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.

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+neurotrophic+factor+gene+expression+profiles+between+neonate+and+adult+rat+spinal+cord+after+injury.">Differences in neurotrophic factor gene expression profiles between neonate and adult rat spinal cord after injury.</a> Exp Neurol. 169 (2), 407-415 (2001).</li><li>Widenfalk, J., Lundstr&#246;mer, K., Jubran, M., Brene, S., Olson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophic+factors+and+receptors+in+the+immature+and+adult+spinal+cord+after+mechanical+injury+or+kainic+acid.">Neurotrophic factors and receptors in the immature and adult spinal cord after mechanical injury or kainic acid.</a> J Neurosci. 21 (10), 3457-3475 (2001).</li><li>Fern&#225;ndez-L&#242;pez, D., Faustino, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+permeability+is+increased+after+acute+adult+stroke+but+not+neonatal+stroke+in+the+rat.">Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat.</a> J Neurosci. 32 (28), 9588-9600 (2012).</li><li>Cusick, C. 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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 border="0" cellpadding="0" cellspacing="0" width="1354">
	<tbody>
		<tr height="20">
			<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>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:271px;">Anesthesia Dish, PYREX&#8482; Crystallizing Dish</td>
			<td style="width:239px;">Corning Life Sciences Glass&#160;</td>
			<td style="width:103px;">3140125</td>
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Covered lead ring</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:103px;">S90139C</td>
			<td style="width:741px;">Lead ring for stablizing flasks in a water bath. It is used inside the anesthesia dish.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Scalpel Blade #11</td>
			<td style="width:239px;">World Precision Instrucments, Inc.</td>
			<td style="width:103px;">500240</td>
			<td style="width:741px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Small Vessel Cauterizer</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:103px;">18000-00</td>
			<td style="width:741px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Micro Hook</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:103px;">10064-14</td>
			<td style="width:741px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Vetbond Suture Glue</td>
			<td style="width:239px;">3M</td>
			<td style="width:103px;">1469SB</td>
			<td style="width:741px;">n-butyl cyanoacrylate adhesive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Lipopolysaccharide</td>
			<td style="width:239px;">Sigma Life Science</td>
			<td style="width:103px;">L4391</td>
			<td style="width:741px;">Lipopolysaccaride from e.coli 0111:B4, gamma irradiated</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:271px;">12x12 inch opaque box</td>
			<td style="width:239px;">Acrylic Display Manufacturing: A division of Piasa Plastics</td>
			<td style="width:103px;">C4022</td>
			<td>Colored Acrylic 5-Sided Cube, 3/16&#34; Colored Acrylic, 12&#34;W x 12&#34;D x 12&#34;H;&#160; http://www.acrylicdisplaymfg.com/html/cubes_19.html</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:271px;">Camera/camcorder</td>
			<td style="width:239px;">JVC</td>
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Covidien Tendersorb&#8482; Underpads</td>
			<td style="width:239px;">Kendall Healthcare Products Co</td>
			<td style="width:103px;">7174</td>
			<td style="width:741px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">WypAll L40</td>
			<td style="width:239px;">Kimberly-Clark Professional</td>
			<td style="width:103px;">5600</td>
			<td style="width:741px;">Any surface with moderate grip will do</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Surface at 45 degree incline</td>
			<td style="width:239px;"></td>
			<td style="width:103px;"></td>
			<td style="width:741px;">We use a cardboard box.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Thin wire from a pipe cleaner</td>
			<td style="width:239px;">Creatology</td>
			<td style="width:103px;">M10314420</td>
			<td style="width:741px;">Any pipe cleaner from any craft store will work.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">50mL conical tube</td>
			<td style="width:239px;">Falcon</td>
			<td style="width:103px;">352070</td>
			<td style="width:741px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Fiberglass Screen Wire</td>
			<td style="width:239px;">New York Wire&#160; www.lowes.com</td>
			<td style="width:103px;">14436</td>
			<td style="width:741px;">Any supplier can be used as long as their screen is 16x16 or 18x16</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Razor blade</td>
			<td style="width:239px;">Fisherbrand</td>
			<td style="width:103px;">12-640</td>
			<td style="width:741px;">A wooden stick applicator or wooden part of a cotton-tipped swab will also work.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:271px;">OPTIX 24-in x 4-ft x 0.22-in Clear Acrylic Sheet to make Clear Acrylic Walkway</td>
			<td style="width:239px;">PLASKOLITE INC</td>
			<td style="width:103px;">1AG2196A</td>
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Protractor</td>
			<td style="width:239px;">Westscott</td>
			<td style="width:103px;">ACM14371</td>
			<td style="width:741px;"></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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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

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under 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 ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...