Altered Tau Kinase Activity in rTg4510 Mice after a Single Interfaced CHIMERA Traumatic Brain Injury.

Traumatic brain injury (TBI) is an established risk factor for neurodegenerative diseases. In this study, we used the Closed Head Injury Model of Engineered Rotational Acceleration (CHIMERA) to investigate the effects of a single high-energy TBI in rTg4510 mice, a mouse model of tauopathy. Fifteen male rTg4510 mice (4 mo) were impacted at 4.0 J using interfaced CHIMERA and were compared to sham controls. Immediately after injury, the TBI mice showed significant mortality (7/15; 47%) and a prolonged duration of loss of the righting reflex. At 2 mo post-injury, surviving mice displayed significant microgliosis (Iba1) and axonal injury (Neurosilver). Western blotting indicated a reduced p-GSK-3ß (S9):GSK-3ß ratio in TBI mice, suggesting chronic activation of tau kinase. Although longitudinal analysis of plasma total tau suggested that TBI accelerates the appearance of tau in the circulation, there were no significant differences in brain total or p-tau levels, nor did we observe evidence of enhanced neurodegeneration in TBI mice compared to sham mice. In summary, we showed that a single high-energy head impact induces chronic white matter injury and altered GSK-3ß activity without an apparent change in post-injury tauopathy in rTg4510 mice.

An Automated Kinematic Measurement System for Sagittal Plane Murine Head Impacts.

Mild traumatic brain injuries are typically caused by nonpenetrating head impacts that accelerate the skull and result in deformation of the brain within the skull. The shear and compressive strains caused by these deformations damage neural and vascular structures and impair their function. Accurate head acceleration measurements are necessary to define the nature of the insult to the brain. A novel murine head tracking system was developed to improve the accuracy and efficiency of kinematic measurements obtained with high-speed videography. A three-dimensional (3D)-printed marker carrier was designed for rigid fixation to the upper jaw and incisors with an elastic strap around the snout. The system was evaluated by impacting cadaveric mice with the closed head impact model of engineered rotational acceleration (CHIMERA) system using an energy of 0.7 J (5.29 m/s). We compared the performance of the head-marker system to the previously used skin-tracking method and documented significant improvements in measurement repeatability (aggregate coefficient of variation (CV) within raters from 15.8 to 1.5 and between raters from 15.5 to 1.5), agreement (aggregate percentage error from 24.9 to 8.7), and temporal response (aggregate temporal curve agreement from 0.668 to 0.941). Additionally, the new system allows for automated software tracking, which dramatically decreases the analysis time required (74% reduction). This novel head tracking system for mice offers an efficient, reliable, and real-time method to measure head kinematics during high-speed impacts using CHIMERA or other rodent or small mammal head impact models.

Extensional Strain Hardening in Highly Entangled Molecular Bottlebrushes.

The highly strained conformation in dense molecular bottlebrushes (DB) has profound effects on the dynamics of these type of macromolecules. Understanding of such effects in both their linear and nonlinear viscoelastic responses is crucial for their design and processing. The nonlinear response of poly(1-octadecene), a highly entangled α-olefin DB with linear side chains sixteen carbons long, is studied here and compared to the nonlinear response of a linear polyolefin (polypropylene) with equivalent linear viscoelastic response. We found that the DB shows remarkably larger extensional strain hardening (SH) and extensibility than the linear polyolefin. The strong SH is attributed to a strain-induced increase in friction drag between adjacent chains resulting from side-chain interdigitation and alignment perpendicular to the flow. The higher extensibility of DBs compared to linear counterparts has been predicted previously [Daniel et al., Nat. Mater. 15, 183 (2015)NMAACR1476-1122] and confirmed here for the first time in DB melts.

Extensional Strain Hardening Induced by π-π Interactions in Barely Entangled Polymer Chains: The Curious Case of Poly(4-vinylbiphenyl).

