Jill Ralston

Diffuse optical monitoring of hemodynamic changes in piglet brain with closed head injury.

We used a nonimpact inertial rotational model of a closed head injury in neonatal piglets to simulate the conditions following traumatic brain injury in infants. Diffuse optical techniques, including diffuse reflectance spectroscopy and diffuse correlation spectroscopy (DCS), were used to measure cerebral blood oxygenation and blood flow continuously and noninvasively before injury and up to 6 h after the injury. The DCS measurements of relative cerebral blood flow were validated against the fluorescent microsphere method. A strong linear correlation was observed between the two techniques (R=0.89, p<0.00001). Injury-induced cerebral hemodynamic changes were quantified, and significant changes were found in oxy- and deoxy-hemoglobin concentrations, total hemoglobin concentration, blood oxygen saturation, and cerebral blood flow after the injury. The diffuse optical measurements were robust and also correlated well with recordings of vital physiological parameters over the 6-h monitoring period, such as mean arterial blood pressure, arterial oxygen saturation, and heart rate. Finally, the diffuse optical techniques demonstrated sensitivity to dynamic physiological events, such as apnea, cardiac arrest, and hypertonic saline infusion. In total, the investigation corraborates potential of the optical methods for bedside monitoring of pediatric and adult human patients in the neurointensive care unit.

Physiological and histopathological responses following closed rotational head injury depend on direction of head motion

Rotational inertial forces are thought to be the underlying mechanism for most severe brain injuries. However, little is known about the effect of head rotation direction on injury outcomes, particularly in the pediatric population. Neonatal piglets were subjected to a single non-impact head rotation in the horizontal, coronal, or sagittal direction, and physiological and histopathological responses were observed. Sagittal rotation produced the longest duration of unconsciousness, highest incidence of apnea, and largest intracranial pressure increase, while coronal rotation produced little change, and horizontal rotation produced intermediate and variable derangements. Significant cerebral blood flow reductions were observed following sagittal but not coronal or horizontal injury compared to sham. Subarachnoid hemorrhage, ischemia, and brainstem pathology were observed in the sagittal and horizontal groups but not in a single coronal animal. Significant axonal injury occurred following both horizontal and sagittal rotations. For both groups, the distribution of injury was greater in the frontal and parietotemporal lobes than in the occipital lobes, frequently occurred in the absence of ischemia, and did not correlate with regional cerebral blood flow reductions. We postulate that these direction-dependent differences in injury outcomes are due to differences in tissue mechanical loading produced during head rotation.

Neurobehavioral Functional Deficits Following Closed Head Injury in the Neonatal Pig

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 (beta-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.

Folic Acid Enhances Early Functional Recovery in a Piglet Model of Pediatric Head Injury

For stroke and spinal cord injury, folic acid supplementation has been shown to enhance neurodevelopment and to provide neuroprotection. We hypothesized that folic acid would reduce brain injury and improve neurological outcome in a neonatal piglet model of traumatic brain injury (TBI), using 4 experimental groups of 3- to 5-day-old female piglets. Two groups were intubated, anesthetized and had moderate brain injury induced by rapid axial head rotation without impact. One group of injured (Inj) animals received folic acid (Fol; 80 µg/kg) by intraperitoneal (IP) injection 15 min following injury, and then daily for 6 days (Inj + Fol; n = 7). The second group of injured animals received an IP injection of saline (Sal) at the same time points (Inj + Sal; n = 8). Two uninjured (Uninj) control groups (Uninj + Fol, n = 8; Uninj + Sal, n = 7) were intubated, anesthetized and received folic acid (80 µg/kg) or saline by IP injection at the same time points as the injured animals following a sham procedure. Animals underwent neurobehavioral and cognitive testing on days 1 and 4 following injury to assess behavior, memory, learning and problem solving. Serum folic acid and homocysteine levels were collected prior to injury and again before euthanasia. The piglets were euthanized 6 days following injury, and their brains were perfusion fixed for histological analysis. Folic acid levels were significantly higher in both Fol groups on day 6. Homocysteine levels were not affected by treatment. On day 1 following injury, the Inj + Fol group showed significantly more exploratory interest, and better motor function, learning and problem solving compared to the Inj + Sal group. Inj + Fol animals had a significantly lower cognitive composite dysfunction score compared to all other groups on day 1. These functional improvements were not seen on day 4 following injury. Axonal injury measured by β-amyloid precursor protein staining 6 days after injury was not affected by treatment. These results suggest that folic acid may enhance early functional recovery in this piglet model of pediatric head injury. This is the first study to describe the application of complex functional testing to assess an intervention outcome in a swine model of TBI.

