This study was supported by grants from National Research Foundation (NRF-2014R1A2A1A11052042; 2015M3A9B4067068), the Ministry of Science and Technology, Republic of Korea, the Korean Health Technology R&#38;D Project (HI16C1012), Ministry of Health &#38; Welfare, Republic of Korea, and the &#8220;Dongwha&#8221; Faculty Research Assistance Program of Yonsei University College of Medicine (6-2016-0126).

All animals were housed in a standard cage (27 &#215; 22.5 &#215; 14 cm3) in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and given food and water ad libitum under alternating 12-h light/dark cycles. The authors followed animal protection regulations, and the experimental procedures were approved by the Institutional Animal Care and Use Committee of Yonsei University College of Medicine (IACUC No. 2010-0252; 2013-0220).
1. Mouse Model of Neonatal HI Brain Injury
<ol>
	<li>Anesthetize the pups with isoflurane.
	<ol>
		<li>Place the pups (less than 5) into an anesthetizing box and close the lid.</li>
		<li>Turn on the anesthetizing system for approximately 15 min; adjust the gas and isoflurane using a table top anesthesia machine. Adjust the oxygen flowmeter to 1.5 L/min. Adjust the isoflurane vaporizer to 3-5% for the induction of anesthesia.</li>
		<li>After 15 min, adjust the isoflurane vaporizer to 1-2% for the maintenance of anesthesia.</li>
	</ol>
	</li>
	<li>Lay a fully anesthetized pup under a dissection microscope (abdomen facing the researcher) and secure it with tape.</li>
	<li>Make a ~0.7-mm incision in the neck using sterilized scissors.</li>
	<li>Carefully remove the adipose tissue using sterilized forceps and expose the unilateral right carotid artery.</li>
	<li>Ligate the unilateral right carotid artery with a 5-0 absorbable suture.</li>
	<li>Suture the incision in the neck with 5-0 suture.</li>
	<li>Place each pup in a 37 &#176;C warm hypoxic chamber for 1 h for recovery. Do not close the chamber lid.</li>
	<li>1 h after the surgery, when the pups are fully awake, close the hypoxia chamber lid and decrease the gas levels to establish hypoxic conditions (8% O2 and 92% N2).</li>
	<li>After 90 min of hypoxia, return the pups to their cages.</li>
	<li>One week after the HI brain injury, repeat step 1.
	<ol>
		<li>After the anesthesia, make an incision in the scalp with sterilized scissors and forceps to identify the brain lesion in the posterolateral area of the right hemisphere. 
		Note: This treatment induces hypoxia in pups. The presence and extent of brain injury in all mice is visually assessed with the naked eye through the semi-transparent skull. As determined by the size or volume of the discoloration (i.e., the brain lesion), pups are classified into groups. If there is no visible cortical injury , the mouse is classified into the &#8220;no cortical injury&#8221; group. If there is a visible cortical injury (i.e., a lesion in the right hemisphere), the mouse is classified into the &#8220;cortical injury&#8221; group. Since classification of the mice into groups is done one week after the operation, the groupings can be modified when the morphologies of the brain samples are clearly defined at the time of sacrifice1,2,3,4.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55838/55838fig1v3.jpg" /> 
Figure 1: Modeling neonatal HI brain injury in mice. 
(A) A seven-day-old mouse pup underwent surgery, and the unilateral right carotid artery was ligated. (B) Pups were placed in a hypoxic chamber for 90 min with 8% O2 and 92% N2. (C, D, and E) The brains with neonatal HI injury showed various severity of damage and were categorized based on the degree of damage. At week 14, the brains were obtained, and the lesions were visualized. (C) Image of a brain classified as a &#34;no cortical injury&#34;. Both (D) and (E) were classified into the &#34;cortical injury&#34; group. (F, G, and H) Representative MRI of (C), (D), and (E) mice, respectively. (F) The damage in the hippocampus is indicated with a yellow arrow, and lesions in the right hemisphere are also indicated with yellow arrows (G and H). Scale bars = 1 mm&#160; <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55838/55838fig1v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Neonatal Behavioral Tests
Note: Here, the behavioral tests were performed at 6 weeks of age.
<ol>
	<li>Passive avoidance task. 
	NOTE: To evaluate memory function based on learning and the avoidance of an aversive stimulus, a two-compartment step-through PAT should be conducted13,14,15,16.
	<ol>
		<li>Place a mouse into the bright compartment of the Plexiglas shuttle box (41.5 &#215; 21 &#215; 35 cm3) of a PAT apparatus.</li>
		<li>After 30 s, open the guillotine door and record the latency time for the mouse to move into the dark compartment (up to 300 s).</li>
		<li>Close the guillotine door when all four limbs of the mouse are fully inside the dark compartment.</li>
		<li>Administer electric foot shock (0.5 mA) for 2 s and return the mouse to its cage.</li>
		<li>Replace the mouse in the bright compartment 24 h after the electric foot shock.</li>
		<li>Open the guillotine door 10 s after the mouse is fully placed in the bright compartment, and record the latency time for the mouse to move into the dark compartment (up to 300 s).</li>
	</ol>
	</li>
	<li>Ladder walking test. 
	NOTE: The ladder rung walking task allows for the discrimination between subtle disturbances of motor function by combining qualitative and quantitative analyses of skilled walking17,18.
	<ol>
		<li>Turn on a video camera.</li>
		<li>Place a mouse on the start panel of the ladder and immediately begin recording.</li>
		<li>Record the video, focusing on the mouse limbs.</li>
		<li>Stop recording when the mouse touches the last panel of the ladder. Repeat the back-and-forth trip four times.</li>
		<li>Analyze the video recording and manually count the number of slips of each forelimb, as follows:
		<ol>
			<li>Play recording of the video on a computer at a slow speed (0.1x) and count the steps manually.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Grip strength test. 
	NOTE: The grip strength test is performed using a grip strength meter, which includes a push-pull strain gauge.
	<ol>
		<li>Fix the grip strength apparatus onto an acrylic panel.</li>
		<li>Place a mouse on the acrylic panel and hold its tail.
		<ol>
			<li>Move the hand holding the tail so that the mouse can reach and grip the metal wire of the apparatus.</li>
		</ol>
		</li>
		<li>Allow multiple trials until the mouse grips a triangular piece of metal wire (2 mm in diameter); the peak force is automatically registered in grams by the apparatus. 
		NOTE: Use the mean peak force of three trials for analysis19,20,21.</li>
	</ol>
	</li>
</ol>