Aromatic π-π interactions between phenyl groups of adjacent chains in poly(4-vinylbiphenyl) (PVBP) have profound effects on the dynamics of this polymer. We report two unexpected nonlinear viscoelastic responses of PVBP when subjected to uniaxial flow. One is the unprecedented observation of extensional strain hardening (SH) in a barely entangled polymer melt. An even more intriguing finding is that SH of lightly (or even barely) entangled melts occurs at strain rates one order of magnitude below the coil-stretch transition predicted by Rouse theory (ϵ[over ˙]_{H}=0.5/τ_{R}).We postulate that this behavior is due to a molecular rearrangement mechanism (supported by x-ray diffraction measurements) that involves flow-induced π-π stacking of the phenyl groups, which results in an enhancement of the friction coefficient between polymer chains.

Spatiotemporal stress and structure evolution in dynamically sheared polymer-like micellar solutions.

The complex, nonlinear flow behavior of soft materials transcends industrial applications, smart material design and non-equilibrium thermodynamics. A long-standing, fundamental challenge in soft-matter science is establishing a quantitative connection between the deformation field, local microstructure and macroscopic dynamic flow properties i.e., the rheology. Here, a new experimental method is developed using simultaneous small angle neutron scattering (SANS) and nonlinear oscillatory shear rheometry to investigate the spatiotemporal microstructure evolution of a polymer-like micellar (PLM) solution. We demonstrate the novelty of nonlinear oscillatory shear experimental methods to create and interrogate metastable material states. These include a precursory state to the shear banded condition as well as a disentangled, low viscosity state with an inhomogeneous supra-molecular microstructure flowing at high shear rates. This new experimental evidence provides insight into the complexities of the shear banding phenomenon often observed in sheared complex fluids and provides valuable data for quantitatively testing non-equilibrium theory.

Tack of Entangled Molecular Bottlebrushes.

Polymer softness (i.e., low elastic modulus) is a known requirement for good tack in adhesives. We assess the tack performance of α-olefin molecular bottlebrushes having an elastic modulus from 4 to 30 times lower than a linear polyolefin (polypropylene). Monotonic increases of the tack parameters are observed as the bottlebrush side chain length (Nsc) increases and the modulus decreases. All-atom molecular dynamics simulations reveal that the monomeric bonding energy increases with Nsc due to a strong van der Waals interaction between the side chains and the aluminum sheet, which overcomes the energy penalty imposed by side-chain bending.

Spontaneous thermoreversible formation of cationic vesicles in a protic ionic liquid.

The search for stable vesicular structures is a long-standing topic of research because of the usefulness of these structures and the scarcity of surfactant systems that spontaneously form vesicles in true thermodynamic equilibrium. We report the first experimental evidence of spontaneous formation of vesicles for a pure cationic double tail surfactant (didodecyldimethylammonium bromide, DDAB) in a protic ionic liquid (ethylammonium nitrate, EAN). Using small and ultra-small angle neutron scattering, rheology and bright field microscopy, we identify the coexistence of two vesicle containing phases in compositions ranging from 2 to 68 wt %. A low density highly viscous solution containing giant vesicles (D ~ 30 µm) and a sponge (L(3)) phase coexists with a dilute high density phase containing large vesicles (D ~ 2.5 µm). Vesicles form spontaneously via different thermodynamic routes, with the same size distribution, which strongly supports that they exist in a true thermodynamic equilibrium. The formation of equilibrium vesicles and the L(3) phase is facilitated by ion exchange between the cationic surfactant and the ionic liquid, as well as the strength of the solvophobic effect in the protic ionic liquid.

Dynamics of melting and recrystallization in a polymeric micellar crystal subjected to large amplitude oscillatory shear flow.

Shear-induced structural transitions of a micellar cubic phase during large amplitude oscillatory shear flow is studied with time-resolved oscillatory rheological small angle neutron scattering. This technique allows us to resolve the structural changes within a cycle of oscillation. By applying a strain rate near the critical melting shear rate, melting and recrystallization occurs in a cyclic mode. The maximum degree of order is observed when the shear stress reaches a plateau value during the large amplitude oscillatory shear cycle, whereas melting is maximized at the strain rate wave peaks. This structural evolution confirms the cyclic mechanism of sticking and sliding of 2D hexagonal close-packed layers [I. W. Hamley et al., Phys. Rev. E 58, 7620 (1998)].

