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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. &#160;&#160;&#160;
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 tabletop 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 &#34;no cortical injury&#34; group. If there is a visible cortical injury (i.e., a lesion in the right hemisphere), the mouse is classified into the &#34;cortical injury&#34; 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 sacrifice.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hypoxic chamber</td>
			<td>Jeung Do Bio &#38; Plant Co</td>
			<td>Experimental Builder</td>
			<td></td>
		</tr>
		<tr>
			<td>Harvard Apparatus Fluovac anesthetizing system</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>
	</tbody>
</table>

establishing a mouse model of neonatal hypoxic-ischemic brain injury

Source: Kim, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55838">Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury.</a> J. Vis. Exp. (2017).
The video demonstrates the procedure for creating a mouse model to study neonatal hypoxic-ischemic brain injury. It outlines the steps for surgically inducing ischemia and subjecting the animal to hypoxia to exacerbate brain damage. Finally, it shows how to assess the resulting brain lesions, ensuring the mouse model is prepared for further scientific investigation.

Establishing a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. ...

Secure an anesthetized mouse pup abdomen-up under a dissection microscope.
Make an incision in the neck.
Retract the adipose tissue to expose the unilateral right carotid artery.
Ligate the artery to restrict blood flow to the right brain hemisphere, simulating an ischemic condition.
Close the incision and transfer the pup to a hypoxic chamber.
Once the pup regains consciousness, close the chamber and lower the oxygen levels to establish a hypoxic condition.&#160;
Decreased blood supply and oxygen levels in the brain result in excitatory neurotransmitter release.
These neurotransmitters induce neuronal hyperexcitability, calcium influx, and calcium-dependent damage to neuronal membranes, cellular proteins, and DNA, leading to neuronal death.
In response, microglia become activated and release pro-inflammatory cytokines, worsening the brain damage.
Allow the pup to recover and grow for a week.
Anesthetize the pup, position it abdomen-down, and make a scalp incision.
Visualize the brain lesion in the right hemisphere through the semi-transparent skull to detect the hypoxic-ischemic injury.

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33 Views. Source: Kim, M., et al. Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury. J. Vis. Exp. (2017). The video demonstrates the procedure for creating a mouse model to study neonatal hypoxic-ischemic brain injury. It outlines the steps for surgically inducing ischemia and subjecting the animal to hypoxia to exacerbate brain damage. Finally, it shows how to assess the resulting brain lesions, ensuring the mouse model is prepared for further scientific investigatio...

Video: Establishing a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury - Concept

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

JoVE Journal

Medicine

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart&#160;catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to&#160;treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right&#160;ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...

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 ...

Establishing a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

Transcript

Establishing a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice