We would like to acknowledge funding from the Mission Connect, a project of TIRR Foundation (CES and ZZK), the Craig Neilsen Foundation (CES) and NSF Graduate Research Fellowship (Grant #: 2011112479 to SAG).

All animal procedures were done in accordance with approved protocols with the internal IACUC at the University of Texas at Austin and under National Institutes of Health (NIH) guidelines.
Animals used in this study had a C3/C4 lateral contusion injury using an Infinite Horizon Impactor, or a C3/C4 lateral hemisection model and these methods can be applied more broadly to other models18. We recommend that researchers test animals for 2 weeks prior to injury, 3 days post injury and regularly (e.g. weekly) for the duration of the study. When performing behavioral tests on post surgical animals, especially at three days post surgery, care must be taken that the animals have recovered sufficiently from the surgery and that the behavioral testing procedures do not cause additional pain or stress to the animals. 
In general, 2-3 weeks of handling and pre-testing may be needed prior to surgery. This involves getting the animal comfortable to the testing environment by handling them in a similar way to how they will be handled during testing as well as allowing the animal to move around freely on the testing surface. It is also important to get the animal used to being held, this can be done by moving the animal on and near the testing surface in non-direct motions to acclimate the animal to handling. It is also important to relax the animals at the beginning and in between test sessions. To relax the animals, hold the animal on a tabletop in a forelimb-only bearing stance, in a "wheelbarrow" position and bounce the animal gently on the tabletop allowing the forelimbs to touch the surface. This teaches the animals to sense below them for a stable surface and is an important and necessary step in the procedure because it ensures that the animals are relaxed before testing begins. 
1. Forelimb Step-alternation Test
<ol>
 <li>Test Summary: Place the animals with both forelimbs touching the tabletop to determine whether they alternate the use of their forelimbs. Move the rat forward, shifting their center of gravity, forcing them to try to step. This test is repeated four times with each animal and if an animal shows ability to alternate at, or higher than, 75%, it is considered to be an alternator. This is a forced motion task, so animals that are considered non-alternators will repeatedly step with the same forelimb.</li>
 <li>Hold an animal on a tabletop in a forelimb-only weight-bearing stance, in a "wheelbarrow" position with their body at nearly 90&deg; from the table.</li>
 <li>Once the animal appears relaxed, push the animal forward to move along the surface of the tabletop.</li>
 <li>Determine and record whether the rat alternates use of the forelimbs while moving across the surface.</li>
 <li>Note and record the forelimb that initiated the movement and whether the animal can alternate or not on the Forelimb Step-Alternation Test Score Sheet (provided).</li>
 <li>Repeat this test at least twice while holding the animal with one hand (right or left hand). Repeat at least twice again while holding the animal in the other hand. </li>
 <li>If rats showed this ability, step-alternation can be tested again by introducing a 5 sec wait after the first step to determine if the pattern of alternation remains after a delay. Note the results and record in the score sheet.</li>
 <li>This test can be used daily, and it is recommended that the animals be tested at least weekly for a repeated measures experiment.</li>
</ol>
Note: It is important to alternate the experimenter's hand position since it may affect the alternation behavior of the test animals. The Step-alternation score sheet provided can be used to note paw position and use during stepping. Although we have not incorporated those data into our scoring system currently (we have only used alternation status to group the lesioned animals in the current study), users can readily include paw position data as part of the analysis depending on the specific project.
2.	Postural Instability Test
<ol>
 <li>Test Summary: For this behavioral test, hold rats in a similar position as for the forelimb alternation test, however, each forelimb should be tested individually. The tabletop should be covered with sandpaper (No. 220) to prevent slipping, bracing, or dragging of the forelimbs during the test.
	 <ol>
		 <li>In this test, the distance it takes for the animal to take a step with the forelimb being examined to regain its balance will be recorded.</li>
 <li>Animals within a study group should be approximately the same weight, size and age as large differences in body weight can result in variation in the baseline distance needed for maintaining center of gravity for a given animal 19. A sham group should be included while using PIT to verify that changes in behavior are due to regeneration or recovery rather than age and weight gain.</li>
		 </ol>
	 </li>
	 <li>Hold an animal on a tabletop in a comfortable stance and allow both forelimbs to reach the surface, the animal should comfortably be in a "wheelbarrow" position with their body at nearly 90&deg; from the table. Note that it is important to hold the animal in a more vertical position. This will allow a more consistent distance needed to trigger a step to regain center of gravity.</li>
 <li>Lightly restrain one forelimb against the animal's torso and align the rat's nose with the zero line as viewed from above.</li>
 <li>Move the rat forward. This will shift the animal's center of gravity forward stimulating the animal to step to regain its balance.</li>
 <li>Record the new position of the nose after the rat steps twice and use the average of these two steps as the distance needed to trigger a step.</li>
 <li>Test each forelimb independently 5x, bringing the animal back to the 0 position and verifying that the animal continues to be relaxed in the experimenter's hands before continuing to ensure consistent results.</li>
 <li>Record on the Postural Instability Test (PIT) Score Sheet, provided. This test can be performed daily, and it is recommended that the animals are tested at least weekly for the duration of the experiment.</li>
</ol>
3.	Pasta Handling Test
<ol>
 <li>Test Summary: Use this test to determine the time it takes to eat a piece of pasta and the paw preference during a pasta-eating session. In this test, use 4.0 cm strands of dry pasta (thin spaghetti; diameter ~1.6 mm). The test should be administered at approximately the same time each testing day. Rodents generally eat dry pasta readily; however, 4-6 hours of food withdrawal is recommended before testing if this does not occur.</li>
 <li>Baseline measurements should be acquired 1-2 weeks prior to injury. Prior to baseline recordings, rats should be given the same type of pasta in their home cage to prepare them for testing. Then, allow the rats to eat pasta in a testing chamber and record the time to eat pasta. Pasta pieces should be placed near the front of the testing chamber where the use of forelimbs can be observed readily. Each pasta-eating session should include eating at least three pieces of pasta. Rats are considered proficient in eating pasta if their pasta-eating time is consistent for at least 3 days.</li>
 <li>After training the rats to eating pasta consistently in the testing chamber, place rats in recording chambers.</li>
 <li>Place pasta pieces onto the floor near the front of the testing chamber and record time to eat a piece of pasta, forelimbs used, and the positions of the forelimbs, as seen on the Pasta Handling Score sheet.</li>
 <li>This test can be performed daily, but it is recommended to perform it weekly while observing the animals' weight as to not over-feed the animals.</li>
</ol>
Note: The Pasta Handling Score sheet provides space to record time to eat pasta and paw use, similar to the data included in this study. We have also included areas to record additional details of paw use during a pasta eating session, such as paw position.

