This research was supported by Chungnam National University, Korea Institute of Oriental Medicine (KIOM) and a grant of the Korea Health Technology R&#38;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &#38; Welfare, Republic of Korea (grant number: HI15C0007).

All experiments were conducted in accordance with the ethical guidelines of the International Association for the Study of Pain and were approved by the Institutional Animal Care and Use Committee at the Chungnam National University (Daejeon, South Korea).
1. Induction of CCI on Sciatic Nerve
<ol>
	<li>House male ICR mice weighing 20 - 25 g under a 12 h light/dark cycle at controlled room temperature (maintained at 20 - 25 &#176;C) and humidity (40 - 60%), with free access to food and water. Allow an acclimation period for mice in the animal care room of at least 1 week before surgery.
	<ol>
		<li>During housing, observe the appearance and behavior of the mice and do not use mice showing abnormal locomotor activity. 
		NOTE: One day prior to the CCI surgery, measure the mechanical sensitivity of the hind paw by applying von Frey filaments and perform the automated gait analysis to obtain normal baseline values. Then, assign mice randomly to control and experimental groups.</li>
	</ol>
	</li>
	<li>On the day of surgery, anesthetize mice by injecting 2,2,2-tribromoethanol (250 mg/kg, intraperitoneal (IP)). 
	NOTE: Wear personal protective equipment such as surgical gown, gloves, and mask.
	<ol>
		<li>Weigh 2.5 g of 2,2,2-tribromoethanol and add 2-methyl-2-buthanol to the final volume of 5 mL. Keep the solution away from light (e.g., use a dark container or wrap in foil).</li>
		<li>Allow reagents to dissolve completely by heating at 40 &#176;C and stirring for 10-30 min.</li>
		<li>Add distilled water to the final volume of 200 mL and stir until mixed well.</li>
		<li>Store aliquots at 4 &#176;C and keep in the dark. After two weeks, the anesthetic should be replaced with new aliquots.</li>
		<li>Inject a volume of 20 &#181;L per 1 g body weight. For example, if the body weight of the mouse is 25 g, give 500 &#181;L of solution. The typical duration of this anesthesia is 1 h. 
		NOTE: This solution will become toxic when exposed to light and/or heat so be careful to avoid exposure to light and heat. An IP injection route is highly recommended.</li>
	</ol>
	</li>
	<li>When the mouse falls into deep anesthesia, place it on the operating table with the dorsal side up (i.e., prone position) and sterilize the outer mid-thigh area of the right side with a 70% alcohol swab. 
	NOTE: Confirm deep anesthesia by checking the lack of response to pinch or pressure stimulus on the hind paws or tail.</li>
	<li>Induce the CCI on the right sciatic nerve of the mouse.
	<ol>
		<li>Make an incision on the skin around the mid-thigh area of 1.0 - 1.5 cm length with a scalpel blade.</li>
		<li>Bluntly dissect the thigh muscles with the micro-mosquito hemostat to expose the sciatic nerve.</li>
		<li>Ligate the exposed sciatic nerve 3 times with 1.0 - 1.5 mm intervals using a 4-0 chromic catgut suture. 
		NOTE: Make loose ligations by tying off the sciatic nerve until mild shaking appears on the ipsilateral hind paw. The sham group of mice received the same surgical opening under similar conditions except without nerve ligation.</li>
	</ol>
	</li>
	<li>Close the surgical opening with 3 - 4 simple interrupted sutures using 5-0 silk and sterilize with povidone iodine for disinfection of the outer area of surgery.</li>
	<li>After surgery, place the mouse in a clean cage on a heating pad. When animals are recovered from anesthesia, return them to their home cage. 
	NOTE: In this study, antibiotics were not used. The CCI+GBP group received IP gabapentin (GBP) at a dose of 50 mg/kg once daily as a positive control.</li>
</ol>
2. Measurement of Mechanical Allodynia (von Frey Test)
NOTE: Assess the frequency of the withdrawal response to the mechanical stimuli by using 1 g of von Frey filament to the plantar surface of the ipsilateral hind paw.
<ol>
	<li>On each test day, bring mice to the behavioral test room and acclimate the mice in their own home cage for at least 30 min before the test. 
	NOTE: Wear personal protective equipment such as surgical gown, gloves, and mask.</li>
	<li>Put the mouse in a transparent acrylic box on the metal mesh floor and acclimate the mouse for 30 min.</li>
	<li>Gently apply 1 g of von Frey filament to the plantar surface of the hind paw until the filament bends.</li>
	<li>Apply filament stimulation to the ipsilateral hind paw 10 times with at least 10 s intervals and record the result. 
	NOTE: In this study, the number of paw withdrawal responses from 10 trials are shown as percentage of paw withdrawal frequency (PWF, %). The von Frey test was performed every two days after CCI surgery.</li>
</ol>
3. Performing Automated Gait Analysis
NOTE: The gait analysis system visualizes each paw print while the animal is walking and analyzes automatically various gait parameters, such as paw print, paw intensity, stride length, stance phase, step sequence, swing, swing speed, etc. In this study, we showed paw print area and single stance after converting the data into the percentage change between contralateral left versus ipsilateral right hind paw. Thus, a result of 50% indicates that the size of the paw print and the duration of paw area in contact with floor, between the left and right sides are same. In addition, lower data values approaching 0% indicate that both the size and duration of contact are decreased in the ipsilateral hind paw as compared to the contralateral side (see panel B of Figure 2 and Figure 3).
<ol>
	<li>For acclimation, keep the mice within the gait analysis device for 10 min once a day beginning 5 days before the CCI surgery. 
	NOTE: Gait analysis test including acclimation should be performed in the dark.</li>
	<li>Bring the mice to the test room for gait analysis and acclimate in their home cage for at least 30 min before the test.
	<ol>
		<li>In the setup tab of the program menu, set the &#34;walkway length&#34; to 30 cm, and set the &#34;maximum run duration&#34; to 5 s and the &#34;maximum run variation&#34; to 50%.</li>
		<li>Select a registered camera from the &#39;setup&#39; tab of the &#39;program&#39; menu.</li>
		<li>Select &#34;open acquisition&#34; from the &#39;acquire&#39; tab of the &#39;program&#39; menu.</li>
		<li>Following the status message, click the &#39;snap background&#39; button to take a background picture of the empty (i.e., blank) walkway.</li>
	</ol>
	</li>
	<li>Click the &#39;Start acquisition&#39; button and place a mouse on the walkway; the recording starts automatically according to the movement of the mouse. 
	NOTE: When the mouse clearly walks across the walkway, the program automatically classifies this move as &#34;Compliant run&#34; with a green icon. If the software failed to detect the foot print, the experimenter will see a red icon and must repeat the recording. At least five successful compliant runs are needed for analysis.</li>
	<li>After the test, select &#39;classify runs&#39; from the acquire tab of the &#39;program&#39; menu.</li>
	<li>Select the runs for analysis and click the &#39;auto classify&#39; button. 
	NOTE: After classification, all statistic parameters are automatically saved and the experimenter can find the results by clicking &#34;view run statistics&#34; on the analyze menu.</li>
</ol>