Physiological and Pathological Responses to Head Rotations in Toddler Piglets

Closed head injury is the leading cause of death in children less than 4 years of age, and is thought to be caused in part by rotational inertial motion of the brain. Injury patterns associated with inertial rotations are not well understood in the pediatric population. To characterize the physiological and pathological responses of the immature brain to inertial forces and their relationship to neurological development, toddler-age (4-week-old) piglets were subjected to a single non-impact head rotation at either low (31.6 +/- 4.7 rad/sec(2), n = 4) or moderate (61.0 +/- 7.5 rad/sec(2), n = 6) angular acceleration in the axial direction. Graded outcomes were observed for both physiological and histopathological responses such that increasing angular acceleration and velocity produced more severe responses. Unlike low-acceleration rotations, moderate-acceleration rotations produced marked EEG amplitude suppression immediately post-injury, which remained suppressed for the 6-h survival period. In addition, significantly more severe subarachnoid hemorrhage, ischemia, and axonal injury by beta-amyloid precursor protein (beta-APP) were observed in moderate-acceleration animals than low-acceleration animals. When compared to infant-age (5-day-old) animals subjected to similar (54.1 +/- 9.6 rad/sec(2)) acceleration rotations, 4-week-old moderate-acceleration animals sustained similar severities of subarachnoid hemorrhage and axonal injury at 6 h post-injury, despite the larger, softer brain in the older piglets. We conclude that the traditional mechanical engineering approach of scaling by brain mass and stiffness cannot explain the vulnerability of the infant brain to acceleration-deceleration movements, compared with the toddler.

Behavioral Deficits and Axonal Injury Persistence after Rotational Head Injury Are Direction Dependent

Pigs continue to grow in importance as a tool in neuroscience. However, behavioral tests that have been validated in the rodent model do not translate well to pigs because of their very different responses to behavioral stimuli. We refined metrics for assessing porcine open field behavior to detect a wide spectrum of clinically relevant behaviors in the piglet post-traumatic brain injury (TBI). Female neonatal piglets underwent a rapid non-impact head rotation in the sagittal plane (n=8 evaluable) or were instrumented shams (n=7 evaluable). Open field testing was conducted 1 day prior to injury (day -1) in order to establish an individual baseline for analysis, and at days +1 and +4 after injury. Animals were then killed on day +6 after injury for neuropathological assessment of axonal injury. Injured piglets were less interested in interacting with environmental stimuli and had a lower activity level than did shams. These data were compared with previously published data for axial rotational injuries in neonatal piglets. Acute behavioral outcomes post-TBI showed a dependence on the rotational plane of the brain injury, with animals with sagittal injuries demonstrating a greater level of inactivity and less random usage of the open field space than those with axial injuries. The persistence of axonal injury is also dependent on the rotational plane, with sagittal rotations causing more prolonged injuries than axial rotations. These results are consistent with animal studies, finite element models, and studies of concussions in football, which have all demonstrated differences in injury severity depending upon the direction of head impact rotation.

Neurocritical Care Monitoring Correlates with Neuropathology in a Swine Model of Pediatric Traumatic Brain Injury

BACKGROUND Small-animal models have been used in traumatic brain injury (TBI) research to investigate the basic mechanisms and pathology of TBI. Unfortunately, successful TBI investigations in small-animal models have not resulted in marked improvements in clinical outcomes of TBI patients. OBJECTIVE To develop a clinically relevant immature large-animal model of pediatric neurocritical care following TBI. METHODS Eleven 4-week-old piglets were randomly assigned to either rapid axial head rotation without impact (n = 6) or instrumented sham (n = 5). All animals had an intracranial pressure monitor, brain tissue oxygen tension (Pbto2) probe, and cerebral microdialysis probe placed in the frontal lobe and data collected for 6 hours following injury. RESULTS Injured animals had sustained elevations in intracranial pressure and lactate-pyruvate ratio (LPR), and decreased Pbto2 compared with sham. Pbto2 and LPR from separate frontal lobes had strong linear correlation in both sham and injured animals. Neuropathologic examination demonstrated significant axonal injury and infarct volumes in injured animals compared with sham at 6 hours postinjury. Averaged over time, Pbto2 in both injured and sham animals had a strong inverse correlation with total injury volume. Average LPR had a strong correlation with total injury volume. CONCLUSION LPR and Pbto2 can be utilized as serial nonterminal secondary markers in our injury model for neuropathology, and as evaluation metrics for novel interventions and therapeutics in the acute postinjury period. This translational model bridges a vital gap in knowledge between TBI studies in small-animal models and clinical trials in the pediatric TBI population. ABBREVIATIONS ANOVA: analysis of variance &bgr;-APP: &bgr;-amyloid precursor protein CPP: cerebral perfusion pressure H&E: hematoxylin and eosin ICP: intracranial pressure LPR: lactate-pyruvate ratio Pbto2: brain tissue oxygen tension TBI: traumatic brain injury

Development of a fluorescent microsphere technique for rapid histological determination of cerebral blood flow.

