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

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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6.0K 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

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

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

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

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

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

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

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 ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

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 ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

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 ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

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

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

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

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