<ol>
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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=Characterization+of+a+neuropathic+pain+model:+sciatic+cryoneurolysis+in+the+rat.">Characterization of a neuropathic pain model: sciatic cryoneurolysis in the rat.</a> Pain. 56 (1), 9-16 (1994).</li><li>Kim, S. H., Chung, J. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peripheral+mononeuropathy+in+rat+that+produces+disorders+of+pain+sensation+like+those+seen+in+man.">A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man.</a> Pain. 33 (1), 87-107 (1988).</li><li>Dixon, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+analysis+of+experimental+observations.">Efficient analysis of experimental observations.</a> Annu Rev Pharmacol Toxicol. 20, 441-462 (1980).</li><li>Bonin, R. P., Bories, C., De Koninck, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+up-down+method+(SUDO)+for+measuring+mechanical+nociception+in+rodents+using+von+Frey+filaments.">A simplified up-down method (SUDO) for measuring mechanical nociception in rodents using von Frey filaments.</a> Mol Pain. 10, 26 (2014).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">Quantitative assessment of tactile allodynia in the rat paw.</a> J Neurosci Methods. 53 (1), 55-63 (1994).</li><li>Chen, H., Du, J., Zhang, Y., Barnes, K., Jia, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+a+Reliable+Gait+Evaluation+Method+for+Rodent+Studies.">Establishing a Reliable Gait Evaluation Method for Rodent Studies.</a> J Neurosci Methods. , (2017).</li><li>Kang, D. W., 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=Antinociceptive+Profile+of+Levo-tetrahydropalmatine+in+Acute+and+Chronic+Pain+Mice+Models:+Role+of+spinal+sigma-1+receptor.">Antinociceptive Profile of Levo-tetrahydropalmatine in Acute and Chronic Pain Mice Models: Role of spinal sigma-1 receptor.</a> Sci Rep. 6, 37850 (2016).</li><li>Huehnchen, P., Boehmerle, W., Endres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+paclitaxel+induced+sensory+polyneuropathy+with+&amp;#34;Catwalk&amp;#34;+automated+gait+analysis+in+mice.">Assessment of paclitaxel induced sensory polyneuropathy with &amp;#34;Catwalk&amp;#34; automated gait analysis in mice.</a> PLoS One. 8 (10), e76772 (2013).</li><li>Vrinten, D. H., Hamers, F. F. <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 (1-2), 203-209 (2003).</li><li>Martinov, T., Mack, M., Sykes, A., Chatterjea, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+changes+in+tactile+sensitivity+in+the+hind+paw+of+mice+using+an+electronic+von+Frey+apparatus.">Measuring changes in tactile sensitivity in the hind paw of mice using an electronic von Frey apparatus.</a> J Vis Exp. (82), e51212 (2013).</li><li>Ferland, C. E., Laverty, S., Beaudry, F., Vachon, P. <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+and+pain+response+of+two+rodent+models+of+osteoarthritis.">Gait analysis and pain response of two rodent models of osteoarthritis.</a> Pharmacol Biochem Behav. 97 (3), 603-610 (2011).</li><li>Mogil, J. 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=Hypolocomotion,+asymmetrically+directed+behaviors+(licking,+lifting,+flinching,+and+shaking)+and+dynamic+weight+bearing+(gait)+changes+are+not+measures+of+neuropathic+pain+in+mice.">Hypolocomotion, asymmetrically directed behaviors (licking, lifting, flinching, and shaking) and dynamic weight bearing (gait) changes are not measures of neuropathic pain in mice.</a> Mol Pain. 6, 34 (2010).</li><li>Ferreira-Gomes, J., Adaes, S., Castro-Lopes, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+movement-evoked+pain+in+osteoarthritis+by+the+knee-bend+and+CatWalk+tests:+a+clinically+relevant+study.">Assessment of movement-evoked pain in osteoarthritis by the knee-bend and CatWalk tests: a clinically relevant study.</a> J Pain. 9 (10), 945-954 (2008).</li></ol>

