Targeting of nanoparticles to the cerebral vasculature after traumatic brain injury.

Traumatic brain injury has faced numerous challenges in drug development, primarily due to the difficulty of effectively delivering drugs to the brain. However, there is a potential solution in targeted drug delivery methods involving antibody-drug conjugates or nanocarriers conjugated with targeting antibodies. Following a TBI, the blood-brain barrier (BBB) becomes permeable, which can last for years and allow the leakage of harmful plasma proteins. Consequently, an appealing approach for TBI treatment involves using drug delivery systems that utilize targeting antibodies and nanocarriers to help restore BBB integrity. In our investigation of this strategy, we examined the efficacy of free antibodies and nanocarriers targeting a specific endothelial surface marker called vascular cell adhesion molecule-1 (VCAM-1), which is known to be upregulated during inflammation. In a mouse model of TBI utilizing central fluid percussion injury, free VCAM-1 antibody did not demonstrate superior targeting when comparing sham vs. TBI brain. However, the administration of VCAM-1-targeted nanocarriers (liposomes) exhibited a 10-fold higher targeting specificity in TBI brain than in sham control. Flow cytometry and confocal microscopy analysis confirmed that VCAM-1 liposomes were primarily taken up by brain endothelial cells post-TBI. Consequently, VCAM-1 liposomes represent a promising platform for the targeted delivery of therapeutics to the brain following traumatic brain injury.

Sex differences in the extent of acute axonal pathologies after experimental concussion.

Although human females appear be at a higher risk of concussion and suffer worse outcomes than males, underlying mechanisms remain unclear. With increasing recognition that damage to white matter axons is a key pathologic substrate of concussion, we used a clinically relevant swine model of concussion to explore potential sex differences in the extent of axonal pathologies. At 24 h post-injury, female swine displayed a greater number of swollen axonal profiles and more widespread loss of axonal sodium channels than males. Axon degeneration for both sexes appeared to be related to individual axon architecture, reflected by a selective loss of small caliber axons after concussion. However, female brains had a higher percentage of small caliber axons, leading to more extensive axon loss after injury compared to males. Accordingly, sexual dimorphism in axonal size is associated with more extensive axonal pathology in females after concussion, which may contribute to worse outcomes.

Concussion leads to widespread axonal sodium channel loss and disruption of the node of Ranvier.

Despite being a major health concern, little is known about the pathophysiological changes that underly concussion. Nonetheless, emerging evidence suggests that selective damage to white matter axons, or diffuse axonal injury (DAI), disrupts brain network connectivity and function. While voltage-gated sodium channels (NaChs) and their anchoring proteins at the nodes of Ranvier (NOR) on axons are key elements of the brain's network signaling machinery, changes in their integrity have not been studied in context with DAI. Here, we utilized a clinically relevant swine model of concussion that induces evolving axonal pathology, demonstrated by accumulation of amyloid precursor protein (APP) across the white matter. Over a two-week follow-up post-concussion with this model, we found widespread loss of NaCh isoform 1.6 (Nav1.6), progressive increases in NOR length, the appearance of void and heminodes and loss of ßIV-spectrin, ankyrin G, and neurofascin 186 or their collective diffusion into the paranode. Notably, these changes were in close proximity, yet distinct from APP-immunoreactive swollen axonal profiles, potentially representing a unique, newfound phenotype of axonal pathology in DAI. Since concussion in humans is non-fatal, the clinical relevance of these findings was determined through examination of post-mortem brain tissue from humans with higher levels of acute traumatic brain injury. Here, a similar loss of Nav1.6 and changes in NOR structures in brain white matter were observed as found in the swine model of concussion. Collectively, this widespread and progressive disruption of NaChs and NOR appears to be a form of sodium channelopathy, which may represent an important substrate underlying brain network dysfunction after concussion.

Traumatic brain injury recapitulates developmental changes of axons.

