script generated for use with Rstudio</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vicon Motus</td>
			<td>Vicon Motus</td>
			<td>Version 9.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism</td>
			<td>GraphPad</td>
			<td>Version 6.00</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 is a schematic representation of the procedure used for kinematic gait analysis. First, reflective markers are made and placed on a mouse at 5 anatomical points. Gait is then recorded while the mouse is walking on a treadmill. Motion analysis software is used to extract kinematic data for subsequent analysis.
Figure 2A-C represent the step cycle of a control CFA mouse for the hip, knee, and ankle joint angles...

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

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 ...

The overall goal of this procedure is to measure functional motor impairments in the experimental autoimmune encephalomyelitis or EAE, mouse model multiple sclerosis using kinematic gait analysis. The application of kinematic gait analysis to mouse-walking behavior has been previously established and described by others. Neurological deficits in an EAE result from neural inflammation and sporadic white matter loss throughout the spinal cord and the cerebellum.Traditionally, motor stability in EAE mice has been assessed using clinical scoring systems, in which mice are assigned a clinical score based on the observer's impression of the severity of motor deficits. Data from clinical scores are ordinal and do not correlate well with the spinal cord histopathology. Kinematic gait analysis has recently been shown to be a better behavioral correlate of white matter loss than clinical scores, in addition to providing an objective description of walking deficits in the EAE mice.This technique involves placing reflective markers on the high limbs of mice and allowing them to walk on a treadmill while being recorded with a high-speed camera. Kinematic parameters are then extracted from the video using motion analysis software. The first step is to make the markers that will be placed on the animal's hind leg.The reflection of light off of these markers allows the coordinates of anatomical points on the leg to be extracted from the videos. To begin, punch the desired number of circles from the sheet of reflective paper. Each mouse requires five markers per recording, two large and three small.Using fine scissors, make a cut extending from the perimeter to the center of the circle. Peel off the paper backing to reveal the adhesive surface using fine forceps. Grip the marker with forceps and using your finger, roll the marker in on itself to form a cone.To make a small marker, make a longer cut and curl the cone more tightly. To make a large marker, make a shorter cut and curl the cone more loosely. Using a hot glue gun, fill the inside of the cone with glue and adhere it to a piece of cardboard.It is important to fully fill the marker with glue to prevent the marker from collapsing when handling during recording. Once the glue is dry, cut the marker off using a scalpel. Ensure that you cut away from your body.The next step is to prepare the animal for recording. To do this, the reflective markers must be adhered to the hind limb of the mouse at the proper anatomical locations. This is conducted under light anesthesia.Place the mouse in an induction chamber and anesthetize with 2.5%isoflurane. Once unconscious, transfer the mouse from the induction chamber to a nose cone or a recirculating water heating pad. Apply a topical lubricant to both eyes.Shave the desired hind limb extending from the ankle to the spine and the bottom of the ribs. In this demonstration, we are recoding movements of the right hind limb, but either or both limbs may be used. Locate the iliac crest by bringing both knees together with your thumb and forefinger and palpating just below the ribs.Mark this location using a permanent marker. Locate the hip by extending the leg and moving it back and forth. Place a marker over the hip joint, which is the point of articulation between the head of the femur and the pelvis.Using a flexible ruler, measure and record the length of the tibia or shank of the mouse. Measure and record the length of the femur or thigh as well. To isolate the fourth toe for marker placement, tape down the rest of the foot.Grasp a small marker with forceps and dip the flat end in glue. Place the small marker on the tip of the fourth toe. Place another small marker on the metatarsophalangeal joint.Place the last small marker on the ankle. Place a large marker on the hip joint directly over the mark on the skin. Place the second large marker on the mark over the iliac crest.Remove the tape from the foot. Place the mouse in a recovery cage and transport immediately to the gait recording room. The next step is recording the mouse walking on a treadmill.This picture is of our treadmill recording setup, showing the light, high-speed camera, and treadmill. Prior to recording the mouse's gait, take a picture of a calibration block with known dimensions on the treadmill. This will allow the pixels in the video to be converted to real measurements.It is critical that the camera angle and position at which the calibration picture is taken remains the same during the recording of walking behavior. Place the mouse in the treadmill. Increase the treadmill's speed gradually to orient the mouse in the correct direction.Slowly accelerate to 20 centimeters per second, which is the ideal running speed for obtaining recordings of steady gait. For accurate analysis, it's best to record eight to 12 step cycles of steady