The authors have nothing to disclose.

Currently, measurement of mechanical allodynia by using von Frey filaments is the most widely used method in pain animal models to demonstrate tactile hypersensitivity. As animal models for neuropathic pain have continued to be developed, the methodology of assessment for sensory function has been also improved8,9,10,15. In those reports, it has been suggested these modifications can provide a more accurate, fast, and user-friendly replacement for older methods. However, data from those methods still may be limited by its subjectivity and the requirement of skill or experience to perform the method. In addition, restraining the animals cannot be avoided when performing the von Frey test, and therefore restraint-related stress of the animals may affect the pain response results.
The automated gait analysis system automatically measures various parameters of animal walking (e.g., total area of paw contact to the floor, angle of paw print, stance time, etc.), and therefore the experimenter can use more objective data for analysis12,13,14,16,17,18. Furthermore, this method can be performed without any stimulation; thus, it is less-invasive and data from the freely moving rodent can be obtained. As shown in results of this study, the foot print size and stance time of the ipsilateral hind paw are reduced by CCI surgery and recovered by GBP treatment; similar results are also found in the von Frey test. Consistent with previous observations14, results of gait analysis in neuropathic pain animal models have a high degree of positive correlation with that from the von Frey test, indicating that the CCI-related changed parameters may reflect the degree of mechanical allodynia, one of the most irritating symptoms of neuropathic pain.
Here, we provide a method on how to operate the automated gait analysis system in detail and a brief comparison of data with the classical von Frey test in CCI neuropathic mice. In conclusion, the automated gait analysis may be the most improved accessible tool for the study of neuropathic pain because of it is user-friendly, objective, and sensitive.

