The authors would like to thank Karin Brenner for her passionate caretaking of the animals. The authors would also like to thank Claudia Keibl, James Ferguson, Gabriele Leinfellner, and Susanne Drechsler for their assistance during the experimental surgeries.

The authors declare that this work was supported by Noldus Inc. by coverage of the open access publication fee. None of the authors received any personal salary or any kind of financial gratuity. The authors have no other competing interests to declare.

Assessment of functional recovery in animal models of PNI and SCI remains challenging due to the great variety of evaluation methods, each with individual advantages and disadvantages. Only few approaches have been tested and validated in multiple models of peripheral and central nervous injuries, although promising new techniques which combine motion tracking and machine learning might potentially propel neurobehavioral research to the next level of functional testing. We are convinced that cutting-edge methods broadly applicable to a wide variety of animal and injury models will soon emerge. In the light of these considerations, one of the advantages of AGA is the possibility to evaluate functional recovery in multiple models of nerve injury using only one device. Since the early 2000s this approach has been used in experimental models of PNI such as the sciatic37, peroneal38, and femoral nerve injury model22 as well as after root avulsion of both the lumbar39 and the brachial plexus40. Various central nervous injuries including spinal cord contusion injury have also been studied with the method41,42. With this paper, we presented a detailed protocol on how to induce three commonly studied nerve injuries as well as how to evaluate functional recovery afterwards. In our opinion, a hands-on-guideline for researchers interested in the area of experimental nerve injury, repair, and regeneration on how to make optimum use of the method&#8217;s advantageous features would be of great help.
Several authors have addressed the potential of AGA to evaluate functional recovery in rodents, highlighting the method&#8217;s advantage to simultaneously assess gait parameters related to motor and sensory reinnervation27,28. Additionally, comparison of data from an experimental paw, e.g., reconstructed nerve injury to an unoperated paw as was shown in both of the models presented allows inclusion of an intra-animal positive control. Inversely, an operated paw without surgical reconstruction or additional treatment could serve as an intra-animal negative control. It was also shown that it is possible to combine AGA with machine learning approaches43. In spite of the method&#8217;s advantages, it also has several limitations and drawbacks, such as the time-consuming training efforts, which are mandatory to accustom the animal to the acquisition procedure28,44. Another limitation of AGA is the maximum size of the animals eligible for testing due to the limited dimensions of the apparatus. Therefore, the use of AGA is currently limited to animals the size of rodents and ferrets45. Additionally, recently emerging neurobehavioral assessment approaches in the field of motion tracking capable of machine-learning may surpass AGA in both comprehensiveness as well as possible applications18,19,46. Most noteworthy, but in accordance with other evaluation methods, it seems that functional recovery as assessed by AGA is strongly limited&#8212;if even occurring&#8212;in models of sciatic nerve neurotmesis47,48. On the other hand, AGA allows for comprehensive evaluation of the course of functional recovery following femoral nerve neurotmesis as shown by our data. With this work, we demonstrated that Paw Print Area is a representative gait parameter assessable via AGA, which is exemplary for the course of functional recovery in the two aforementioned peripheral nerve injury models presented by us. While functional recovery ad integrum was observable after autograft repair of the femoral nerve, AGA parameters were still significantly changed from baseline at the end of the observation period following autograft repair of the sciatic nerve. It is noteworthy in this context that limb contractures are a common phenomenon in rats with sciatic nerve injury and caution is necessary not to confuse these signs of muscular imbalance and paralysis with the proceeding functional recovery32. This on the one hand underlines the AGA method&#8217;s inability to detect significant functional recovery following neurotmesis injury in this model. On the other hand, it raises the question whether it is feasible to evaluate the sciatic nerve injury model of the rat, which is still the most commonly used experimental nerve repair model, by means of gait analysis in general in case the nerve injury is more severe than axonotmesis48. Troubleshooting details are provided in Supplementary File 1.
