Name: sagittal plane kinematic gait analysis in c57bl/6 mice subjected to mog35-55 induced experimental autoimmune encephalomyelitis
Uploaded: 2017-11-04T13:00:00.000+00:00
Duration: 13 min 2 s
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 the circle.</li>
	<li>Remove the paper backing of the marker to reveal the adhesive surface. Using fine forceps, grip the marker firmly and curl it in on itself using your finger to form a cone shape. To make a small marker, curl the cone tightly. To make a large marker, curl the cone loosely.</li>
	<li>Using a hand-held glue gun, fill the inside of the cone-shaped marker with glue while gripping the tip of the cone with forceps and adhere the marker to a flat piece of cardboard. The glue will prevent the marker from collapsing and bending during recording to ensure optimal reflection of light. When the glue is dry (approximately 10 min), remove the marker from the cardboard with a scalpel (Figure 1A).</li>
</ol>
2. Prepare the Animal for Recording
<ol>
	<li>Anesthetize the mouse with isoflurane gas (2.5%; 2 liters/min O2) by placing the mouse into an induction chamber. Once the mouse is unconscious, place it into a nose cone positioned on top of a recirculating water heating blanket. The purpose of anesthetization is to immobilize the mouse for marker placement; the procedure is not painful. Therefore, depth of anesthesia does not need to be assessed.</li>
	<li>Apply a topical eye lubricant to both eyes.</li>
	<li>Shave the desired hindlimb using electric clippers. Begin at the ankle and extend to the spine and bottom of the ribs; ensure no fur is left as this will impair marker adhesion. 
	Note: Here, the right hindlimb was recorded; however, either hindlimb can be used.</li>
	<li>Using a permanent marker, indicate the location of the iliac crest and the hip joint. The iliac crest is just below the bottom of the ribs and is easily palpable by bringing the knees together under the mouse&#39;s body. 
	Note: The hip joint can be found by flexing and extending the leg to find the articulation point between the pelvis and femur.</li>
	<li>Using fine forceps, grasp the pointed end of a small marker and dip the base in fast-acting adhesive glue, or an equivalent alternative. Place the marker on the tip of the fourth digit and hold in place for 2-3 s to allow the glue to dry. Place the other two small markers on the metatarsophalangeal joint and ankle in the same manner (Figure 1B).</li>
	<li>Place large markers on the iliac crest and hip joint (Figure 1B) in the same manner as the small markers.</li>
	<li>Remove the mouse from the nose cone and immediately transfer to the recording room using a transfer cage. Place the mouse on the stationary treadmill and allow for full recovery from anesthesia.</li>
</ol>
3. Gait Recording
<ol>
	<li>Prior to recording the mouse&#39;s gait, take a picture of a calibration block with known dimensions on the treadmill. 
	Note: This will allow the pixels in the video to be converted to real measurements. The camera should be placed approximately 120 cm from the treadmill.
	<ol>
		<li>Position the camera at the same height and level as the treadmill. Maintain the same camera position for the recordings following the calibration image.</li>
	</ol>
	</li>
	<li>Once the mouse has fully recovered from anesthesia, turn the treadmill to a low speed (5 cm/s) to let the mouse begin walking. Ensure that the treadmill belt direction is such that the markers on the mouse are facing towards the camera.</li>
	<li>Increase the treadmill speed gradually up to 20 cm/s; this is the ideal speed for a consistent gait in most healthy mice. 
	Note: Although it is ideal to have all mice walking at the same speed, some may be unable to consistently reach this speed.
	<ol>
		<li>If the mouse is unable to walk at 20 cm/s, reduce the speed as necessary and be sure to make a note of this. Reduce the speed of the treadmill until consistent step cycles are achieved. 
		Note: Later data analysis can adjust for differences in speeds.</li>
	</ol>
	</li>
	<li>Begin recording the video once the mouse is walking steadily (i.e., walking at a consistent pace, not rearing or weaving side to side). Continue recording until 8 to 12 consecutive step cycles have been recorded. For every video, record the speed of the treadmill and the side of the mouse recorded.</li>
	<li>Once recording is complete, turn off the treadmill and return the mouse to its cage. Clean the treadmill thoroughly between recordings as scents left behind by other mice may alter the behavior of incoming mice. To reduce stress and skin damage, do not remove the markers; allow the mice to remove them on their own.</li>
</ol>
4. Analysis
<ol>
	<li>Process the videos using motion analysis software. 
	Note: In our experiments, we used custom scripts designed for imaging and statistical software (see Table of Materials) that were written by Dr. Nicolas Stifani. The following steps are performed using the selected motion analysis software.
	<ol>
		<li>Extract the pixel coordinates of the markers from the videos, and using the calibration video, transform the pixel values to centimeters and calculate the joint angles at each frame.</li>
		<li>Identify the beginning and end of each step cycle, thereby obtaining information about step duration and length.</li>
		<li>Normalize the step cycle duration to 200 normalized frames, such that swing and stance are represented by 100 frames, respectively.</li>
	</ol>
	</li>
	<li>Using the normalized frames, calculate kinematic parameters for data analysis using spreadsheet software (see Table of Materials).
	<ol>
		<li>To establish the average angle of a particular joint, take the mean of all angles within a normalized frame as: 
		<img src="/files/ftp_upload/56032/56032eq1v4.jpg" /> 
		Note: Here x represents the angle value at a given normalized frame, and n represents the normalized frame number.</li>
		<li>To establish the range of motion of a particular joint for a given mouse, subtract the smallest angle from the largest angle in a set of normalized frames as follows: 
		Range of motion = Anglemaximum - Angleminimum. 
		Note: Here Anglemaximum and Angleminimum are the greatest and smallest angles achieved within the normalized step cycle, respectively.</li>
		<li>To establish RMS difference, first subtract the average angle of each experimental time-point from the baseline recording. Next, square each difference, take the mean of all squared values and square root the mean. The equation is as follows: 
		<img src="/files/ftp_upload/56032/56032eq3v4.jpg" /> 
		Note: Here <img src="/files/ftp_upload/56032/56032eq4.jpg" /> represents the average angle from the baseline recording; y represents the average angle from each experimental time-point; n and represents the number of normalized frames. Root mean squared (RMS) difference is a measurement used to assess deviation in gait from baseline recordings.</li>
	</ol>
	</li>
	<li>Use scientific graphing and statistics software to analyze and present the data (see Table of Materials).</li>
</ol>