Pathological changes of nervous system induced by trauma, metabolic dysfunction, inflammation, infection, ischemia, or autoimmune disease occasionally result in neuropathic pain, which is defined as a pain arising as a direct consequence of a lesion or disease affecting the somatosensory system1. Neuropathic pain is usually unbearable and unfortunately, conventional analgesics generally do not produce sufficient pain relief2. A major feature of neuropathic pain contains spontaneous and stimulation-evoked (i.e., allodynia and hyperalgesia) pains. Allodynia is a nociceptive response that occurs to normally non-painful stimuli, such as light touch or warm stimulation. Hyperalgesia indicates an enhanced pain response to noxious mechanical and/or thermal stimuli3. Although these two symptoms both critically impair the patient&#39;s quality of life, mechanical allodynia evoked by gentle tactile stimulation is the most aggravating symptom because soft contact is difficult to avoid in everyday life.
To investigate the underlying mechanism and new analgesics for the treatment of neuropathic pain, the precise measurement of the pain response is essential. Numerous neuropathic pain animal models have developed nociceptive responses on the hind paw area because of its high accessibility4,5,6,7. Thus, most pain response assessments have been performed on the plantar or dorsal surface of the hind paw by applying mechanical stimuli using special instruments, such as von Frey filaments. One of the most frequently used method is the up and down method described by Dixon8 and the later modified versions9,10. However, very skilled, experienced experimenters are required to perform the von Frey test and the results may be subjective.
The automated gait analysis system can investigate neurological and neuromuscular disorders by measuring various parameters of walking in freely moving rodents. In a variety of nerve injury animal models, the degree of nociception and the antinociceptive effect of several treatments can be evaluated without adding a pain stimulation11,12,13,14. This analysis system can detect static and dynamic gait parameters, such as: paw print area (the area of the complete paw print that contacts with the floor), paw intensity (the average intensity of the contacting paw area), stride length (the distance between successive placements of the same paw), stance phase (the duration of ground contact for a single hind paw), step sequence (the order in which the four paws are placed on the floor), swing (the duration of the swing phase), and swing speed (computed from stride length and swing duration and expressed as pixels per second). This paper demonstrates the use an analysis system and provides a brief comparison of data with the von Frey test using chronic constriction injury (CCI) neuropathic mice.

<table>
	<tbody>
		<tr>
			<td>0.9% Saline</td>
			<td>JW Pharmaceutical</td>
			<td>N/A</td>
			<td>Vehicle for drugs</td>
		</tr>
		<tr>
			<td>1ml syringe</td>
			<td>BD Plastipak</td>
			<td>300013</td>
			<td>Injecting device</td>
		</tr>
		<tr>
			<td>2, 2, 2-tribromoethanol (97% purity)</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>2-methyl-2-buthanol (99% purity)</td>
			<td>Sigma</td>
			<td>152463</td>
			<td>Solvent for 2, 2, 2-tribromoethanol</td>
		</tr>
		<tr>
			<td>Catwalk Automated gait analysis system</td>
			<td>Noldus</td>
			<td>N/A</td>
			<td>Automatic analysis software of aniaml gait</td>
		</tr>
		<tr>
			<td>Chromic catgut (4-0 thickness)</td>
			<td>AILEE</td>
			<td>C442</td>
			<td>Ligature to make chronic constriction injury on the sciatic nerve</td>
		</tr>
		<tr>
			<td>Gabapentin</td>
			<td>Sigma</td>
			<td>Y0001280</td>
			<td>Analgeisc, Used as a positive control drug in this study</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>F.S.T</td>
			<td>11051-10</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>DAESHIN ELECTRONICS</td>
			<td>M-303AT</td>
			<td>Regulation of body temperature</td>
		</tr>
		<tr>
			<td>ICR Mouse</td>
			<td>Samtaco</td>
			<td>N/A</td>
			<td>Experimental animal</td>
		</tr>
		<tr>
			<td>Mersilk (3-0 thickness)</td>
			<td>ETHICON</td>
			<td>W598H</td>
			<td>Suture material for surgical closure of skin</td>
		</tr>
		<tr>
			<td>Micro-Mosquito</td>
			<td>F.S.T</td>
			<td>13010-12</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Micro-scissors</td>
			<td>F.S.T</td>
			<td>14090-09</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>F.S.T</td>
			<td>12002-12</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Povidone Iodine</td>
			<td>Firson</td>
			<td>N/A</td>
			<td>Disinfectant to prevent infection after surgery</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>F.S.T</td>
			<td>10010-00 (#10)</td>
			<td>Surgical instrument to make an incision</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>F.S.T</td>
			<td>10003-12 (#3)</td>
			<td>Surgical instrument to make an incision</td>
		</tr>
		<tr>
			<td>Von-Frey filaments</td>
			<td>North Coast</td>
			<td>NC12775-99</td>
			<td>Measurement device to test sensory function for mechanical stimulation</td>
		</tr>
	</tbody>
</table>

automated gait analysis in mice with chronic constriction injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