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No conflicts of interest declared.

Cervical spinal cord injury (cSCI) can result in devastating and life altering injuries in patients. There are a number of cervical spinal cord injury models developed in rodents that are used to study plasticity of the neural substrates and therapeutic agents. Developing sensitive, effective, reproducible and easy to use behavioral tests to assess functional deficits and recovery after cSCI is an important goal. Here, we describe in detail the use of three such behavioral tests: limb alternation, postural instability and pasta handling.
Spinal cord injury in rodents, as in the human population, is a heterogeneous injury. A very similar injury can induce a variety of behavioral deficits. Therefore, it is important to determine the severity of the lesions in injured animals. We found that the limb alternation task is a very effective way of determining if the contralesional side is damaged after a unilateral injury (hemisection or contusion type injury) to the cervical spinal cord 19. Data from our group showed that the use of this limb alternation task can determine the severity of the injury within the first week after injury. In addition, the more severely injured animals (non-alternators) had a significantly different recovery profile compared to alternators (using the forelimb locomotor scale). Therefore, the limb alternation task is extremely useful in grouping the injured rats into alternators and non-alternators, particularly if the study includes chronic treatment to ensure there is an equal distribution of lesion severity in each treatment group.
It is recommended that the forelimb-alternation test be used in combination with other tasks that can examine the use of animals' forelimbs (such as the forelimb locomotor scale or the cylinder paw preference test). While administering this test it is imperative that the experimenter perform the test the same number of times with each hand (e.g. twice with the left hand and twice with the right) to minimize the effect of experimenter hand position on the rats' behavior. This test is only effective if the animal is relaxed in the experimenter's hands; the 2-3 weeks of handling suggested should afford relaxation. Further, once the animal is relaxed, the experimenter should hold the animal at nearly 90&deg; in a wheelbarrow position to prevent within-animal variation.
The forelimb-alternation test can also provide insight to cross-spinal neural connections when animals are unable to step individually with a paw but are able to perform an alternation task. For normal quadrupedal locomotion in animals such as rats, many brain regions as well as local central pattern generators (CPGs) along the length of the spinal cord are involved. Specifically for forelimb rhythmic movement, the cervical spinal segments, C3-C6 are thought to be important22. Previously, we described anatomical differences between alternators and non-alternators in lesioned animals (a lateral hemisection at C3/C4 was performed) and found more damage to the contralesional dorsal column and corticospinal tract in non-alternating animals18. It is thought that for forelimb movement, rhythmic pattern generator is present at each level of the cervical hemi-segment and that the cross-cord inhibitor connections are responsible for alternation23. Our previous anatomical findings also implies that damage to the contralesional side of the spinal cord may cause disruption to the rhythmic motor output (such as alternation), whereas if the contralesional of the spinal cord is more intact, the rhythmic motor outputs are more likely to function normally. We did not observe significant damage to any contralesional medial ventral portions of the cords and hence that analysis was not performed.
The postural instability test described here is a very useful test because it can detect changes in both forelimb (ipsi- and contralesional) caused by a unilateral lesion. It is important to note that this test should be administered only when the animal is relaxed in the experimenter's hands. In our experience, this can take daily handling of the animals for 2-3 weeks. Once the experimenter and the animal are comfortable, this test is very sensitive and can be used to obtain highly consistent displacement data for each forelimb. If the animal is tense, the experimenter can gently hold the animal and move them up off of and down onto the table until the animal is comfortable and understands that the table is a safe location. Once the animal is relaxed, it is important to hold the animals in a wheelbarrow position at nearly 90&deg; for consistent results. This test is easy to administer and provides insight beyond other behavioral tests by providing information about compensation and deficit with one simple task.
