Name: automated gait analysis to assess functional recovery in rodents with peripheral nerve or spinal cord contusion injury
Uploaded: 2020-10-06T15:00:00
Duration: 6 min 31 s
Description: 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

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 (<img alt="Equation 1" src="/files/ftp_upload/61852/61852equ01.jpg" />)</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 &#38; Probe</td>
			<td>F.S.T</td>
			<td>10090-13</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Sprague Dawley rat (<img alt="Equation 1" src="/files/ftp_upload/61852/61852equ01.jpg" />)</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 (Video) | JoVE

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.

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

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