We have performed the von Frey test and automated gait analysis in CCI mice until 10 days after CCI surgery. For statistical analysis, repeated measures of two-way analysis of variance (ANOVA) determines the overall effects, and Dunnett&#39;s post-hoc analysis was performed to determine the p-value among the experimental groups.
The results shown in Figure 1 indicate the time course of the classical von Frey test data. The PWF (%) of CCI mice was increased 2 days after CCI surgery and sustained for 10 days as compared with the Sham group. In the CCI positive control group, GBP (50 mg/kg, IP, once daily for 10 days starts 1 day after surgery) significantly alleviated CCI-enhanced PWF. In the Sham group of panel A, PWF (%) was 8.00 &#177; 5.83 on the timepoint pre-surgery (Pre), 6.00 &#177; 2.45 on day 2, 4.00 &#177; 2.45 on day 4, 2.00 &#177; 2.00 on day 6, 4.00 &#177; 22.45 on day 8, and 6.00 &#177; 2.45 on day 10. In the CCI group, PWF (%) was 2.86 &#177; 1.84 on Pre, 61.43 &#177; 5.95 on day 2, 68.57 &#177; 4.59 on day 4, 72.86 &#177; 5.22 on day 6, 75.71 &#177; 6.49 on day 8, and 75.71 &#177; 3.69 on day 10. In CCI+GBP group, PWF (%) was 2.50 &#177; 1.43 on Pre, 57.50 &#177; 5.22 on day 2, 50.00 &#177; 4.04 on day 4, 45.00 &#177; 4.81 on day 6, 46.25 &#177; 5.65 on day 8, and 47.50 &#177; 4.88 on day 10. In panel B, the area under curve (AUC) was 23.00 &#177; 6.00 in Sham, 317.86 &#177; 17.04 in CCI, and 223.75 &#177; 17.05 in CCI+GBP group.
The results shown in Figure 2 indicate a time course change of the paw print area after CCI surgery. Data are shown as the percent change of the paw print area between the left and right hind paws. If the paw print area is the same at the left and right sides in a normal condition, the data will be 50%. CCI significantly reduced paw print area of the ipsilateral hind paw 2 days after surgery and was maintained for 10 days. GBP remarkably recovered the reduced paw print area. In the Sham group of panel B, the paw print area (%) was 50.13 &#177; 2.13 on Pre, 50.30 &#177; 1.45 on day 2, 53.24 &#177; 0.80 on day 4, 50.30 &#177; 1.68 on day 6, 52.08 &#177; 1.79 on day 8, and 49.66 &#177; 2.24 on day 10. In the CCI group, the paw print area (%) was 50.71 &#177; 3.17 on Pre, 0 &#177; 0 on day 2, 0 &#177; 0 on day 4, 8.86 &#177; 3.27 on day 6, 6.6 &#177; 3.20 on day 8, and 10.30 &#177; 5.60 on day 10. In the CCI+GBP group, the paw print area (%) was 55.59 &#177; 2.01 on Pre, 4.65&#177;4.17 on day 2, 15.18&#177;5.57 on day 4, 20.20 &#177; 4.00 on day 6, 26.01 &#177; 5.53 on day 8, and 28.40 &#177; 6.04 on day 10.
The results shown in Figure 3 indicate the time course change of a single stance after CCI surgery. Data were calculated and shown as the percent change (similar to the paw print area data). CCI reduced the single stance of the ipsilateral side and GBP significantly recovered it. In the Sham group of panel B, the single stance (%) was 49.31 &#177; 2.15 on Pre, 50.71 &#177; 0.67 on day 2, 50.76 &#177; 0.44 on day 4, 50.60 &#177; 1.11 on day 6, 51.50 &#177; 0.96 on day 8, and 49.00 &#177; 2.35 on day 10. In the CCI group, the single stance (%) was 50.36 &#177; 3.17 on Pre, 0 &#177; 0 on day 2, 0 &#177; 0 on day 4, 11.5 &#177; 3.25 on day 6, 13.61 &#177; 5.04 on day 8, and 12.94 &#177; 6.40 on day 10. In the CCI+GBP group, the single stance (%) was 52.35 &#177; 0.91 on Pre, 5.44 &#177; 4.87 on day 2, 18.66 &#177; 4.33 on day 4, 25.48 &#177; 4.10 on day 6, 30.26 &#177; 2.17 on day 8, and 32.24 &#177; 4.95 on day 10.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56402/56402fig1.jpg" /> 
Figure 1: Mechanical allodynia assessed by von Frey test in CCI mice. (A) The paw withdrawal frequency (PWF, %) in the von Frey test was increased 2 days after CCI surgery and sustained for 10 days as compared with the Sham group. Gabapentin (GBP, 50 mg/kg, IP) significantly reversed the CCI-induced mechanical allodynia. (B) Accumulated data of PWF are shown as area under curve. **p &#60;0.01 and ***p &#60;0.001 versus CCI group. Error bars indicate mean &#177; SD. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56402/56402fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56402/56402fig2.jpg" /> 
Figure 2: Paw print area by automated gait analysis in CCI mice. (A) Captured images of hind paws from the groups of Sham, CCI, and CCI+GBP (gabapentin, 50 mg/kg, IP) by analysis software. White rectangles are indicators of hind paw detected by the analysis system. Note the smallest paw size of CCI and this reduction was partly recovered by GBP treatment. (B) Time course of the paw print area (%). Data are calculated as the percent change of print area between the left and right hind paw (e.g., 50% indicates the same paw print areas in the left and right sides). *p &#60;0.05 and **p &#60;0.01 versus CCI group. Error bars indicate mean &#177; SD.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56402/56402fig3.jpg" /> 
Figure 3: Single stance by automated gait analysis in CCI mice. (A) Captured and converted images of single stance highlighted by different colors for each paw. Note that the pink trace for the ipsilateral hind paw is shortened in the CCI group and gabapentin (GBP, 50 mg/kg, IP) significantly recovered it similarly to the Sham level. (B) Time course of single stance (%). Data are summarized as a line graph after calculating percent change of single stance between the left and right hind paw (e.g., 50% indicates the same single stance in the left and right sides). *p &#60;0.05, **p &#60;0.01 and ***p &#60;0.001 versus CCI group. Error bars indicate mean &#177; SD. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56402/56402fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Automated Gait Analysis in Mice with Chronic Constriction Injury at JoVE.com