During development, half of brain white matter axons are maintained for growth, while the remainder undergo developmental axon degeneration. After traumatic brain injury (TBI), injured axons also appear to follow pathways leading to either degeneration or repair. These observations raise the intriguing, but unexamined possibility that TBI recapitulates developmental axonal programs. Here, we examined axonal changes in the developing brain in young rats and after TBI in adult rat. Multiple shared changes in axonal microtubule (MT) through tubulin post-translational modifications and MT associated proteins (MAPs), tau and MAP6, were found in both development and TBI. Specifically, degenerating axons in both development and TBI underwent phosphorylation of tau and excessive tubulin tyrosination, suggesting MT instability and depolyermization. Conversely, nearby axons without degenerating morphologies, had increased MAP6 expression and maintenance of tubulin acetylation, suggesting enhanced MT stabilization, thereby supporting survival or repair. Quantitative proteomics revealed similar signaling pathways of axon degeneration and growth/repair, including protein clusters and networks. This comparison approach demonstrates how focused evaluation of developmental processes may provide insight into pathways initiated by TBI. In particular, the data suggest that TBI may reawaken dormant axonal programs that direct axons towards either degeneration or growth/repair, supporting further study in this area.

Ultrasensitive Single Extracellular Vesicle Detection Using High Throughput Droplet Digital Enzyme-Linked Immunosorbent Assay.

Extracellular vesicles (EVs) have attracted enormous attention for their diagnostic and therapeutic potential. However, it has proven challenging to achieve the sensitivity to detect individual nanoscale EVs, the specificity to distinguish EV subpopulations, and a sufficient throughput to study EVs among an enormous background. To address this fundamental challenge, we developed a droplet-based optofluidic platform to quantify specific individual EV subpopulations at high throughput. The key innovation of our platform is parallelization of droplet generation, processing, and analysis to achieve a throughput (∼20 million droplets/min) more than 100× greater than typical microfluidics. We demonstrate that the improvement in throughput enables EV quantification at a limit of detection = 9EVs/µL, a >100× improvement over gold standard methods. Additionally, we demonstrate the clinical potential of this system by detecting human EVs in complex media. Building on this work, we expect this technology will allow accurate quantification of rare EV subpopulations for broad biomedical applications.

Modeling links softening of myelin and spectrin scaffolds of axons after a concussion to increased vulnerability to repeated injuries.

Damage to the microtubule lattice, which serves as a rigid cytoskeletal backbone for the axon, is a hallmark mechanical initiator of pathophysiology after concussion. Understanding the mechanical stress transfer from the brain tissue to the axonal cytoskeleton is essential to determine the microtubule lattice's vulnerability to mechanical injury. Here, we develop an ultrastructural model of the axon's cytoskeletal architecture to identify the components involved in the dynamic load transfer during injury. Corroborative in vivo studies were performed using a gyrencephalic swine model of concussion via single and repetitive head rotational acceleration. Computational analysis of the load transfer mechanism demonstrates that the myelin sheath and the actin/spectrin cortex play a significant role in effectively shielding the microtubules from tissue stress. We derive failure maps in the space spanned by tissue stress and stress rate to identify physiological conditions in which the microtubule lattice can rupture. We establish that a softer axonal cortex leads to a higher susceptibility of the microtubules to failure. Immunohistochemical examination of tissue from the swine model of single and repetitive concussion confirms the presence of postinjury spectrin degradation, with more extensive pathology observed following repetitive injury. Because the degradation of myelin and spectrin occurs over weeks following the first injury, we show that softening of the myelin layer and axonal cortex exposes the microtubules to higher stress during repeated incidences of traumatic brain injuries. Our predictions explain how mechanical injury predisposes axons to exacerbated responses to repeated injuries, as observed in vitro and in vivo.

COllaborative Neuropathology NEtwork Characterizing ouTcomes of TBI (CONNECT-TBI).