We also provided exemplary data on use of the method to evaluate locomotor function in rats with Spinal Cord Injury, which is possible without any required changes of the hardware setup or acquisition procedure. The same principle applies to other rodent models of central nervous injury (CNI)26,49,50 and root avulsion injury. In contrast to isolated PNIs, injuries of the spinal cord are far more complex in their pathophysiological consequences, as a multitude of highly important structures are damaged, involving efferent pathways such as the corticospinal and rubrospinal tracts and afferent pathways such as the dorsal columns and spinothalamic tracts35. The challenge to adequately assess these pathological changes is reflected in the comprehensive armamentarium of behavioral tests, such as the Basso, Beattie, and Bresnahan (BBB) score36. The gait parameter Base of Support has been reported to increase following central nervous injuries, most probably to account for a resulting instable gait. Base of Support was significantly changed from baseline from WPO10 until WPO14 in our model, supporting our presumption that this parameter allows assessment of the course of functional recovery by AGA following thoracic spinal cord contusion injury.
We are convinced that AGA is a feasible tool to evaluate functional recovery in rodents with injuries of the nervous system. Nevertheless, we advise to reflect the observed changes of gait carefully and thoroughly in each respective experimental setup. Alterations in gait parameters, e.g., an increase in Print Area following an immediate postoperative decrement or a decrease in Swing Time proceeding an immediate postoperative elevation of this parameter, over the course of the observation period do not inevitably relate to functional recovery. Instead these changes can also be related to a possible functional adaption to maintain an inconspicuous gait, given that rats are a prey species and try to avoid showing pain or disability to potential predators51. It is, therefore, recommended to use automated gait analysis as a complementary tool to relate changes of gait to other outcome measures of peripheral nerve injury and regeneration21. As mentioned previously, we also believe that it should be carefully reflected if rodents with sciatic nerve neurotmesis should be investigated by means of AGA as our finding strongly indicates that functional recovery is severely limited in this case.
As shown in our work, AGA&#8217;s main asset is the possibility to study both motor and sensory reinnervation in a multitude of experimental PNI models as well as CNI while requiring only one setup. Therefore, the method is, in our opinion, a highly valuable tool for comprehensive neurobehavioral testing. One of AGA&#8217;s assets, which is the possibility to study motor and sensory reinnervation in various animal models of PNI and CNI while requiring only one setup, is in our opinion the method&#8217;s main advantage in comparison to other evaluation methods to study functional recovery, such as walking track analysis52, Von Frey testing53, or gait kinematics16. The potential to simultaneously evaluate changes of gait which do either correlate with results of electrophysiological investigations of reinnervated muscle22 or evaluation methods for sensory function54 is promising in regard to future applications of the method. We therefore recommend using AGA to investigate functional recovery in rodent models of forelimb PNI, such as the ulnar, radial, or median nerve, or experimental nerve transfer models55, which remain unstudied with this method yet.
We hereby provide a detailed protocol on how to use automated gait analysis to study functional recovery in three rodent models of nerve injury. While the method requires careful consideration of various key aspects such as adequate training and meticulous hard- and software calibration, it is a feasible and valuable complementary tool to evaluate nerve regeneration in rodent models of central and peripheral nerve injury.