walking behavior. This is an example of consistent walking.The following is the same video shown at half speed. Because mice walk so quickly, it can be useful to view the videos at slower speeds to count step cycles and to better appreciate the gait pattern. This is one example of an EAE mouse walking on the treadmill.This mouse is unable to support its body weight as its pelvis is very low to the ground. Additionally, it is having difficulty lifting its foot off the ground during the swing phase. This is an example of another EAE mouse that has less severe motor deficits.The EAE mouse is walking on its toes with reduced movement at the ankle joint. This gives the mouse an uneven gait. The following are examples of behaviors that will compound analysis.Recordings, including these behaviors, should not be used. Lagging occurs when the mouse stops walking and travels to the back of the treadmill, but then resumes walking. This may occur in any mouse, but will occur more frequently at lower speeds.Rearing occurs when the mouse shifts its weight on its back hind limbs and raises its head and upper body. This behavior is common in anxious mice. The following are examples of poor lighting that will lead to issues with analysis of the markers'position in time and space.If the lighting is too dim, markers may not reflect enough light to be recognized by the computer software. In this video, the toe markers are not easily visible due to insufficient lighting. If the lighting is too bright, objects other than the markers may reflect too much light and be recognized as a marker.In this video, the toe markers appear to be merging with the reflective parts of the treadmill. Data from the reflective markers on mice walking can be used to create stick models of the leg from which kinematic parameters can be extracted. This is a recording of a mouse walking with a stick model of the leg superimposed.Note that in this video, there is a sixth marker on the knee, which is not necessary because the location of the knee can be triangulated from the positions of the hip and ankle joints, and the measured lengths of the femur and tibia. Because markers over the knee joint are often inaccurate due to skin slippage, triangulation is the preferred method. A mouse's step cycle can be divided into two main phases.The stance phase and the swing phase. This stick diagram can be used to qualitatively illustrate the movement of the hind limb in time and space. An example of this could be to assess the movement of the hind limb at different times points throughout the study.This example shows that the hind leg is compressed during the stance phase, indicating that the mouse has difficulty supporting its body weight. This is reflected by increased flexion of the knee and ankle joints. At the later time point, this has partially recovered.This video illustrates the relationship between the walking behavior and joint angles over time. The hip, knee, and ankle angle waveforms can be extracted from each recording. The joint angles can then be averaged over eight to 12 consecutive step cycles, resulting in an average step cycle that can be used for further analysis.This graph represents a knee joint waveform averaging from 10 consecutive step cycles. The data has been normalized, so that the length of the stance and swing phases are 100 frames respectively. The clear background represents the swing phase and the green background represents the stance phase.For this mouse, the walking behavior from week to week was very consistent and the step-cycle waveforms from each week overlapped considerably. However, there can also be substantial variability in gait in healthy mice as seen in this graph. The degree of variability shown here is acceptable and within the range of what might be expected from a mouse.This graph represents the knee step cycle from the EAE mouse recorded on three consecutive weeks. There is a slight change in the shape of the step cycle at week two and a substantial deviation by week three, which the mouse's knee is much more flexed and does not extend during walking. There are numerous parameters that can be measured using this technique.We'll briefly describe three. The average angle is obtained by averaging all angles throughout the normalized step cycle. In this instance, the average angle decreases throughout the study, suggesting that the mice are not extending their knees as much as normal.The range of motion is obtained by subtracting the smallest angle from the largest angle in the normalized step cycle. This parameter can give you insight into joint flexibility, rigidity, or weakness. In this example, the knee range of motion decreases throughout the study indicating that the mice are not able to move their knee normally, possibly due to muscle weakness.Root-Mean-Square difference is a method used to measure deviation of step-cycle waveforms from the baseline recording. This parameter tells you how much deviation there is from the initial recording. Kinematic gait analysis is a valuable technique that can be used to sensitively detect and describe changes in gait.The application of kinematic gait analysis to EAE studies may be a valuable tool in understanding the functional consequences of spinal cord pathology in this model. It may facilitate discovery of novel treatments for multiple sclerosis. Additionally, kinematic gait analysis is not limited to the context of EAE.This technique has previously been used in mouse models with spinal cord injury, amyotrophic lateral sclerosis, Huntington's disease and stroke, and can be applied to other mouse models with neurological disorders, including Parkinson's disease.

