Name: paw-dragging: a novel, sensitive analysis of the mouse cylinder test
Uploaded: 2015-04-29T16:00:00.000+00:00
Duration: 9 min 14 s
Description: Watch this Scientific Journal Video about Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test at JoVE.com

We thank Mr. John Crowell and Mr. Terry Upshall for their technical expertise and assistance with the photography and videography. This work was supported by operating grants to JLV from the Canadian Institutes of Health Research and the Research and Development Corporation of Newfoundland and a Heart &#38; Stroke Foundation of Canada Canadian Partnership for Stroke Recovery Catalyst grant. RBR was a recipient of a Keith Griffiths Memorial Heart &#38; Stroke Foundation Graduate Scholarship.

Ethics statement: All experiments were approved by Memorial University of Newfoundland&#39;s Animal Care Ethics Committee according to the guidelines of the Canadian Council on Animal Care.
1. Mice
<ol>
	<li>Use adult mice. In this study, adult male FVBN mice (n=10) between 2-4 months were used. House mice on a 12:12 hr reverse light-dark cycle and provide standard rodent chow and water ad libitum.</li>
</ol>
2. Materials Required for the Cylinder Test
<ol>
	<li>Obtain a table with a transparent top to film the cylinder test. The dimensions of the table are irrelevant, the top must be plexiglass or glass, and there must be enough room to position a mirror below the table. This allows for the mouse to be videotaped from below. Use a mirror below the table to reflect the image through the cylinder. As an alternative, use an upside-down camera if available. The dimensions of the table used in this protocol are 54 x 56 x 66.5cm (w x l x h) with a 51 x 51cm top (w x l).</li>
	<li>Obtain a mirror. The dimensions of the mirror used in this protocol are 34 x 58cm (w x l).</li>
	<li>Obtain a transparent/plexiglass cylinder for the mouse to perform in. The dimensions of the cylinder used in this protocol are 17.5cm high, 8.8cm I.D, 9.5cm O.D with a wall thickness of 0.35cm. A taller cylinder may be required for more active mouse strains.</li>
	<li>Place the cylinder on the tabletop and film the reflection in the mirror below.</li>
	<li>A videocamera and tripod are required. Record videos at approximately 650Kb/s, which is approximately 190Mb per 5 min of cylinder video. Ensure that the camera has a zoom functionality to ensure that the cylinder encompasses the entire field of view. 
	NOTE: The videocamera used in this protocol is a Sony DCR-SR42, 40x optical zoom, 2,000x digital zoom, 680kpix which uses standard definition, NTSC interlaced video.)</li>
	<li>Obtain software for analysis &#8211; a media player with video support and playback speed modulation. The media player used in this protocol is the VLC Media Player v2.1.2.</li>
	<li>Obtain a computer with an operating system capable of running the media player and a monitor.</li>
	<li>Electronic storage for videos is required. Download videos to an external hard drive or copy it onto DVDs for long-term storage. 
	NOTE: At 190Mb per video, 84 sessions will fit on a 16 GB SD card and 168 sessions will fit on a 32GB SD card. Due to the relative inexpensiveness of SD media, and the uncertainty in how much time some mice require to complete 20 rears, a 32 GB card is recommended. In the current study, the videos were copied from the camera to a 2TB external hard drive and then recopied onto DVDs as backup.</li>
</ol>
3. Experimental Setup of the Cylinder Test
<ol>
	<li>Fasten mirror below the table at a 45 degree angle to the tabletop. Do this using two support brackets attached to the table legs to support the top and bottom of the mirror, respectively. Bracket locations are noted in a side-view of the table (Figure 1A) and a face-on view of the table (Figure 1B).</li>
	<li>Place cylinder on the center of the table. Draw four equidistant lines where the cylinder sits on the table with a black marker (Sharpie) so that the cylinder can be lifted and returned to the same position (Figure 2). Drawing them on the underside of the transparent tabletop allows the tabletop to be cleaned between animal tests without dissolving the marker ink.</li>
