Sarah Sullivan

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.

Finite Element Model Predictions of Intracranial Hemorrhage from Non-Impact, Rapid Head Rotations in the Piglet

Clinicians are charged with the significant task of distinguishing between accidental and inflicted head trauma. Oftentimes this distinction is straightforward, but many times probabilities of injuries from accidental scenarios are unknown making the differential diagnosis difficult. For example, it is unknown whether intracranial hemorrhage (IH) can occur at a location other than a focal contact site following a low height fall. To create a foundation for predicting regional IH in infants, we sought to identify the biomechanical response and injury threshold best able to predict IH in 3–5 day old piglets. First, finite element (FE) model simulations of in situ animal studies were performed to ascertain the optimal representation of the pia‐arachnoid complex, cerebrospinal fluid and cortical vasculature (PCC) for predicting brain strain and brain/skull displacement. Second, rapid head rotations resulting in various degrees of IH were simulated (n = 24) to determine the biomechanical predictor and injury threshold most closely correlated with IH. FE models representing the PCC with either spring connectors or solid elements between the brain and skull resulted in peak brain strain and brain/skull displacement similar to measured values in situ. However, when predicting IH, the spring connector representation of the PCC had the best predictive capability for IH with a sensitivity of 80% and a specificity of 85% when ≥1% of all spring connectors had at least a peak strain of 0.31 mm/mm. These findings and reported methodology will be used in the development of a human infant FE model to simulate real‐world falls and identify injury thresholds for predicting IH in infants.

Influences of developmental age on the resolution of diffuse traumatic intracranial hemorrhage and axonal injury

This study investigated the age-dependent injury response of diffuse traumatic axonal injury (TAI) and regional subdural and subarachnoid intracranial hemorrhage (ICH) in two pediatric age groups using a porcine head injury model. Fifty-five 5-day-old and 40 four-week-old piglets-which developmentally correspond to infants and toddlers, respectively-underwent either a sham injury or a single rapid non-impact rotational injury in the sagittal plane and were grouped by post-TBI survival time (sham, 3-8 h, one day, 3-4 days, and 5-6 days). Both age groups exhibited similar initial levels of ICH and a significant reduction of ICH over time (p<0.0001). However, ICH took longer to resolve in the five-day-old age group. At 5-6 days post-injury, ICH in the cerebrum had returned to sham levels in the four-week-old piglets, while the five-day-olds still had significantly elevated cerebral ICH (p=0.012). Both ages also exhibited similar resolution of axonal injury with a peak in TAI at one day post-injury (p<0.03) and significantly elevated levels even at 5-6 days after the injury (p<0.008), which suggests a window of vulnerability to a second insult at one day post-injury that may extend for a prolonged period of time. However, five-day-old piglets had significantly more TAI than four-week-olds overall (p=0.016), which presents some evidence for an increased vulnerability to brain injury in this age group. These results provide insight into an optimal window for clinical intervention, the period of increased susceptibility to a second injury, and an age dependency in brain injury tolerance within the pediatric population.

Establishing a Clinically Relevant Large Animal Model Platform for TBI Therapy Development: Using Cyclosporin A as a Case Study

We have developed the first immature large animal translational treatment trial of a pharmacologic intervention for traumatic brain injury (TBI) in children. The preclinical trial design includes multiple doses of the intervention in two different injury types (focal and diffuse) to bracket the range seen in clinical injury and uses two post‐TBI delays to drug administration. Cyclosporin A (CsA) was used as a case study in our first implementation of the platform because of its success in multiple preclinical adult rodent TBI models and its current use in children for other indications. Tier 1 of the therapy development platform assessed the short‐term treatment efficacy after 24 h of agent administration. Positive responses to treatment were compared with injured controls using an objective effect threshold established prior to the study. Effective CsA doses were identified to study in Tier 2. In the Tier 2 paradigm, agent is administered in a porcine intensive care unit utilizing neurological monitoring and clinically relevant management strategies, and intervention efficacy is defined as improvement in longer term behavioral endpoints above untreated injured animals. In summary, this innovative large animal preclinical study design can be applied to future evaluations of other agents that promote recovery or repair after TBI.

