Diagnosis of traumatic brain injury using miRNA signatures in nanomagnetically isolated brain-derived extracellular vesicles.

The accurate diagnosis and clinical management of traumatic brain injury (TBI) is currently limited by the lack of accessible molecular biomarkers that reflect the pathophysiology of this heterogeneous disease. To address this challenge, we developed a microchip diagnostic that can characterize TBI more comprehensively using the RNA found in brain-derived extracellular vesicles (EVs). Our approach measures a panel of EV miRNAs, processed with machine learning algorithms to capture the state of the injured and recovering brain. Our diagnostic combines surface marker-specific nanomagnetic isolation of brain-derived EVs, biomarker discovery using RNA sequencing, and machine learning processing of the EV miRNA cargo to minimally invasively measure the state of TBI. We achieved an accuracy of 99% identifying the signature of injured vs. sham control mice using an independent blinded test set (N = 77), where the injured group consists of heterogeneous populations (injury intensity, elapsed time since injury) to model the variability present in clinical samples. Moreover, we successfully predicted the intensity of the injury, the elapsed time since injury, and the presence of a prior injury using independent blinded test sets (N = 82). We demonstrated the translatability in a blinded test set by identifying TBI patients from healthy controls (AUC = 0.9, N = 60). This approach, which can detect signatures of injury that persist across a variety of injury types and individual responses to injury, more accurately reflects the heterogeneity of human TBI injury and recovery than conventional diagnostics, opening new opportunities to improve treatment of traumatic brain injuries.

Relationship between structural modeling and hyperelastic material behavior: application to CNS white matter.

Recent measurements of the material properties of brain tissue allow an examination of the underlying microstructural basis in both physiological and pathophysiological conditions. The purpose of this study is to develop a mathematical relationship between microstructurally based models of the central nervous system (CNS) white matter and equivalent hyperelastic material models. For simplicity, time dependent material behavior is not included in this formulation. The microstructural representation is used to formulate structural property relationships for highly oriented white matter, and is mathematically compared to one isotropic and two anisotropic hyperelastic formulations. For the anisotropic characterizations, the population of axons in the white matter is assumed to align along one preferred direction of the material, yielding a transversely isotropic formulation. Relatively simple strain-energy functions incorporating material anisotropy provide sufficient flexibility to model the nonlinear behavior predicted from structurally based models, although the tangential stiffness of the hyperelastic approaches does not follow completely the behavior of the structurally based formulations. This analysis is an initial step towards linking microstructural aspects of the tissue to material models commonly used for large deformations, and may be an important step in relating predicted tissue deformation to the deformation and stress of cellular and subcellular structures.

Dynamic stretch correlates to both morphological abnormalities and electrophysiological impairment in a model of traumatic axonal injury.

In this investigation, the relationships between stretch and both morphological and electrophysiological signs of axonal injury were examined in the guinea pig optic nerve stretch model. Additionally, the relationship between axonal morphology and electrophysiological impairment was assessed. Axonal injury was produced in vivo by elongating the guinea pig optic nerve between 0 and 8 mm (Ntotal = 70). Morphological damage was detected using neurofilament immunohistochemistry (SMI 32). Electrophysiological impairment was determined using changes in visual evoked potentials (VEPs) measured prior to injury, every 5 min for 40 min following injury, and at sacrifice (72 h). All nerves subjected to ocular displacements greater than 6 mm demonstrated axonal swellings and retraction bulbs, while nerves subjected to displacements below 4 mm did not show any signs of morphological injury. Planned comparisons of latency shifts of the N35 peak in the VEPs showed that ocular displacements greater than 5 mm produced electrophysiological impairment that was significantly different from sham animals. Logit analysis demonstrated that less stretch was required to elicit electrophysiological changes (5.5 mm) than morphological signs of damage (6.8 mm). Moreover, Student t tests indicated that the mean latency shift measured in animals exhibiting morphological injury was significantly greater than that calculated from animals lacking morphological injury (p < 0.01). These data show that distinct mechanical thresholds exist for both morphological and electrophysiological damage to the white matter. In a larger context, the distinct injury thresholds presented in the report will aid in the biomechanical assessment of animate models of head injury, as well as assist in extending these findings to predict the conditions that cause white matter injury in humans.