Injuries of the peripheral and central nervous system are often studied in rodents, resulting in a great amount of comparative data regarding the course of nerve injury, repair, or neuroprotection to counteract further secondary injuries and regeneration1,2,3. The outcome of experimental treatment strategies in rodent models can be assessed by a variety of techniques such as histology, immunohistochemistry, electrophysiology, and imaging techniques such as X-ray microtomography (&#956;CT) scans, but the most important criterion to determine the success of a treatment is&#8212;like in human patients&#8212;the degree of functional recovery4,5. The first studies investigating locomotor performance in rodents date back to the 1940s6,7,8. Rats and mice were subject to a great amount of studies investigating their locomotor behavior in the following decades9,10,11. Nowadays, a broad range of assessment techniques for rodent models of peripheral and central nerve injuries exist, ranging from walking track analysis with ink and paper12,13,14 over ankle and gait kinematics15,16,17 to machine-learning enhanced methods, which allow for the complex estimation of gait, limb, and joint trajectories18,19.
Computerized Automated Gait Analysis (AGA) is used to evaluate locomotor function following peripheral and central nervous injuries and potential experimental treatment of such injuries. The device mainly consists of a glass walkway and a light source that illuminates the rodent&#8217;s paw prints in correlation with the pressure exceeded by them. This data is then computerized to calculate a broad array of static and dynamic parameters. According to Deumens, these parameters can be further subdivided into the categories of general parameters, pain-related parameters as well as coordination-related parameters of gait20 (Table 1). The feasibility of AGA to detect changes in gait behavior has been proven in various animal models of peripheral nerve injury (PNI)21, such as the sciatic nerve20, femoral nerve22, and median nerve23,24. It is also routinely used to assess locomotor function in rats with central nervous injuries, e.g., stroke25 or spinal cord contusion26. The method&#8217;s advances lie in the great amount of comparable data and its possibility to record a plethora of parameters related to gait27. This paper aims to provide researchers interested in animal models of PNI and spinal cord injury (SCI) with a detailed and hands-on guideline to assess locomotor function in such models.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Category</td>
			<td>Parameter</td>
			<td colspan="2">Description</td>
		</tr>
		<tr>
			<td rowspan="4">General parameters of gait</td>
			<td>Print Area (distance unit)</td>
			<td colspan="2">Area of the paw print</td>
		</tr>
		<tr>
			<td>Print Length (distance unit)</td>
			<td colspan="2">Length of the paw print</td>
		</tr>
		<tr>
			<td>Base of Support (BoS) (distance unit)</td>
			<td colspan="2">Distance between the two hind-or front paws</td>
		</tr>
		<tr>
			<td>Stride Length (distance unit)</td>
			<td colspan="2">Distance between two consecutive placements of a paw</td>
		</tr>
		<tr>
			<td rowspan="3">Pain-related parameters of gait</td>
			<td>Swing Time (s)</td>
			<td colspan="2">Duration of the swing phase</td>
		</tr>
		<tr>
			<td>Stand Time (s)</td>
			<td colspan="2">Duration of the stance phase</td>
		</tr>
		<tr>
			<td>Mean Paw Print Intensity (arbitrary unit)</td>
			<td colspan="2">Mean iIntensity of the paw print during the stance phase</td>
		</tr>
		<tr>
			<td rowspan="3">Coordination-related parameters of gait</td>
			<td>Normal Step Sequence Patterns (NSSP)</td>
			<td colspan="2">Specific sequences of paw placements during a step cycle</td>
		</tr>
		<tr>
			<td>Phase Dispersions (%)</td>
			<td colspan="2">Temporal differences between the step cycles of two specific paws</td>
		</tr>
		<tr>
			<td>Regularity Index (RI) (%)</td>
			<td colspan="2">Quantification of interlimb coordination by dividing the amount of flawless NSSP times 4 by the overall number of paw placement during one step cycle</td>
		</tr>
	</tbody>
</table>
Table 1: Parameters of gait assessable with the automated gait analysis. The categories in which the parameters are classified are chosen according to Deumens et al.20.