	<li>Attach the camera and tripod. Aim the camera at the mirror so that the picture is looking directly through the barrel of the cylinder. Ensure that the full inner wall of the cylinder is visible and unobstructed by the base (Figure 3). See a side-on view of the setup, including the relative angle of the camera and mirror setup, from above (Figure 4A) and from the level of the table, including a mouse rearing (Figure 4B).</li>
	<li>Prepare cue cards to identify each mouse prior to filming. Ensure that the cards typically include an identification number for each mouse, the time-point of the test (e.g., 3 days after treatment) and the date of the filming session. Do not include the treatment group on the cue card to ensure the experimenter is blinded.</li>
	<li>Film in standard indoor lighting conditions as this level of light is required to clearly see the mouse movements around the cylinder. 
	NOTE: If available filming in the dark with a red light camera may suffice, however one would first need to test whether paw touches and drags are clearly visible for quantification.</li>
</ol>
4. Execution
<ol>
	<li>Start filming. Display the appropriate mouse cue card in front of camera lens.</li>
	<li>Lower the mouse into the cylinder from the open top immediately after filming the cue card.</li>
	<li>Begin filming the mouse. Minimize noise during this time, as mice may lose interest in exploring if startled.</li>
	<li>Observe the mice rear to explore the cylinder. Capture the video until the mouse performs a minimum of twenty rears. 
	NOTE: A rear occurs when both forepaws lose contact with the floor and the mouse stands on its hind legs.</li>
	<li>Wipe tabletop and cylinder with an appropriate cleaning solution between each mouse to sanitize and remove mouse scents.</li>
	<li>Freezing is when mice stop exploring and remain quietly sitting on all fours for approximately 5 min. If mice freeze before twenty rears occur, it may be necessary to remove them from the cylinder for 10-20 min before resuming the test. If mice do not perform twenty rears, they are removed from the study. 
	NOTE: In our experience, mice have never needed to be excluded due to failure to explore the cylinder.</li>
</ol>
5. Evaluation of the Cylinder Test using Paw-Dragging Analysis
<ol>
	<li>Play back the video at a rate of between 0.25x and 0.67x regular speed depending on how quickly the mouse explores the cylinder. Use a media player that offers slower playback speeds.</li>
	<li>Quantify the total number of paw touches. Paw touches occur when the mouse rears (Figure 5A), touches the side of the cylinder (Figure 5B), subsequently dismounts with both paws simultaneously (Figure 5C) and lands (Figure 5D). The paw may or may not contact the cylinder wall with a full palm, but some contact with the cylinder wall must occur. Assess paw touches by counting the number of times the mouse makes contact (no matter how brief) with the cylinder wall with either or both forepaws while standing on its hind limbs during a rear. 
	Note that contact with the cylinder wall is only counted as a &#8220;paw touch&#8221; or &#8220;paw-drag&#8221; if the mouse is in a rear position &#8211; standing on its hind limbs with both forepaws off of the tabletop. If the mouse remains in a 3-point stance &#8211; both hind limbs and one forepaw on the tabletop and proceeds to touch the wall with the free paw &#8211; this is not counted as a paw touch. Mice may rear and touch the cylinder wall with a single forepaw and this is counted as a paw touch. Note: a mouse may also move its body around the cylinder during a rear, making more than two contacts. These contacts are tallied &#8211; one for each left forepaw touch and one for each right forepaw touch.</li>
	<li>Quantify the number of paw-drags. Paw-dragging behaviour is distinct from normal paw touches.
