Name: a battery of motor tests in a neonatal mouse model of cerebral palsy
Uploaded: 2016-11-03T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy at JoVE.com

We would like to thank everyone at Shriners Hospital Pediatric Research Center, in particular Dr. Mickey Seltzer, of whom without his support, this work would not have been funded. In addition, we would like to thank Isha Srivastava, who contributed to early data collection and Amy He, who helped with the figures. This study was funded by Shriners Hospitals for Children. No funding source played a role in experimental design or decision to submit the paper for publication.

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The authors declare that they have no competing financial interests.

Using animal models to study human diseases is only relevant if there is overlap between the cellular and molecular response between human and rodent and that the behavioral tests performed have direct relevance to human symptoms. One of the major issues with pediatric disease studies is that many researchers use adult rodents to create the model, as well as adult rodent behavioral evaluation, without considering the developmental differences that may be important for the disease process. Because of these issues, it is important that research on pediatric disease use not only the appropriate adjusted developmental time-points (e.g., human CNS development at 28 - 32 weeks is equivalent to a post-natal day 2 - 7 day rodent) 7, but also behavioral tests that will examine appropriate motor, sensory or reflexive developmental behaviors. Thus, as each new neonatal disease model is developed, it must be rigorously tested to assure that the cellular and behavioral responses will provide the most appropriate translatable data between rodent and human.
Cerebral palsy is a motor disorder, which persist into adulthood. One problem with many of the cerebral palsy models available today is the lack of repeatable, standardized motor testing that can correlate with the deficits seen in pediatric patients. In this new model, which combines hypoxia, ischemia and inflammation in a neonatal mouse, motor behavior was evaluated using a battery of tests specific for neonatal mice. In order to decrease the subjectivity and increase the quantitative reporting, several tests have been modified to include very specific, but easy to evaluate measures that can be standardized. In addition, front- and hindlimb evaluations can be performed separately, and left/right differences can be determined. This battery of tests is specific for neonatal mice up to two weeks of age.
This CP model demonstrates difficulty in walking (ambulation, hindlimb foot angle), as well limb-specific weakness (four limb suspension, hindlimb suspension), and deficits in developmental reflexes (grasping reflex). Although in this study only one timepoint was examined, these deficits can be tracked over time.
There are other batteries of tests that can be used on the neonate, such as the Fox&#39;s battery of tests or Heyser&#39;s Assessment of Developmental Milestones 15. However, these tests compare the neonate to the adult, whose responses may not be the same because the neonate is still developing. Fox&#39;s battery and Heyser&#39;s Assement tests rely on observational subjective information with dichotomous (yes or no) assessment, rather than objective data (angle, posture based on strength, etc). Because of the subjectiveness of these tests, many scientists have adapted, added, or removed criteria, thus making their results incomparable to others and limiting the usefulness of the data in terms of establishing a baseline deficit for a particular disease or disorder. By establishing one set of standardized motor tests that are qualitative and specifically designed to test neonates, results from individual research groups can be accurately and reliably reported and compared.