Rats readily eat dry pasta after training in front of the camera. Therefore, pasta handling is a relatively easy test to administer. In this study, we were recording and using data for limb use (left, right or both) and time to eat pasta only. A more detailed analysis including paw adjustments during pasta eating has been described previously in animals with injuries such as unilateral ischemic lesions and unilateral striatal dopamine depletion 20,21,24 . Therefore, it is possible to develop and use a more sensitive analysis from the pasta eating test after cSCI. We included paw position on the scoring sheet, as this may reveal handedness or ability with each hand. Further, we are noting the grip of each paw; some animals rest their paw on one side of the pasta, using it possibly as a support, rather than grip the pasta during eating. If animals are hesitant to eat pasta, a food deprivation step can be introduced to the protocol prior to testing.

Cervical spinal cord injury (cSCI) is the most prevalent form of SCI in patients, representing about 62% of all SCIs (<a href="http://www.spinalcord.uab.edu" target="_blank">http://www.spinalcord.uab.edu</a>). Injuries to the cervical spinal cord can result in impairments in the upper extremities as well as breathing. A recent study in patients with cSCI suggests that partial or full function of the arm and/or hand is considered to be a major priority 1. Therefore, developing cSCI models and associated behavioral assays that are easy, sensitive and reliable to use is an important goal. There are several useful behavioral tests to assess forelimb function after cSCI in rodents 1-17, however, many of these tests are difficult to use and require specialized equipment. Pasta handling tests have been developed by other groups18,19, but these tests require that the animals be filmed in specific pasta eating cages, which requires a long training period, while the method we propose can be performed in the animals&#39; home cage. Previously described pasta tests require significant analysis time and often expensive software, unlike the test described here. The goal of our method is to develop and use behavioral tests that are easy to use, reliable, and do not require expensive equipment. Specifically, the forelimb step-alternation test described here allows researchers to estimate lesion volume early after injury, providing a means to create equivalent treatment groups before a chronic treatment is administered. In this study, we describe in detail three novel behavioral tests: (1) forelimb step-alternation, (2) postural instability, and (3) pasta handling.
Determining the right behavioral tests to use can be difficult. Indeed, we feel that no one behavioral test can assess the functioning of the forelimbs adequately. Therefore, we suggest using a combination of a few behavioral tests to assess forelimb function after SCI. Categorically speaking, one should use an open-field locomotor type task (e.g. forelimb locomotor scale, cylinder) where animals are assessed on the use of their forelimb during normal locomotion, and a task specific test, where animals are asked to perform a specific task involving the use of their forelimb (eg., pasta eating, postural instability, grip strength, etc.). The behavioral tests described here in detail are useful for determining lesion severity at ~ one week post lesion (forelimb step-alternation, postural instability, pasta handling), the effects of a unilateral lesion on the contralesional forelimb (forelimb step-alternation, postural instability) and fine motor movement of the forelimbs (pasta handling). Knowing the severity and effects of the lesion used can also dictate the appropriate behavioral tests. If fine motor movements of the wrist are not possible after lesion, using a test that measures and quantifies the use of forelimb paws is inappropriate. For example, many of the previously described pasta handling tests look more in depth at the individual movements of digits, however, we found with the severity and location of our injury, this was irrelevant and instead observed differences in the overall placement of the limb.
Researchers may be tempted to conduct many behavioral tests and use a sample of them for demonstration based on the results, however, it is recommended that for each experimental model being used a pilot study is performed to determine appropriate behavioral tests for the lesion. Therefore, it is important to note that the user should have at least some prior knowledge of the type of deficit and extent of a particular lesion type before deciding on behavioral tests to be employed. Assessing lesion severity based on behavioral performance also allows treatment groups to be created with an equal distribution of lesion severity before treatment is administered.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Material/ Equipment</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments/Description</td>
 </tr>
 <tr>
 <td>Sand paper</td>
 <td>3M</td>
 <td>5097</td>
 <td>3M Gold Fre-Cut Sandpaper was used. 
 Any 220-grit sandpaper would work well </td>
 </tr>
 <tr>
 <td>Dry Pasta</td>
 <td>Skinner</td>
 <td></td>
 <td>Skinner Thin Spaghetti was used. It is available at Walmart</td>
 </tr>
 </tbody>
</table>