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The authors declare that they have nothing to disclose.

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 motor deficits. The rotarod test improves on some limitations of clinical scoring, but only measures general motor coordination and does not measure specific aspects of walking.
By comparison, kinematic gait analysis provides sensitive measures about specific aspects of locomotion, including the range of motion and average angles at various joints. Subtle deficits in hip and knee joint movements for MOG35-55 EAE mice have been detected at DPI9, approximately 5-9 days before the onset of clinical symptoms or rotarod deficits26. These deficits persisted despite a complete remission of clinical signs and were observed in the absence of rotarod deficits26. Importantly, impaired ankle movements as measured by RMS difference correlated extremely well with white matter loss in the spinal cord26.
Several methodological points deserve specific mention: 1) Accurate and consistent placement of the joint markers is crucial - the hip joint and iliac crest must be carefully identified by palpitation; 2) It is necessary to obtain recordings from 8-12 step cycles. Averaging these step cycles produces a representative average step cycle that can be further analyzed; 3) Optimal lighting conditions must be established to ensure that the markers are clearly visible in the recordings. If markers are not illuminated properly, this can make digitizing the videos a laborious process as many motion analysis programs will be unable to track the markers, necessitating manual tracking.
One additional limitation of this technique is that it is labor intensive. For example, to record and analyze data from a group of 10 mice, we estimate the total process takes approximately 7.0-9.0 hours (h). Making 50 markers (5 per mouse) takes about 2.0 h. Recording mouse walking behavior can be done either alone or in a pair. Working alone, it takes about 25 min per mouse, while working in a pair takes around 10 min per mouse; therefore, recording 10 mice may take from 1.5 h (pair) to 4.0 h (solo). Lastly, data analysis and graphing take approximately 3.5 h. Although this technique is labor intensive, we feel that the potential insights into disease mechanisms offered by kinematic gait analysis justifies this investment. Having good behavioral correlates of disease pathology is useful as serial measurements can be taken from a live mouse non-invasively. Given the near perfect correlation between ankle kinematics and lumbar spinal cord white matter loss26, this method may be used to ascertain the temporal profile of demyelination and remyelination in EAE mice over the course of an experiment, allowing recovery to be assessed.
Gait analysis is complicated by severe paralysis that restricts movement of the hindlimbs. However, even severely paralyzed mice (clinical score &#62;3.0) are often able to ambulate to some extent. In these cases, the forelimbs are used to pull the animal forward, and some hindlimb movement occurs which can be measured by kinematic gait analysis. Even in these severe cases, it is still possible to measure recovery of hindlimb function over time. Only in very severe cases (20% of animals with clinical scores &#62;3.5 at peak disease, DPI 16-23) have we been unable to obtain useful recordings of hindlimb movement. Nevertheless, these animals usually regain some hindlimb function by DPI 30, allowing meaningful recordings to be obtained at that time point.
A future application of this technique is coupling kinematic data with simultaneous electromyographic recordings of the hindlimb during locomotion. This technique has been done in mouse models of ALS and SCI and can be used to elucidate the relationship between muscle activity, innervation, and gait. This technique could also be coupled with more targeted models of MS and demyelination that may produce more discrete gait deficits, including focal EAE models29,30 or cuprizone-induced demyelination31.
The techniques we have described for the measurement of joint movements in EAE mice can also be applied to other disorders that impair gait. Distinct changes in gait have been reported for mouse models of PD, SCI, ALS, and stroke8,9,10,11,13,14. For example, rodent models of PD are characterized by reduced stride length and velocity, resulting in elevated cadence to maintain walking speed32. Kinematic gait analysis therefore provides powerful behavioral tools to elucidate disease mechanisms and identify potential treatments using these models.