Efforts to characterize the late effects of traumatic brain injury (TBI) have been in progress for some time. In recent years much of this activity has been directed towards reporting of chronic traumatic encephalopathy (CTE) in former contact sports athletes and others exposed to repetitive head impacts. However, the association between TBI and dementia risk has long been acknowledged outside of contact sports. Further, growing experience suggests a complex of neurodegenerative pathologies in those surviving TBI, which extends beyond CTE. Nevertheless, despite extensive research, we have scant knowledge of the mechanisms underlying TBI-related neurodegeneration (TReND) and its link to dementia. In part, this is due to the limited number of human brain samples linked to robust demographic and clinical information available for research. Here we detail a National Institutes for Neurological Disease and Stroke Center Without Walls project, the COllaborative Neuropathology NEtwork Characterizing ouTcomes of TBI (CONNECT-TBI), designed to address current limitations in tissue and research access and to advance understanding of the neuropathologies of TReND. As an international, multidisciplinary collaboration CONNECT-TBI brings together multiple experts across 13 institutions. In so doing, CONNECT-TBI unites the existing, comprehensive clinical and neuropathological datasets of multiple established research brain archives in TBI, with survivals ranging minutes to many decades and spanning diverse injury exposures. These existing tissue specimens will be supplemented by prospective brain banking and contribute to a centralized route of access to human tissue for research for investigators. Importantly, each new case will be subject to consensus neuropathology review by the CONNECT-TBI Expert Pathology Group. Herein we set out the CONNECT-TBI program structure and aims and, by way of an illustrative case, the approach to consensus evaluation of new case donations.

Mechanisms of Local Stress Amplification in Axons near the Gray-White Matter Interface.

Diffuse axonal injury is a primary neuropathological feature of concussion and is thought to greatly contribute to the classical symptoms of decreased processing speed and memory dysfunction. Although previous studies have investigated the injury biomechanics at the micro- and mesoscale of concussion, few have addressed the multiscale transmission of mechanical loading at thresholds that can induce diffuse axonal injury. Because it has been recognized that axonal pathology is commonly found at anatomic interfaces across all severities of traumatic brain injury, we combined computational, analytical, and experimental approaches to investigate the potential mechanical vulnerability of axons that span the gray-white tissue interface. Our computational models predict that material heterogeneities at the gray-white interface lead to a highly nonuniform distribution of stress in axons, which was most amplified in axonal regions near the interface. This mechanism was confirmed using an analytical model of an individual fiber in a strained bimaterial interface. Comparisons of these collective data with histopathological evaluation of a swine model of concussion demonstrated a notably similar pattern of axonal damage adjacent to the gray-white interface. The results suggest that the tissue property mismatch at the gray-white matter interface places axons crossing this region at greater risk of mechanical damage during brain tissue deformation from traumatic brain injury.

Dorsal root ganglion axons facilitate and guide cortical neural outgrowth: In vitro modeling of spinal cord injury axonal regeneration.

BACKGROUND: Spinal cord injury (SCI) patients represent a heterogeneous group, with injuries ranging from partial compression to complete transection. Patients with complete injuries are unlikely to exhibit recovery and suffer from paralysis as well as the loss of bowel and bladder function. One treatment option is the formation of a bridge through a lesion site, whereby transplanted cells or biocompatible scaffolds guide the regenerating axons across the site of injury. Moreover, the viability of transplanted dorsal root ganglia (DRGs) into rat spinal cord has been previously demonstrated. OBJECTIVE: We aim to demonstrate the feasibility of using DRG axons as a bridging tool to help guide the axonal growth of cortical neurons. METHODS: Cortical neurons were isolated from embryonic rats and two aggregated populations were cultured at increasing distances in isolation and in a co-culture with DRG explants. Growth rates of the sprouting axons and connections between the two populations were observed over a period of twelve days. RESULTS: DRG explants demonstrated the ability to grow robust axonal connections that can connect two explants separated by up to 10 mm, however, CNAs could not achieve connections in distances greater than 2 mm. The co-culture of CNAs with DRG explants facilitated axonal growth between two populations of CNAs at distances they cannot otherwise traverse. CONCLUSIONS: Our findings support the use of DRG axons to facilitate the growth of cortical neurons in a process of axon-facilitated axon regeneration. We believe these results could have implications for the treatment of SCI.

Newfound effect of N-acetylaspartate in preventing and reversing aggregation of amyloid-beta in vitro.

