Name: low-cost protocol of footprint analysis and hanging box test for mice applied the chronic restraint stress
Uploaded: 2019-01-23T15:00:00.000+00:00
Duration: 6 min 38 s
Description: Watch this Scientific Journal Video about Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress at JoVE.com

This work was supported by JSPS (Japan Society for the Promotion of Science) KAKENHI (Grant-in-Aid for Scientific Research C), Grant number 18K07373 (H.S.) and Subsidies for Private Universities.

All animal experiments were conducted in a humane manner. The Institutional Animal Experiment Committee of Jichi Medical University approved the study. The study was conducted in accordance with the Institutional Regulation for Animal Experiment and Fundamental Guideline for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the MEXT of Japan. Mice used in this protocol have been described previously21.
1. Hanging Box Test
<ol>
	<li>Record the weight of each mouse. Mark the tail by marking pen for individual discrimination (e.g., a line, double lines, and triple lines). 
	NOTE: Growth curves are used for an index of general health22.</li>
	<li>Place the mice in the experimental room at least 30 min before the behavioral test. Set the hanging box, which consists of a clear box (25 x 25 x 40 cm3) with a rotatable mesh lid on the top (Figure 1). The mesh lid can be rotated along a central bar so that the top is flipped 180 degrees.</li>
	<li>Put a mouse in the center of the mesh lid. Carefully turn the mesh lid up side down.</li>
	<li>Measure the fall latency (hanging time) of the mouse from the mesh lid. 
	NOTE: If the mouse does not fall within 5 min, record the latency as 5 min.</li>
	<li>Return the mouse to the home cage. Clean the hanging box with 70% ethanol after every test.</li>
</ol>
2. Footprint Analysis
NOTE: Following the hanging box test, perform the footprint analysis.
<ol>
	<li>Set up the Runway (Figure 2A).
	<ol>
		<li>Cut a piece of white paper (29.7 cm x 42 cm x 0.09 mm) longitudinally into three lengths of equal width. Set a piece of the white paper (9.9 cm x 42 cm) on the table.</li>
		<li>Put the dark goal box at the distal end of the paper. Put other boxes (approximately the same length as that of the paper) with the walls on both sides of the runway, preventing the escape of mice.</li>
		<li>Put black ink and red ink into separate Petri dishes (35 mm in diameter).</li>
	</ol>
	</li>
	<li>Training session. 
	NOTE: Perform the training session only at 4 weeks of age.
	<ol>
		<li>Put a mouse on the proximal end of the paper (Face the head toward the goal box). Let the mouse walk from the proximal end to the goal box. Remove the mouse from the goal box. If the mouse stops on the paper, gently push the mouse to the goal box by finger.</li>
		<li>Hold the mouse by grasping the scruff between the thumb and forefinger to limit the movement of forelimbs. Then, grasp the back and the tail between the ball of the thumb and the other fingers to limit the movement of hindlimbs. 
		NOTE: Insufficient holding of a mouse results in blots of ink on clothing.</li>
		<li>Immerse the bottoms of forelimbs in red ink and the bottoms of hindlimbs in black ink. Immediately put the mouse on the proximal end of the paper (Face the head toward the goal box). Let the mouse walk from the proximal end to the goal box. If the mouse stops on the paper, gently push the mouse to the goal box by finger.</li>
		<li>Remove the mouse from the goal box. Go to the test session.</li>
	</ol>
	</li>
	<li>Test Session.
	<ol>
		<li>Following the training session, set up the runway for footprints with a new cut piece of white paper.</li>
		<li>Hold the mouse by grasping the scruff between the thumb and forefinger to limit the movement of forelimbs. Then, grasp the back and the tail between the ball of the thumb and the other fingers to limit the movement of hindlimbs.</li>
		<li>Immerse the bottoms of forelimbs in red ink and the bottoms of hindlimbs in black ink. Immediately put the mouse on the proximal end of the paper. Let the mouse walk from the proximal end to the goal box. 
		NOTE: Because mice prefer the dark, walking becomes steadier as the mouse approaches the dark goal box. If the mouse stops on the paper, gently push the mouse to the goal box by finger. Then, if reliable footprints are not obtained for analysis (see step 2.4. Analysis of footprints for details) because the mouse stopped, retry the test session.</li>