<table><tbody><tr><td>0.9% Saline</td><td>B. Braun Austria</td><td>3570410</td><td>Vehicle for drug delivery</td></tr><tr><td>1 ml syringe</td><td>B. Braun Austria</td><td>9161708V</td><td>Injecting device</td></tr><tr><td>10 ml syringe</td><td>B. Braun Austria</td><td>4606728 V</td><td>Injecting device</td></tr><tr><td>1-Propanol, 2-Propanol, Hexetidin</td><td>Gebro Pharma</td><td>N/A</td><td>Alcoholic skin disinfection</td></tr><tr><td>23-gauge (G) canula</td><td>B. Braun Austria</td><td>4657667</td><td>Canula for s.c. injection</td></tr><tr><td>26-gauge (G) canula</td><td>B. Braun Austria</td><td>4657683</td><td>Canula for s.c. injection</td></tr><tr><td>5 ml syringe</td><td>B. Braun Austria</td><td>4606710 V</td><td>Injecting device</td></tr><tr><td>Buprenorphine hydrochloride</td><td>Sigma</td><td>B9275</td><td>Analgetic agent</td></tr><tr><td>Burrs for Micro Drill</td><td>F.S.T</td><td>19007-29</td><td>Drilling of a hole inside the lamina</td></tr><tr><td>Caprofen</td><td>Zoetis Austria</td><td>N/A</td><td>Analgetic agent</td></tr><tr><td>Catwalk Automated gait analysis system</td><td>Noldus</td><td>N/A</td><td>Automatic analysis software of animal gait</td></tr><tr><td>Cauterizer Kit</td><td>F.S.T</td><td>18010-00</td><td>Cauterization of vessels during surgery</td></tr><tr><td>Enrofloxacin</td><td>Bayer Austria</td><td>N/A</td><td>Antibiotic</td></tr><tr><td>Ethilon (10-0)</td><td>ETHICON</td><td>2810G</td><td>Suture material for neurrorhaphy</td></tr><tr><td>Ethilon (11-0)</td><td>ETHICON</td><td>EH7465G</td><td>Suture material for neurrorhaphy</td></tr><tr><td>Eye ointment</td><td>Fresenius Kabi Austria</td><td>4302436</td><td>Eye protection during anesthesia</td></tr><tr><td>Friedman-Pearson Rongeurs</td><td>F.S.T</td><td>16221-14</td><td>Surgical instrument</td></tr><tr><td>Gabapentin</td><td>Wedgewood Pharmacy</td><td>N/A</td><td>Analgetic agent</td></tr><tr><td>Goldstein retractor</td><td>F.S.T</td><td>17003-03</td><td>Retraction of tissues during surgery</td></tr><tr><td>Hair trimmer</td><td>Aescular</td><td>N/A</td><td>Hair trimmer for shaving of the operation site prior to surgery</td></tr><tr><td>Heating Pad for rodents</td><td>ALA Scientific Instruments</td><td>N/A</td><td>Regulation of body temperature</td></tr><tr><td>Impactor</td><td>Precision Systems and Instrumentation</td><td>N/A</td><td>Induction of spinal cord contusion</td></tr><tr><td>Lewis rat (&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/61852/61852equ01.jpg&quot;/&gt;)</td><td>Janvier</td><td>N/A</td><td>Experimental animal</td></tr><tr><td>Magnetic Fixator Retraction System</td><td>F.S.T</td><td>18200-50</td><td>Retraction of tissues during surgery</td></tr><tr><td>Metzenbaum Baby Scissors</td><td>F.S.T</td><td>14019-13</td><td>Surgical instrument</td></tr><tr><td>Micro Drill</td><td>Word Precision Instruments</td><td>503599</td><td>Instrument for bone drilling</td></tr><tr><td>Micro Needle holder</td><td>F.S.T</td><td>12076-12</td><td>Surgical instrument</td></tr><tr><td>Micro-scissors (curved)</td><td>F.S.T</td><td>15023-10</td><td>Surgical instrument</td></tr><tr><td>Micro-scissors (straight)</td><td>F.S.T</td><td>15007-08</td><td>Surgical instrument</td></tr><tr><td>Mirror Finish Forceps</td><td>F.S.T</td><td>11251-23</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>Operating microscope</td><td>Leica</td><td>M651 MSD</td><td>Magnification of the operative site</td></tr><tr><td>Povidone Iod</td><td>B. Braun Melsungen</td><td>N/A</td><td>Non-alcoholic skin disinfectant</td></tr><tr><td>Pulse Oximeter</td><td>STARR Life Sciences</td><td>N/A</td><td>Surveillance of heart rate and oxygen saturation</td></tr><tr><td>Rodent thermometer</td><td>BIOSEB</td><td>BIO-TK8851</td><td>Surveillance of body temperature</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>Sevoflurane Inhalation Vapour, Liquid (100%)</td><td>Baxter</td><td>HDG9117A</td><td>Anesthetic</td></tr><tr><td>Spatula &amp;amp; Probe</td><td>F.S.T</td><td>10090-13</td><td>Surgical instrument</td></tr><tr><td>Sprague Dawley rat (&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/61852/61852equ01.jpg&quot;/&gt;)</td><td>Janvier</td><td>N/A</td><td>Experimental animal</td></tr><tr><td>Sterila gauze 5x5cm</td><td>EVAC MEDICAL</td><td>E010.03.00215</td><td>Sterile gauze compress</td></tr><tr><td>Tissue Forceps</td><td>F.S.T</td><td>11021-12</td><td>Surgical instrument</td></tr><tr><td>Vicryl (4-0)</td><td>ETHICON</td><td>V3040H</td><td>Suture material for subcutaneous sutures</td></tr><tr><td>Vicryl (5-0)</td><td>ETHICON</td><td>V303H</td><td>Suture material for subcutaneous sutures</td></tr><tr><td>Vicryl cutting needle (4-0)</td><td>ETHICON</td><td>V392ZH</td><td>Suture material for skin sutures</td></tr><tr><td>Vicryl cutting needle (5-0)</td><td>ETHICON</td><td>V391H</td><td>Suture material for skin sutures</td></tr></tbody></table>