assessing forelimb function after unilateral cervical sci using novel tasks: limb step-alternation, postural instability and pasta handling

Cervical spinal cord injury (cSCI) can cause devastating neurological deficits, including impairment or loss of upper limb and hand function. A majority of the spinal cord injuries in humans occur at the cervical levels. Therefore, developing cervical injury models and developing relevant and sensitive behavioral tests is of great importance. Here we describe the use of a newly developed forelimb step-alternation test after cervical spinal cord injury in rats. In addition, we describe two behavioral tests that have not been used after spinal cord injury: a postural instability test (PIT), and a pasta-handling test. All three behavioral tests are highly sensitive to injury and are easy to use. Therefore, we feel that these behavioral tests can be instrumental in investigating therapeutic strategies after cSCI.

We recommend that animals be tested regularly throughout the experiment in a repeated measures-type experiment for improved analysis and to analyze improvement over time.
Forelimb Step-alternation Test
This test is designed to determine an animal's ability to alternate the use of its forelimbs. Using the forelimb step-alternation test, only 50% of the animals were able to alternate the use of their forelimbs after cSCI 19 at 12-16 weeks post injury (Figure 1A). Next we introduced a delay (5 sec) by holding the animal stationary after the first step before moving the animal forward for the second step. Only 50% of the animals that were alternating the use of their limbs (25% of the total lesioned group) were able to alternate after a 5-sec delay (Figure 1B). We correlated these findings with anatomical studies and the analysis revealed that alternators have significantly larger area of spared corticospinal tract [F(1,1) = 5.56, p &#60; 0.05] and dorsal column [F(1,1) = 19.2, p &#60; 0.003] on the contralesional side 19. These data indicate that the forelimb step alternation test can predict the severity of the lesion as well as unilaterality of the lesions. This test can result in nominal categorical data that should be analyzed with a repeated measures analysis. In this example, it was not a repeated measure, we used one time-point to illustrate the difference between groups. Each animal will be categorized as an &#34;alternator&#34; or a &#34;non-alternator&#34;, separating the animals into two groups of injuries (moderate vs. severe, respectively).
Postural Instability Test
Unilateral lesions of the spinal cord can cause changes not only to the impaired and/or affected limb, but also to the non-impaired limb. The postural instability test (PIT) was previously described and used in animals with a rodent model of Parkinson&#39;s disease 18 and in a rodent model of cSCI 19. We used PIT after cSCI and found that there was a significant effect of lesion status [F(1,1) = 8.17, p &#60; 0.01]. In addition, forelimb displacement (distance to regain center of gravity) was significantly larger in the ipsilesional side (right forelimb) of injured animals compared to animals with sham surgery (6.00 &#177; 0.24 cm versus 8.00 &#177; 0.10 cm, p &#60; 0.0001; Figure 2). The displacement distance on the contralesional forelimb was significantly smaller in lesioned animals compared to sham-operated animals (6.00 &#177; 0.25 cm for sham versus 4.00 &#177; 0.10 cm for cSCI, p &#60; 0.0001; Figure 2). This result indicates that contralesional forelimb also resulted in changes due to lesion to the opposite side (i.e. changes in postural adjustment by the contralesional forelimb to regain center of gravity). The data from this test will be quantitative and should be analyzed using a repeated measures ANOVA to examine changes in groups throughout an experiment.
Pasta Handling Test