		<li>Return the mouse to the home cage from the goal box. Clean the goal box with 70% ethanol after each test session. Air-dry the foot-printed paper.</li>
	</ol>
	</li>
	<li>Analysis of footprints
	<ol>
		<li>Obtain three measurements of each parameter (stride lengths of forelimbs and hindlimbs, front and hind base widths, overlap between forelimb and hindlimb, Figure 2B) with a ruler from foot-printed paper. 
		NOTE: Because footprints of proximal and distal ends frequently show large variations because of stopping or running, choose the part with a steady gait pattern of footprints. The middle part of the foot-printed paper will usually be suitable for the analysis.
		<ol>
			<li>For the stride length, measure the distances between the same parts of the paw (e.g., paw pad or toe).</li>
			<li>For the front base width, draw a line between consecutive right (or left) front footprints. Then, measure the length of the vertical line from the pad of the left (or right) front footprint to the line drawn between the right (or left) footprints.</li>
			<li>For the hind base width, draw a line between consecutive right (or left) hind footprints. Then, measure the length of the vertical line from the pad of the left (or right) hind footprint to the line drawn between the right (or left) footprints.</li>
			<li>For overlap, measure the distance between pads of left (or right) front and hind footprints.</li>
		</ol>
		</li>
		<li>Average the three measurements for each individual. Use the individual average of each parameter for the statistical analysis.
		<ol>
			<li>For the stride length, use the average of the individual averages of the left and right strides.</li>
			<li>For asymmetry of stride length, use the absolute value of the difference between individual averages of left limb and right limb stride length.</li>
			<li>For the statistical analysis of the other parameters (front base width, hind base width, and overlap), use the individual average directly.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Restraint Stress Loading
<ol>
	<li>Preparation of Restraint Tubes. 
	<ol>
		<li>Make 16 holes (approximately 2 mm in diameter) in a 50 ml conical tube (30 mm in diameter x 115 mm in length) along the scale marks (5, 10, 15, 20, 25, 30, 35, 40 mL) and the backside of each scale mark by square drill (Figure 3). Make a hole on the tip of the 50 mL conical tube (approximately 5 mm in diameter) for breathing by cutting off the tip. Make a hole (approximately 4 mm in diameter) in the tube cap to pass the tail of mice.</li>
	</ol>
	</li>
	<li>Stress Loading
	<ol>
		<li>Place the mice in the experimental room.</li>
		<li>Hold a mouse by grasping the scruff between the thumb and the forefinger. Enter the mouse into the restraint tube from the head. Pass the tail through the hole in the cap. Close the cap. 
		NOTE: Limit the forelimb movement, because mice reject entering the tube by the forelimbs.</li>
		<li>Keep the mouse enclosed for 2 h on a desk at room temperature. Remove the mouse from the restraint tube and return to the home cage. 
		NOTE: The restraint tubes can be reused after a wash and dry.</li>
	</ol>
	</li>
</ol>
4. Experimental Schedule (Figure 4):
<ol>
	<li>Perform the hanging box test and the footprint analysis on the same day at 4 weeks of age (see step 1. Hanging box test and step 2. Footprint analysis for details) as a baseline measurement on all mice prior to the grouping into &#8216;stress group&#8217; and a &#8216;non-stress group&#8217;. 
	NOTE: About 8-10 mice in 2-3 litters may be suitable to use in an experiment. Footprint analysis at 4 weeks of age consists of training and test sessions.</li>
	<li>Randomly divide the mice into a &#8216;stress group&#8217; and a &#8216;non-stress group&#8217;. 
	NOTE: When the mice are used consisting of several litters, divide littermates evenly into both groups. Number in each group consists of about 4-5 mice.</li>
	<li>Apply the restraint stress to the &#8216;stress group&#8217; 6 times over the course of two weeks (see step 3. Restraint stress loading for details). 
	NOTE: 6 times of restraint are applied every two weeks, followed by a hanging box test and footprint analysis from 6-12 weeks of age. Do not apply the restraint stress on the test day of the hanging box test and the footprint analysis.</li>
	<li>Perform the hanging box test and the test session of footprint analysis on the same day at 6, 8, 10, and 12 weeks of age.</li>
</ol>

