Neurobehavioral functional deficits following closed head injury in the neonatal pig

Abstract

Neurobehavioral deficits in higher cortical systems have not been described previously in a large animal model of diffuse brain injury. Anesthetized 3–5 day old piglets were subjected to either mild (142 rad/s) or moderate (188 rad/s) rapid non-impact axial rotations of the head. Multiple domains of cortical function were evaluated 5 times during the 12 day post-injury period using tests of neurobehavioral function devised for piglets. There were no observed differences in neurobehavioral outcomes between mild injury pigs (N = 8) and instrumented shams (N = 4). Moderately injured piglets (N = 7) had significantly lower interest in exploring their environment and had higher failure rates in visual-based problem solving compared to instrumented shams (N = 5) on days 1 and 4 after injury. Neurobehavioral functional deficits correlated with neuropathologic damage in the neonatal pigs after inertial head injury. Injured axons detected by immunohistochemistry (β-APP) were absent in mild injury and sham piglets, but were observed in moderately injured piglet brains. In summary, we have developed a quantitative battery of neurobehavioral functional assessments for large animals that correlate with neuropathologic axonal damage and may have wide applications in the fields of cardiac resuscitation, stroke, and hypoxic–ischemic brain injury.

Introduction

Traumatic brain injury (TBI) is the most common cause of death in childhood. Brain injuries resulting in hospitalization or death occur in at least 150,000 children per year, at a rate of over 200 per 100,000 children (Fisher, 1997). Widespread axonal injury is one of most commonly observed pathologies in pediatric brain injury. The long term morbidity and mortality associated with traumatic brain injury has in part been attributed to axonal injury (Babikian et al., 2005). Outcomes of children with diffuse axonal injury can vary from little or no cognitive deficits to life-long cognitive, behavioral, and motor disabilities.

Acute injury to the developing brain has been the focus of research involving numerous animal models(Adelson et al., 2001, Prins and Hovda, 2003, Prins et al., 1996, Raghupathi et al., 2004). A critical link in successful translational research is the development of animal models of brain injury that can provide a platform to correlate meaningful functional outcome measures with well-characterized histological and molecular substrates. Increasingly, piglets have been used to model acute damage to the developing brain resulting from asphyxic circulatory arrest (Brambrink et al., 1999), cardiopulmonary bypass (Kurth et al., 1999, Schultz et al., 2004), and traumatic brain injury (Armstead, 2002, Raghupathi and Margulies, 2002, Raghupathi et al., 2004). Moreover, the histopathology and acute cerebral physiologic responses seen in piglets in these injury paradigms closely resemble that seen in human infants (Hagberg et al., 2002, Martin et al., 1997).

Previous survival studies of acute brain injury in piglets have incorporated relatively simplistic neurological functional outcome scoring measures. Typically a series of 4 or 5 categories of neurological function are used (e.g. mental status, cranial nerves, sensorimotor function, feeding), each of which is assigned a graded score of 1–4 representing the degree of abnormality, yielding a final score ranging from a possible total of 9 for the simplest grading scheme (Midulla et al., 1994), to 150 for a more complicated neurological deficit score (Agnew et al., 2003). These grading systems have shown only limited correlation with histopathologic abnormalities (Priestley et al., 2001), but their sensitivity for assessment of complex behavior or cortical functions is poor, and none have been well validated with long-term survival studies.

In contrast, there are numerous rodent models of acute and chronic degenerative diseases in the young and mature brain, in which standardized and sensitive behavioral outcome measures have been successfully correlated with regional neuropathology due in large part, to an extensive literature in rodent behavioral neuroscience (Almli et al., 2000, Bona et al., 1997, Dixon et al., 2003, Young et al., 1986). Research in swine behavior is historically limited to veterinary science communities, with a focus on neuro-development rather than neuropathology. Results indicate that even newborn piglets are readily amenable to quantitative behavioral assessment, displaying sophisticated social learning skills, highly developed sensory discrimination capabilities for auditory, olfactory, visual and tactile modalities, and the ability to be trained in maze-learning tasks (De Jong et al., 2000, Hammell et al., 1975, Puppe et al., 1999).

Previously we presented our acute (6 h survival) non-impact neonatal (3–5 day old) piglet model of closed head injury, with a markedly lower density of injured axons after a single rapid axial rotation of 160–190 rad/s (moderate) compared to a single severe axial rotation of 240–260 rad/s (Raghupathi et al., 2004). Furthermore, we observed an increase in distribution of injured axons following two consecutive mild axial accelerations of 130–150 rad/s spaced 15 min apart compared to single accelerations at 6 h survival (Raghupathi et al., 2004). Our goal was to develop reliable quantitative functional neurobehavioral assessments for brain injury in piglets. In this communication, we present our double mild (≈ 140 rad/s) and single moderate (≈ 190 rad/s) velocity rotations in a survival piglet model and a battery of novel behavior, cognitive, and motor tests for piglets with a sensitivity to detect differences in injury level and correlating with differences in histopathology. Functional data from piglets experiencing mild and moderate non-impact rapid head rotations were compared to instrumented shams over a 12 day period, revealing transient and persistent deficits after moderate but not mild brain injury that correlated with histopathology.