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.

12 rats underwent experimental peripheral nerve surgery. Sciatic nerve resection (Figure 2A) was performed in 7 rats, while femoral nerve neurotmesis (Figure 2B) was induced in 5 rats. In all animals, the nerve defect was reconstructed by means of an autologous nerve graft. Spinal cord contusion injury (Figure 2C) at level Th11 was induced in 6 rats, resulting in a total of 18 rats.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61852/61852fig02.jpg" /> 
Figure 2: Operative sites after nerve reconstruction.&#160;Nerve reconstruction with autografts in the sciatic nerve (A) and femoral nerve (B) as well as after spinal cord contusion injury (C). <a href="https://www.jove.com/files/ftp_upload/61852/61852fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
All animals recovered well from surgery and no cases of self-mutilation occurred. One animal of the sciatic nerve injury group developed strong contractures of the right hind paw during the course of the postoperative observation period and had to be excluded from further data analysis.
Sciatic nerve neurotmesis 
Since the sciatic nerve provides muscular and sensory innervation to the majority of the hindlimb, its resection results in a severe impairment of locomotor function. Following injury, rats use the heel of the paw for weight support only (Figure 3B&#8211;E) and the limb is moved in a sweeping circumductory movement. Therefore, locomotor changes assessed via AGA become apparent by means of a significantly reduced Print Area (Figure 4A) and significantly increased Swing Time (Figure 4B). Both parameters were still significantly altered in comparison to Pre-OP measurements as at the end of the observation period. Noteworthy, one animal developed strong contractures of the right hind paw starting at postoperative week (WPO) 10. This resulted in an increase of the Print Area of the right hind paw to more than 150% in comparison to the left paw at WPO12 (Figure 5). As this was an extremum in comparison to all other animals assessed in this study, we excluded this animal from data analysis in regard to Print Area.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61852/61852fig03.jpg" /> 
Figure 3: Representative paw prints prior to and following critical size resection of the right sciatic nerve and autograft repair.&#160;Note the strong decrease in Print Area following nerve injury (B) as compared to preoperatively (A). Despite a slight increment in Print Area during the course of the observation period (C&#8211;E) the paw prints of the right hind limb remained notably changed from baseline recordings. <a href="https://www.jove.com/files/ftp_upload/61852/61852fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61852/61852fig04.jpg" /> 
Figure 4: The course of functional recovery after critical size resection and autograft repair of the sciatic nerve.&#160;Print Area Ratio (A) and Swing Time Ratio (B) were statistically changed significantly from Pre-OP values immediately after sciatic nerve resection. While Print Area remained significantly decreased compared to baseline until WPO10, Swing Time was still significantly increased to Pre-OP values at WPO12. *: p &#60; 0.05 as compared to Pre-OP, **: p &#60; 0.01 as compared to Pre-OP. Error bars indicate mean &#177; standard error of the mean (SEM). <a href="https://www.jove.com/files/ftp_upload/61852/61852fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61852/61852fig05.jpg" /> 
Figure 5: Boxplot of the course of Print Area following sciatic nerve injury.&#160;Note the extremum (red ellipse) at WPO12, which is explained by the fact that one animal developed strong contractures of the right hind paw starting from WPO10. The animal was therefore excluded from the statistical analysis displayed in Figure 4. <a href="https://www.jove.com/files/ftp_upload/61852/61852fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Femoral nerve neurotmesis 
Femoral nerve resection results in denervation of the quadriceps muscle of the thigh33,34. In consequence, knee extension is impaired, resulting in hyperflexion of the ankle joint with consecutive lifting of the paw&#8217;s heel. Therefore, the respective paw&#8217;s Print Area (Figure 6B), is strongly reduced after surgery. Print Area of the left hind paw is increased due to a compensatory shifting of weight to the left. This should be kept in mind, as this phenomenon directly influences the calculated ratio between the &#8220;experimental&#8221; and &#8220;control&#8221; paw. Starting from WPO4 reinnervation of the quadriceps by the regenerating femoral nerve leads to reversal of these changes resulting in increased Paw Print Area of the right hind paw (Figure 7A). As the quadriceps muscle of the thigh also plays a role in the swing phase of the respective paw, Swing Time (Figure 7B) is greatly prolonged in rats with femoral nerve injury. Mirroring the return of Print Area, Swing Time decreases as the regenerating femoral nerve reaches the quadriceps muscle of the thigh. At WPO10, both parameters of gait returned to baseline, signaling full functional recovery.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61852/61852fig06.jpg" /> 
