Brain injury and neurofunctional deficit in neonatal mice with hypoxic-ischemic encephalopathy

Abstract

Birth asphyxia accounts for the majority of developmental motor and cognitive deficits. Studies were undertaken to develop a reproducible murine model of perinatal hypoxic-ischemic encephalopathy (HIE) which would permit both anatomic and neurofunctional quantification of injury. Short-term neurofunctional outcomes consisted of three developmental reflexes (righting, cliff aversion and geotaxis) assessed in 7-day-old mouse pups 24 h after unilateral carotid artery ligation followed by inhalation of 8% oxygen. Cerebral infarct volume was dependent on duration of hypoxia, being ≈2.5-fold greater with longer (60 min) versus shorter (30 min) hypoxia exposure (P=0.001). All three sensorimotor neonatal reflexes assessed at 24 h after HIE injury correlated significantly with long-term neurofunction evaluated using a water-maze test of navigational learning and memory assessed 8 weeks later in the same animals. Cerebral atrophy, a delayed consequence of HIE in this model, also correlated strongly with water-maze performance (r=0.86, P=0.002). These data demonstrate for the first time that murine neonatal sensorimotor reflex performance can be rigorously quantified in the acute phase of perinatal HIE and has strong predictive value not only for anatomic extent of cerebral injury, but also for long-term neurofunctional outcome.

Introduction

Perinatal hypoxic-ischemic encephalopathy (HIE) occurs in 0.2–0.4% of full-term neonates, and 25% of the survivors exhibit permanent neuropsychological deficits. Because of the large numbers of individuals affected and the substantial burdens associated with perinatal HIE, there is an urgent need to develop models which provide insights into the molecular mechanisms involved. In the most widely accepted rodent model of perinatal HIE, the dominant outcome measure of brain injury is based on histopathological changes reflecting neuronal cell loss [1], [18], [28], [30]. However, demonstrating short- and long-term functional brain recovery is of paramount clinical importance for evaluation of mechanisms of HIE and assessment of therapeutic effects. One potentially valuable approach in neonatal mice subjected to HIE could be the testing of early developmental sensorimotor reflexes as well as neurobehavioral paradigms for both short- and long-term assessment of neurofunctional outcome.

Evaluation of neurological deficit following stroke has been performed using a number of behavioral tasks in adult mice and rats [2], [12], [13], [14], [25], [35]. In juvenile and adult rats which had been subjected to a hypoxic-ischemic insult in the perinatal period, locomotor ability, learning, and navigational memory are impaired [4], [15], [17]. However, there are no studies which have examined neurological deficits at discrete developmental stages following HIE in newborn mice. It is important to develop neurofunctional assessment tools in mice subjected to perinatal HIE, since genetically modified mice could be used to determine precise molecular mechanisms of brain injury.

Recognizing the importance of using a murine system to study HIE, several investigators have been successful in adapting the rat model of perinatal HIE [24] for use in mice [6], [21], [31]. However, given the technical challenges of this model, there has been little standardization of protocol and no neurofunctional assessment. For instance, mice are used at various developmental ages, and the hypoxic exposure is of variable severity and duration. Consequently, the reported variability and perioperative mortality can be extremely high (up to 50%). The current study was undertaken to develop a reproducible, standardized short- and long-term neurofunctional assessment methodology for use in mice subjected to HIE which permits definition of the relationship between degree of ischemic insult and severity of anatomic and neurofunctional deficit.