Although N-acetylaspartate (NAA) has long been recognized as the most abundant amino acid in neurons by far, its primary role has remained a mystery. Based on its unique tertiary structure, we explored the potential of NAA to modulate aggregation of amyloid-beta (Aß) peptide 1-42 via multiple corroborating aggregation assays along with electron microscopy. Thioflavin-T fluorescence assay demonstrated that at physiological concentrations, NAA substantially inhibited the initiation of Aß fibril formation. In addition, NAA added after 25 min of Aß aggregation was shown to break up preformed fibrils. Electron microscopy analysis confirmed the absence of mature fibrils following NAA treatment. Furthermore, fluorescence correlation spectroscopy and dynamic light scattering measurements confirmed significant reductions in Aß fibril hydrodynamic radius following treatment with NAA. These results suggest that physiological levels of NAA could play an important role in controlling Aß aggregation in vivo where they are both found in the same neuronal compartments.

Newfound sex differences in axonal structure underlie differential outcomes from in vitro traumatic axonal injury.

Since traumatic axonal injury (TAI) is implicated as a prominent pathology of concussion, we examined potential sex differences in axon structure and responses to TAI. Rat and human neurons were used to develop micropatterned axon tracts in vitro that were genetically either male or female. Ultrastructural analysis revealed for the first time that female axons were consistently smaller with fewer microtubules than male axons. Computational modeling of TAI showed that these structural differences place microtubules in female axons at greater risk of failure during trauma under the same applied loads than in male axons. Likewise, in an in vitro model of TAI, dynamic stretch-injury to axon tracts induced greater pathophysiology of female axons than male axons, including more extensive undulation formations resulting from mechanical breaking of microtubules, and greater calcium influx shortly after the same level of injury. At 24h post-injury, female axons exhibited significantly more swellings and greater loss of calcium signaling function than male axons. Accordingly, sexual dimorphism of axon structure in the brain may also contribute to more extensive axonal pathology in females compared to males exposed to the same mechanical injury.

Brain-on-a-chip microsystem for investigating traumatic brain injury: Axon diameter and mitochondrial membrane changes play a significant role in axonal response to strain injuries.

Diffuse axonal injury (DAI) is a devastating consequence of traumatic brain injury, resulting in significant axon and neuronal degeneration. Currently, therapeutic options are limited. Using our brain-on-a-chip device, we evaluated axonal responses to DAI. We observed that axonal diameter plays a significant role in response to strain injury, which correlated to delayed elasticity and inversely correlated to axonal beading and axonal degeneration. When changes in mitochondrial membrane potential (MMP) were monitored an applied strain injury threshold was noted, below which delayed hyperpolarization was observed and above which immediate depolarization occurred. When the NHE-1 inhibitor EIPA was administered before injury, inhibition in both hyperpolarization and depolarization occurred along with axonal degeneration. Therefore, axonal diameter plays a significant role in strain injury and our brain-on-a-chip technology can be used both to understand the biochemical consequences of DAI and screen for potential therapeutic agents.

An organotypic uniaxial strain model using microfluidics.

Traumatic brain injuries are the leading cause of disability each year in the US. The most common and devastating consequence is the stretching of axons caused by shear deformation that occurs during rotational acceleration of the brain during injury. The injury effects on axonal molecular and functional events are not fully characterized. We have developed a strain injury model that maintains the three dimensional cell architecture and neuronal networks found in vivo with the ability to visualize individual axons and their response to a mechanical injury. The advantage of this model is that it can apply uniaxial strains to axons that make functional connections between two organotypic slices and injury responses can be observed in real-time and over long term. This uniaxial strain model was designed to be capable of applying an array of mechanical strains at various rates of strain, thus replicating a range of modes of axonal injury. Long term culture, preservation of slice and cell orientation, and slice-slice connection on the device was demonstrated. The device has the ability to strain either individual axons or bundles of axons through the control of microchannel dimensions. The fidelity of the model was verified by observing characteristic responses to various strain injuries which included axonal beading, delayed elastic effects and breakdown in microtubules. Microtubule breakdown was shown to be dependent on the degree of the applied strain field, where maximal breakdown was observed at peak strain and minimal breakdown is observed at low strain. This strain injury model could be a powerful tool in assessing strain injury effects on functional axonal connections.

REVERSAL OF FIBRONECTIN-INDUCED HIPPOCAMPAL DEGENERATION WITH ENCAPSULATED MESENCHYMAL STROMAL CELLS.

