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

Summary

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.

Abstract

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'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 ("no cortical injury" group) or presence ("cortical injury" 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.

Introduction

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 rats^7,8. However, since rats and mice are slightly different^9,10, 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 P0^11,12. 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.

Protocol

All animals were housed in a standard cage (27 × 22.5 × 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

1. Anesthetize the pups with isoflurane.
   1. Place the pups (less than 5) into an anesthetizing box and close the lid.
   2. 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.
   3. After 15 min, adjust the isoflurane vaporizer to 1-2% for the maintenance of anesthesia.
2. Lay a fully anesthetized pup under a dissection microscope (abdomen facing the researcher) and secure it with tape.
3. Make a ~0.7-mm incision in the neck using sterilized scissors.
4. Carefully remove the adipose tissue using sterilized forceps and expose the unilateral right carotid artery.
5. Ligate the unilateral right carotid artery with a 5-0 absorbable suture.
6. Suture the incision in the neck with 5-0 suture.
7. Place each pup in a 37 °C warm hypoxic chamber for 1 h for recovery. Do not close the chamber lid.
8. 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).
9. After 90 min of hypoxia, return the pups to their cages.
10. One week after the HI brain injury, repeat step 1.
    1. 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 “no cortical injury” group. If there is a visible cortical injury (i.e., a lesion in the right hemisphere), the mouse is classified into the “cortical injury” 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^1,2,3,4.

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 "no cortical injury". Both (D) and (E) were classified into the "cortical injury" 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  Please click here to view a larger version of this figure.

2. Neonatal Behavioral Tests

Note: Here, the behavioral tests were performed at 6 weeks of age.

1. 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 conducted^13,14,15,16.
   1. Place a mouse into the bright compartment of the Plexiglas shuttle box (41.5 × 21 × 35 cm³) of a PAT apparatus.
   2. 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).
   3. Close the guillotine door when all four limbs of the mouse are fully inside the dark compartment.
   4. Administer electric foot shock (0.5 mA) for 2 s and return the mouse to its cage.
   5. Replace the mouse in the bright compartment 24 h after the electric foot shock.
   6. 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).
2. 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 walking^17,18.
   1. Turn on a video camera.
   2. Place a mouse on the start panel of the ladder and immediately begin recording.
   3. Record the video, focusing on the mouse limbs.
   4. Stop recording when the mouse touches the last panel of the ladder. Repeat the back-and-forth trip four times.
   5. Analyze the video recording and manually count the number of slips of each forelimb, as follows:
      1. Play recording of the video on a computer at a slow speed (0.1x) and count the steps manually.
3. Grip strength test.
   NOTE: The grip strength test is performed using a grip strength meter, which includes a push-pull strain gauge.
   1. Fix the grip strength apparatus onto an acrylic panel.
   2. Place a mouse on the acrylic panel and hold its tail.
      1. Move the hand holding the tail so that the mouse can reach and grip the metal wire of the apparatus.
   3. 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 analysis^19,20,21.

Results

All data are expressed as the mean ± 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 <0.05 was considered statistically significant.

The brains with neonatal HI injury showed different severity of damage and were categorized accordingly (Figure 1C-E). T...

Discussion

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...

Materials

List of materials used in this article

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