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Research

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Neuroscience

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 ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

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.

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 ...

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 ...

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics ...

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

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.

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different ...

Adolescent Social Stress Paradigm in C57BL&#47;6 Mice for Behavioral Research

Social Defeat Stress Model for Adolescent C57BL&#47;6 Male and Female Mice

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female rodents is needed to understand its effects on ongoing brain development and behavioral outcomes. The chronic social defeat stress paradigm (CSDS) has been widely used to model social stress in adult C57BL/6 male mice by leveraging on the aggressive behavior displayed by an adult male rodent to an intruder invading its territory. An advantage of this paradigm is that it allows to categorize defeated mice into resilient and susceptible groups based on their individual differences in social behavior 24 h after the last defeat session. Implementing this model in adolescent C57BL/6 mice has been challenging because adult or adolescent mice do not typically attack early adolescent male or female mice and because adolescence is a short period of life, encompassing discreet temporal windows of vulnerability. This limitation was overcome by adapting an accelerated version of the CSDS to be used for adolescent male and female mice. This 4-day stress paradigm with 2 physical attack sessions per day uses a C57BL/6 male adult to prime the CD-1 mouse for aggressiveness such that it readily attacks the male or female adolescent mouse. This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Adolescent exposure to AcSD induces social avoidance 24 h later in both males and females, but only in a subset of defeated mice. This vulnerability occurs despite the number of attacks being consistent across sessions between resilient and susceptible groups. The AcSD model is short enough to allow exposure during discrete periods within adolescence, allows the segregation of mice according to the presence or absence of social avoidance behavior, and is the first model available to study social defeat stress in adolescent C57BL/6 female mice.

Author Spotlight: Understanding Adolescent Social Adversity Effects on Neurodevelopment in Mice

We developed an accelerated social defeat stress model for adolescent C57BL/6 mice, which works in both males and females and allows exposure during discrete adolescent periods. Exposure to this model induces social avoidance, but only in a subset of defeated male and female mice.

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female ...

Preparation of MOG35-55 Emulsion for Immunization of Mouse

Preparation of MOG35-55 Emulsion for Immunization of Mouse  

To begin, prepare the myelin oligodendrocyte glycoprotein peptide at 35 to 55, or MOG 35 to 55 solution, in a glass beaker. To do so, add the liquid component to the beaker, followed by adding the appropriate mycobacterium tuberculosis strain. Place the glass beaker containing the solution in an ice bath.Using a glass syringe with an 18-gauge needle, initiate the emulsification process, and continue emulsifying the solution for a minimum of 15 minutes. To assess the quality of the emulsion, dispense a drop of the emulsion into a transparent container filled with water. Consider the emulsion ready if the drop maintains its structure and remains intact.Transfer the emulsion directly into the one-milliliter syringes designated for immunization purposes. Store the filled syringes at four degrees Celsius until they're ready for use.

Immunization of Mouse with MOG35-55 to Induce Experimental Autoimmune Encephalomyelitis

Immunization of Mouse with MOG35-55 to Induce Experimental Autoimmune Encephalomyelitis  

Begin the immunization of the mouse by performing a PT intravenous injection into its lateral caudal vein. To do so, gently plug the tail of the properly anesthetized mouse with an ethanol solution, which acts as a vasodilator to make the veins visible. Focusing on one of the two lateral veins of the tail, use 0.5 milliliter syringe fitted with a 30 gauge needle to inject 500 nanograms of PT.Next, proceed to administer subcutaneous injections of MOG 35 to 55 emulsion previously prepared in one milliliter syringes.Using the syringes with 26 gauge needles, perform two subcutaneous injections of 100 microliters each under the rostral part of the flanks. Then administer one injection of 100 microliters at the base of the tail.