Figure 6: Representative paw prints.&#160;Representative paw prints prior to (A) and following (B&#8211;E) right femoral nerve resection and autograft repair. Print Area of RH decreased strongly at WPO2 (B), while an increase in Print Area of the left hind paw (LH) due to increased weight load became visible. RH Print Area started to increase starting from WPO6 (C) accompanied by a decrease in Print Area of LH. At WPO8 (D) and WPO10 (E) Print Area of RH recovered back close to preoperative levels. (Adapted with permission from Heinzel et al.22, licensed under CC BY 4.0.) <a href="https://www.jove.com/files/ftp_upload/61852/61852fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61852/61852fig07.jpg" /> 
Figure 7: The course of functional recovery after 7-mm resection and autograft repair of the femoral nerve.&#160;The course of Print Area Ratio (A) and Swing Time Ratio (B) revealed a strong change immediately after femoral nerve resection, but values recovered back to preoperative values at WPO8. #: p &#60; 0.05. Error bars indicate mean &#177; SEM. (Adapted with permission from Heinzel et al.22, licensed under CC BY 4.0.) <a href="https://www.jove.com/files/ftp_upload/61852/61852fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Spinal cord contusion 
Gait analysis revealed markedly altered paw prints after thoracic spinal cord contusion injury (Figure 8), most noteworthy a decrement in Print Area and a marked internal rotation of the hind paws at WPO2 (Figure 8B). Noteworthy, the paw rotation is also implemented as an assessable feature in the BBB, underscoring the applicability of computerized gait analysis to evaluate changes of gait which were originally evaluated with Open Field testing. Regarding the course of the individual gait parameters, spinal cord contusion at the Th11 level resulted in a decrease of the Print Area Ratio (Figure 9A) and increment of Swing Time Ratio (Figure 9B). Both the parameters trended toward baseline levels during the further course of the observation period, but there were no statistically significant changes observable. The coordination-related parameter Regularity Index (Figure 9C) also decreased at WPO2, but the degree varied greatly between animals. It also trended toward preoperative values until WPO16. Base of Support of the hind paws (Figure 9D), a general parameter of gait according to Deumens, showed a marked increase, which was statistically significant from WPO10 until WPO14. It trended toward baseline levels at WPO16 and was no longer significantly altered from the Pre-OP value at this time point.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61852/61852fig08.jpg" /> 
Figure 8: Representative paw prints of the two hind paws.&#160;Paw prints preoperatively (A) and following thoracic spinal cord contusion injury (B&#8211;F). Note the reduction in print area starting from WPO2 (B) accompanied by a notable internal rotation of the paws. During the course of the observation period (C&#8211;F) an increment of the Print Area is observable as well as clearance of the internal rotation. <a href="https://www.jove.com/files/ftp_upload/61852/61852fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61852/61852fig09.jpg" /> 
Figure 9: Spinal cord contusion at the Th 11 level.&#160;Spinal cord contusion at Th 11 resulted in observable alterations of Print Area Ratio (A) and Swing Time (B) and the Regularity Index (C), but these changes were not statistically significant. Following injury, Base of Support of the hind paws showed a marked increase compared to baseline, what was statistically significant at WPO10 until WPO14. *: p &#60; 0.05 as compared to Pre-OP. Error bars indicate mean &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/61852/61852fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Troubleshooting details. <a href="https://cloudflare.jove.com/files/ftp_upload/61852/Supplementary_File_1.docx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury at JoVE.com