<ol>
	<li>Warner, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+disorders.">Movement disorders.</a> Practical Guide to Neurogenetics. , (2008).</li><li>Brashear, A., DeLeon, D., Bressman, S. B., Thyagarajan, D., Farlow, M. R., Dobyns, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid-onset+dystonia-parkinsonism+in+a+second+family.">Rapid-onset dystonia-parkinsonism in a second family.</a> Neurology. 48 (4), 1066-1069 (1997).</li><li>Linazasoro, G., Indakoetxea, B., Ruiz, J., Van Blercom, N., Lasa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+sporadic+rapid-onset+dystonia-parkinsonism.">Possible sporadic rapid-onset dystonia-parkinsonism.</a> Movement Disorders. 17 (3), 608-609 (2002).</li><li>Svetel, M., Ozelius, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid-onset+dystonia-parkinsonism:+case+report.">Rapid-onset dystonia-parkinsonism: case report.</a> Journal of Neurology. 257 (3), 472-474 (2010).</li><li>Vrinten, D. H., Hamers, F. F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="CatWalk"+automated+quantitative+gait+analysis+as+a+novel+method+to+assess+mechanical+allodynia+in+the+rat;+a+comparison+with+von+Frey+testing.">"CatWalk" automated quantitative gait analysis as a novel method to assess mechanical allodynia in the rat; a comparison with von Frey testing.</a> PAIN. 102 (1), 203-209 (2003).</li><li>Berryman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DigigaitTM+quantitation+of+gait+dynamics+in+rat+rheumatoid+arthritis+model.">DigigaitTM quantitation of gait dynamics in rat rheumatoid arthritis model.</a> Journal of Musculoskeletal and Neuronal Interactions. 9 (2), 89-98 (2009).</li><li>Beare, J. E., Morehouse, J. R., 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=Gait+analysis+in+normal+and+spinal+contused+mice+using+the+TreadScan+system.">Gait analysis in normal and spinal contused mice using the TreadScan system.</a> Journal of Neurotrauma. 26 (11), 2045-2056 (2009).</li><li>Rushton, R., Steinberg, H., Tinson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+single+experience+on+subsequent+reactions+to+drugs.">Effects of a single experience on subsequent reactions to drugs.</a> British Journal of Pharmacology and Chemotherapy. 20, 99-105 (1963).</li><li>Lee, C. C., Peters, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxicity+and+behavioral+effects+of+thiram+in+rats.">Neurotoxicity and behavioral effects of thiram in rats.</a> Environmental health perspectives. 17, 35-43 (1976).</li><li>van der Zee, C. E., Schuurman, T., Traber, J., Gispen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+administration+of+nimodipine+accelerates+functional+recovery+following+peripheral+nerve+damage+in+the+rat.">Oral administration of nimodipine accelerates functional recovery following peripheral nerve damage in the rat.</a> Neuroscience Letters. 83 (1-2), 143-148 (1987).</li><li>Leroy, T., Stroobants, S., Aerts, J. -. M., D'Hooge, R., Berckmans, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+analysis+of+altered+gait+in+arylsulphatase+A-deficient+mice+in+the+open+field.">Automatic analysis of altered gait in arylsulphatase A-deficient mice in the open field.</a> Behavior Research Methods. 41 (3), 787-794 (2009).</li><li>Sango, K., McDonald, M. P., 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=Mice+lacking+both+subunits+of+lysosomal+beta-hexosaminidase+display+gangliosidosis+and+mucopolysaccharidosis.">Mice lacking both subunits of lysosomal beta-hexosaminidase display gangliosidosis and mucopolysaccharidosis.</a> Nature Genetics. 14 (3), 348-352 (1996).</li><li>Deacon, R. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+Strength+of+Mice.">Measuring the Strength of Mice.</a> Journal of Visualized Experiments. (76), e2610 (2013).</li><li>Djamshidian, A., Lees, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+stress+trigger+Parkinson's+disease?.">Can stress trigger Parkinson's disease?.</a> Journal of Neurology, Neurosurgey, and Psychiatry. 85 (8), 879-882 (2014).</li><li>Brashear, A., Dobyns, W. 