Developing new models of pediatric injury or disease using rodents is often difficult due to the amazing ability of both rats and mice to rapidly recover from neurological injury. Therefore, in order to validate any new pediatric disease model, thoroughly examining the cellular and molecular changes must go hand-in-hand with behavioral outcomes. In many ways, functional behavioral recovery may be more important than underlying cellular changes in terms of therapeutic or translational relevance. As researchers learn more about injury in the adult and neonate, it is clear that their responses are very different and cannot be extrapolated between the two. For example, neonatal mice display different levels of nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and glial cell line-derived neurotrophic factor following spinal cord injury 1,2. Additionally, neonates have significant blood-brain barrier leakage after stroke 3, demonstrate cortical neuron rearrangement after peripheral nerve injury 4, and have a delayed or slowed astrogliosis following spinal cord injury and hypoxia-ischemia 5,6. Therefore, it is important that translational pediatric research use developmentally equivalent models and that those models are evaluated for both cellular/molecular changes and age-appropriate behavioral tests.
Cerebral Palsy (CP) is a motor disorder that affects 3:1000 live births annually (NIH). Children with CP exhibit a range of symptoms and co-morbid conditions, depending on the severity of the disease. Difficulty with movement and coordination are the most common signs, along with delays in reaching motor developmental milestones. Other signs include abnormal muscle tone (either increased or decreased), reduced fine motor skills, difficulty walking, excessive drooling and swallowing, and speech delays (NIH). The underlying cause of CP is believed to be a lack of oxygen and/or blood flow to the brain during the pre- or peripartum period, or up to one-year post-partum. In addition, inflammation is now believed to be a key component in the development of CP.
The majority of CP cases are associated with white matter damage around the ventricles, known as periventricular leukomalacia (PVL). This neurological hallmark suggests that the initial insult leading to CP occurs during the period of brain development when the oligodendrocytes are most vulnerable to insult. The period of rapid oligodendrocyte growth in a human, also the period when oligodendrocytes are the most susceptible to injury, is between 24 - 32 weeks gestation. In the rodent, the equivalent period is post-natal days 2 - 7 7, and is when CP is induced in this model.
The neonatal mouse model of CP that was used to conduct the tests outlined here combines hypoxia and ischemia with inflammation to create an injury that better mimics the neurodegeneration seen in human CP. This model addresses some of the major shortcomings observed in other animal models of CP, which lack distinct motor deficits that resembles human CP patients, as well as distinct white matter damage. Previous studies by a collaborator using the same model have demonstrated that the addition of inflammation enhances white matter damage, thus better emulating the PVL seen in children with CP 8. Building on the previous data, this paper presents a comprehensive battery of neonatal motor tests in order to evaluate changes in motor behavior as the animal ages.