	<ol>
		<li>If the paw contacts the cylinder wall with a full open palm (Figure 6B), it will slowly fall away from the wall, often with a slight tremor. The movement begins with the digits dragging against the cylinder wall either in a medial or downward direction, (Figure 6C) before falling away completely (Figure 6D). The mouse will then dismount with its unaffected paw (Figure 6E) before landing on all fours (Figure 6F). This is considered a paw-drag and should be counted in a tally.</li>
		<li>If the paw does not contact the cylinder wall with a fully open palm, it will graze the cylinder wall with its digits before falling away from the cylinder wall. Similarly, a mouse may drag its paw against the cylinder wall but not release it entirely before dismounting. These are both considered paw-drags as well as touches and should be counted as both in a tally.</li>
		<li>The paw may also drag along the cylinder wall while a mouse explores the cylinder. In this case, the paw will follow the twisting of the mouse&#8217;s torso as it explores left or right of its original position (Figure 7A-D) before dismounting (Figure 7E). This is not considered a paw-drag, as it depends on the mouse randomly choosing a direction to explore and does not depend on which cortical hemisphere was damaged.</li>
	</ol>
	</li>
	<li>Paw-drags are expressed as a percentage of paw-drags per total number of paw touches during a session. Express the number of paw-drags as a percentage of total paw contacts for each forelimb separately.</li>
	<li>Touches resulting in a paw-drag count as a paw-drag and a touch simultaneously. Thus, if a mouse drags its paw each time its paw contacts the cylinder wall, the paw-dragging percentage is expressed as 100%.</li>
</ol>
6. Additional Experimental Design Suggestions
<ol>
	<li>To minimize extraneous variables:
	<ol>
		<li>Test the mice at the same time on each testing day. Test the mice during their wake cycle. Keeping mice on a 12 hr reverse light cycle facilitates performance.</li>
		<li>Mice may be reluctant to explore the cylinder if stressed either by noise or a novel environment. Testing mice in their animal holding room or a room they have been familiarized with reduces stress. This can occur if the room is noisy, if the mouse has been jostled prior to entering the cylinder or due to habituation. 
		NOTE: Testing should be performed once before experimental manipulation to serve as a baseline reading. After manipulation, testing days are at the experimenter&#8217;s discretion, though it is advised to avoid an excessive number of exposures to the cylinder over a short period of time.</li>
		<li>Mice may become reluctant to rear after 6-7 exposures to the cylinder. For the current study, mice were tested in the cylinder for a total of seven times, prior to ischemia and on days 1, 3, 7, 14, 21 and 28 post-surgery. 
		NOTE: In this study we used the FVBN mouse strain. We have previously tested C57Bl/6 mice in the cylinder and observed paw-dragging behaviour following an ET-1 ischemic injury (data not shown). C57BL/6 mice were more active than FVBN mice when rearing and frequently jumped onto the rim of the cylinder before climbing out. Taller cylinders should be used if mice attempt to escape by jumping.</li>
	</ol>
	</li>
</ol>
7. Endothelin-1 Surgery and Infarct Volume Measurements
<ol>
	<li>Perform Endothelin-1 surgery and infarct volume measurements according to previously published protocols4 . To target the anterior forelimb motor cortex, each mouse should receive three ET-1 injections at the following coordinates: (i) +0.7 anteriorposterior (AP)/1.5 medial-lateral (ml)/-1.2 dorsal-ventral (DV), (ii) +0.4 AP/1.25 ml/- 1.2 DV and (iii) +0.1 AP/1.75 ml/-1.2 DV4 .</li>
</ol>
8. Statistical Analysis
<ol>
	<li>A two-way repeated measures analysis of variance (ANOVA) is recommended to analyze the percent of paw-dragging for the affected and unaffected paws across different time points.</li>
</ol>

