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).
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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 observ...

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 border="0" cellpadding="0" cellspacing="0" width="1008">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:363px;">Hypoxic chamber</td>
			<td style="width:203px;">Jeung Do Bio &#38; Plant Co</td>
			<td colspan="2" style="width:443px;">Experimental Builder&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PAT apparatus&#160;</td>
			<td>Jeung Do Bio &#38; Plant Co</td>
			<td colspan="2">Experimental Builder&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">The ladder rung walking&#160;</td>
			<td>Jeung Do Bio &#38; Plant Co</td>
			<td colspan="2">Experimental Builder&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">SDI Grip Strength System&#160;</td>
			<td colspan="2">San Diego Instruments Inc.</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:363px;">Grip-Strength Meter</td>
			<td>Ugo Basile&#160;</td>
			<td align="right">47200</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Harvard Apparatus Fluovac anesthetizing system&#160;</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Anesthetizing box</td>
			<td></td>
			<td></td>
			<td>acryl box</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">I-Fran Liquid (Isofluorane)</td>
			<td>Hana Pharm. Co., Ltd.</td>
			<td></td>
			<td>General Anesthetics ( isoflurane 100ml)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CD-1 mice</td>
			<td>Orient Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Blue Nylon Mono Non-Absorbbable suture 5-0 50cm</td>
			<td>Ailee Co., Ltd.</td>
			<td>NB 521</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">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...

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 ...

The overall goal of the hypoxic-ischemic neonatal brain injury surgery, followed by cognitive and motor testing, is to have a mouse model to study such injuries and related brain damage. This method can help answer key questions about neonatal hypoxic-ischemic brain injury, such as how such injuries affect motor and cognitive functions. The main advantage of this technique is that it is well-known and is commonly used.It is also a preclinical model for cerebral palsy and neonatal strokes. Generally, individuals new to this method will struggle because the unilateral right carotid artery is difficult to identify. Demonstrating the procedure, along with myself, will be Yoon-Kyum Shin, a graduate student from our laboratory.After anesthetizing at least five P-seven mouse pups, position a pup under a dissection microscope with the abdomen up. Then, clean the neck with alcohol. To begin the surgery, use scissors to make a 0.7-millimeter incision into the neck.Then, carefully remove the adipose tissue, using forceps, until the unilateral right carotid artery is exposed. Next, using a 5-0 suture, ligate the unilateral right carotid artery. Now, suture the incision in the neck with 5-0 nylon, and sterilize the incision.Then, allow the pup to recover in a 37-degree Celsius hypoxic chamber with the lid open for about an hour. Place another surgically manipulated mouse into the hypoxic chamber to recover. Once the pups are all fully awake, close the chamber lid and decrease the gas levels to establish hypoxic conditions.After 90 minutes of hypoxia, return the brain-injured pups to their cages. A week later, prepare the pups for surgery, as before, to look for evidence of the injury. Make an incision in the scalp to identify the brain lesion in the posterolateral area of the right hemisphere.Look through the semi-transparent skull for evidence of a cortical injury and a hippocampal injury. Thus, categorize the pups as injured or uninjured controls. After checking, close the scalp with suture, and sterilize the incision.When the mice are six weeks old, evaluate their memory based on the learning and avoidance of an adversive stimulus. Use a commercial two-compartment, step-through passive avoidance apparatus for this task. To begin, place a mouse into the bright compartment of the box for 30 seconds.Then, open the door between the compartments, and record the animal's latency to enter the dark compartment. Allow up to 300 seconds to perform the task. When all four limbs of the mouse are fully inside the dark compartment, close the door.Then, administer a 1/2 milliamp electric foot shock for two seconds, and return the mouse to its cage. 24 hours later, repeat the test. This time, allow the mouse a full 300 seconds to move into the dark chamber, and do not shock the mouse.Perform the ladder walking test on the six-week-old mice to discriminate between subtle disturbances of motor function, using both qualitative and quantitative analyses. First, set up the ladder apparatus, and then set up a video camera to record the limb movements of a mouse on the apparatus. Next, place a mouse on the start panel, and start making a recording with the video camera.Make certain that the video shows the legs in good focus. When the mouse touches the last panel of the ladder apparatus, stop the recording. Then, move the mouse back to the starting panel, and record the mouse crossing the apparatus again.Do this a total of four times per mouse on each trial date. Later, when analyzing the video, manually count the number of slips made by each limb while playing the video at 1/10 speed. When a limb slides between the bars, this constitutes a slip.When the mice are six weeks old, assess their grip strength. Use a push/pull strain gauge, outfitted to a two-millimeter diameter triangular wire, all fixed to a panel. Position the mouse on the panel, holding it by its tail.Then, allow the mouse to just reach the metal wire with its forelimbs. As soon as mice grip the wire, gently pull its tail until it releases its grasp. The apparatus will register the peak force on the wire in grams.Repeat this test five times before giving the mouse a rest. During the grip strength test, there are noticeable movements of accurate performances. Try to find those movements over five efforts before resting the mouse, for at least one minute to reduce its muscle fatigue.After the mouse rests for at least one minute, repeat the five-trial series again. Then, give the mouse another rest, and perform the series one last time for that test day. From the data, use the average of the three largest scores for either forelimb or for both forelimbs for analysis.After harvesting the brains of 14-week-old hypoxia-ischemia injured animals, the brains were evaluated morphologically as having no cortical injury, a mild injury, or a severe injury. The same animals were scanned by an MRI a week earlier, which was also very revealing. Even the morphologically normal brain showed mild injury in the hippocampus.At six weeks of age, five weeks post-injury, the mice were assessed with various behavioral tasks. Hypoxia-ischemia injured mice were cognitively impaired on the 24-hour passive avoidance task. Because only the right hemisphere was damaged, the mice showed hemiplegic motor functions.This was revealed by the difference in the percentage of slips on the transverse rungs of the ladder for each forelimb. Moreover, since grip strength involves the motor cortex in the brain, the normal and cortical injury groups showed differences in grip power. No difference was observed between the normal controls and categorically uninjured mice, but the injured mice were clearly impaired.After watching this video, you should have a good understanding of how to ligate the unilateral right carotid artery to generate a neonatal hypoxic-ischemic mice and how to perform follow-up behavioral analysis. It is important to note that even when there is no visible damage seen on the cortical brain seven days after injury, an MRI may reveal a hippocampal injury. Also, it is important to remember to treat the pups gently and complete the surgery as quickly as possible.Pups have very weak and thin skins, so be very careful.

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12.1K Views.  Yonsei University College of Medicine. The overall goal of the hypoxic-ischemic neonatal brain injury surgery, followed by cognitive and motor testing, is to have a mouse model to study such injuries and related brain damage. This method can help answer key questions about neonatal hypoxic-ischemic brain injury, such as how such injuries affect motor and cognitive functions. The main advantage of this technique is that it is well-known and is commonly used.It is also a preclinical model for cerebral palsy and neonatal strok...