A new strategy to produce sustained growth of central nervous system axons: continuous mechanical tension.

Although a primary strategy to repair spinal cord and other nerve injuries is to bridge the damage with axons, producing axons of sufficient length and number has posed a significant challenge. Here, we explored the ability of integrated central nervous system (CNS) axons to grow long distances in response to continuous mechanical tension. Neurons were plated on adjacent membranes and allowed to integrate, including the growth of axons across a 50-microm border between the two membranes. Using a microstepper motor system, we then progressively separated the two membranes further apart from each other at the rate of 3.5 microm every 5 min. In the expanding gap, we found thick bundles comprised of thousands of axons that responded to this tensile elongation by growing a remarkable 1 cm in length by 10 days of stretch. This is the first evidence that the center portion of synapsed CNS axons can exhibit sustained "stretch-induced growth." This may represent an important growth mechanism for the elongation of established white matter tracts during development. We also found by doubling the stretch rate to 7 microm/5 min that the axon bundles could not maintain growth and disconnected in the center of the gap by 3 days of stretch, demonstrating a tolerance limit for the rate of axonal growth. We propose that this newfound stretch-induced growth ability of integrated CNS axons may be exploited to produce transplant materials to bridge extensive nerve damage.

Numerical model and experimental validation of microcarrier motion in a rotating bioreactor.

The equations of motion for microcarriers in a rotating bioreactor have been formulated and trajectories obtained using numerical techniques. An imaging system was built to validate the results by direct observation of microcarrier trajectories in the rotating frame of reference. The microcarrier motion observed by this imaging system was in excellent agreement with the numerical predictions of that motion. In the rotating frame of reference, microcarriers with density greater than the surrounding fluid medium followed a circular motion relative to the culture medium combined with a persistent migration and eventual collision with the outer wall of the reactor. However, for microcarrier density less the fluid medium, their circular motion migrated toward the central region of the reactor. When multiple microcarrier beads that are lighter than water are inserted into the reactor, the centrally directed migration results in the formation of clusters that are stabilized by tissue bridges formed by osteoblasts seeded onto the microcarriers. This system offers unique opportunities to monitor tissue synthesis on microcarriers using real-time optical techniques and to optimize the bioreactor operating conditions for exploiting this technology to study early bone tissue synthesis in vitro.

TUNEL-positive staining of surface contusions after fatal head injury in man.

In frontal lobe contusions obtained post mortem from 18 patients who survived between 6 h and 10 days after head injury, DNA fragmentation associated with either apoptotic and/or necrotic cell death was identified by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labelling (TUNEL) histochemical technique. Additional histological techniques were also used to identify regional and temporal patterns of tissue damage. TUNEL-positive cells were present in both the grey and white matter of the contusion, where they peaked in number between 25 and 48 h, and were still identifiable at 10 days post injury. Fewer TUNEL-positive cells were observed in grey than in white matter; and most TUNEL-positive neurons in the grey matter demonstrated the morphological features of necrosis. However, the morphology of some TUNEL-stained neurons, and of TUNEL-stained oligodendroglia and macrophages in white matter was suggestive of apoptosis. Apoptosis was not seen in age- and sex-matched controls, none of whom had died from intracranial pathology or had pre-existing neurological disease. These findings suggest that multiple cell types in frontal lobe contusions exhibit DNA fragmentation and that both necrosis and apoptosis are likely to contribute to post-traumatic pathology. These findings provide further evidence that the observations made in animal models of traumatic brain injury have fidelity with clinical head injury.

Immediate coma following inertial brain injury dependent on axonal damage in the brainstem.

OBJECT: Immediate and prolonged coma following brain trauma has been shown to result from diffuse axonal injury (DAI). However, the relationship between the distribution of axonal damage and posttraumatic coma has not been examined. In the present study, the authors examine that relationship. METHODS: To explore potential anatomical origins of posttraumatic coma, the authors used a model of inertial brain injury in the pig. Anesthetized miniature swine were subjected to a nonimpact-induced head rotational acceleration along either the coronal or axial plane (six pigs in each group). Immediate prolonged coma was consistently produced by head axial plane rotation, but not by head coronal plane rotation. Immunohistochemical examination of the injured brains revealed that DAI was produced by head rotation along both planes in all animals. However, extensive axonal damage in the brainstem was found in the pigs injured via head axial plane rotation. In these animals, the severity of coma was found to correlate with both the extent of axonal damage in the brainstem (p < 0.01) and the applied kinetic loading conditions (p < 0.001). No relationship was found between coma and the extent of axonal damage in other brain regions. CONCLUSIONS: These results suggest that injury to axons in the brainstem plays a major role in induction of immediate posttraumatic coma and that DAI can occur without coma.

Dynamic mechanical stretch of organotypic brain slice cultures induces differential genomic expression: relationship to mechanical parameters.

Although the material properties of biological tissues are reasonably well established, recent studies have suggested that the biological response of brain tissue and its constituent cells may also be viscoelastic and sensitive to both the magnitude and rate of a mechanical stimulus. Given the potential involvement of changes in gene expression in the pathogenic sequelae after head trauma, we analyzed the expression of 22 genes related to cell death and survival and found that a number of these genes were differentially regulated after mechanical stretch of an organotypic brain slice culture. Twenty-four hours after stretch, the expression of BDNF, NGF, and TrkA was significantly increased, whereas that of bcl-2, CREB, and GAD65 was significantly decreased (MANOVA followed by ANOVA, p < 0.05). Expression of CREB and GAD65 was negatively correlated with strain, whereas expression of APP695 was negatively correlated with strain rate (all p < 0.05). This study demonstrates that a subset of genes involved in cell death and survival are differentially regulated after dynamic stretch in vitro and that the expression of specific genes is correlated with mechanical parameters of that stretch.

Traumatic injury induces differential expression of cell death genes in organotypic brain slice cultures determined by complementary DNA array hybridization.

The expression of a large panel of selected genes hypothesized to play a central role in post-traumatic cell death was shown to be differentially altered in response to a precisely controlled, mechanical injury applied to an organotypic slice culture of the rat brain. Within 48 h of injury, the expression of nerve growth factor messenger RNA was significantly increased whereas the levels of bcl-2, alpha-subunit of calcium/calmodulin-dependent protein kinase II, cAMP response element binding protein, 65,000 mol. wt isoform of glutamate decarboxylase, 1beta isoform of protein kinase C, and ubiquitin messenger RNA were significantly decreased. Because the expression levels of a number of other messenger RNAs such as the neuron-specific amyloid precursor protein, beta(2) microglobulin, bax, bcl(xl), brain-derived neurotrophic factor, cyclooxygenase-2, interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, receptor tyrosine kinase A, and receptor tyrosine kinase B were unaffected, these selective changes may represent components of an active and directed response of the brain initiated by mechanical trauma. Interpretation of these co-ordinated alterations suggests that mechanical injury to the central nervous system may lead to disruption of calcium homeostasis resulting in altered gene expression, an impairment of intracellular cascades responsible for trophic factor signaling, and initiation of apoptosis via multiple pathways. An understanding of these transcriptional changes may contribute to the development of novel therapeutic strategies to enhance beneficial and blunt detrimental, endogenous, post-injury response mechanisms.

Tissue-level thresholds for axonal damage in an experimental model of central nervous system white matter injury.