<ol>
	<li>Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., Bland, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke,+cortical+ablation,+parkinsonism+and+spinal+cord+injury.">CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.</a> Neuropharmacology. 39, 777-787 (2000).</li><li>Kolb, 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=Growth+factor-stimulated+generation+of+new+cortical+tissue+and+functional+recovery+after+stroke+damage+to+the+motor+cortex+of+rats.">Growth factor-stimulated generation of new cortical tissue and functional recovery after stroke damage to the motor cortex of rats.</a> J Cereb Blood Flow Metab. 27 (5), 983-997 (2007).</li><li>Windle, 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=An+analysis+of+four+different+methods+of+producing+focal+cerebral+ischemia+with+endothelin-1+in+the+rat.">An analysis of four different methods of producing focal cerebral ischemia with endothelin-1 in the rat.</a> Exp Neurol. 201 (2), 324-334 (2006).</li><li>Roome, R. 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=A+reproducible+Endothelin-1+model+of+forelimb+motor+cortex+stroke+in+the+mouse.">A reproducible Endothelin-1 model of forelimb motor cortex stroke in the mouse.</a> J Neurosci Methods. 233, 34-44 (2014).</li><li>Tennant, K. A., Jones, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorimotor+behavioral+effects+of+endothelin-1+induced+small+cortical+infarcts+in+C57BL/6+mice.">Sensorimotor behavioral effects of endothelin-1 induced small cortical infarcts in C57BL/6 mice.</a> J Neurosci Methods. 181 (1), 18-26 (2009).</li><li>Wang, Y., Jin, K., Greenberg, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+associated+with+endothelin-induced+cortical+infarction+in+the+mouse.">Neurogenesis associated with endothelin-induced cortical infarction in the mouse.</a> Brain Res. 1167, 118-122 (2007).</li><li>Baskin, Y. K., Dietrich, W. D., Green, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+effective+behavioral+tasks+for+evaluating+sensorimotor+dysfunction+following+traumatic+brain+injury+in+mice.">Two effective behavioral tasks for evaluating sensorimotor dysfunction following traumatic brain injury in mice.</a> J Neurosci Methods. 129 (1), 87-93 (2003).</li><li>Li, X., 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=Chronic+behavioral+testing+after+focal+ischemia+in+the+mouse:+functional+recovery+and+the+effects+of+gender.">Chronic behavioral testing after focal ischemia in the mouse: functional recovery and the effects of gender.</a> Exp Neurol. 187 (1), 94-104 (2004).</li><li>Clarke, J., Ploughman, M., Corbett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+qualitative+and+quantitative+analysis+of+skilled+forelimb+reaching+impairment+following+intracerebral+hemorrhage+in+rats.">A qualitative and quantitative analysis of skilled forelimb reaching impairment following intracerebral hemorrhage in rats.</a> Brain Res. , 204-212 (2007).</li><li>Montoya, C. P., Campbell-Hope, L. J., Pemberton, K. D., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'staircase+test':+a+measure+of+independent+forelimb+reaching+and+grasping+abilities+in+rats.">The 'staircase test': a measure of independent forelimb reaching and grasping abilities in rats.</a> J Neurosci Methods. 36 (2-3), 219-228 (1991).</li><li>Farr, T. D., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+qualitative+impairments+in+skilled+reaching+in+the+mouse+(Mus+musculus)+after+a+focal+motor+cortex+stroke.">Quantitative and qualitative impairments in skilled reaching in the mouse (Mus musculus) after a focal motor cortex stroke.</a> Stroke. 33 (7), 1869-1875 (2002).</li><li>Tennant, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vermicelli+and+capellini+handling+tests:+simple+quantitative+measures+of+dexterous+forepaw+function+in+rats+and+mice.">The vermicelli and capellini handling tests: simple quantitative measures of dexterous forepaw function in rats and mice.</a> J Vis Exp. (41), (2010).</li><li>Clarkson, A. N., 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=AMPA+receptor-induced+local+brain-derived+neurotrophic+factor+signaling+mediates+motor+recovery+after+stroke.">AMPA receptor-induced local brain-derived neurotrophic factor signaling mediates motor recovery after stroke.</a> J Neurosci. 31 (10), 3766-3775 (2011).</li><li>Clarkson, A. N., Huang, B. S., Macisaac, S. E., Mody, I., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+excessive+GABA-mediated+tonic+inhibition+promotes+functional+recovery+after+stroke.">Reducing excessive GABA-mediated tonic inhibition promotes functional recovery after stroke.</a> Nature. 468 (7321), 305-309 (2010).</li></ol>

