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,<....

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 anesthet....

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