In vivo, tissue-level, mechanical thresholds for axonal injury were determined by comparing morphological injury and electrophysiological impairment to estimated tissue strain in an in vivo model of axonal injury. Axonal injury was produced by dynamically stretching the right optic nerve of an adult male guinea pig to one of seven levels of ocular displacement (Nlevel = 10; Ntotal = 70). Morphological injury was detected with neurofilament immunohistochemical staining (NF68, SM132). Simultaneously, functional injury was determined by the magnitude of the latency shift of the N35 peak of the visual evoked potentials (VEPs) recorded before and after stretch. A companion set of in situ experiments (Nlevel = 5) was used to determine the empirical relationship between the applied ocular displacement and the magnitude of optic nerve stretch. Logistic regression analysis, combined with sensitivity and specificity measures and receiver operating characteristic (ROC) curves were used to predict strain thresholds for axonal injury. From this analysis, we determined three Lagrangian strain-based thresholds for morphological damage to white matter. The liberal threshold, intended to minimize the detection of false positives, was a strain of 0.34, and the conservative threshold strain that minimized the false negative rate was 0.14. The optimal threshold strain criterion that balanced the specificity and sensitivity measures was 0.21. Similar comparisons for electrophysiological impairment produced liberal, conservative, and optimal strain thresholds of 0.28, 0.13, and 0.18, respectively. With these threshold data, it is now possible to predict more accurately the conditions that cause axonal injury in human white matter.

Defining brain mechanical properties: effects of region, direction, and species.

No regional or directional large-deformation constitutive data for brain exist in the current literature. To address this deficiency, the large strain (up to 50%) directional properties of gray and white matter were determined in the thalamus, corona radiata, and corpus callosum. The constitutive relationships of all regions and directions are well fit by an Ogden hyperelastic relationship, modified to include dissipation. The material parameter alpha, representing the non-linearity of the tissue, was not significantly sensitive to region, direction, or species. The average value of the material parameter mu, corresponding to the shear modulus of the tissue, was significantly different for each region, demonstrating that brain tissue is inhomogeneous. In each region, mu, obtained in 2 orthogonal directions, was compared. Consistent with local neuroarchitecture, gray matter showed the least amount of anisotropy and corpus callosum exhibited the greatest degree of anisotropy. Finally, human temporal lobe gray matter properties were determined and compared to porcine thalamic properties. The results show significant regional inhomogeneity at large strains and significant anisotropy in each region tested. The extent of regional anisotropy correlated with the degree of alignment in the local neuroarchitecture. These large strain, regional and directional data should enhance the biofidelity of computational models and provide important information regarding the mechanisms of traumatic brain injury.

Accumulation of amyloid beta and tau and the formation of neurofilament inclusions following diffuse brain injury in the pig.

Brain trauma in humans increases the risk for developing Alzheimer disease (AD) and may induce the acute formation of AD-like plaques containing amyloid beta (A beta). To further explore the potential link between brain trauma and neurodegeneration, we conducted neuropathological studies using a pig model of diffuse brain injury. Brain injury was induced in anesthetized animals via nonimpact head rotational acceleration of 110 degrees over 20 ms in the coronal plane (n = 15 injured, n = 3 noninjured). At 1, 3, 7, and 10 days post-trauma, control and injured animals were euthanized and immunohistochemical analysis was performed on brain sections using antibodies specific for A beta, beta-amyloid precursor protein (betaPP), tau, and neurofilament (NF) proteins. In addition to diffuse axonal pathology, we detected accumulation of A beta and tau that colocalized with immunoreactive betaPP and NF in damaged axons throughout the white matter in all injured animals at 3-10 days post-trauma. In a subset of brain injured animals, diffuse A beta-containing plaque-like profiles were found in both the gray and white matter, and accumulations of tau and NF rich inclusions were observed in neuronal perikarya. These results show that this pig model of diffuse brain injury is characterized by accumulations of proteins that also form pathological aggregates in AD and related neurodegenerative diseases.

Evolution of neurofilament subtype accumulation in axons following diffuse brain injury in the pig.