<table><tbody><tr><td></td><td></td><td></td><td></td></tr><tr><td>C57BL/6 mice</td><td>Charles River Laboratories</td><td>STRAIN CODE: 027&amp;nbsp;</td><td>C57BL/6NCrl is the exact strain we use</td></tr><tr><td>Anesthesia Dish, PYREX&amp;trade; Crystallizing Dish</td><td>Corning Life Sciences Glass&amp;nbsp;</td><td>3140125</td><td>Capacity: 25.03 oz. (740ml); Dia. x H: 4.92 x 2.55 in. (125 x 65mm). However, any small round glass container will work. A 2 cup capacity pyrex food storage bowl with flat bottom will also work and is much cheaper (Pyrex model number: 6017399).</td></tr><tr><td>Covered lead ring</td><td>Fisher Scientific</td><td>S90139C</td><td>Lead ring for stablizing flasks in a water bath. It is used inside the anesthesia dish.</td></tr><tr><td>Scalpel Blade #11</td><td>World Precision Instrucments, Inc.</td><td>500240</td><td></td></tr><tr><td>Small Vessel Cauterizer</td><td>Fine Science Tools</td><td>18000-00</td><td></td></tr><tr><td>Micro Hook</td><td>Fine Science Tools</td><td>10064-14</td><td></td></tr><tr><td>Vetbond Suture Glue</td><td>3M</td><td>1469SB</td><td>n-butyl cyanoacrylate adhesive</td></tr><tr><td>Lipopolysaccharide</td><td>Sigma Life Science</td><td>L4391</td><td>Lipopolysaccaride from E.coli 0111:B4, gamma irradiated</td></tr><tr><td>12 x 12 inch opaque box</td><td>Acrylic Display Manufacturing: A division of Piasa Plastics</td><td>C4022</td><td>Colored Acrylic 5-Sided Cube, 3/16&quot; Colored Acrylic, 12&quot;W x 12&quot;D x 12&quot;H;&amp;nbsp; http://www.acrylicdisplaymfg.com/html/cubes_19.html</td></tr><tr><td>Camera/camcorder</td><td>JVC</td><td>GC-PX100BUS</td><td>Any camcorder that works well in low light and can be imported and edited. We use the JVC GC-PX100 Full HD Everio Camcorder.</td></tr><tr><td>Covidien Tendersorb&amp;trade; Underpads</td><td>Kendall Healthcare Products Co</td><td>7174</td><td></td></tr><tr><td>WypAll L40</td><td>Kimberly-Clark Professional</td><td>5600</td><td>Any surface with moderate grip will do</td></tr><tr><td>Surface at 45 degree incline</td><td></td><td></td><td>We use a cardboard box.</td></tr><tr><td>Thin wire from a pipe cleaner</td><td>Creatology</td><td>M10314420</td><td>Any pipe cleaner from any craft store will work.</td></tr><tr><td>50mL conical tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Fiberglass Screen Wire</td><td>New York Wire&amp;nbsp; www.lowes.com</td><td>14436</td><td>Any supplier can be used as long as their screen is 16 x 16 or 18 x 16</td></tr><tr><td>Razor blade</td><td>Fisherbrand</td><td>12-640</td><td>A wooden stick applicator or wooden part of a cotton-tipped swab will also work.</td></tr><tr><td>OPTIX 24-in x 4-ft x 0.22-in Clear Acrylic Sheet to make Clear Acrylic Walkway</td><td>PLASKOLITE INC</td><td>1AG2196A</td><td>Clear acrylic (1/8&quot; thick) with sides and a top to limit exploration. We bought a sheet of acrylic from a local hardware store and had them cut it to size. (2) 2&quot; x 2&quot;; (3) 2&quot; x 18&quot;; (1) 2&quot; x 15.5&quot;; (1) 2&quot; x 3&quot;. Using clear tape, tape all sides together, with the 15.5&quot; piece on top. Tape the 3&quot; piece to the end of the 15.5&quot; piece to create a flap/entryway for the mice. Alternatively, part or all of the walkway can be glued together, and only taping on the top pieces. This design will allow for the walkway to be opened for easy cleaning.</td></tr><tr><td>Protractor</td><td>Westscott</td><td>ACM14371</td><td></td></tr></tbody></table>