Pasta handling was designed to test the skilled use of the forelimbs while eating a piece of dry pasta. A similar test has been used previously to detect deficits in skilled use of forelimbs in animal models of unilateral stroke and Parkinson&#39;s disease 20,21 and after cSCI 19. At chronic time points after injury (12 weeks post-injury), all sham animals ate pasta using both paws during the test period. In the lesioned group, we found that overall time to eat a piece of pasta is similar to the control group (Figure 3). However, more alternators (milder injury; 10 out of 15) were able to use their ipsilesional forelimb compared to non-alternators (severely injured group; 1 out of 7; Figure 3). The pasta handling test provides data that is both continuous and quantitative (time to eat pasta), and categorical (paw use) and should be analyzed appropriately.
<img alt="Figure 1" src="/files/ftp_upload/50955/50955fig1.jpg" /> 
Figure 1. Chronic assessment of forelimb function using the forelimb step-alternation test revealed two different groups of animals with different lesion severity. Animals showed a difference in behavior during forward stepping using both paws. Only 50% of the lesioned animals alternated paws while stepping (alternators; 5 out of 10 lesioned rats), whereas the others showed a lack of step-alternation by taking two contralesional steps in a row (non-alternators; 5 out of 10 lesioned rats). More animals displayed this tendency to take multiple contralesional steps if a 5-sec delay was introduced between steps (8 out of 10 lesioned rats).
<img alt="Figure 2" src="/files/ftp_upload/50955/50955fig2.jpg" /> 
Figure 2. Chronic forelimb behavioral assessment using the postural instability test (PIT). As expected, the ipsilesional forelimb in animals with cervical spinal cord injury (cSCI) had significantly larger displacement distance than animals with sham surgery (6 cm versus 8 cm; *p&#60;0.0001). In addition, the displacement distance on the contralesional forelimb was significantly smaller in lesioned animals compared to sham operated animals (6 cm versus 4 cm; *p&#60;0.0001). This may suggest enhanced function of the contralesional limb during PIT in lesioned animals.
<img alt="Figure 3" src="/files/ftp_upload/50955/50955fig3.jpg" /> 
Figure 3. Forelimb assessment using the pasta handling and eating test showed significant impairments in lesioned non-alternators. The animals were given a standard piece of pasta. Paw use and time to eat pasta were recorded. The results indicated that a low number of alternators (33%) exclusively used their contralesional forelimb (indicating more severe impairment) compared to non-alternators (86%). In addition, animals in all three groups (sham, lesioned alternators, and lesioned non-alternators) took a similar length of time to eat the pasta (p&#60;0.05). <a href="https://www.jove.com/files/ftp_upload/50955/50955fig3large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about Assessing Forelimb Function after Unilateral Cervical SCI using Novel Tasks: Limb Step-alternation, Postural Instability and Pasta Handling at JoVE.com

Assessing Forelimb Function after Unilateral Cervical SCI using Novel Tasks: Limb Step-alternation, Postural Instability and Pasta Handling

Three new behavioral tests (forelimb step-alternation, postural instability test, pasta handling test) for evaluating forelimb function after cervical spinal cord injury in rodents are described.

Cervical spinal cord injury (cSCI) can cause devastating neurological deficits, including impairment or loss of upper limb and hand function. A majority ...

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Research

JoVE Journal

Behavior

21.7K Views.  The University of Texas at Austin. Three new behavioral tests (forelimb step-alternation, postural instability test, pasta handling test) for evaluating forelimb function after cervical spinal cord injury in rodents are described.

