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 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-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. 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.

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

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for infants with the neurological sequela of prematurity. Ferrets are ideal candidates for modeling the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32&#8211;36 weeks&#39; gestation. We describe an injury model in the P17 ferret, where lipopolysaccharide administration is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This simulates the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of neonates who develop brain injury. Injured animals display a range of gross injury severity, with morphological changes in the brain including narrowing of multiple cortical gyri and associated sulci. Injured animals also show slowed reflex development, slower and more variable speed of locomotion in an automated catwalk, and decreased exploration in an open field. This model provides a platform in which to test putative therapies for infants with neonatal encephalopathy associated with inflammation and HI, study mechanisms of injury that affect cortical development, and investigate pathways that provide resilience in unaffected animals.

The method describes inflammation-sensitized hypoxic-ischemic and hyperoxic brain injury in the P17 ferret to model the complex interaction between prolonged inflammation and oxidative brain injury experienced in a number of late preterm infants.

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...