Structural transitions of CTAB micelles in a protic ionic liquid.

Micellar solutions of hexadecyltrimethylammonium bromide (CTAB) in a protic ionic liquid, ethylammonium nitrate (EAN), are studied by shear rheology, polarizing optical microscopy (POM), conductivity measurements, and small angle neutron scattering (SANS). Three concentration regimes are examined: A dilute regime (with concentrations [CTAB] < 5 wt %) consisting of noninteracting spherical micelles, a semidilute regime (5 wt % ≤ [CTAB] ≤ 45 wt %) where micelles interact via electrostatic repulsions, and a concentrated regime (45 wt % < [CTAB] ≤ 62 wt %) where a reversible, temperature-dependent isotropic (L(1)) to hexatic (Hex) phase transition is observed. The L(1)-Hex transition, which has been predicted but not previously observed, is characterized by (1) a sharp increase in the shear viscosity, (2) the formation of focal conical birefringence textures (observed by POM), and (3) enhancement of the crystalline order, evidenced by the appearance of Bragg reflections in the SANS profiles. Ionic conductivity is not sensitive to the L(1)-Hex transition, which corroborates the absence of topological transitions.

Measurement of geometrical parameters in cocontinuous polymer blends: 3D versus 2D image analysis.

Formulae of stereology are used to estimate 3D geometrical parameters of cocontinuous structures measured from 2D micrographs of polymer blends. 3D images of symmetric and nonsymmetric polymer blends made of fluorescently labelled polystyrene and styrene-ran-acrylonitrile copolymer were obtained with laser scanning confocal microscopy. Geometrical parameters of the blend interface, specifically volume fraction, surface area per unit volume (S (V) ) and average of local mean curvature were measured directly from the 3D images and compared to the values estimated from analysis of a number of 2D slices combined with stereological relations. When the total length of phase boundary considered in the analysis of the 2D slices (L(Tot) ) was at least 6000 times bigger than the characteristic length of the microstructure (S(-1) (V) ), the standard deviation for all the parameters measured became negligible. However, considerable discrepancies between the average values computed from 3D and 2D images were observed for any value of L(Tot) . The mean curvature distribution was also measured from both the 3D images and the 2D slices. The distribution was estimated from the 2D slices but with a width about 2.4 times that of the true value obtained from the 3D images.

Development of a novel, sensitive translational immunoassay to detect plasma glial fibrillary acidic protein (GFAP) after murine traumatic brain injury.

BACKGROUND: Glial fibrillary acidic protein (GFAP) has emerged as a promising fluid biomarker for several neurological indications including traumatic brain injury (TBI), a leading cause of death and disability worldwide. In humans, serum or plasma GFAP levels can predict brain abnormalities including hemorrhage on computed tomography (CT) scans and magnetic resonance imaging (MRI). However, assays to quantify plasma or serum GFAP in preclinical models are not yet available. METHODS: We developed and validated a novel sensitive GFAP immunoassay assay for mouse plasma on the Meso Scale Discovery immunoassay platform and validated assay performance for robustness, precision, limits of quantification, dilutional linearity, parallelism, recovery, stability, selectivity, and pre-analytical factors. To provide proof-of-concept data for this assay as a translational research tool for TBI and Alzheimer's disease (AD), plasma GFAP was measured in mice exposed to TBI using the Closed Head Impact Model of Engineered Rotational Acceleration (CHIMERA) model and in APP/PS1 mice with normal or reduced levels of plasma high-density lipoprotein (HDL). RESULTS: We performed a partial validation of our novel assay and found its performance by the parameters studied was similar to assays used to quantify human GFAP in clinical neurotrauma blood specimens and to assays used to measure murine GFAP in tissues. Specifically, we demonstrated an intra-assay CV of 5.0%, an inter-assay CV of 7.2%, a lower limit of detection (LLOD) of 9.0 pg/mL, a lower limit of quantification (LLOQ) of 24.8 pg/mL, an upper limit of quantification (ULOQ) of at least 16,533.9 pg/mL, dilution linearity of calibrators from 20 to 200,000 pg/mL with 90-123% recovery, dilution linearity of plasma specimens up to 32-fold with 96-112% recovery, spike recovery of 67-100%, and excellent analyte stability in specimens exposed to up to 7 freeze-thaw cycles, 168 h at 4 °C, 24 h at room temperature (RT), or 30 days at - 20 °C. We also observed elevated plasma GFAP in mice 6 h after TBI and in aged APP/PS1 mice with plasma HDL deficiency. This assay also detects GFAP in serum. CONCLUSIONS: This novel assay is a valuable translational tool that may help to provide insights into the mechanistic pathophysiology of TBI and AD.

Direct observation of flow-concentration coupling in a shear-banding fluid.

We present the first direct evidence of flow-concentration coupling in a shear-banding fluid using the first measurements of scanning narrow-aperture flow ultrasmall-angle neutron scattering. Transmission measurements in the flow-gradient shear plane determine the spatial concentration profile of the material. Shear banding in a model fluid composed of wormlike micelles near an equilibrium isotropic-nematic transition is shown to be concomitant with shear-induced demixing, providing new insight into the nonequilibrium phase behavior of the fluid. The new experimental approach and analysis apply generally to any fluid, and will enable more rigorous testing of theories for shear-banding and shear-induced phase separation.

Direct measurement of interface anisotropy of bicontinuous structures via 3D image analysis.

A very important morphological parameter in two-phase fluids is the interface anisotropy, which can be quantified using the interface tensor, q(ij). However, the computation of this tensor for complex interfaces is not straightforward. A novel method (the local cross product method, LCPM) to compute the interface tensor of two-phase fluids using 3D imaging coupled with differential geometry is presented here. The method was used to evaluate the degree of anisotropy of phase separated systems with bicontinuous morphologies subjected to uniaxial and shear deformation fields. A model bicontinuous structure (i.e., the gyroid surface) was used to assess the accuracy and precision of the method. The method was then used to track the anisotropy changes of an immiscible polymer blend with cocontinuous morphology, during uniaxial deformation and subsequent retraction. It was found that the dependence of the anisotropy on the Hencky strain of both the gyroid surface and the cocontinuous blend follow the same trend. The retraction of the blend after uniaxial extension is accompanied by an exponential decay of the second invariant of q(ij), which obeys the relation: |II(q)|/Q(2) approximately e(-0.129t).

Local and Global Stretching of Polymer Chains during Startup of Extensional Flow.

The nonlinear rheological response to extensional flows in entangled polymers is related to the segmental chain stretching and to the chemical identity of the monomeric units. The latter has a strong effect on the drag coefficients, and therefore, quantification of molecular conformation changes in the subnanometer scale (at the monomer level) are crucial to fully understand nonlinear viscoelastic behavior in polymer melts. We report in situ time-resolved extensional rheo-small-angle neutron scattering (tEr-SANS) and wide-angle X-ray scattering (tEr-WAXS) during startup of uniaxial flow on a monodisperse polystyrene melt. Flow-induced segmental alignment was quantified with tEr-SANS, whereas local alignment of the backbone-backbone and phenyl-phenyl interactions were measured with tEr-WAXS. Linear relations between the three alignment factors and stress were observed at low stresses, which confirmed the validity of simple stress-SANS and stress-WAXS rules (SSR and SWR, respectively). Significant differences in SSR and SWR coefficients, as well as the stress values for failure of the two rules suggest very different correlations between global (at the segmental level) and local (at the monomer level) conformations with stress.

Increased severity of the CHIMERA model induces acute vascular injury, sub-acute deficits in memory recall, and chronic white matter gliosis.