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.

<table><tbody><tr><td>Camera</td><td>Nikon</td><td>Nikon D750</td><td>Used to film the video</td></tr><tr><td>Reflective tape</td><td>B&amp;amp;L Engineering</td><td>MKR-Tape-2</td><td></td></tr><tr><td>Fine scissors</td><td>Fine Science Tools</td><td>15023-10</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Glue gun</td><td>Craftsmart</td><td>E231647</td><td></td></tr><tr><td>scalpel handle #4</td><td>Roboz</td><td>R5-9884</td><td></td></tr><tr><td>Scalpel Blade No.10</td><td>Feather</td><td>2020-12</td><td></td></tr><tr><td>C57BL/6 mice</td><td>Charles River Laboratories</td><td></td><td></td></tr><tr><td>Anesthetic machine</td><td>EZ Anesthesia</td><td>EZ-AF9000 Auto Flow System</td><td></td></tr><tr><td>Recirculating water heating blanket</td><td>Androit</td><td>HTP-1500</td><td></td></tr><tr><td>topical eye lubricant</td><td>Refresh</td><td>DIN00210889</td><td></td></tr><tr><td>Shaver</td><td>Oster</td><td>78997-010</td><td></td></tr><tr><td>High speed camera</td><td>Fastec</td><td>Fastec IL3-100</td><td></td></tr><tr><td>High power light</td><td>Smith Victor Corporation</td><td>Model 700 SG (600 Watt quartz light, 120 Volts)</td><td></td></tr><tr><td>Light Stand</td><td>Promaster</td><td>LS1</td><td></td></tr><tr><td>Treadmill</td><td>Custom built at the Zoological Institute, University of Cologne</td><td></td><td></td></tr><tr><td>Microsoft Excel 2016</td><td>Microsoft</td><td>Version 2016</td><td></td></tr><tr><td>KinemaJ</td><td>Nicolas Stifani</td><td></td><td>This is a script generated for use with ImageJ</td></tr><tr><td>KinemaR</td><td>Nicolas Stifani</td><td></td><td>This is a script generated for use with Rstudio</td></tr><tr><td>Vicon Motus</td><td>Vicon Motus</td><td>Version 9.00</td><td></td></tr><tr><td>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 recorded at three consecutive recording sessions spaced one week apart. The overlap between the waveforms shows minimal deviation in the step cycles from sessions 1-3. Figure 2D-F represent the step cycle of a second control CFA mouse that displayed greater walking variability from recording sessions 1-3. Although the step cycles are shifted along the y-axis, the shape of the waveforms remains consistent between recordings. This level of variability is typical for mouse walking.
Figure 3A-C represent the step cycle of a mouse with EAE recorded on three consecutive recording sessions. There are minimal changes in gait from the first to the second recording session, but by the third session, gait has been profoundly altered at all three joints. For the hip, a significant flattening over the step cycle has occurred, indicating a substantial loss of movement. The knee has become more flexed and less able to extend and support the animal&#39;s body weight. Movements at the ankle joint were also substantially altered. Foot dorsiflexion and plantar flexion are delayed during the swing (white panel) and stance (green panel) phases, respectively. These deficits are indicative of muscle weakness at this joint as the animal is impaired in its ability to both raise its foot during the swing phase, and propel the body forward during the stance phase.
The following data presented in Figure 4 were republished from Fiander et al. (2017)26 with permission. The data were analyzed using one-way repeated measures ANOVA with Holm-Sidak multiple comparisons test to compare all time points to baseline26. The average angle (Figure 4A and Figure 4D), range of motion (Figure 4B and Figure 4E) and RMS difference (Figure 4C and Figure 4F) were calculated at each time-point to quantify gait deficits (n=8 per group). In the present EAE experiment, the onset of clinical scores was DPI 14, which is after the second week of recording. CFA mice showed no change in average knee angle (Figure 4A) or knee RMS difference (Figure 4C), but did exhibit a small increase in knee range of motion [F(2,7) = 5.871, p = 0.0083], on both DPI 16 and 30 relative to the baseline (Figure 4B). This small change may reflect pain resulting from the CFA injection. In contrast to the CFA animals, there were large changes at the knee joint for EAE animals for the average angle [F(6,7) = 11.08, p &#60; 0.0001] (Figure 4D), range of motion [F(6,7) = 14.42, p &#60; 0.0001] (Figure 4E) and RMS difference (Figure 4F). The average angle was significantly reduced, indicating that EAE mice had their knees more flexed during walking. This may be indicative of muscle weakness, as the animals were unable to extend their knee joints to support their body weight. The range of motion was also decreased, again likely due to an inability of the animals to extend the knee joint. The significant increase in knee RMS difference indicates that the movements of the knee joint in EAE mice were substantially different from their baseline recording.