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=The+phenotypic+spectrum+of+rapid-onset+dystonia-parkinsonism+(RDP)+and+mutations+in+the+ATP1A3.">The phenotypic spectrum of rapid-onset dystonia-parkinsonism (RDP) and mutations in the ATP1A3.</a> Brain. 130 (Pt 3), 828-835 (2007).</li><li>Kirshenbaum, G. S., Saltzman, K., Rose, B., Petersen, J., Vilsen, B., Roder, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+neuronal+Na+,K+-ATPase+activity+in+Atp1a3+heterozygous+mice+increases+susceptibility+to+depression-like+endophenotypes+by+chronic+variable+stress.">Decreased neuronal Na+,K+-ATPase activity in Atp1a3 heterozygous mice increases susceptibility to depression-like endophenotypes by chronic variable stress.</a> Genes, Brain and Behavior. 10 (5), 542-550 (2011).</li><li>DeAndrade, M. P., Yokoi, F., van Groen, T., Lingrel, J. B., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Atp1a3+mutant+mice+as+a+model+of+rapid-onset+dystonia+with+parkinsonism.">Characterization of Atp1a3 mutant mice as a model of rapid-onset dystonia with parkinsonism.</a> Behavioral Brain Research. 216 (2), 659-665 (2011).</li><li>Sugimoto, H., Ikeda, K., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterozygous+mice+deficient+in+Atp1a3+exhibit+motor+deficits+by+chronic+restraint+stress.">Heterozygous mice deficient in Atp1a3 exhibit motor deficits by chronic restraint stress.</a> Behavioral Brain Research. 272, 100-110 (2014).</li><li>Zimprich, A., Garrett, L., 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+robust+and+reliable+non-invasive+test+for+stress+responsivity+in+mice.">A robust and reliable non-invasive test for stress responsivity in mice.</a> Frontiers in Behavioral Neuroscience. 8, 125 (2014).</li><li>Buynitsky, T., Mostofsky, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restraint+stress+in+biobehavioral+research:+recent+developments.">Restraint stress in biobehavioral research: recent developments.</a> Neuroscience and Biobehavioral Reviews. 33 (7), 1089-1098 (2009).</li><li>Ikeda, K., Satake, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+inhibitory+neurotransmission+in+the+cerebellar+cortex+of+Atp1a3-deficient+heterozygous+mice.">Enhanced inhibitory neurotransmission in the cerebellar cortex of Atp1a3-deficient heterozygous mice.</a> The Journal of Physiology. 591 (13), 3433-3449 (2013).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+functions.">Motor functions.</a> What's Wrong with My Mouse?. , (2007).</li><li>. R: A language and environment for statistical computing Available from: <a target="_blank" href="https://www.R-project.org/">https://www.R-project.org/</a> (2014)</li><li>Wils, H., Kleinberger, G., 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=TDP-43+transgenic+mice+develop+spastic+paralysis+and+neuronal+inclusions+characteristic+of+ALS+and+frontotemporal+lobar+degeneration.">TDP-43 transgenic mice develop spastic paralysis and neuronal inclusions characteristic of ALS and frontotemporal lobar degeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (8), 3858-3863 (2010).</li><li>Eilam, R., Peter, Y., 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=Selective+loss+of+dopaminergic+nigro-striatal+neurons+in+brains+of+Atm-deficient+mice.">Selective loss of dopaminergic nigro-striatal neurons in brains of Atm-deficient mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (21), 12653-12656 (1998).</li><li>Lin, C. -. H., Tallaksen-Greene, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurological+abnormalities+in+a+knock-in+mouse+model+of+Huntington's+disease.">Neurological abnormalities in a knock-in mouse model of Huntington's disease.</a> Human Molecular Genetics. 10 (2), 137-144 (2001).</li><li>Dang, M. T., Yokoi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+characterization+of+Dyt1+&#916;GAG+knock-in+mouse+as+a+model+for+early-onset+dystonia.">Generation and characterization of Dyt1 &#916;GAG knock-in mouse as a model for early-onset dystonia.</a> Experimental Neurology. 196 (2), 452-463 (2005).</li><li>Glynn, D., Drew, C. J., Reim, K., Brose, N., Morton, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profound+ataxia+in+complexin+I+knockout+mice+masks+a+complex+phenotype+that+includes+exploratory+and+habituation+deficits.">Profound ataxia in complexin I knockout mice masks a complex phenotype that includes exploratory and habituation deficits.