Traumatic brain injury (TBI) is a leading cause of death and disability in modern societies. Diffuse axonal and vascular injury are nearly universal consequences of mechanical energy impacting the head and contribute to disability throughout the injury severity spectrum. CHIMERA (Closed Head Impact Model of Engineered Rotational Acceleration) is a non-surgical, impact-acceleration model of rodent TBI that reliably produces diffuse axonal injury characterized by white matter gliosis and axonal damage. At impact energies up to 0.7 joules, which result in mild TBI in mice, CHIMERA does not produce detectable vascular or grey matter injury. This study was designed to expand CHIMERA's capacity to induce more severe injuries, including vascular damage and grey matter gliosis. This was made possible by designing a physical interface positioned between the piston and animal's head to allow higher impact energies to be transmitted to the head without causing skull fracture. Here, we assessed interface-assisted single CHIMERA TBI at 2.5 joules in wild-type mice using a study design that spanned 6 h-60 d time points. Injured animals displayed robust acute neurological deficits, elevated plasma total tau and neurofilament-light levels, transiently increased proinflammatory cytokines in brain tissue, blood-brain barrier (BBB) leakage and microstructural vascular abnormalities, and grey matter microgliosis. Memory deficits were evident at 30 d and resolved by 60 d. Intriguingly, white matter injury was not remarkable at acute time points but evolved over time, with white matter gliosis being most extensive at 60 d. Interface-assisted CHIMERA thus enables experimental modeling of distinct endophenotypes of TBI that include acute vascular and grey matter injury in addition to chronic evolution of white matter damage, similar to the natural history of human TBI.

Enhanced Foamability with Shrinking Microfibers in Linear Polymer.

Strain hardening has important roles in understanding material structures and polymer processing methods, such as foaming, film forming, and fiber extruding. A common method to improve strain hardening behavior is to chemically branch polymer structures, which is costly, thus preventing users from controlling the degree of behavior. A smart microfiber blending technology, however, would allow cost-efficient tuning of the degree of strain hardening. In this study, we investigated the effects of compounding polymers with microfibers for both shear and extensional rheological behaviors and characteristics and thus for the final foam morphologies formed by batch physical foaming with carbon dioxide. Extensional rheometry showed that compounding of in situ shrinking microfibers significantly enhanced strain hardening compared to compounding of nonshrinking microfibers. Shear rheometry with linear viscoelastic data showed a greater increase in both the loss and storage modulus in composites with shrinking microfibers than in those with nonshrinking microfibers at low frequencies. The batch physical foaming results demonstrated a greater increase in the cell population density and expansion ratio with in situ shrinking microfibers than with nonshrinking microfibers. The enhancement due to the shrinkage of compounded microfibers decreasing with temperature implies that the strain hardening can be tailored by changing processing conditions.

Repetitive closed-head impact model of engineered rotational acceleration (CHIMERA) injury in rats increases impulsivity, decreases dopaminergic innervation in the olfactory tubercle and generates white matter inflammation, tau phosphorylation and degeneration.

Traumatic brain injury (TBI) affects at least 3 M people annually. In humans, repetitive mild TBI (rmTBI) can lead to increased impulsivity and may be associated with chronic traumatic encephalopathy. To better understand the relationship between repetitive TBI (rTBI), impulsivity and neuropathology, we used CHIMERA (Closed-Head Injury Model of Engineered Rotational Acceleration) to deliver five TBIs to rats, which were continuously assessed for trait impulsivity using the delay discounting task and for neuropathology at endpoint. Compared to sham controls, rats with rTBI displayed progressive impairment in impulsive choice. Histological analyses revealed reduced dopaminergic innervation from the ventral tegmental area to the olfactory tubercle, consistent with altered impulsivity neurocircuitry. Consistent with diffuse axonal injury generated by CHIMERA, white matter inflammation, tau immunoreactivity and degeneration were observed in the optic tract and corpus callosum. Finally, pronounced grey matter microgliosis was observed in the olfactory tubercle. Our results provide insight into the mechanisms by which rTBI leads to post-traumatic psychiatric-like symptoms in a novel rat TBI platform.

CHIMERA repetitive mild traumatic brain injury induces chronic behavioural and neuropathological phenotypes in wild-type and APP/PS1 mice.