The data in Figure 5 were analyzed using one-way repeated measures ANOVA with the Holm-Sidak multiple comparisons test that compared gait parameter values at clinical scores of 0.5 - 3.5 to those detected at a clinical score of 0. Correlational analysis was also performed using Spearman rho (&#961;). The average knee angle (Figure 5A), range of motion (Figure 5B), and RMS difference (Figure 5C) were strongly correlated with clinical scores (p &#60; 0.001). These correlations between joint movements and classical clinical scoring substantiate the validity of kinematic gait analysis to assess motor deficits for EAE mice. Knee range of motion (Figure 5A) and RMS difference (Figure 5C) were significantly decreased beginning at a clinical score of 2.0 (p&#60;0.05). These findings suggest that impaired knee movements do not contribute to motor deficits detected by clinical scores lower than 2.0. However, knee average angle (Figure 5B) was decreased beginning at a clinical score of 1.0 (p&#60;0.05). This suggests that for knee movement, average angle is the most sensitive of the three measures.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56032/56032fig1v2.jpg" /> 
Figure 1: Schematic for kinematic gait recording with mice. Once reflective markers are made, they are placed on the iliac crest, hip joint, ankle, metatarsophalangeal joint, and the tip of the fourth digit. Gait is recorded by a high-speed camera while the mouse is walking on a treadmill. Motion analysis software is used to extract gait parameters for subsequent analysis. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56032/56032fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56032/56032fig2v2.jpg" /> 
Figure 2: Example of step cycle waveforms in two control mice that received CFA 
The white and green backgrounds represent the swing and stance phase, respectively. For mouse 1, the hip (A), knee (B), and ankle (C) step cycle waveforms overlap each other across 3 consecutive recording sessions spaced one week apart. For mouse 2, the hip (D), knee (E), and ankle (F) step cycle waveforms deviate slightly from each other due to the inherent variability in walking behavior. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56032/56032fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56032/56032fig3v2.jpg" /> 
Figure 3: Step cycle waveforms in mice with EAE. The white and green backgrounds represent swing and stance phase, respectively, for three consecutive recording sessions spaced a week apart. By the 3rd recording session, the hip (A), knee (B), and ankle (C) waveforms are greatly changed due to EAE disease progression. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56032/56032fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56032/56032fig4v2.jpg" /> 
Figure 4: Average angle, range of motion, and root mean squared are used to analyze kinematic data. Average angle, range of motion, and RMS differences were calculated to quantify motor deficits in EAE mice. The average knee angle (A), range of motion (B), and RMS (C) for CFA mice remained relatively constant. Mice with EAE showed impaired knee average angle (D), range of motion (E), and RMS (F). Data are expressed as mean &#177; standard deviation; * p&#60;0.05, ** p&#60;0.01, *** p&#60;0.001, difference from day post immunization (DPI) -2; # p &#60; 0.05, difference from peak deficit. Reprinted from reference 26 with permission from original publishers. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56032/56032fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56032/56032fig5v2.jpg" /> 
Figure 5. Average knee angle, range of motion and RMS difference correlate with clinical score 
Correlation analysis was performed between three kinematic measures of knee movements and clinical scores to compare the two methods. The average knee angle (A), range of motion (B), and RMS difference (C) were strongly correlated with clinical scores. The knee range of motion and RMS difference decreased beginning at a clinical score of 2.0, while average knee angle was reduced earlier to a clinical score of 1.0. Data are expressed as mean &#177; standard deviation; * p&#60; 0.05 difference from clinical score 0.0. For Spearman rho (&#961;), *** p&#60; 0.001. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56032/56032fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