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.

Noninvasive metrics for identification of brain injury deficits in piglets.

Balance and bispectral index metrics were evaluated in piglets following focal and diffuse brain injury. A significant decrease in bispectral index existed at 24 hours after diffuse brain injury, but not after focal injury. Postural sway increased at 1–6 hours after both focal and diffuse injuries.

Biofidelic neck influences head kinematics of parietal and occipital impacts following short falls in infants

Falls are a major cause of traumatic head injury in children. Understanding head kinematics during low height falls is essential for evaluating injury risk and designing mitigating strategies. Typically, these measurements are made with commercial anthropomorphic infant surrogates, but these surrogates are designed based on adult biomechanical data. In this study, we improve upon the state-of-the-art anthropomorphic testing devices by incorporating new infant cadaver neck bending and tensile data. We then measure head kinematics following head-first falls onto 4 impact surfaces from 3 fall heights with occipital and parietal head impact locations. The biofidelic skull compliance and neck properties of the improved infant surrogate significantly influenced the measured kinematic loads, decreasing the measured impact force and peak angular accelerations, lowering the expected injury risk. Occipital and parietal impacts exhibited distinct kinematic responses in primary head rotation direction and the magnitude of the rotational velocities and accelerations, with larger angular velocities as the head rebounded after occipital impacts. Further evaluations of injury risk due to short falls should take into account the impact surface and head impact location, in addition to the fall height.

Alterations in Daytime and Nighttime Activity in Piglets after Focal and Diffuse Brain Injury

We have developed and implemented a noninvasive, objective neurofunctional assessment for evaluating the sustained effects of traumatic brain injury (TBI) in piglets with both diffuse and focal injury types. Derived from commercial actigraphy methods in humans, this assessment continuously monitors the day/night activity of piglets using close-fitting jackets equipped with tri-axial accelerometers to monitor movements of the thorax. Acceleration metrics were correlated (N = 7 naïve piglets) with video images to define values associated with a range of activities, from recumbancy (rest) to running. Both focal (N = 8) and diffuse brain injury (N = 9) produced alterations in activity that were significant 4 days post-TBI. Compared to shams (N = 6) who acclimated to the animal facility 4 days after an anesthesia experience by blurring the distinction between day and night activity, post-TBI time-matched animals had larger fractions of inactive periods during the daytime than nighttime, and larger fractions of active time in the night were spent in high activity (e.g., constant walking, intermittent running) than during the day. These persistent disturbances in rest and activity are similar to those observed in human adults and children post-TBI, establishing actigraphy as a translational metric, used in both humans and large animals, for assessment of injury severity, progressions, and intervention.

Establishing a Clinically Relevant Large Animal Model Platform for TBI Therapy Development

We have developed the first immature large animal translational treatment trial of a pharmacologic intervention for traumatic brain injury (TBI) in children. The preclinical trial design includes multiple doses of the intervention in two different injury types (focal and diffuse) to bracket the range seen in clinical injury and uses two post‐TBI delays to drug administration. Cyclosporin A (CsA) was used as a case study in our first implementation of the platform because of its success in multiple preclinical adult rodent TBI models and its current use in children for other indications. Tier 1 of the therapy development platform assessed the short‐term treatment efficacy after 24 h of agent administration. Positive responses to treatment were compared with injured controls using an objective effect threshold established prior to the study. Effective CsA doses were identified to study in Tier 2. In the Tier 2 paradigm, agent is administered in a porcine intensive care unit utilizing neurological monitoring and clinically relevant management strategies, and intervention efficacy is defined as improvement in longer term behavioral endpoints above untreated injured animals. In summary, this innovative large animal preclinical study design can be applied to future evaluations of other agents that promote recovery or repair after TBI.