Mesenchymal stromal cells (MSC) can promote tissue protection following injury, in part by modulating inflammatory cell responses. The aim of this study was to investigate the potential tissue protective properties of encapsulated MSCs (eMSC) in an organotypic injury model induced by fibronectin culture. MSC were encapsulated in alginate beads containing a network of nanopores, which segregate the cells from the extracapsular milieu, while still permitting diffusion into and out of the capsule. An increase in blood brain barrier permeability during pathological conditions permits the influx of blood plasma constituents that can be quite harmful to surrounding tissues. In particular, increased concentrations of fibronectin have been shown in a number of diseases and CNS traumas, co-localizing in areas of activated microglia. We observed over a 14-day period, a consistent increase in OHC degradation in the presence of fibronectin measured by a significant decrease in slice area, the breakdown in OHC pyramidal layers, and consistent cell death over the culture period. Microglial ionized calcium-binding adapter molecule 1 (IBA-1) expression remained elevated throughout the culture period with the majority found within the pyramidal layers. When eMSC were added to the cultures, a significant decrease in OHC degradation was observed as reflected by a reduction in OHC area shrinkage and in the amount of cell death. In the presence of eMSC, pyramidal layer structure was maintained and axonal extension from the periphery of the OHCs was observed. Therefore, MSC, delivered in a nanoporous alginate matrix, can modulate responses to injury by reversing fibronectin-induced OHC degradation.

In vitro models of traumatic brain injury.

In vitro models of traumatic brain injury (TBI) are helping elucidate the pathobiological mechanisms responsible for dysfunction and delayed cell death after mechanical stimulation of the brain. Researchers have identified compounds that have the potential to break the chain of molecular events set in motion by traumatic injury. Ultimately, the utility of in vitro models in identifying novel therapeutics will be determined by how closely the in vitro cascades recapitulate the sequence of cellular events that play out in vivo after TBI. Herein, the major in vitro models are reviewed, and a discussion of the physical injury mechanisms and culture preparations is employed. A comparison between the efficacy of compounds tested in vitro and in vivo is presented as a critical evaluation of the fidelity of in vitro models to the complex pathobiology that is TBI. We conclude that in vitro models were greater than 88% predictive of in vivo results.

Nerve growth factor-induced migration of endothelial cells.

Nerve growth factor (NGF) is a well known neurotropic and neurotrophic agonist in the nervous system, which recently was shown to also induce angiogenic effects in endothelial cells (ECs). To measure NGF effects on the migration of cultured ECs, an important step in neoangiogenesis, we optimized an omnidirectional migration assay using human aortic endothelial cells (HAECs) and validated the assay with human recombinant basic fibroblast growth factor (rhbFGF) and human recombinant vascular endothelial growth factor (rhVEGF). The potencies of nerve growth factor purified from various species (viper, mouse, and recombinant human) to stimulate HAEC migration was similar to that of VEGF and basic fibroblast growth factor (bFGF) (EC50 of approximately 0.5 ng/ml). Recombinant human bFGF was significantly more efficacious than either viper NGF or rhVEGF, both of which stimulated HAEC migration by approximately 30% over basal spontaneous migration. NGF-mediated stimulation of HAEC migration was completely blocked by the NGF/TrkA receptor antagonist K252a [(8R*,9S*,11S*)-(/)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,-8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(c,d,e)trindene-1-one] (30 nM) but not by the VEGF/Flk receptor antagonist SU-5416 [3-[(2,4-dimethylpyrrol-5-yl) methylidenyl]-indolin-2-one] (250 nM), indicating a direct effect of NGF via TrkA receptor activation on HAEC migration. Viper NGF stimulation of HAEC migration was additively increased by either rhVEGF or rhbFGF, suggesting a potentiating interaction between their tyrosine kinase receptor signaling pathways. Viper NGF represents a novel pharmacological tool to investigate possible TrkA receptor subtypes in endothelial cells. The ability of NGF to stimulate migration of HAEC cells in vitro implies that this factor may play an important role in the cardiovascular system besides its well known effects in the nervous system.