Automated Gait Analysis in Mice with Chronic Constriction Injury

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

automated-gait-analysis-in-mice-with-chronic-constriction-injury

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.0K Views.  Chungnam National University. The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

Video: Automated Gait Analysis in Mice with Chronic Constriction Injury

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

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

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also demonstrate that traumatic brain injuries cause changes to the gut microbiota. However, mechanisms underlying the bidirectional regulation of the brain-gut axis remain unknown. Currently, few models exist for studying the changes in gut microbiota after traumatic brain injury. Therefore, the presented study combines protocols for inducing traumatic brain injury using a lateral fluid percussion device and analysis of caecum samples following injury for investigating alterations in the gut microbiome. Alterations of the gut microbiota composition after traumatic brain injury are determined using 16S-rDNA sequencing. This protocol provides an effective method for studying the relationships between enteric microorganisms and traumatic brain injury.

Presented here is a protocol to induce diffuse traumatic brain injury using a lateral fluid percussion device followed by the collection of the caecum content for gut microbiome analysis.

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also ...

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

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

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

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

Motor Dual-Tasks for Gait Analysis and Evaluation in Post-Stroke Patients

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask gait analysis consisted of a single walking task (Task 0), a simple motor dual-task (water-holding, Task 1), and a complex motor dual-task (crossing obstacles, Task 2). The task of crossing obstacles was considered to be equivalent to the combination of a simple walking task and a complex motor task as it involved more nervous system, skeletal movement, and cognitive resources. To eliminate heterogeneity in the results of the gait analysis of the stroke patients, the dual-task gait cost values were calculated for various kinematic parameters. The major differences were observed in the proximal joint angles, especially in the angles of the trunk, pelvis, and hip joints, which were significantly larger in the dual motor tasks than in the single walking task. This research protocol aims to provide a basis for the clinical diagnosis of gait function and an in-depth study of motor control in stroke patients with motor control deficits through the analyses of dual-motor walking tasks.

This paper presents a protocol specifically for dual motor task gait analysis in stroke patients with motor control deficits.

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask ...

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...