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 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.

<table border="0" cellpadding="0" cellspacing="0" width="893">
	<tbody>
		<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...

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

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

Assessing Burrowing, Nest Construction, and Hoarding in Mice

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening of possible treatments for AD currently largely focuses on cognitive testing, which frequently demands expensive equipment and lots of experimenter time. However, human episodic memory (the most severely affected aspect of memory in AD) is different to rodent memory, which seems to be largely non-episodic. Therefore the present ways of screening for new AD treatments for AD in rodents are intrinsically unlikely to succeed. A new approach to preclinical screening would be to characterise the ADL of mice. Fortuitously, several such assays have recently been developed at Oxford, and here the three most sensitive and well-characterised are presented.
Burrowing was first developed in Oxford13. It evolved from a need to develop a mouse hoarding paradigm. Most published rodent hoarding paradigms required a distant food source to be linked to the home cage by a connecting passage. This would involve modifying the home cage as well as making a mouse-proof connecting passage and food source. So it was considered whether it would be possible to put the food source inside the cage. It was found that if a container was placed on the floor it was emptied by the next morning., The food pellets were, however, simply deposited in a heap at the container entrance, rather than placed in a discrete place away from the container, as might be expected if the mice were truly hoarding them. Close inspection showed that the mice were performing digging ("burrowing") movements, not carrying the pellets in their mouths to a selected place as they would if truly hoarding them.6



Food pellets are not an essential substrate for burrowing; mice will empty tubes filled with sand, gravel, even soiled bedding from their own cage. Moreover, they will empty a full tube even if an empty one is placed next to it8.



Several nesting protocols exist in the literature. The present Oxford one simplifies the procedure and has a well-defined scoring system for nest quality5. 



A hoarding paradigm was later developed in which the mice, rather than hoarding back to the real home cage, were adapted to living in the "home base" of a hoarding apparatus. This home base was connected to a tube made of wire mesh, the distal end of which contained the food source. This arrangement proved to yield good hoarding behaviour, as long as the mice were adapted to living in the "home base" during the day and only allowed to enter the hoarding tube at night.

Burrowing, nesting, and hoarding are species-typical activities that mice readily perform in the laboratory. This article describes how they can be easily and cheaply assessed. These protocols are extremely sensitive to mouse strain, brain lesions and diseases. Moreover they constitute &#x201C;environmental enrichment&#x201D; for the mice, and embody the &#x201C;Refinement&#x201D; aspect of the &#x201C;3 Rs&#x201D;18.

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening ...

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

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 ...

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

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

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

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

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

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 ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...