a battery of motor tests in a neonatal mouse model of cerebral palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Mice were tested from P7 (24 hr following surgery) to P13 (1 week following surgery), using different mice for each time-point so that learning a testing paradigm was not a confounding variable. P8 was selected as representative results, as mice showed the greatest deficits at this time point.
Transition from Crawling to Walking is Delayed in CP Neonatal Mice
Human CP patients have gait abnormalities, ranging from toe-walking to a scissored gait. As this CP model displays gait deficits similar to humans, ambulation was assessed. Mice were scored on gait symmetry and limb-paw movement during a straight walk. At 48 hr following surgery (PND 8), CP mice had less symmetric limb movement and a &#34;crawling&#34; gait as compared to their sham counterparts (average ambulation score: CP 1.083 &#177; 0.6337, n = 12 vs sham 1.639 &#177; 0.4859, n = 9; p &#60; 0.05, Figure 10). By one week, both CP and sham mice have transitioned to walking (data not shown).
<img alt="Figure 10" src="/files/ftp_upload/53569/53569fig10.jpg" /> 
Figure 10. CP Mouse Pups do not Ambulate as well as Shams. Sham mice (black bar) have a mean score of 1.639 &#177; 0.4859 (n = 9), meaning their ambulatory development falls between asymmetric limb movement and slow crawling. CP mice (gray bar) receive an average score of 1.083 &#177; 0.6337 (n = 12), meaning their ambulation is less developed and tend to have asymmetric limb movement. Data are expressed as mean &#177; SEM; * is p &#60; 0.05.<a href="https://www.jove.com/files/ftp_upload/53569/53569fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Hindlimb Foot Angle is Increased in CP
In addition to ambulation, hindlimb foot-angle was assessed. Eight-day-old sham mouse pups walk with their hindpaws facing forward, compared to HIL mice, who have splayed hindpaws when walking in a straight line (Figure 2; average angle: CP 77.48 &#177; 9.848, n = 9, vs sham 54.54 &#177; 8.043, n = 11; p &#60; 0.0001, Figure 11). This increased angle correlates with gait instability, in that the pups need to increase the angle of their rear paws in order to stabilize their gait and assist with balance and coordination.
<img alt="Figure 11" src="/files/ftp_upload/53569/53569fig11.jpg" /> 
Figure 11. CP Mouse Pups Splay their Hindpaws When Walking. CP mice (black bars) have an average angle between their hindlimbs of 77.48 &#177; 8.043 (n = 11), while sham mice (gray bars) have an average angle of 54.54 &#177; 9.848 (n = 9). Data are expressed as mean &#177; SEM; **** is p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/53569/53569fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
CP Mice do not Show Deficits when Surface Righting
The surface righting test was included as some CP patients have impaired trunk control (Heyrman et al., 2013). Additionally, the vestibular system is necessary to detect the need for righting and there are vestibular deficits in some CP patients23. CP mice do not show significant deficits when righting as compared to sham controls (data not shown).
CP Mice Perform the Same as Sham in Negative Geotaxis Testing
Negative geotaxis is used to test motor coordination in young pups. Mice are challenged by being place facing downhill on a sloped surface. Delay or failure to orient uphill could indicate deficits in coordination, balance, or vestibular input. CP mice show no deficits when challenged with negative geotaxis as compared to sham mice (data not shown). Additionally, CP mice did not show a preference to turn toward one side versus another when re-orienting.
Front-Limb Suspension Test is Appropriate for Mice Older than 10 Days
CP patients have decreased muscle tone and deficits in fine motor skills, such as grasping. To test weakness in this mouse model, we used a front-limb suspension test. Furthermore, this model uses unilateral ischemic injury and sided-ness could be determined using this suspension test. This test is better for mice older than 10 days 15. At 8 days old, two days following injury, there were no significant differences between CP and sham mice (data not shown).
Hindlimb Strength is Decreased in CP Mice