The purpose of this study was to develop a more efficient fluorescent microsphere method to facilitate the rapid use of the histological technique and to enable its use in large tissue regions. Using fluorescent plate/slide imaging technology and automated detection and analysis software, we were able to rapidly image, detect, and count 3 separate microsphere colors in 200 microm thick tissue sections from piglet brain. In resting newborn piglets (n=6) on isoflurane anesthesia, we measured a median total cerebral blood flow (CBF) of 105 ml/min/100g (range 27-206 ml/min/100 g). Compared with other FM analysis methods, our method reduces the time required to determine blood flow, improves accuracy in lipid-rich tissues and large tissue regions and, unlike the radiolabeled microsphere method, can be combined with histological analysis.

Improved Behavior, Motor, and Cognition Assessments in Neonatal Piglets

The alterations of animal behavior after traumatic brain injury (TBI) can be subtle, and their quantitative characterization can present significant methodological challenges. Meeting these challenges is a critical need, because quantitative measures are required in studies that compare the efficacy of different clinical interventions. We developed a battery of assessments to quantify behavioral, motor, and cognitive changes in neonatal piglets with good sensitivity and specificity to the detection of persistent deficits that correlate with axonal injury severity after a rapid non-impact head rotation with a diffuse pattern of axonal injury. The battery of measures developed included open field behaviors of sniffing and moving a toy, locomotion measures of Lempel-Ziv complexity and the probability of remaining in the current location, and a novel metric for evaluating motor performance. Our composite porcine disability score was able to detect brain injury with a sensitivity of 100% and specificity of 85.7% at day +4 post-injury for n=8 injured and n=7 sham piglets and significantly correlated with the percent axonal injury in these animals (day +4: ρ=0.76, p=0.0011). A significant improvement over our previous assessments, this new porcine disability score has potential use in a wide variety of porcine disease and injury models.

Mitochondrial bioenergetic alterations after focal traumatic brain injury in the immature brain.

Traumatic brain injury (TBI) is one of the leading causes of death in children worldwide. Emerging evidence suggests that alterations in mitochondrial function are critical components of secondary injury cascade initiated by TBI that propogates neurodegeneration and limits neuroregeneration. Unfortunately, there is very little known about the cerebral mitochondrial bioenergetic response from the immature brain triggered by traumatic biomechanical forces. Therefore, the objective of this study was to perform a detailed evaluation of mitochondrial bioenergetics using high-resolution respirometry in a high-fidelity large animal model of focal controlled cortical impact injury (CCI) 24h post-injury. This novel approach is directed at analyzing dysfunction in electron transport, ADP phosphorylation and leak respiration to provide insight into potential mechanisms and possible interventions for mitochondrial dysfunction in the immature brain in focal TBI by delineating targets within the electron transport system (ETS). Development and application of these methodologies have several advantages, and adds to the interpretation of previously reported techniques, by having the added benefit that any toxins or neurometabolites present in the ex-vivo samples are not removed during the mitochondrial isolation process, and simulates the in situ tricarboxylic acid (TCA) cycle by maximizing key substrates for convergent flow of electrons through both complexes I and II. To investigate alterations in mitochondrial function after CCI, ipsilateral tissue near the focal impact site and tissue from the corresponding contralateral side were examined. Respiration per mg of tissue was also related to citrate synthase activity (CS) and calculated flux control ratios (FCR), as an attempt to control for variability in mitochondrial content. Our biochemical analysis of complex interdependent pathways of electron flow through the electron transport system, by most measures, reveals a bilateral decrease in complex I-driven respiration and an increase in complex II-driven respiration 24h after focal TBI. These alterations in convergent electron flow though both complex I and II-driven respiration resulted in significantly lower maximal coupled and uncoupled respiration in the ipsilateral tissue compared to the contralateral side, for all measures. Surprisingly, increases in complex II and complex IV activities were most pronounced in the contralateral side of the brain from the focal injury, and where oxidative phosphorylation was increased significantly compared to sham values. We conclude that 24h after focal TBI in the immature brain, there are significant alterations in cerebral mitochondrial bioenergetics, with pronounced increases in complex II and complex IV respiration in the contralateral hemisphere. These alterations in mitochondrial bioenergetics present multiple targets for therapeutic intervention to limit secondary brain injury and support recovery.