Although accumulation of neurofilament (NF) proteins in axons has been recognized as a prominent feature of brain trauma, the temporal course of the accumulation of specific NF subtypes has not been well established. In the present study, 17 miniature swine were subjected to nonimpact inertial brain injury. At 3 hours (h), 6 h, 24 h, 3 days, 7 days, and 10 days post-trauma, immunohistochemical analysis was performed to determine axonal accumulation of NF-light (NF-L), the rod and sidearm domains and sidearm phosphorylation states of NF-medium (NF-M), and heavy (NF-H). We found that NF-L accumulation was easily identified in damaged axons by 6 h post-trauma, but NF-M and H accumulation was not clearly visualized until 3 days following injury. In addition, the axonal accumulation of NF-M and H appeared to be primarily comprised of the sidearm domains. While the accumulating NF was found to be predominantly dephosphorylated, we also detected accumulation of phosphorylated NF. Finally, we found that developing axonal pathology may proceed either towards axotomy with discrete terminal bulb formation or towards the development of varicose swellings encompassing long portions of axons. These findings suggest that there is a differential temporal course in NF subtype disassembly, dephosphorylation, and accumulation in axons following initial brain trauma and that these processes occur in morphologically distinct phenotypes of maturing axonal pathology.

Diffuse axonal pathology detected with magnetization transfer imaging following brain injury in the pig.

This study was designed to evaluate with magnetization transfer imaging (MTI) and conventional magnetic resonance (MR) imaging the manifestation of diffuse axonal injury (DAI) in an animal model of injury via nonimpact coronal plane rotational acceleration. A second objective was to investigate the diagnostic use of quantitative MTR imaging based on statistical parameters in a single subject, as opposed to grouped analysis. Seven mini-swine were subjected to brain trauma known to produce isolated DAI and to MR imaging at two time points. Following sacrifice, the brains were harvested for histopathologic examination. Magnetization transfer ratio (MTR) maps were generated for double-blinded comparison of regions with abnormal MTR values and regions with documented DAI. Positive and negative predictive values for MTR detection of DAI were 67 and 56%, respectively, and in acute studies alone, 89 and 61%. Gains in sensitivity over conventional imaging for detection of DAI were demonstrated.

High tolerance and delayed elastic response of cultured axons to dynamic stretch injury.

Although axonal injury is a common feature of brain trauma, little is known of the immediate morphological responses of individual axons to mechanical injury. Here, we developed an in vitro model system that selectively stretches axons bridging two populations of human neurons derived from the cell line N-Tera2. We found that these axons demonstrated a remarkably high tolerance to dynamic stretch injury, with no primary axotomy at strains <65%. In addition, the axolemma remained impermeable to small molecules after injury unless axotomy had occurred. We also found that injured axons exhibited the behavior of "delayed elasticity" after injury, going from a straight orientation before injury to developing an undulating course as an immediate response to injury, yet gradually recovering their original orientation. Surprisingly, some portions of the axons were found to be up to 60% longer immediately after injury. Subsequent to returning to their original length, injured axons developed swellings of appearance remarkably similar to that found in brain-injured humans. These findings may offer insight into mechanical-loading conditions leading to traumatic axonal injury and into potential mechanisms of axon reassembly after brain trauma.

Experimental investigation of cerebral contusion: histopathological and immunohistochemical evaluation of dynamic cortical deformation.

We used a new approach, termed dynamic cortical deformation (DCD), to study the neuronal, vascular, and glial responses that occur in focal cerebral contusions. DCD produces experimental contusion by rapidly deforming the cerebral cortex with a transient, nonablative vacuum pulse of short duration (25 milliseconds) to mimic the circumstances of traumatic injury. A neuropathological evaluation was performed on brain tissue from adult rats sacrificed 3 days following induction of either moderate (4 psi, n = 6) or high (8 psi, n = 6) severity DCD. In all animals, DCD produced focal hemorrhagic lesions at the vacuum site without overt damage to other regions. Examination of histological sections showed localized gross tissue and neuronal loss in the cortex at the injury site, with the volume of cell loss dependent upon the mechanical loading (p < 0.001). Axonal pathology shown with neurofilament immunostaining (SMI-31 and SMI-32) was observed in the subcortical white matter inferior to the injury site and in the ipsilateral internal capsule. No axonal injury was observed in the contralateral hemisphere or in any remote regions. Glial fibrillary acidic protein (GFAP) immunostaining revealed widespread reactive astrocytosis surrounding the necrotic region in the ipsilateral cortex. This analysis confirms that rapid mechanical deformation of the cortex induces focal contusions in the absence of primary damage to remote areas 3 days following injury. Although it is suggested that massive release of neurotoxic substances from a contusion may cause damage throughout the brain, these data emphasize the importance of combined injury mechanisms, e.g. mechanical distortion and excitatory amino acid mediated damage, that underlie the complex pathology patterns observed in traumatic brain injury.