Human CP patients often need braces or assistive walking devices due to lack of motor control and strength. In order to compare the rodent CP model to humans, hindlimb strength was assessed using the hindlimb suspension test. When suspended from the side of a conical tube, CP mice showed hindlimb weakness, as demonstrated by a decrease in hanging score (hindlimb hanging score: CP 3.468 &#177; 0.5561, n = 13, vs sham 3.891 &#177; 0.1329, n = 13; p &#60; 0.05, Figure 12). No difference was observed in hindlimb suspension time (data not shown). Thus, similar to human CP patients, CP mice demonstrate hindlimb (leg) weakness.
<img alt="Figure 12" src="/files/ftp_upload/53569/53569fig12.jpg" /> 
Figure 12. Sham Mice are Slightly but Significantly Stronger in their Hindlimbs than CP Mice. At an average hanging score of 3.891 &#177; 0.1329 (n = 13), sham mice (black bar) show more hindlimb separation, and therefore a stronger hindlimb stance, when hanging on the edge of a tube than CP mice (gray bar) with an average hanging score of 3.468 &#177; 0.5561 (n = 13). Data are expressed as mean &#177; SEM; * is p &#60; 0.05. <a href="https://www.jove.com/files/ftp_upload/53569/53569fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Grip Strength is Decreased Following CP Injury 
Grasping with all four paws is important for a rodent in terms of climbing and running across uneven surfaces. Grip requires significant sustained strength, rather than dexterity or linear force, mainly in the digits and paws 24. Mice were required to hold their body weight on an inverted wire mesh screen. CP mice were not able maintain their grip and these mice fell at significantly lower angles (four limb average angle: CP 75.627 &#177; 24.48, n = 11, vs sham 96.57 &#177; 10.836, n = 9; p &#60; 0.05, Figure 13). This data shows that there is a significant deficit in grip strength in CP mice.
<img alt="Figure 13" src="/files/ftp_upload/53569/53569fig13.jpg" /> 
Figure 13. CP Mice have Weaker Grip than Shams. Sham mice (black bar) can grasp to an average inverted angle of 96.57 &#177; 10.836 (n = 9). CP mice (gray bar) can only reach an inverted angle of 75.627 &#177; 24.48 (n = 11). Data are expressed as mean &#177; SEM; * is p &#60; 0.05. <a href="https://www.jove.com/files/ftp_upload/53569/53569fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Grasping Reflex Deficits are Apparent in CP Mice
Along with gross motor deficits, fine motor movements are also impaired in CP patients 25,26. The grasping reflex in humans is present at birth and disappears around 5 - 6 months. However, changes in the grasping reflex, such as exaggerated velocity or strength of grasping, failure to grasp, or the reemergence of the grasping reflex after 6 months of age, all indicate damage to the nervous system. To compare grasping in the CP model, reflexive grasping deficits were determined.
At 48 hr after injury, CP mice demonstrate a decrease in grasping reflex (average paws grasped at 48 hr: CP 2.429 &#177; 0.9376, n = 14, vs sham 3.214 &#177; 0.8018, n = 14; p &#60; 0.05, Figure 14A). There was a slight, but not significant increase in right paw preference in the forepaws (data not shown). There was a significant right paw preference in the hindpaws (CP 75.0 &#177; 42.74, n = 14, vs sham 17.86 &#177; 54.09, n = 14; p &#60; 0.005, Figure 14B). One week after injury, CP mice show grasping deficits (average paws grasped at 1 week: CP 2.75 &#177; 1.035, n = 8, vs sham 3.80 &#177; 0.6325, n = 10; p &#60; 0.05, Figure 14C), with no notable paw preference.
<img alt="Figure 14" src="/files/ftp_upload/53569/53569fig14.jpg" /> 
Figure 14. CP Mice have Grasping Deficits, in the Hindpaws, Contralateral to the Injured Brain Region. (A) 48 hr following injury (PND 8), CP mice (gray bar) grasp a stick with, on average, fewer paws than sham animals (black bar). (B) CP mice (gray bar) display a preference for grasping with the right hindpaw (contralateral to injury) as opposed to using the left hindpaw (ipsilateral to injury). Sham mice (black bar) do not display this right paw preference. Right paw preference is calculated as ([right paw - left paw]/[right paw + left paw + both paws] * 100). (C) One week following injury, CP mice (gray bar) still show grasping deficits as compared to shams (black bar). Data are expressed as mean &#177; SEM; * is p &#60; 0.05, ** is p &#60; 0.005. <a href="https://www.jove.com/files/ftp_upload/53569/53569fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
CP Mice turn away from the Edge During Cliff Aversion
The cliff aversion test relies on the inherent fear of the mice to turn away from a steep cliff and head towards safety. Although some CP patients have vestibular difficulties, as well as impaired motor control, the CP mice did not show any deficits on this test.