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The authors have no competing interests.

In this study, we induced HI brain injury in a neonatal P7-10 CD-1 mouse and identified the brain lesion with relevant cognitive and motor deficits. During this procedure, occlusion of the unilateral right carotid artery was critical. In this step, the artery could be damaged and torn. Most pups who experienced an artery tear died. Conversely, if researchers ligated another blood vein instead of the unilateral right carotid artery, the brain of the pup was only mildly damaged, and no significant phenotype could be observed24.
In this study, due to variations in mice and lesion volume, the brains were categorized into several groups (Figure 1C-H). Several mice with mildly injured brains had damage only in the hippocampus and not in the cortical region (Figure 1F)13. Conversely, several mice with severely damaged brains lost most of the right hemisphere, and the cortices were severely damaged (Figure 1G and H). Therefore, researchers should identify the size of the lesion one week after the procedure19,25. Since brains were evaluated using MRI scans, the determination of the volume and size of a lesion was more reliable. Therefore, we recommend that researchers evaluate the brains using MRI, although visual inspection with the naked eye is also feasible.
Cerebral palsy commonly occurs during early childhood, with an incidence rate of approximately two patients per 1,000 children5. Since the neonatal HI mouse model could be a representative model of cerebral palsy or neonatal stroke4,11,26, the baseline information from this study can be used in preclinical research on cerebral palsy or neonatal stroke.
Neurobehavioral assessments are useful to identify the phenotypes of cognitive and motor deficits13. The neurobehavioral assessments introduced in this study are also adaptable and are commonly used for other neurodegenerative diseases, such as Huntington&#39;s, Parkinson&#39;s, and so on. Researchers should be aware that, during PAT, subjects receive an electric shock. Therefore, PAT should be performed last, so the electric shock does not affect the other behavioral assessments.
For further study, researchers need to study a sham-operated group in comparison with the HI group. For a specific control group, researchers can make an incision on the neck and close the incision without any artery ligation. To mimic the HI operation, these pups should be put into the hypoxic chamber, but without hypoxia, for the same amount of time as an HI group before being returned to their cages.

Neonatal HI brain injury occurs during early childhood (approximately two patients per 1,000 children)1,2,3,4,5. Studies regarding neonatal HI brain injury are important, and using an established neonatal HI brain injury mouse model can facilitate in vivo preclinical research on HI brain injury.
Traditional HI models are used on adult rats6. For the neonate model, the Rice-Vannucci method is commonly used on P7 rats7,8. However, since rats and mice are slightly different9,10, even though they are both rodents, we performed a modified Rice-Vannucci method on CD-1 pups at P7-10, based on previous studies that showed that P7-10 is the period featuring immature oligodendrocytes, corresponding to human term P011,12. The neonatal HI mouse model is established through both the ligation of the unilateral carotid artery and the exposure of the mice to hypoxia with 8% oxygen in P7-10 pups.
The mice subjected to the procedure showed various degrees of brain lesions in the posterolateral area of the right hemisphere. To identify the cognitive and motor deficits, neurobehavioral assessments based on the PAT, ladder walking test, and grip strength test were performed. The differences between non-operated (i.e., normal) and HI mice were analyzed. This work presents basic information on the HI mouse model. The MRI images demonstrate the different phenotypes, separated according to the severity of brain damages using motor and cognitive tests.

