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Unlabelled: Cardiac arrest (CA) results in a sepsis-like syndrome with activation of the innate immune system and increased mitochondrial bioenergetics., Objective: To determine if platelet mitochondrial respiration increases following CA in a porcine pediatric model of asphyxia-associated ventricular fibrillation (VF) CA, and if this readily obtained biomarker is associated with decreased brain mitochondrial respiration. CA protocol: 7 min of asphyxia, followed by VF, protocolized titration of compression depth to systolic blood pressure of 90 mmHg and vasopressor administration to a coronary perfusion pressure greater than 20 mmHg., Primary Outcome: platelet integrated mitochondrial electron transport system (ETS) function evaluated pre- and post-CA/ROSC four hours after return of spontaneous circulation (ROSC). Secondary outcome: correlation of platelet mitochondrial bioenergetics to cerebral bioenergetic function. Platelet maximal oxidative phosphorylation (OXPHOSCI+CII), P < 0.02, and maximal respiratory capacity (ETSCI+CII), P < 0.04, were both significantly increased compared to pre-arrest values. This was primarily due to a significant increase in succinate-supported respiration through Complex II (OXPHOSCII, P < 0.02 and ETSCII, P < 0.03). Higher respiration was not due to uncoupling, as the LEAKCI + CII respiration (mitochondrial respiration independent of ATP-production) was unchanged after CA/ROSC. Larger increases in platelet mitochondrial respiratory control ratio (RCR) compared to pre-CA RCR were significantly correlated with lower RCRs in the cortex (P < 0.03) and hippocampus (P < 0.04) compared to sham respiration. Platelet mitochondrial respiration is significantly increased four hours after ROSC. Future studies will identify mechanistic relationships between this serum biomarker and altered cerebral bioenergetics function following cardiac arrest.

Unique from other brain disorders, traumatic brain injury (TBI) generally results from a discrete biomechanical event that induces rapid head movement. The large size and high organization of the human brain makes it particularly vulnerable to traumatic injury from rotational accelerations that can cause dynamic deformation of the brain tissue. Therefore, replicating the injury biomechanics of human TBI in animal models presents a substantial challenge, particularly with regard to addressing brain size and injury parameters. Here we present the historical development and use of a porcine model of head rotational acceleration. By scaling up the rotational forces to account for difference in brain mass between swine and humans, this model has been shown to produce the same tissue deformations and identical neuropathologies found in human TBI. The parameters of scaled rapid angular accelerations applied for the model reproduce inertial forces generated when the human head suddenly accelerates or decelerates in falls, collisions, or blunt impacts. The model uses custom-built linkage assemblies and a powerful linear actuator designed to produce purely impulsive non-impact head rotation in different angular planes at controlled rotational acceleration levels. Through a range of head rotational kinematics, this model can produce functional and neuropathological changes across the spectrum from concussion to severe TBI. Notably, however, the model is very difficult to employ, requiring a highly skilled team for medical management, biomechanics, neurological recovery, and specialized outcome measures including neuromonitoring, neurophysiology, neuroimaging, and neuropathology. Nonetheless, while challenging, this clinically relevant model has proven valuable for identifying mechanisms of acute and progressive neuropathologies as well as for the evaluation of noninvasive diagnostic techniques and potential neuroprotective treatments following TBI.

