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 ...

<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. 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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 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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Plexi-glass cylinder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>17.5cm high, 9.5cm outer diameter, 8.8cm inner diameter, wall thickness 0.35cm (or 3.5 mm)</td>
		</tr>
		<tr>
			<td>viewing table</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>54x56x66.5cm (width x length x height), top of table is a 51x51cm sheet of plexiglass.</td>
		</tr>
		<tr>
			<td>mirror</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>34x58cm 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)&#160;</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

22.9K 조회수.  Memorial University of Newfoundland.  실린더 시험의 고전 앞다리 비대칭 분석 일상적 뇌 손상 또는 뇌졸중 다음 래트에서 행동 결손을 평가하기 위해 사용된다; 그러나, 쥐에서 일관된 적자를 감지하지 못한다. 이 연구는 발 드래그 동작을 정량화하는 것은 쥐의 뇌 손상에 더 민감 분석 것을 보여줍니다. 