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

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 ...

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6.1K Views.  Eberhard Karls University. 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.

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

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for quantifying locomotor deficits in rodents is to measure minute disturbances of unconstrained gait overground (e.g., manual BBB score or automated CatWalk). However, cortical inputs are not required for the generation of basic locomotion produced by the spinal central pattern generator (CPG). Thus, unconstrained walking tasks test locomotor deficits due to motor cortical impairment only indirectly. In this study, we propose a novel, precise foot-placement locomotor task that evaluates cortical inputs to the spinal CPG. An instrumented peg-way was used to impose symmetrical and asymmetrical locomotor tasks mimicking lateralized movement deficits. We demonstrate that shifts from equidistant inter-stride lengths of 20% produce changes in the forelimb stance phase characteristics during locomotion with preferred stride length. Furthermore, we propose that the asymmetric walkway allows for measurements of behavioral outcomes produced by cortical control signals. These measures are relevant for the assessment of impairment after cortical damage.

Here, we present a protocol to quantify precise stepping in rodents. Cortical and the spinal central pattern generator signals are required for precise foot-placement during obstructed locomotion. We report here the novel constrained walking task that directly examines precise stepping behavior.

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for ...

Behavior

Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER): A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function in many areas of research, the available commercial options have several limitations such as high cost and lack of accessible, open code. To address these issues, we developed PrAnCER, Paw-Print Analysis of Contrast-Enhanced Recordings, for automated quantification of gait. The contrast-enhanced recordings are produced by using a translucent floor that obscures objects not in contact with the surface, effectively isolating the rat&#8217;s paw prints as it walks. Using these videos, our simple software program reliably measures a variety of spatiotemporal gait parameters. To demonstrate that PrAnCER can accurately detect changes in motor function, we employed a haloperidol model of Parkinson&#8217;s disease (PD). We tested rats at two doses of haloperidol: high dose (0.30 mg/kg) and low dose (0.15 mg/kg). Haloperidol significantly increased stance duration and hind paw contact area in the low dose condition, as might be expected in a PD model. In the high dose condition, we found a similar increase in contact area but also an unexpected increase in stride length. With further research, we found that this increased stride length is consistent with the bracing-escape phenomenon commonly observed at higher doses of haloperidol. Thus, PrAnCER was able to detect both expected and unexpected changes in rodent gait patterns. Additionally, we confirmed that PrAnCER is consistent and accurate when compared with manual scoring of gait parameters.

Paw-Print Analysis of Contrast-Enhanced Recordings PrAnCER: A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

We describe a novel gait analysis system, Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER), an open-access automated system for the quantification of gait characteristics in rats that utilizes a novel semitransparent floor to automatically quantify gait. This system was validated using the haloperidol model of Parkinson&#8217;s Disease.

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function ...

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 ...

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 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.

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 ...

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-S&#233;quard syndrome&#160;is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of&#160;proprioception&#160;on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-S&#233;quard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human ...

Medicine

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are valuable tools to understand the neural mechanisms involved in locomotor recovery after SCI and to design therapies for clinical populations. There are several experimental SCI models including contusion, compression, and transection injuries that are used in a wide variety of species. A hemisection involves the unilateral transection of the spinal cord and disrupts all ascending and descending tracts on one side only. Spinal hemisection produces a highly selective and reproducible injury in comparison to contusion or compression techniques that is useful for investigating neural plasticity in spared and damaged pathways associated with functional recovery. We present a detailed step-by-step protocol for performing a thoracic hemisection at the T8 vertebral level in the rat that results in an initial paralysis of the hindlimb on the side of the lesion with graded spontaneous recovery of locomotor function over several weeks. We also provide a locomotor scoring protocol to assess functional recovery in the open-field. The locomotor assessment provides a linear recovery profile and can be performed both early and repeatedly after injury in order to accurately screen animals for appropriate time points in which to conduct more specialized behavioral testing. The hemisection technique presented can be readily adapted to other transection models and species, and the locomotor assessment can be used in a variety of SCI and other injury models to score locomotor function.

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve grafts. Due to their limited availability and associated donor side morbidity, many studies in the field of nerve regeneration focus on alternative techniques to bridge a segmental nerve gap. In order to investigate the outcomes of surgical or pharmacological experimental treatment options, the rat sciatic nerve model is often used as a bioassay. There are a variety of outcome measurements used in rat models to determine the extent of nerve regeneration. The maximum output force of the target muscle remains the most relevant outcome for clinical translation of experimental therapies. Isometric force measurement of tetanic muscle contraction has previously been described as a reproducible and valid technique for evaluating motor recovery after nerve injury or repair in both rat and rabbit models. In this video, we will provide a step-by-step instruction of this invaluable procedure for assessment of functional recovery of the tibialis anterior muscle in a rat sciatic nerve defect model using optimized parameters. We will describe the necessary pre-surgical preparations in addition to the surgical approach and dissection of the common peroneal nerve and tibialis anterior muscle tendon. The isometric tetanic force measurement technique will be detailed. Determining the optimal muscle length and stimulus pulse frequency is explained and measuring the maximum tetanic muscle contraction is demonstrated.

Evaluation of motor recovery remains the benchmark outcome measure in experimental peripheral nerve studies. The isometric tetanic force measurement of the tibialis anterior muscle in the rat is an invaluable tool to assess functional outcomes after reconstruction of sciatic nerve defects. The methods and nuances are detailed in this article.

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve ...

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 ...