Name: a battery of motor tests in a neonatal mouse model of cerebral palsy
Uploaded: 2016-11-03T12:30:00
Duration: 10 min 2 s
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.

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			<td height="20" style="height:20px;width:271px;">C57BL/6 mice</td>
			<td style="width:239px;">Charles River Laboratories</td>
			<td style="width:103px;">STRAIN CODE: 027&#160;</td>
			<td style="width:741px;">C57BL/6NCrl is the exact strain we use</td>
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			<td height="60" style="height:60px;width:271px;">Anesthesia Dish, PYREX&#8482; Crystallizing Dish</td>
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			<td style="width:741px;">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>
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			<td height="20" style="height:20px;width:271px;">Covered lead ring</td>
			<td style="width:239px;">Fisher Scientific</td>
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			<td style="width:741px;">Lead ring for stablizing flasks in a water bath. It is used inside the anesthesia dish.</td>
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			<td style="width:741px;">Lipopolysaccaride from e.coli 0111:B4, gamma irradiated</td>
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			<td style="width:103px;">GC-PX100BUS</td>
			<td style="width:741px;">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>
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			<td style="width:741px;">We use a cardboard box.</td>
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			<td style="width:741px;">A wooden stick applicator or wooden part of a cotton-tipped swab will also work.</td>
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			<td style="width:741px;">Clear acrylic (1/8&#34; 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&#34;x2&#34;; (3) 2&#34;x 18&#34;; (1) 2&#34;x15.5&#34;; (1) 2&#34;x3&#34;. Using clear tape, tape all sides together, with the 15.5&#34; piece on top. Tape the 3&#34; piece to the end of the 15.5&#34; 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>
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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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