sagittal-plane-kinematic-gait-analysis-c57bl6-mice-subjected-to-mog35

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

8.5K Views.  Dalhousie University. 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.

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

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Immunology and Infection

3D Kinematic Gait Analysis for Preclinical Studies in Rodents

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of complex algorithms and mathematical modeling that accompany 3D data collection and analysis procedures. This work provides a simple, user-friendly, step-by-step detailed methodology for 3D kinematic gait analysis during treadmill locomotion in healthy and neurotraumatic rats using a six-camera motion capture system. Also provided are details on 1) calibration of the system in an experimental set-up customized for quadrupedal locomotion, 2) data collection for treadmill locomotion in adult rats using markers positioned on all four limbs, 3) options available for video tracking and processing, and 4) basic 3D kinematic data generation and visualization and quantification of data using the built-in data collection software. Finally, it is suggested that the utility of this motion capture system be expanded to studying a variety of motor behaviors before and after neurotrauma.

Presented here is a protocol to collect and analyze three-dimensional kinematics of quadrupedal locomotion in rodents for preclinical studies.

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of ...

Behavior

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

Developmental Biology

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

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 ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Quantifying Locomotor Dysfunction in Spinal Cord Injury Mice Using MouseWalker

Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and supra-spinal circuits. After a thoracic spinal cord injury, communication with upstream circuits is impaired. This, in turn, leads to a loss of coordination, with limited recovery potential. Hence, to better evaluate the degree of recovery after the administration of drugs or therapies, there is a necessity for new, more detailed, and accurate tools to quantify gait, limb coordination, and other fine aspects of locomotor behavior in animal models of spinal cord injury. Several assays have been developed over the years to quantitatively assess free-walking behavior in rodents; however, they usually lack direct measurements related to stepping gait strategies, footprint patterns, and coordination. To address these shortcomings, an updated version of the MouseWalker, which combines a frustrated total internal reflection (fTIR) walkway with tracking and quantification software, is provided. This open-source system has been adapted to extract several graphical outputs and kinematic parameters, and a set of post-quantification tools can be to analyze the output data provided. This manuscript also demonstrates how this method, allied with already established behavioral tests, quantitatively describes locomotor deficits following spinal cord injury.

Author Spotlight: Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury  

An experimental pipeline to quantitatively describe the locomotor pattern of freely walking mice using the MouseWalker (MW) toolbox is provided, ranging from initial video recordings and tracking to post-quantification analysis. A spinal cord contusion injury model in mice is employed to demonstrate the usefulness of the MW system.

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and ...

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 ...

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...

Biology

Generation of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Source: Tietz, S. M. et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59249">Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo.</a> J. Vis. Exp.&#160; (2019)
This video demonstrates the method of inducing experimental autoimmune encephalomyelitis (EAE) by injecting myelin-derived peptides with an adjuvant to activate autoreactive T cells, which migrate to the brain. Pertussis toxin is administered to increase blood-brain barrier permeability, allowing these T cells and other immune cells to enter the brain. These immune cells cause demyelination, leading to impaired nerve transmission and development of EAE.

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

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

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Myelin Oligodendrocyte Glycoprotein (MOG_35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice