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><li>Loeser, J. D., Treede, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Kyoto+protocol+of+IASP+Basic+Pain+Terminology%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">The Kyoto protocol of IASP Basic Pain Terminology.</a> Pain. 137 (3), 473-477 (2008).</li>
<li>Colloca, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuropathic+pain%5BTitle%5D)+AND+%22Nat+Rev+Dis+Primers%22%5BJournal%5D)">Neuropathic pain.</a> Nat Rev Dis Primers. 3, 17002(2017).</li>
<li>Vranken, J. H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/20021340">Mechanisms and treatment of neuropathic pain.</a> Cent Nerv Syst Agents Med Chem. 9 (1), 71-78 (2009).</li>
<li>DeLeo, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+neuropathic+pain+model%3A+sciatic+cryoneurolysis+in+the+rat%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+experimental+model+for+peripheral+neuropathy+produced+by+segmental+spinal+nerve+ligation+in+the+rat%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat.</a> Pain. 50 (3), 355-363 (1992).</li>
<li>Seltzer, Z., Dubner, R., Shir, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+behavioral+model+of+neuropathic+pain+disorders+produced+in+rats+by+partial+sciatic+nerve+injury%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury.</a> Pain. 43 (2), 205-218 (1990).</li>
<li>Bennett, G. J., Xie, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+peripheral+mononeuropathy+in+rat+that+produces+disorders+of+pain+sensation+like+those+seen+in+man%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/7387124">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/pubmed?term=((A+simplified+up-down+method+%28SUDO%29+for+measuring+mechanical+nociception+in+rodents+using+von+Frey+filaments%5BTitle%5D)+AND+%22Mol+Pain%22%5BJournal%5D)">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/pubmed?term=((Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">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/pubmed?term=((Establishing+a+Reliable+Gait+Evaluation+Method+for+Rodent+Studies%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">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/pubmed?term=((Antinociceptive+Profile+of+Levo-tetrahydropalmatine+in+Acute+and+Chronic+Pain+Mice+Models%3A+Role+of+spinal+sigma-1+receptor%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">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/pubmed?term=((Assessment+of+paclitaxel+induced+sensory+polyneuropathy+with+%26%2334%3BCatwalk%26%2334%3B+automated+gait+analysis+in+mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Assessment of paclitaxel induced sensory polyneuropathy with &#34;Catwalk&#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/pubmed?term=((%27CatWalk%27+automated+quantitative+gait+analysis+as+a+novel+method+to+assess+mechanical+allodynia+in+the+rat%3B+a+comparison+with+von+Frey+testing%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">'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/pubmed?term=((Measuring+changes+in+tactile+sensitivity+in+the+hind+paw+of+mice+using+an+electronic+von+Frey+apparatus%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Gait+analysis+and+pain+response+of+two+rodent+models+of+osteoarthritis%5BTitle%5D)+AND+%22Pharmacol+Biochem+Behav%22%5BJournal%5D)">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/pubmed?term=((Hypolocomotion%2C+asymmetrically+directed+behaviors+%28licking%2C+lifting%2C+flinching%2C+and+shaking%29+and+dynamic+weight+bearing+%28gait%29+changes+are+not+measures+of+neuropathic+pain+in+mice%5BTitle%5D)+AND+%22Mol+Pain%22%5BJournal%5D)">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/pubmed?term=((Assessment+of+movement-evoked+pain+in+osteoarthritis+by+the+knee-bend+and+CatWalk+tests%3A+a+clinically+relevant+study%5BTitle%5D)+AND+%22J+Pain%22%5BJournal%5D)">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 ...

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 ...

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

Research

JoVE Journal

Neuroscience

10.1K 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 ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

3D Kinematic Gait Analysis for Preclinical Studies in Rodents

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of complex algorithms and mathematical modeling that accompany 3D data collection and analysis procedures. This work provides a simple, user-friendly, step-by-step detailed methodology for 3D kinematic gait analysis during treadmill locomotion in healthy and neurotraumatic rats using a six-camera motion capture system. Also provided are details on 1) calibration of the system in an experimental set-up customized for quadrupedal locomotion, 2) data collection for treadmill locomotion in adult rats using markers positioned on all four limbs, 3) options available for video tracking and processing, and 4) basic 3D kinematic data generation and visualization and quantification of data using the built-in data collection software. Finally, it is suggested that the utility of this motion capture system be expanded to studying a variety of motor behaviors before and after neurotrauma.

Presented here is a protocol to collect and analyze three-dimensional kinematics of quadrupedal locomotion in rodents for preclinical studies.

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of ...

Behavior

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

Developmental Biology

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 ...

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 ...

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 ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

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 ...

Quantifying Locomotor Dysfunction in Spinal Cord Injury Mice Using MouseWalker

Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and supra-spinal circuits. After a thoracic spinal cord injury, communication with upstream circuits is impaired. This, in turn, leads to a loss of coordination, with limited recovery potential. Hence, to better evaluate the degree of recovery after the administration of drugs or therapies, there is a necessity for new, more detailed, and accurate tools to quantify gait, limb coordination, and other fine aspects of locomotor behavior in animal models of spinal cord injury. Several assays have been developed over the years to quantitatively assess free-walking behavior in rodents; however, they usually lack direct measurements related to stepping gait strategies, footprint patterns, and coordination. To address these shortcomings, an updated version of the MouseWalker, which combines a frustrated total internal reflection (fTIR) walkway with tracking and quantification software, is provided. This open-source system has been adapted to extract several graphical outputs and kinematic parameters, and a set of post-quantification tools can be to analyze the output data provided. This manuscript also demonstrates how this method, allied with already established behavioral tests, quantitatively describes locomotor deficits following spinal cord injury.

Author Spotlight: Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury  

An experimental pipeline to quantitatively describe the locomotor pattern of freely walking mice using the MouseWalker (MW) toolbox is provided, ranging from initial video recordings and tracking to post-quantification analysis. A spinal cord contusion injury model in mice is employed to demonstrate the usefulness of the MW system.