</a> Human Molecular Genetics. 14 (16), 2369-2385 (2005).</li><li>Becker, E. B. E., Oliver, P. L., 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+point+mutation+in+TRPC3+causes+abnormal+Purkinje+cell+development+and+cerebellar+ataxia+in+moonwalker+mice.">A point mutation in TRPC3 causes abnormal Purkinje cell development and cerebellar ataxia in moonwalker mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (16), 6706-6711 (2009).</li><li>Heck, D. H., Zhao, Y., Roy, S., LeDoux, M. S., Reiter, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cerebellar+function+in+Ube3a-deficient+mice+reveals+novel+genotype-specific+behaviors.">Analysis of cerebellar function in Ube3a-deficient mice reveals novel genotype-specific behaviors.</a> Human Molecular Genetics. 17 (14), 2181-2189 (2008).</li><li>Kirshenbaum, G. S., Dawson, 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=Alternating+hemiplegia+of+childhood-related+neural+and+behavioural+phenotypes+in+Na+,K+-ATPase+&#945;3+missense+mutant+mice.">Alternating hemiplegia of childhood-related neural and behavioural phenotypes in Na+,K+-ATPase &#945;3 missense mutant mice.</a> PLoS ONE. 8 (3), e60141 (2013).</li><li>Klein, A., Wessolleck, J., Papazoglou, A., Metz, G. A., Nikkhah, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Walking+pattern+analysis+after+unilateral+6-OHDA+lesion+and+transplantation+of+foetal+dopaminergic+progenitor+cells+in+rats.">Walking pattern analysis after unilateral 6-OHDA lesion and transplantation of foetal dopaminergic progenitor cells in rats.</a> Behavioral Brain Research. 199 (2), 317-325 (2009).</li><li>Geldenhuys, W. J., Guseman, T. L., Pienaar, I. S., Dluzen, D. E., Young, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+biomechanical+analysis+of+gait+changes+in+the+MPTP+mouse+model+of+Parkinson's+disease.">A novel biomechanical analysis of gait changes in the MPTP mouse model of Parkinson's disease.</a> PeerJ. 3 (Pt 7), e1175 (2015).</li><li>Cecchi, M., Khoshbouei, H., Morilak, 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=Modulatory+effects+of+norepinephrine,+acting+on+alpha1+receptors+in+the+central+nucleus+of+the+amygdala,+on+behavioral+and+neuroendocrine+responses+to+acute+immobilization+stress.">Modulatory effects of norepinephrine, acting on alpha1 receptors in the central nucleus of the amygdala, on behavioral and neuroendocrine responses to acute immobilization stress.</a> Neuropharmacology. 43 (7), 1139-1147 (2002).</li><li>Chu, X., Zhou, Y., 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=24-hour-restraint+stress+induces+long-term+depressive-likephenotypes+in+mice.">24-hour-restraint stress induces long-term depressive-likephenotypes in mice.</a> Scientific Reports. 6, 32935 (2016).</li><li>Freeman, M. L., Sheridan, B. S., Bonneau, R. H., Hendricks, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychological+Stress+Compromises+CD8++T+cell+control+of+latent+herpes+simplex+virus+type+1+infections.">Psychological Stress Compromises CD8+ T cell control of latent herpes simplex virus type 1 infections.</a> The Journal of Immunology. 179 (1), 322-328 (2007).</li><li>Lauretti, E., Di Meco, A., Merali, S., Pratic&#242;, D. <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+stress+exaggerates+motor+deficit+and+neuroinflammation+in+the+MPTP+mouse+model+of+Parkinson's+disease.">Chronic behavioral stress exaggerates motor deficit and neuroinflammation in the MPTP mouse model of Parkinson's disease.</a> Translational Psychiatry. 6, e733 (2016).</li><li>Quartermain, D., Stone, E. A., Charbonneau, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stress+disrupts+risk+assessment+behavior+in+mice.">Acute stress disrupts risk assessment behavior in mice.</a> Physiology and Behavior. 59 (4-5), 937-940 (1996).</li><li>Bannon, 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> The Behavioural effects of stress and aluminum toxicity on a mouse model of amyotrophic lateral sclerosis Parkinsonism-dementia complex. , 1-186 (2015).</li></ol>