<table><tbody><tr><td>Hypoxic chamber</td><td>Jeung Do Bio &amp;amp; Plant Co</td><td>Experimental Builder</td><td></td></tr><tr><td>PAT apparatus</td><td>Jeung Do Bio &amp;amp; Plant Co</td><td>Experimental Builder</td><td></td></tr><tr><td>The ladder rung walking</td><td>Jeung Do Bio &amp;amp; Plant Co</td><td>Experimental Builder</td><td></td></tr><tr><td>SDI Grip Strength System</td><td>San Diego Instruments Inc.</td><td></td><td></td></tr><tr><td>Grip-Strength Meter</td><td>Ugo Basile</td><td>47200</td><td></td></tr><tr><td>Harvard Apparatus Fluovac anesthetizing system&amp;nbsp;</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>Anesthetizing box</td><td></td><td></td><td>acryl box</td></tr><tr><td>I-Fran Liquid (Isofluorane)</td><td>Hana Pharm. Co., Ltd.</td><td></td><td>General Anesthetics ( isoflurane 100ml)</td></tr><tr><td>CD-1 mice</td><td>Orient Co., Ltd.</td><td></td><td></td></tr><tr><td>Blue Nylon Mono Non-Absorbbable suture 5-0 50cm</td><td>Ailee Co., Ltd.</td><td>NB 521</td><td></td></tr><tr><td>IBM SPSS Statistics</td><td>IBM</td><td>Ver. 23</td><td></td></tr></tbody></table>

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.

All data are expressed as the mean &#177; standard error of the mean (SEM). The comparison of variables between the two groups was conducted using an independent or paired t-test on SPSS statistics software. A p-value &#60;0.05 was considered statistically significant.
The brains with neonatal HI injury showed different severity of damage and were categorized accordingly (Figure 1C-E). The brains were obtained at week 14, and the lesions were visualized. Figure 1C shows a brain classified as a &#34;no cortical injury&#34; brain, Figure 1D shows a brain classified as a mild injury, and Figure 1E shows a severely damaged brain. Both (D) mild and (E) severe injuries were classified into the &#34;cortical injury&#34; group. After the HI operation, 13-week-old mice were imaged using MRI, and the results (Figure 1F-H) are representative images of (C), (D), and (E) injuries, respectively. Even though there was no significant lesion on the morphology of the brain, the MRI image showed hippocampal injury (Figure 1F). Damage to the hippocampus (Figure 1F, indicated with a yellow arrow) is slightly apparent in the mildly injured brain. In a severely damaged brain, the mouse lost most of the right hemisphere (Figure 1G and H, indicated with a yellow arrow).
Since the brains with HI injury showed hippocampal injury (Figure 1F-H), the mice with HI injury exhibited memory deficits compared to the normal mice. PAT performance is closely related to hippocampal damage13,15,16,19. Figure 2 shows that the mice with HI injury had more cognitive deficits than the normal mice13, as assessed in the PAT (normal n = 10; HI n = 9). A statistically significant difference was observed between the baseline and the 24-h memory test in normal mice, as shown in Figure 2A (*p = 0.003 based on a paired t-test). Figure 2B shows the changes in cognitive function in the HI injury mice compared to normal mice (delta (&#916;) is the difference between baseline and the 24-h test)13.
Because only the right hemisphere was damaged, the neonatal HI brain injury mice showed hemiplegic motor functions. The difference in the percentage of slips on the transverse rungs of the ladder relative to the total number of steps taken by each forelimb was used to compare normal mice with neonatal HI brain injury mice17,19. Figure 3 shows that the slip rate of the contralateral forelimb in the HI brain injury mice was significantly higher than that in the normal mice (normal n = 19; HI n = 18; *p = 0.010 based on an independent t-test)22, but no difference was observed in the ipsilateral forelimb(p = 0.798 based on an independent t-test).
Moreover, since the grip strength involves the motor cortex in the brain, the normal and cortical injury groups showed differences in grip power. Although the results from the grip strength test showed no difference between the normal and no cortical injury mice (Figure 4A; normal n = 4; no cortical injury n = 12), the graph shows that the grip power of the contralateral forelimb was significantly weaker in the cortical injury mice than in the normal mice (Figure 4B; normal n = 4; cortical injury n = 36; *p = 0.036 based on an independent t-test)21,22,23.