BACKGROUND: The annual incidence of traumatic brain injury (TBI) in the United States is over 2.5 million, with approximately 3-5 million people living with chronic sequelae. Compared with moderate-severe TBI, the long-term effects of mild TBI (mTBI) are less understood but important to address, particularly for contact sport athletes and military personnel who have high mTBI exposure. The purpose of this study was to determine the behavioural and neuropathological phenotypes induced by the Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA) model of mTBI in both wild-type (WT) and APP/PS1 mice up to 8 months post-injury. METHODS: Male WT and APP/PS1 littermates were randomized to sham or repetitive mild TBI (rmTBI; 2 × 0.5 J impacts 24 h apart) groups at 5.7 months of age. Animals were assessed up to 8 months post-injury for acute neurological deficits using the loss of righting reflex (LRR) and Neurological Severity Score (NSS) tasks, and chronic behavioural changes using the passive avoidance (PA), Barnes maze (BM), elevated plus maze (EPM) and rotarod (RR) tasks. Neuropathological assessments included white matter damage; grey matter inflammation; and measures of Aß levels, deposition, and aducanumab binding activity. RESULTS: The very mild CHIMERA rmTBI conditions used here produced no significant acute neurological or motor deficits in WT and APP/PS1 mice, but they profoundly inhibited extinction of fear memory specifically in APP/PS1 mice over the 8-month assessment period. Spatial learning and memory were affected by both injury and genotype. Anxiety and risk-taking behaviour were affected by injury but not genotype. CHIMERA rmTBI induced chronic white matter microgliosis, axonal injury and astrogliosis independent of genotype in the optic tract but not the corpus callosum, and it altered microgliosis in APP/PS1 amygdala and hippocampus. Finally, rmTBI did not alter long-term tau, Aß or amyloid levels, but it increased aducanumab binding activity. CONCLUSIONS: CHIMERA is a useful model to investigate the chronic consequences of rmTBI, including behavioural abnormalities consistent with features of post-traumatic stress disorder and inflammation of both white and grey matter. The presence of human Aß greatly modified extinction of fear memory after rmTBI.

Iono-Elastomer-Based Wearable Strain Sensor with Real-Time Thermomechanical Dual Response.

An ultrastretchable iono-elastomer with resistance sensitive to both elongation strain and temperature has been developed by hierarchical self-assembly of an end functionalized triblock copolymer in a protic ionic liquid (ethylammonium nitrate) followed by cross-linking. Small-angle X-ray scattering experiments in situ with uniaxial elongation reveal a nanoscale microstructural transition of the hierarchically self-assembled cross-linked micelles that is responsible for the material's remarkable mechanical and ionic conductivity responses. The results show that the intermicelle distance extends along the deformation direction while the micelles organize into a long-range ordered face-centered-cubic structure during the uniaxial elongation. Besides good cyclability and resistance to selected physical damage, the iono-elastomer simultaneously achieves an unprecedented combination of high stretchability (340%), highly linear resistance vs elongation strain ( R2 = 0.998), and large temperature gauge factor (Δ R/ R = 3.24%/°C@30 °C). Human subject testing demonstrates that the iono-elastomer-based wearable thermomechanical sensor is able to effectively and accurately register both body motion and skin temperature simultaneously.

Ultrastretchable Iono-Elastomers with Mechanoelectrical Response.

The emerging technologies involving wearable electronics require new materials with high stretchability, resistance to high loads, and high conductivities. We report a facile synthetic strategy based on self-assembly of concentrated solutions of end-functionalized PEO106-PPO70-PEO106 triblock copolymer in ethylammonium nitrate into face-centered cubic micellar crystals, followed by micelle corona cross-linking to generate elastomeric ion gels (iono-elastomers). These materials exhibit an unprecedented combination of high stretchability, high ionic conductivity, and mechanoelectrical response. The latter consists of a remarkable and counterintuitive increase in ion conductivity with strain during uniaxial extension, which is reversible upon load release. Based on in situ SAXS measurements of reversible crystal structure transformations during deformation, we postulate that the origin of the conductivity increase is a reversible formation of ion nanochannels due to a novel microstructural rearrangement specific to this material.