There has been recent dramatic increase in the use of sensors affixed to the heads or helmets of athletes to measure the biomechanics of head impacts that lead to concussion. The relationship between injury and linear or rotational head acceleration measured by such sensors can be quantified with an injury risk curve. The utility of the injury risk curve relies on the accuracy of both the clinical diagnosis and the biomechanical measure. The focus of our analysis was to demonstrate the influence of three sources of error on the shape and interpretation of concussion injury risk curves: sampling variability associated with a rare event, concussion under-reporting, and sensor measurement error. We utilized Bayesian statistical methods to generate synthetic data from previously published concussion injury risk curves developed using data from helmet-based sensors on collegiate football players and assessed the effect of the three sources of error on the risk relationship. Accounting for sampling variability adds uncertainty or width to the injury risk curve. Assuming a variety of rates of unreported concussions in the non-concussed group, we found that accounting for under-reporting lowers the rotational acceleration required for a given concussion risk. Lastly, after accounting for sensor error, we find strengthened relationships between rotational acceleration and injury risk, further lowering the magnitude of rotational acceleration needed for a given risk of concussion. As more accurate sensors are designed and more sensitive and specific clinical diagnostic tools are introduced, our analysis provides guidance for the future development of comprehensive concussion risk curves., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Determining depth of anesthesia (DOA) is a clinical challenge in veterinary medicine, yet it is critical for the appropriate oversight of animals involved in potentially painful experimental procedures. Here, we investigated various parameters used to monitor conscious awareness during surgical procedures and refined the application of noxious stimuli to anesthetized animals. Specifically we used a common stimulus, a compressive toe pinch (TP), to determine physiologic changes that accompanied a positive or negative motion response in isoflurane-anesthetized piglets. A positive response was defined as any reflexive withdrawal, whereas a negative response was defined as the absence of motion after stimulation. We also assessed the utility of the bispectral index (BIS) for its ability to predict a motion response to TP. The average of BIS values over 1 min (BISmean) was recorded before and after TP. In piglets with a positive response to TP, heart rate (HR), but not blood pressure (BP), increased significantly, but receiver operating characteristic (ROC) analysis revealed that HR was not a sensitive, specific predictor of TP motion response. Both before and after TP, BISmean was a strong predictor of a positive motion response. We conclude that HR and noninvasive BP changes are not clinically reliable indicators of anesthetic depth when assessed immediately after a peripherally applied compressive force as an indicator stimulus; however, BISmean and response TP are acceptable for assessing DOA in piglets maintained under isoflurane anesthesia.

A systematic correlation between finite element models (FEMs) and histopathology is needed to define deformation thresholds associated with traumatic brain injury (TBI). In this study, a FEM of a transected piglet brain was used to reverse engineer the range of optimal shear moduli for infant (5 days old, 553-658 Pa) and 4-week-old toddler piglet brain (692-811 Pa) from comparisons with measured in situ tissue strains. The more mature brain modulus was found to have significant strain and strain rate dependencies not observed with the infant brain. Age-appropriate FEMs were then used to simulate experimental TBI in infant (n=36) and preadolescent (n=17) piglets undergoing a range of rotational head loads. The experimental animals were evaluated for the presence of clinically significant traumatic axonal injury (TAI), which was then correlated with FEM-calculated measures of overall and white matter tract-oriented tissue deformations, and used to identify the metric with the highest sensitivity and specificity for detecting TAI. The best predictors of TAI were the tract-oriented strain (6-7%), strain rate (38-40 s(-1), and strain times strain rate (1.3-1.8 s(-1) values exceeded by 90% of the brain. These tract-oriented strain and strain rate thresholds for TAI were comparable to those found in isolated axonal stretch studies. Furthermore, we proposed that the higher degree of agreement between tissue distortion aligned with white matter tracts and TAI may be the underlying mechanism responsible for more severe TAI after horizontal and sagittal head rotations in our porcine model of nonimpact TAI than coronal plane rotations.

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., (© 2015 International Society of Neuropathology.)

Mechanical ventilation with high tidal volumes has been associated with pulmonary alveolar flooding. Understanding the mechanisms underlying cyclic stretch-induced increases in alveolar epithelial permeability may be important in designing preventive measures for acute lung injury. In this work, we assessed whether cyclic stretch leads to the generation of reactive oxygen species in type I-like alveolar epithelial cells, which increase monolayer permeability via activation of NF-κB and extracellular signal-regulated kinase (ERK). We cyclically stretched type I-like rat primary alveolar epithelial cells at magnitudes of 12, 25, and 37% change in surface area (ΔSA) for 10 to 120 minutes. High levels of reactive oxygen species and of superoxide and NO specifically were detected in cells stretched at 37% ΔSA for 10 to 120 minutes. Exogenous superoxide and NO stimulation increased epithelial permeability in unstretched cells, which was preventable by the NF-κB inhibitor MG132. The cyclic stretch-induced increase in permeability was decreased by the superoxide scavenger tiron and by MG132. Furthermore, tiron had a dramatic protective effect on in vivo lung permeability under mechanical ventilation conditions. Cyclic stretch increased the activation of the NF-κB signaling pathway, which was significantly decreased with the ERK inhibitor U0126. Altogether, our in vitro and in vivo data demonstrate the sensitivity of permeability to stretch- and ventilation-induced superoxide production, suggesting that using antioxidants may be helpful in the prevention and treatment of ventilator-induced lung injury.

Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type.