Watch this Scientific Journal Video about A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy at JoVE.com

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

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Research

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Medicine

24.0K Views.  Lewiz Katz School of Medicine at Temple University. Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

Video: A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Behavior

Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore by rearing and touching the walls of the cylinder with their forelimb paws for postural support. Following ischemic injury to the forelimb sensorimotor cortex, rats rely more heavily on their unaffected forelimb paw for postural support resulting in fewer touches with their affected paw which is termed forelimb asymmetry. In contrast, focal ischemic damage in the mouse brain fails to result in comparable consistent deficits in forelimb asymmetry. While forelimb asymmetry deficits are infrequently observed, mice do demonstrate a novel behaviour post stroke termed &#8220;paw-dragging&#8221;. Paw-dragging is the tendency for a mouse to drag its affected paw along the cylinder wall rather than directly push off from the wall when dismounting from a rear to a four-legged stance. We have previously demonstrated that paw-dragging behaviour is highly sensitive to small cortical ischemic injuries to the forelimb motor cortex. Here we provide a detailed protocol for paw-dragging analysis. We define what a paw-drag is and demonstrate how to quantify paw-dragging behaviour. The cylinder test is a simple and inexpensive test to administer and does not require pre-training or food deprivation strategies. In using paw-dragging analysis with the cylinder test, it fills a niche for predicting cortical ischemic injuries such as photothrombosis and Endothelin-1 (ET-1)-induced ischemia &#8211; two models that are ever-increasing in popularity and produce smaller focal injuries than middle cerebral artery occlusion. Finally, measuring paw-dragging behaviour in the cylinder test will allow studies of functional recovery after cortical injury using a wide cohort of transgenic mouse strains where previous forelimb asymmetry analysis has failed to detect consistent deficits.

Classical forelimb asymmetry analysis of the cylinder test is routinely used to assess behavioural deficits in rats following brain injury or stroke; however, it fails to detect consistent deficits in mice. This study demonstrates that quantifying paw-dragging behaviour is a more sensitive analysis of brain injury in mice.

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore ...

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

Neuroscience

Neurodevelopmental Reflex Testing in Neonatal Rat Pups

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are also referred to as primitive reflexes. They are sensitive and consistent with later outcomes. Abnormal reflexes are described as an absence, persistence, reappearance, or latency of reflexes, which are predictive indices of infants that are at high risk for neurodevelopmental disorders. Animal models of neurodevelopmental disabilities, such as cerebral palsy, often display aberrant developmental reflexes, as would be observed in human infants. The techniques described assess a variety of neurodevelopmental reflexes in neonatal rats. Neurodevelopmental reflex testing offers the investigator a testing method that is not otherwise available in such young animals. The methodology presented here aims to assist investigators in examining developmental milestones in neonatal rats as a method of detecting early-onset brain injury and/or determining the effectiveness of therapeutic interventions. The methodology presented here aims to provide a general guideline for investigators.

Behavioral testing is the gold standard for determining outcomes following brain injury, and can identify the presence of developmental disabilities in infants and children. Neurodevelopmental reflexes are an early indicator of these abnormalities. A host of easily accomplished developmental reflex tests in the neonatal rodent were developed and described here.

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are ...

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the animal&#39;s wellbeing and health. There are numerous behavioral tests that may be applied. We selected a test for general locomotion, the open field test (OFT); a test for muscular strength, the mesh test (MT); and a test for coordination, the rotarod test (RR). Testing can be accomplished on a weekly or monthly basis. As a test for general locomotion, the OFT works by objectively monitoring movement parameters while the mouse is in an open field apparatus. The field is generally a 2&#39; x 2&#39; box, and the movements are recorded through laser sensing or through video capture. The mouse is placed in the center of the open field and allowed to move freely for the test. The MT uses the latency for a mouse to fall off an inverted screen as a measure of muscular strength. A mouse is placed on a screen, which is inverted over a clear box, and is timed for their latency to fall. Three trials are performed, with the best of the three trials scored for that day. A score of 60 s is the maximum time a mouse is left inverted. Mice are given a 5-min rest period between mesh test trials. Lastly, an accelerated protocol on the RR assesses motor coordination and endurance. During a trial, a mouse walks on a rotating rod as it increases in speed from 4 rpm to 40 rpm over 5 min. The trial ends when the mouse touches the magnetized pressure sensor upon falling. Each mouse undergoes three trials, and the best trial is scored for that day. This combined behavioral data allows for the global assessment of mobility, coordination, strength, and movement of the test animals. At least two out of the three behavioral testing measures must show improvement for an animal to qualify as having overall improved motor function.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Immunology and Infection

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal ...

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.