The authors have nothing to disclose.

The footprint analysis and the hanging box test are simple and inexpensive behavioral tests for the motor function of mice. The neurobehavioral phenotypes in several mouse models have been successfully detected by these tests. For example, shortened stride length in amyotrophic lateral sclerosis24, increased length of asymmetrical stride in ataxia-telangiectasia25, increased length of overlap in Huntington&#39;s disease26 and dystonia<sup class="xref...

Movement disorders are defined as disturbances of the nervous system showing an excess or paucity of voluntary or automatic movements1. In particular, gait disturbance is frequently documented among patients with movement disorders2,3,4. Therefore, gait analysis is a suitable behavioral test for the validation of animal models of movement disorders. In mice, automated gait analyses have been performed for walking at natural speed5 and at adjustable speeds by treadmill6,7. These analyses provide quantitative results of gait automatically. An alternative method to detect gait disturbance is called footprint analysis. After labeling the bottoms of the feet with ink, mice walk on paper, and the footprints are analyzed. Initially, Vaseline and powdered charcoal were used to visualize the footprint8, and then were replaced by ink on polygraph paper9 and photographic developer on photographic paper10. A cheaper and less toxic method using ink and paper than the other methods remains to date11. Footprint analysis is less expensive compared with automated analysis5,6,7 and would be useful to evaluate the movement disorders in mouse models for the researchers without abundant research funds.
The hanging box test is a kind of four limb hanging tests using wire cage lid12 and wire mesh screen13. The box is an apparatus with rotatable mesh lid on the top of box along a center bar. In addition to gait analysis, the test can be inexpensively and easily performed. Therefore, we conducted the hanging box test to evaluate grip strength and balance, in addition to the footprint analysis in this protocol.
Stress induces the symptoms of movement disorders14,15. Motor deficits are often induced by several chronic stresses even when no spontaneous motor phenotype is observed in the mouse models of a movement disorder16,17,18. Restraint is one of the commonly used methods for stress loading in mice, because the animal is not physically harmed19 and cost is less compared with other methods such as electric shock with dedicated apparatus and forced running with use of a treadmill. Restraint by a tube, which is performed by confining a mouse in a holed 50 mL conical tube, is easier than other methods such as wire mesh strainer, taped limb, and wrapping of animal with gauze (reviewed20). In this paper, we summarize the protocols of footprint analysis and the hanging box test after restraint by a tube. This protocol would help us to use mouse models of movement disorders without spontaneous motor phenotype.