Computer finite element model (FEM) simulations are often used as a substitute for human experimental head injury studies to enhance our understanding of injury mechanisms and develop prevention strategies. While numerous adult FEM of the head have been developed, there are relatively few pediatric FEM due to the paucity of material property data for children. Using radiological serial images of infants (<6 wks old) and recent published material property data of infant skull and suture, we developed a FEM of the infant head to study skull fracture from occipital impacts. Here we determined the relative importance of brain material properties and anatomical variations in infant suture and scalp tissue on principal stress (sigma(p)) estimates in the skull of the model using parametric simulations of occipital impacts from 0.3m falls onto concrete. Decreasing the brain stiffness of pediatric brain tissue by a factor of two to simulate the softer adult brain properties we reported previously did not affect sigma(p). Using adult brain stiffness reported by others (4 times higher than our pediatric values) increased sigma(p) in skull by 38%. Interestingly, the precision used to model compressibility of the brain (0.49-0.4999) significantly varied sigma(p) 30-77%, underscoring the influence of the brain properties in models of fracture in the highly deformable infant skullcase. Suture thickness, small anatomical variations in suture width and the exclusion of scalp did not affect sigma(p) of the skull; however, unusually large sutures (10 mm) in young infants significantly lowered sigma(p). Validation of this model against published infant cadaver drop studies found good agreement with the prediction of fracture for falls onto hard surfaces. More biomechanical data from impacts onto softer surfaces is needed before skull fracture predictions can be made in these scenarios. In summary, the pediatric FEM response is not sensitive to small variations in anatomy or brain modulus, large deviations will significantly influence principal stress estimates and the prediction of skull fracture.

Finite element (FE) models are used to identify head injury mechanisms and design new and improved injury prevention schemes. Although brain-skull boundary conditions strongly influence the model mechanical responses, limited experimental data are available to develop an informed representation. We hypothesize that the spinal cord tension and gravity contribute to the pons displacement in vivo. Static high-resolution T1-weighted sagittal MR images of the inferior portion of the head in neutral and flexion positions were acquired in 15 human volunteers in both supine and prone postures. Boundaries of the pons and clivus were extracted with a gradient-based algorithm, and the pontes were fitted into ellipses. Assuming rigid body motion of the skull, image pairs in different postures were co-registered with an autocorrelation technique. By comparing images before and after the motion, we found that while the rotation of the pons is negligible relative to the skull, the pons displaces significantly at the foramen magnum, on the order of approximately 2 mm. When the spinal cord tension and gravity act in concert, the pons moves caudally; when opposed, superiorly, such that the influence of gravity on the pons is six times that of the spinal cord tension. Based on these findings, we recommend that the brainstem-skull interface be treated as a sliding (with or without friction) boundary condition in FE models of the human head.

While a number of whole cell mechanical models have been proposed, few, if any, have focused on the relationship among plasma membrane tension, plasma membrane unfolding, and plasma membrane expansion and relaxation via lipid insertion. The goal of this communication is to develop such a model to better understand how plasma membrane tension, which we propose stimulates Na(+)-K(+)-ATPase activity but possibly also causes cell injury, may be generated in alveolar epithelial cells during mechanical ventilation. Assuming basic relationships between plasma membrane unfolding and tension and lipid insertion as the result of tension, we have captured plasma membrane mechanical responses observed in alveolar epithelial cells: fast deformation during fast cyclic stretch, slower, time-dependent deformation via lipid insertion during tonic stretch, and cell recovery after release from stretch. The model estimates plasma membrane tension and predicts Na(+)-K(+)-ATPase activation for a specified cell deformation time course. Model parameters were fit to plasma membrane tension, whole cell capacitance, and plasma membrane area data collected from the literature for osmotically swollen and shrunken cells. Predictions of membrane tension and stretch-stimulated Na(+)-K(+)-ATPase activity were validated with measurements from previous studies. As a proof of concept, we demonstrate experimentally that tonic stretch and consequent plasma membrane recruitment can be exploited to condition cells against subsequent cyclic stretch and hence mitigate stretch-induced responses, including stretch-induced cell death and stretch-induced modulation of Na(+)-K(+)-ATPase activity. Finally, the model was exercised to evaluate plasma membrane tension and potential Na(+)-K(+)-ATPase stimulation for an assortment of traditional and novel ventilation techniques.