Immediate in vivo response of the cortex and the blood-brain barrier following dynamic cortical deformation in the rat.

Although it is known that the brain can be injured by mechanical forces initiated at the moment of impact during trauma, it is not clear how the physical response of the brain dictates the injury patterns that occur in experimental models of traumatic brain injury. In this study, we investigated the mechanical response of the brain to a technique that creates a focal injury in the rat brain. Using a transient vacuum pulse applied to the exposed cortical surface, we found that the displacement of the cortex and the extent of in vivo blood-brain barrier breakdown were related significantly to the vacuum pressure level. The relationship between the response of the cortex and injury pattern points towards a new opportunity for control of the distribution and extent of injury patterns in animal models through a precise understanding of the model biomechanics, as well as potential improvements in means of preventing traumatic brain injury.

In vitro central nervous system models of mechanically induced trauma: a review.

Injury is one of the leading causes of death among all people below the age of 45 years. In the United States, traumatic brain injury (TBI) and spinal cord injury (SCI) together are responsible for an estimated 90,000 disabled persons annually. To improve treatment of the patient and thereby decrease the associated mortality, morbidity, and cost, several in vivo models of central nervous system (CNS) injury have been developed and characterized over the past two decades. To complement the ability of these in vivo models to reproduce the sequelae of human CNS injury, in vitro models of neuronal injury have also been developed. Despite the inherent simplifications of these in vitro systems, many aspects of the posttraumatic sequelae are faithfully reproduced in cultured cells, including ultrastructural changes, ionic derangements, alterations in electrophysiology, and free radical generation. This review presents a number of these in vitro systems, detailing the mechanical stimuli, the types of tissue injured, and the in vivo injury conditions most closely reproduced by the models. The data generated with these systems is then compared and contrasted with data from in vivo models of CNS injury. We believe that in vitro models of mechanical injury will continue to be a valuable tool to study the cellular consequences and evaluate the potential therapeutic strategies for the treatment of traumatic injury of the CNS.

Magnetic resonance spectroscopy of diffuse brain trauma in the pig.

The acute metabolic events linked to the evolution of selective axonal pathology in the white matter following diffuse brain injury have not previously been evaluated due to the paucity of relevant experimental models. Here, we utilized a new model of inertial brain injury in the pig that selectively damages axons in the white matter, and applied proton and phosphorous magnetic resonance spectroscopy (MRS) to noninvasively monitor the temporal course of metabolic changes following trauma. Evaluating four pigs with MRS prior to injury, within 1 h and 3 and 7 days postinjury, we found that widespread axonal injury was produced in the absence of changes in pH, PCr/Pi, or the concentrations of ATP, and lactate. However, we did observe an acute 60% loss of intracellular Mg2+ levels, which gradually resolved by 7 days postinjury. In addition, we found that the levels of the neuron marker, N-acetylaspartate (NAA), acutely dropped 20% and remained persistently decreased for at least 7 days postinjury. Moreover, the changes in Mg2+ and NAA were found with MRS in the absence of abnormalities with conventional magnetic resonance imaging (MRI). These results show that (1) profound alterations in intracellular metabolism occur acutely following diffuse axonal pathology in the white matter, but in the absence of indicators of ischemia, and (2) axonal pathology may be evaluated with high sensitivity utilizing noninvasive MRS techniques.