Video: Assessing Forelimb Function after Unilateral Cervical SCI using Novel Tasks: Limb Step-alternation, Postural Instability and Pasta Handling

Compensatory Limb Use and Behavioral Assessment of Motor Skill Learning Following Sensorimotor Cortex Injury in a Mouse Model of Ischemic Stroke

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. The present protocol describes a skilled motor task for use in mouse models of stroke. The Pasta Matrix Reaching Task functions as a versatile and sensitive behavioral assay that permits experimenters to collect accurate outcome data and manipulate limb use to mimic human clinical phenomena including compensatory strategies (i.e., learned non-use) and focused rehabilitative training. When combined with neuroanatomical tools, this task also permits researchers to explore the mechanisms that support behavioral recovery of function (or lack thereof) following stroke. The task is both simple and affordable to set up and conduct, offering a variety of training and testing options for numerous research questions concerning functional outcome following injury. Though the task has been applied to mouse models of stroke, it may also be beneficial in studies of functional outcome in other upper extremity injury models.

Mouse models have become increasingly popular in studies of behavioral neuroscience. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. This protocol describes the Pasta Matrix Reaching Task, which is a skilled motor task for use in mouse models of stroke.

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models ...

A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan (IBB) Forelimb Scale

Several experimental models of cervical spinal cord injury (SCI) have been developed recently to assess the consequences of damage to this level of the spinal cord (Pearse et al., 2005, Gensel et al., 2006, Anderson et al., 2009), as the majority of human SCI occur here (Young, 2010; www.sci-info-pages.com). Behavioral deficits include loss of forelimb function due to damage to the white matter affecting both descending motor and ascending sensory systems, and to the gray matter containing the segmental circuitry for processing sensory input and motor output for the forelimb. Additionally, a key priority for human patients with cervical SCI is restoration of hand/arm function (Anderson, 2004). Thus, outcome measures that assess both proximal and distal forelimb function are needed. Although there are several behavioral assays that are sensitive to different aspects of forelimb recovery in experimental models of cervical SCI (Girgis et al., 2007, Gensel et al., 2006, Ballerman et al., 2001, Metz and Whishaw, 2000, Bertelli and Mira, 1993, Montoya et al., 1991, Whishaw and Pellis, 1990), few techniques provide detailed information on the recovery of fine motor control and digit movement.
The current measurement technique, the Irvine, Beatties and Bresnahan forelimb scale (IBB), can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation. The IBB was generated by observing recovery after a unilateral C6 SCI, and involves video recording of animals eating two differently shaped cereals (spherical and doughnut) of a consistent size. These videos were then used to assess features of forelimb use, such as joint position, object support, digit movement and grasping technique.
The IBB, like other forelimb behavioral tasks, shows a consistent pattern of recovery that is sensitive to injury severity. Furthermore, the IBB scale could be used to assess recovery following other types of injury that impact normal forelimb function.

A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan IBB Forelimb Scale

Here we will describe a rodent behavioral assay that can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation.

Several experimental models of cervical spinal cord injury (SCI) have been developed recently to assess the consequences of damage to this level of the ...

Neuroscience

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 ...

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Medicine

The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity largely use video analysis of reaching or food manipulation. While these tasks are easy to implement and are robust across disease models, they are subjective and laborious for the experimenter. Automating traditional tasks or creating new automated tasks can make the tasks more efficient, objective, and quantitative. Since rats are less dexterous than primates, central nervous system (CNS) injury produces more subtle deficits in dexterity, however, supination is highly affected in rodents and crucial to hand function in primates. Therefore, we designed a semi-automated task that measures forelimb supination in rats. Rats are trained to reach and grasp a knob-shaped manipulandum and turn the manipulandum in supination to receive a reward. Rats can acquire the skill within 20 &plusmn; 5 days. While the early part of training is highly supervised, much of the training is done without direct supervision. The task reliably and reproducibly captures subtle deficits after injury and shows functional recovery that accurately reflects clinical recovery curves. Analysis of data is performed by specialized software through a graphical user interface that is designed to be intuitive. We also give solutions to common problems encountered during training, and show that minor corrections to behavior early in training produce reliable acquisition of supination. Thus, the knob supination task provides efficient and quantitative evaluation of a critical movement for dexterity in rats.

This manuscript describes a semi-automated task that quantifies supination in rats. Rats reach, grasp, and supinate a spherical manipulandum. The rat is rewarded with a pellet if the turn angle exceeds a criterion set by the user. This task increases throughput, sensitivity to injury, and objectivity compared to traditional tasks.

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity ...

A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. &#160;However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). &#160;Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. &#160;This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. &#160;We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. &#160;Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. &#160;Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. &#160;This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.&#160; Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing ...

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Biology

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 ...

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 ...

Quantifying Locomotor Dysfunction in Spinal Cord Injury Mice Using MouseWalker

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

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

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

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