The objective of this study was to define the constitutive response of brainstem undergoing finite shear deformation. Brainstem was characterized as a transversely isotropic viscoelastic material and the material model was formulated for numerical implementation. Model parameters were fit to shear data obtained in porcine brainstem specimens undergoing finite shear deformation in three directions: parallel, perpendicular, and cross sectional to axonal fiber orientation and determined using a combined approach of finite element analysis (FEA) and a genetic algorithm (GA) optimizing method. The average initial shear modulus of brainstem matrix of 4-week old pigs was 12.7 Pa, and therefore the brainstem offers little resistance to large shear deformations in the parallel or perpendicular directions, due to the dominant contribution of the matrix in these directions. The fiber reinforcement stiffness was 121.2 Pa, indicating that brainstem is anisotropic and that axonal fibers have an important role in the cross-sectional direction. The first two leading relative shear relaxation moduli were 0.8973 and 0.0741, respectively, with corresponding characteristic times of 0.0047 and 1.4538 s, respectively, implying rapid relaxation of shear stresses. The developed material model and parameter estimation technique are likely to find broad applications in neural and orthopaedic tissues.

Finite element models are increasingly important in understanding head injury mechanisms and designing new injury prevention equipment. Although boundary conditions strongly influence model responses, only limited quantitative data are available. While experimental studies revealed some motion between brain and skull, little data exists regarding the base of the skull. Using magnetic resonance images (MRI) of the caudal brain regions, we measured in vivo, quasi-static angular displacement of the cerebellum (CB) and brainstem (BS) relative to skull, and axial displacement of BS at the foramen magnum in supine human subjects (N=5). Images were obtained in flexion (7 degrees - 54 degrees ) and neutral postures using SPAMM tagging technique (N=47 pairs). Rigid body skull rotation angle from neutral posture (theta, degrees) was determined by extracting the edge feature points of the skull, and rotating and displacing the coordinates in one image until they matched those in the other. Tissue rotation was obtained by comparing tag lines in image pairs before and after flexion, and the motion of BS and CB were expressed relative to skull rotation and displacement. During flexion, the CB rotated in the flexion direction, exceeding the skull rotation, but relative BS rotations were negligible. Meanwhile, the BS moved caudally toward the foramen magnum. With a flexion angle of 54 degrees , the 95% confidence interval for the relative CB rotation was 2.7 degrees - 4.3 degrees , and 0.8 - 1.6mm for the relative BS axial displacement. Albeit quasi-static, this study provides important data that can be implemented to create more life-like boundary conditions in human finite element models.

Mechanical ventilation with high tidal volumes has been shown to contribute to the formation or worsening of interstitial and alveolar edema. Previously we showed that application of large biaxial deformations in vitro perturbs the concentration and distribution of functional tight junction proteins in alveolar epithelial cells. Using a novel method, we determined that applied epithelial strain increases paracellular permeability in a dose- and rate-dependent manner. Primary rat alveolar epithelial cells were subjected to 12%, 25%, or 37% change in surface area (Delta SA) cyclic equibiaxial stretch for 1 h. Cells were also stretched noncyclically at 25% Delta SA for 1 h. During the experimental period, a fluorescently tagged ouabain derivative was added to the apical fluid. Evidence of binding indicated functional failure of the paracellular transport barrier. The percentage of field area stained was quantified from microscopic images. There was no significant evidence of basolateral fluorescent staining at 12% Delta SA or at 25% Delta SA applied cyclically or statically. However, cyclic stretch at 37% Delta SA resulted in significantly more staining than in unstretched cells (P < 0.0001) or those stretched at either 12% (P < 0.0001) or 25% cyclic (P < 0.0005) or static (P < 0.05) Delta SA. These results suggest that large cyclic tidal volumes may increase paracellular permeability, potentially resulting in alveolar flooding.

The large strain mechanical properties of adult porcine gray and white matter brain tissues were measured in shear and confirmed in compression. Consistent with local neuroarchitecture, gray matter showed the least amount of anisotropy, and corpus callosum exhibited the greatest degree of anisotropy. Mean regional properties were significantly distinct, demonstrating that brain tissue is inhomogeneous. Fresh adult human brain tissue properties were slightly stiffer than adult porcine properties but considerably less stiff than the human autopsy data in the literature. Mixed porcine gray/white matter samples were obtained from animals at "infant" and "toddler" stages of neurological development, and shear properties compared to those in the adult. Only the infant properties were significantly different (stiffer) from the adult.