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55838/55838fig2v2.jpg" /> 
Figure 2: PAT in neonatal HI brain injury and normal mice. 
(A) The latency time in the bright compartment was measured and compared between neonatal HI brain injury and normal mice (n = 9 and n = 10, respectively). (B) The measurement at the moment of the electronic shock was considered the baseline, and long-term memory was evaluated 24 h after the electric shock. Delta (&#916;) 24-h latency was the difference between the function evaluated at 24 h and at baseline. *p&#60;0.05; all data are expressed as the mean &#177; SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55838/55838fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55838/55838fig3v2.jpg" /> 
Figure 3: Forelimb slip rate in the ladder walking test. 
The rates of contralateral and ipsilateral forelimb slip were evaluated between the normal and HI brain injury mice (n = 19 and n = 18, respectively). *p&#60;0.05; all data are expressed as the mean &#177; SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55838/55838fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55838/55838fig4v2.jpg" /> 
Figure 4: Grip strength test in neonatal HI brain injury and normal mice. 
Grip power of the contralateral forelimb was evaluated and compared between (A) normal, no cortical injury and (B) cortical injury mice (n = 4, n = 12, n = 36). *p&#60;0.05; all data are expressed as the mean &#177; SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55838/55838fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury at JoVE.com

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

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 ...

neurobehavioral-assessments-mouse-model-neonatal-hypoxic-ischemic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.2K Views.  Yonsei University College of Medicine. Wij unilaterale halsslagader occlusie uitgevoerd op postnatale dag 7-10 CD-1 pups om een neonatale hypoxische ischemische (HI) model te maken van de muis en onderzocht de effecten van HI hersenletsel. We studeerde neuro functies in deze muizen in vergelijking met normale muizen niet bediende.

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

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

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 ...

Behavior

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

Biology

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 ...

Medicine

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

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.

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, ...

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

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying neonatal seizures and hypoxia ischemia. This manuscript describes a method for continuous video electroencephalogram (EEG) monitoring in neonatal mice to detect seizures and analyze EEG background during hypoxia ischemia. Use of video and EEG in conjunction allows description of seizure semiology and confirmation of seizures. This method also allows analysis of power spectrograms and EEG background pattern trends over the experimental time period. In this hypoxia ischemia model, the method allows EEG recording prior to injury to obtain a normative baseline and during injury and recovery. Total monitoring time is limited by the inability to separate pups from the mother for longer than four hours. Although, we have used a model of hypoxic-ischemic seizures in this manuscript, this method for neonatal video EEG monitoring could be applied to diverse disease and seizure models in rodents.

This manuscript describes a method for continuous video EEG recordings using multiple depth electrodes in neonatal mice undergoing hypoxia-ischemia.

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term ...

CHILD Model &#8212; Recapitulating Juvenile Concussion and Long-Term Outcomes in Mice

A Pediatric Concussion Model in Mice: Closed Head Injury with Long-Term Disorders (CHILD)

Concussions and mild traumatic brain injuries (mTBI) during childhood represent a significant health endangerment, with many patients later in life exhibiting debilitating physiological, neurological, and psychosocial outcomes. The cellular and molecular pathophysiological mechanisms are relatively unknown, and this significant gap effectively precludes investigations into specific therapeutic strategies to mitigate chronic sequelae. No single animal concussion model recapitulates all the features reported in human subjects, and current models are designed to answer specific research questions. We set out to develop a juvenile concussion that recapitulates clinical early, and long-term symptomology encountered, termed Closed Head Injury with Long-term Disorder (CHILD). In this model, a concussive force via an electromagnetic impactor is delivered to the head of a lightly anesthetized unrestrained post-natal day 17 (P17) mouse, allowing free rotation of the head. Mice are placed on a tightly stretched tin foil across a stereotactic device, and the impactor is carefully aligned with the targeted cortical region. The electromagnetic impactor facilitates the selection of impact depth, dwell, and duration to determine injury severity. A strength of this model is that it allows head rotation, an important clinical feature. After impact, mice are immediately monitored for righting time and time to explore their environment, followed by their return to their dam. Increasing the severity of concussion results in intracranial bleeds in a subset of mice. Mice can be routinely monitored for behavior and neuroimaging over their lifespan, as desired. Various injury severities mimic the heterogeneous nature of juvenile concussions. The current standard CHILD model exhibits no skull fracture, no observable conventional neuroimaging change (similar to clinical), but leads to progressive and persistent neuronal death, altered diffusion MRI, modified neuronal activity and plasticity, increased gliosis, and progressive behavioral perturbations with age. In summary, this CHILD model mimics the early and long-term features observed in many clinical concussion patients.

Pediatric concussions can result in long-term sequelae with physiological and psychological perturbations. This pediatric concussion mouse model results in long-term neuroinflammation, white matter alterations, and neuronal modifications with behavioral dysfunction. The model can be adapted to differential impact locations or repeated concussions to assess lifetime changes.

Concussions and mild traumatic brain injuries (mTBI) during childhood represent a significant health endangerment, with many patients later in life ...

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

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