<table><tbody><tr><td>Hanging box</td><td>O&amp;rsquo;hara &amp;amp; Co.</td><td>http://ohara-time.co.jp/products/wire-hanging-test/</td><td></td></tr><tr><td>Marking pen</td><td>ZEBRA</td><td>MO-120-MC-BK</td><td></td></tr><tr><td>Goal box</td><td>O&amp;rsquo;hara &amp;amp; Co.</td><td>http://ohara-time.co.jp/products/balanced-beam-test/</td><td>Accessory for apparatus of balanced beam test</td></tr><tr><td>Boxes</td><td>O&amp;rsquo;hara &amp;amp; Co.</td><td>-</td><td>Side wall of runway</td></tr><tr><td>Black ink</td><td>Shin-asahi</td><td>-</td><td></td></tr><tr><td>Red ink</td><td>Maruyamakogyo</td><td>BC-6</td><td></td></tr><tr><td>Disposable Petri Dish</td><td>Corning</td><td>351008</td><td>Petri dishes (35 mm in diameter)</td></tr><tr><td>Askul Multipaper Super White J Monochrome A3</td><td>Askul</td><td>701-712</td><td>White paper (29.7 cm x 42 cm x 0.09mm)</td></tr><tr><td>50 mL Conical tube</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>Square drill</td><td>KAKURI Corporation</td><td>DIY FACTORY (K32-0313)</td><td></td></tr></tbody></table>

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 heterozygous male mice of Atp1a3 (Atp1a3+/−) that are the mouse model for rapid onset dystonia parkinsonism and wild-type littermates were used in this protocol. Atp1a3+/− showed significantly shorter stride lengths of forelimb and hindlimb than those of the wild type at 4 weeks of age (Figure 5A and Figure 5B, open circle and square). 'Stressed' Atp1a3+/−...

Watch this Scientific Journal Video about Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress at JoVE.com

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

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 ...

low-cost-protocol-footprint-analysis-hanging-box-test-for-mice

Research

JoVE Journal

Behavior

18.3K Views.  Jichi Medical University. 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.

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

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression have been reported, the unpredictable chronic mild stress (UCMS) protocol is an established translationally-relevant model for inducing behavioral symptoms commonly associated with clinical depression, such as anhedonia, altered grooming behavior, and learned helplessness in rodents. The UCMS protocol also induces physiological (e.g., hypercortisolemia, hypertension) and neurological (e.g., anhedonia, learned helplessness) changes that are clinically associated with depression. Importantly, UCMS-induced depressive symptoms can be ameliorated through chronic, but not acute, treatment with common SSRIs. As such, the UCMS protocol offers many advantages over acute stress protocols or protocols that utilize more extreme stressors. Our protocol involves randomized, daily exposures to 7 distinct stressors: damp bedding, removal of bedding, cage tilt, alteration of light/dark cycles, social stresses, shallow water bath, and predator sounds/smells. By subjecting rodents 3-4 hr daily to these mild stressors for 8 weeks, we demonstrate both significant behavioral changes and poor health outcomes to the cardiovascular system. This approach allows for in-depth interrogation of the neurological, behavioral, and physiological alterations associated with chronic stress-induced depression, as well as for testing of new potential therapeutic agents or intervention strategies.

The unpredictable chronic mild stress (UCMS) protocol is a validated method for studying behavioral and physiological changes associated with chronic stress and depressive symptoms. Eight weeks of imposition of the UCMS protocol induces behavioral changes and poor health outcomes in rodents of either gender.

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

The Tail Suspension Test

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test.

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that ...

Neuroscience

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, therapeutic treatments for depression or research on depression is scarce. Thus, in the present paper, we propose a mouse model of depression induced by movement restriction. Chronic mild stress (CMS) is a well-known technique to induce depressive-like behavior. However, it necessitates a complex procedure consisting of a combination of various mild stresses. In contrast, chronic immobilization stress (CIS) is a readily accessible chronic stress model, modified from a restraint model that induces depressive behavior by restricting movement using a restrainer for a certain period. To evaluate the depressive-like behaviors, the sucrose preference test (SPT), the tail suspension test (TST), and the ELISA assay to measure stress marker corticosterone levels are combined in the present experiment. The described protocols illustrate the induction of CIS and evaluation of the changes in behavior and physiological factors for the validation of depression.