The authors have no competing financial interests.

The key points to establish when quantifying paw-dragging behaviour in the cylinder test are the following: i) quantify the number of paw-drags versus total paw touches for each paw before brain injury to establish a baseline; ii) quantify the number of paw-drags versus total paw touches for each paw following the ischemic injury; and iii) discriminate between a paw-drag and the lateral motion of the paw along the cylinder wall during lateral rotation of the mouse&#8217;s torso.
Paw-dragging i...

The goal of neural regeneration strategies is to demonstrate both tissue repair and functional recovery. Functional recovery is typically evaluated with behavioural tests that measure functional deficits, in this case involving motor skills that are associated with damage to the specific brain regions. Traumatic brain injury or ischemic damage to the sensorimotor forelimb area of the cortex can be demonstrated by a number of behavioural tests. One such test, the cylinder test is used extensively in rats to assess functional deficits in forelimb activity1. The test has a low set-up cost requiring only a cylinder, camera and table with a transparent top. It is easy to administer as it is based on the natural exploratory behaviour of rodents, so pre-training and food deprivation or rewards are not required. Despite these numerous advantages, the cylinder test is under-utilized to assess forelimb deficits in mice following focal injuries to the forelimb sensorimotor cortex, which we attribute to the analysis of mouse behaviour in the cylinder test. Forelimb asymmetry is the classical measure of analysis for the cylinder test. When placed in the cylinder, rodents naturally explore the walls of the cylinder by rearing onto their hind limbs and touching the cylinder walls with their forelimb paws for postural balance. The number of paw touches with the wall with each forelimb is easily quantified by filming rodents during this exploration of the cylinder. Forelimb asymmetry occurs when the affected forelimb paw makes fewer touches with the wall than the unaffected forelimb paw and is indicative of damage to the contralateral sensorimotor cortex. In rats, intra-cortical injections of the vasoconstrictive agent, Endothelin (ET-1), into the forelimb sensorimotor cortex causes a focal ischemic lesion which results in behavioural deficits in the contralateral forelimb. Deficits in contralateral forelimb use are readily detected as changes in forelimb asymmetry in the cylinder test in rats1-3. In contrast to rats however, changes in forelimb asymmetry are variable and less consistent in mice following comparable ET-1 injections4-6. Here we demonstrate a novel analysis of forelimb behaviour in the cylinder test &#8211; analysis of paw-dragging behaviour. We have previously shown that paw-dragging analysis is a more sensitive measure of damage to the forelimb sensorimotor cortex in mice than the classical forelimb asymmetry analysis and therefore is applicable to a variety of focal cortical injury models.
Examination of how the forepaw contacts the cylinder wall following ischemic damage to the forelimb sensorimotor cortex revealed a novel behaviour in mice - paw-dragging4. A paw-drag occurs when a mouse stands on its rear legs to explore the cylinder wall then drags its affected (contra-lesional) paw along the cylinder wall towards its midline or down the wall while its unaffected forepaw provides postural support against the wall. Paw-drags rarely occur in uninjured mice therefore the appearance of a paw-drag is a positive indicator of injury to the forelimb sensorimotor cortex4. We have previously quantified paw-dragging behaviour in mice following ET-1 ischemic damage to the forelimb sensorimotor cortex and have shown sustained paw-dragging behaviour in mice up to two weeks post-stroke4. Here we show that paw-dragging behaviour is sustained up to four weeks post-stroke. Analysis of paw-dragging behaviour provides a novel and sensitive tool for assessing focal ischemic damage to the forelimb sensorimotor cortex in mice. Its inexpensive set-up, ease of administration and scoring make this a simple, yet useful tool to rapidly assess forelimb behavioural deficits in mice.

<table><tbody><tr><td>Plexi-glass cylinder</td><td></td><td></td><td>17.5 cm high, 9.5 cm outer diameter, 8.8 cm inner diameter, wall thickness 0.35 cm (or 3.5 mm)</td></tr><tr><td>viewing table</td><td></td><td></td><td>54x56x66.5 cm (width x length x height), top of table is a 51x51 cm sheet of plexiglass.</td></tr><tr><td>mirror</td><td></td><td></td><td>34x58 cm mirror</td></tr><tr><td>video camera</td><td>Sony</td><td>DCR-SR42</td><td>Video camera with onboard storage, SD functionality, 40x optical zoom</td></tr><tr><td>computer</td><td>Dell</td><td>Optiplex 760</td><td>Processor: Intel, 3.0 GHz, Memory 4.00GB (RAM)&amp;nbsp;</td></tr><tr><td>computer monitor</td><td>Samsung</td><td>S22C350H</td><td></td></tr><tr><td>Excel (Microsoft Office Professional Plus)</td><td>Microsoft</td><td>v14.0.7106.5003</td><td></td></tr><tr><td>VLC Media Player</td><td>Video LAN</td><td>v2.1.2</td><td>Media player with playback speed modulation and video support</td></tr><tr><td>External Hard Drive</td><td>Western Digital</td><td>WDBAAU0020HBK-01</td><td>2 TB</td></tr></tbody></table>

paw-dragging: a novel, sensitive analysis of the mouse cylinder test

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore by rearing and touching the walls of the cylinder with their forelimb paws for postural support. Following ischemic injury to the forelimb sensorimotor cortex, rats rely more heavily on their unaffected forelimb paw for postural support resulting in fewer touches with their affected paw which is termed forelimb asymmetry. In contrast, focal ischemic damage in the mouse brain fails to result in comparable consistent deficits in forelimb asymmetry. While forelimb asymmetry deficits are infrequently observed, mice do demonstrate a novel behaviour post stroke termed &#8220;paw-dragging&#8221;. Paw-dragging is the tendency for a mouse to drag its affected paw along the cylinder wall rather than directly push off from the wall when dismounting from a rear to a four-legged stance. We have previously demonstrated that paw-dragging behaviour is highly sensitive to small cortical ischemic injuries to the forelimb motor cortex. Here we provide a detailed protocol for paw-dragging analysis. We define what a paw-drag is and demonstrate how to quantify paw-dragging behaviour. The cylinder test is a simple and inexpensive test to administer and does not require pre-training or food deprivation strategies. In using paw-dragging analysis with the cylinder test, it fills a niche for predicting cortical ischemic injuries such as photothrombosis and Endothelin-1 (ET-1)-induced ischemia &#8211; two models that are ever-increasing in popularity and produce smaller focal injuries than middle cerebral artery occlusion. Finally, measuring paw-dragging behaviour in the cylinder test will allow studies of functional recovery after cortical injury using a wide cohort of transgenic mouse strains where previous forelimb asymmetry analysis has failed to detect consistent deficits.