This article provides a simplified and standardized protocol for induction of depressive-like behavior in chronically immobilized mice by using a restrainer. In addition, behavior and physiological techniques to verify induction of depression are explained.

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, ...

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

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

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

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

Handling Techniques to Reduce Stress in Mice

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects on&#160;animal well-being and can also be a confounding factor for experimental variables such as anxiety measures. Over the years, handling techniques that minimize handling-related stress have been developed with a particular focus on rats, and little attention to mice. However, it has been shown that mice can be habituated to manipulations using handling techniques. Habituating mice to handling reduces stress, facilitates routine handling, improves animal wellbeing, decreases data variability, and improves experimental reliability. Despite beneficial effects of handling, the tail-pick up approach, which is particularly stressful, is still widely used. This paper provides a detailed description and demonstration of a newly developed mouse-handling technique intended to minimize the stress experienced by the animal during human interaction. This manual technique is performed over 3 days (3D-handling technique) and focuses on the animal&#39;s capacity to habituate to the experimenter. This study also shows the effect of previously established tunnel handling techniques (using a polycarbonate tunnel) and the tail-pick up technique. Specifically studied are their effects on anxiety-like behaviors, using behavioral tests (Elevated-Plus Maze and Novelty Suppressed Feeding), voluntary interaction with experimenters and physiological measurement (corticosterone levels). The 3D-handling technique and the tunnel handling technique reduced anxiety-like phenotypes. In the first experiment, using 6-month-old male mice, the 3D-handling technique significantly improved experimenter interaction. In the second experiment, using 2.5-month-old female, it reduced corticosterone levels. As such, the 3D-handling is a useful approach in scenarios where interaction with the experimenter is required or preferred, or where tunnel handling may not be possible during the experiment.

This paper describes a handling technique in mice, the 3D-handling technique, which facilitates routine handling by reducing anxiety-like behaviors and presents details on two existing related techniques (tunnel and tail handling).

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects ...

Impact of Restraint Stress on Physiology and Neurobiology in Rodent Models

Restraint to Induce Stress in Mice and Rats

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the restraint stress procedure. Restraint stress has been used for decades to investigate changes in physiology, genetics, neurobiology, immunology, and other systems impacted by stress. Due to the ease of performing the procedure, low cost, and numerous modifications to scale for the intensity and duration of stress exposure, a vast literature of studies has used restraint stress in mice and rats. As one example, this study presents previously published data showing the impact of restraint stress in transgenic mice on plasma corticosterone levels and optogenetically-induced norepinephrine release. Acute restraint stress increased plasma corticosterone levels, yet this effect was blunted in mice following repeat restraint stress. However, stimulated norepinephrine release in the bed nucleus of the stria terminalis was increased only in the repeat restraint stress group. These data highlight important considerations of restraint parameters on dependent measures. Additional descriptions of restraint stress in rats are also included for comparison. Finally, the influence of the parameters of the restraint (e.g., acute vs. chronic) and characteristics of the animal subjects (strain, sex, age) are discussed.

This article covers the procedures to induce stress responses using physical restraint stress in mice and rats. Additional considerations that should be observed when selecting and using restraint stress in rodent models are discussed.

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the ...

Assessing Gait by Footprint Analysis: A Method to Test Motor Coordination in Mice

Source: Sugimoto, H. and Kawakami, K. <a href="https://www.jove.com/video/59027/low-cost-protocol-footprint-analysis-hanging-box-test-for-mice">Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress.</a>&#160;J. Vis. Exp. (2019).
In this video, we describe the footprint testing procedure and related analysis to assess gait and motor coordination in mice.

Source: Sugimoto, H. and Kawakami, K. Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress.&#160;J. ...