We have previously demonstrated that paw-dragging behaviour appears following a focal ischemic injury to the forelimb sensorimotor cortex and is a positive indicator of damage4. Intra-cortical injections of ET-1 into the forelimb sensorimotor cortex were used to induce an ischemic lesion (Figure&#160;8A,B). This study examined whether paw-dragging behaviour extended for longer than 14 days post-injury for its potential use to assess functional recovery. Mice were tested in the cylinder test on...

Watch this Scientific Journal Video about Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test at JoVE.com

Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

Classical forelimb asymmetry analysis of the cylinder test is routinely used to assess behavioural deficits in rats following brain injury or stroke; however, it fails to detect consistent deficits in mice. This study demonstrates that quantifying paw-dragging behaviour is a more sensitive analysis of brain injury in mice.

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore ...

paw-dragging-a-novel-sensitive-analysis-of-the-mouse-cylinder-test

Research

JoVE Journal

Behavior

23.4K Views.  Memorial University of Newfoundland. Classical forelimb asymmetry analysis of the cylinder test is routinely used to assess behavioural deficits in rats following brain injury or stroke; however, it fails to detect consistent deficits in mice. This study demonstrates that quantifying paw-dragging behaviour is a more sensitive analysis of brain injury in mice.

Video: Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Neuroscience

Assessing Functional Performance in the Mdx Mouse Model

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions (e.g. forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories.

Assessing Functional Performance in the Mdx Mouse Model

The primary outcome measure in clinical trials for neuromuscular disorders is generally improved muscle function. Therefore, assessing the effect of potential therapeutic compounds on muscle performance pre clinically in mouse models is of great importance. We here describe several functional tests to address this.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential ...

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 ...

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important behavioral test to determine whether the mice mimic the symptoms of patients. Motor deficits are often induced by stress when no spontaneous motor phenotype is observed in the mouse models. Therefore, gait analysis followed by stress loading would be a sensitive method for evaluating the motor phenotype in mouse models. However, researchers face the requirement of an expensive apparatus to obtain quantitative results automatically from gait analysis. For stress, stress loading by simple methods without expensive apparatuses required for electric shock and forced running is desirable. Therefore, we introduce a simple and low-cost protocol consisting of footprint analysis with paper and ink, hanging box test to evaluate motor function, and stress loading defined by restraint with a conical tube. The motor deficits of mice were successfully detected by this protocol.

The low-cost protocol consisting of footprint analysis and hanging box test after restraint stress is useful for evaluating the movement disorders of mouse model.

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Assessment of Mouse Judgment Bias through an Olfactory Digging Task

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This phenomenon has long been observed in humans, with individuals in positive states responding to ambiguity 'optimistically' and those in negative states instead showing 'pessimism'. Researchers aiming to assess animal affect have taken advantage of these differential responses, developing tasks to assess judgment bias as an indicator of affective state. These tasks are becoming increasingly popular across diverse species and fields of research. However, for laboratory mice, the most widely used vertebrates in research and a species heavily relied upon to model affective disorders, only one JB task has been successfully validated as sensitive to changes in affective state. Here, we provide a detailed description of this novel murine JB task, and evidence of its sensitivity to mouse affect. Though refinements are still necessary, assessment of mouse JB opens the door for answering both practical questions regarding mouse welfare, and fundamental questions about the impact of affective state in translational research.

This article provides a detailed description of a novel mouse judgment bias protocol. Evidence of this olfactory digging task's sensitivity to affective state is also demonstrated and its utility across diverse research fields is discussed.

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This ...

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

Medicine

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...

Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

Summary

Explore More Videos

Chapters in this video

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Assessing Functional Performance in the Mdx Mouse Model

Measuring Motor Coordination in Mice

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Assessment of Mouse Judgment Bias through an Olfactory Digging Task

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice