Temporal and structural sensitivities of major biomarkers for detecting neuropathology after traumatic brain injury in the mouse.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality, especially in teenagers to young adults. In recent decades, different biomarkers and/or staining protocols have been employed to evaluate the post-injury development of pathological structures, but they have produced many contradictory findings. Since correctly identifying the underlying neuroanatomical changes is critical to advancing TBI research, we compared three commonly used markers for their ability to detect TBI pathological structures: Fluoro-Jade C, the rabbit monoclonal antibody Y188 against amyloid precursor protein and the NeuroSilver kit were used to stain adjacent slices from naïve or injured mouse brains harvested at different time points from 30 min to 3 months after lateral fluid percussion injury. Although not all pathological structures were stained by all markers at all time points, we found damaged neurons and deformed dendrites in gray matter, punctate and perivascular structures in white matter, and axonal blebs and Wallerian degeneration in both gray and white matter. The present study demonstrates the temporal and structural sensitivities of the three biomarkers: each marker is highly effective for a set of pathological structures, each of which in turn emerges at a particular time point. Furthermore, the different biomarkers showed different abilities at detecting identical types of pathological structures. In contrast to previous studies that have used a single biomarker at a single time range, the present report strongly recommends that a combination of different biomarkers should be adopted and different time points need to be checked when assessing neuropathology after TBI.

Neuronal somatic plasmalemmal permeability and dendritic beading caused by head rotational traumatic brain injury in pigs-An exploratory study.

Closed-head traumatic brain injury (TBI) is induced by rapid motion of the head, resulting in diffuse strain fields throughout the brain. The injury mechanism(s), loading thresholds, and neuroanatomical distribution of affected cells remain poorly understood, especially in the gyrencephalic brain. We utilized a porcine model to explore the relationships between rapid head rotational acceleration-deceleration loading and immediate alterations in plasmalemmal permeability within cerebral cortex, sub-cortical white matter, and hippocampus. To assess plasmalemmal compromise, Lucifer yellow (LY), a small cell-impermeant dye, was delivered intraventricularly and diffused throughout the parenchyma prior to injury in animals euthanized at 15-min post-injury; other animals (not receiving LY) were survived to 8-h or 7-days. Plasmalemmal permeability preferentially occurred in neuronal somata and dendrites, but rarely in white matter axons. The burden of LY+ neurons increased based on head rotational kinematics, specifically maximum angular velocity, and was exacerbated by repeated TBI. In the cortex, LY+ cells were prominent in both the medial and lateral gyri. Neuronal membrane permeability was observed within the hippocampus and entorhinal cortex, including morphological changes such as beading in dendrites. These changes correlated with reduced fiber volleys and synaptic current alterations at later timepoints in the hippocampus. Further histological observations found decreased NeuN immunoreactivity, increased mitochondrial fission, and caspase pathway activation in both LY+ and LY- cells, suggesting the presence of multiple injury phenotypes. This exploratory study suggests relationships between plasmalemmal disruptions in neuronal somata and dendrites within cortical and hippocampal gray matter as a primary response in closed-head rotational TBI and sets the stage for future, traditional hypothesis-testing experiments.

Detection and verification of neurodegeneration after traumatic brain injury in the mouse: Immunohistochemical staining for amyloid precursor protein.

Previous studies of human traumatic brain injury (TBI) have shown diffuse axonal injury as varicosities or spheroids in white matter (WM) bundles when using immunoperoxidase-ABC staining with 22C11, a mouse monoclonal antibody against amyloid precursor protein (APP). These findings have been interpreted as TBI-induced axonal pathology. In a mouse model of TBI however, when we used immunofluorescent staining with 22C11, as opposed to immunoperoxidase staining, we did not observe varicosities or spheroids. To explore this discrepancy, we performed immunofluorescent staining with Y188, an APP knockout-validated rabbit monoclonal that shows baseline immunoreactivity in neurons and oligodendrocytes of non-injured mice, with some arranged-like varicosities. In gray matter after injury, Y188 intensely stained axonal blebs. In WM, we encountered large patches of heavily stained puncta, heterogeneous in size. Scattered axonal blebs were also identified among these Y188-stained puncta. To assess the neuronal origin of Y188 staining after TBI we made use of transgenic mice with fluorescently labeled neurons and axons. A close correlation was observed between Y188-stained axonal blebs and fluorescently labeled neuronal cell bodies/axons. By contrast, no correlation was observed between Y188-stained puncta and fluorescent axons in WM, suggesting that these puncta in WM did not originate from axons, and casting further doubt on the nature of previous reports with 22C11. As such, we strongly recommend Y188 as a biomarker for detecting damaged neurons and axons after TBI. With Y188, stained axonal blebs likely represent acute axonal truncations that may lead to death of the parent neurons. Y188-stained puncta in WM may indicate damaged oligodendrocytes, whose death and clearance can result in secondary demyelination and Wallerian degeneration of axons. We also provide evidence suggesting that 22C11-stained varicosities or spheroids previously reported in TBI patients might be showing damaged oligodendrocytes, due to a cross-reaction between the ABC kit and upregulated endogenous biotin.

Maintenance of a Lateral Fluid Percussion Injury Device.

Traumatic brain injury (TBI) accounts for roughly 2.5 million emergency room visits and hospitalizations annually and is a leading cause of death and disability in children and young adults. TBI is caused by a sudden force applied to the head and, to better understand human TBI and its underlying mechanisms, experimental injury models are necessary. Lateral fluid percussion injury (LFPI) is a commonly used injury model due to similarities in the pathological changes found in human TBI compared to LFPI, including hemorrhages, vascular disruption, neurological deficits, and neuron loss. LFPI employs a pendulum and a fluid-filled cylinder, the latter having a moveable piston at one end, and a Luer lock connection to stiff, fluid-filled tubing at the other end. Preparation of the animal involves performing a craniectomy and attaching a Luer hub over the site. The next day, the tubing from the injury device is connected to the Luer hub on the animal's skull and the pendulum is raised to a specified height and released. The impact of the pendulum with the piston generates a pressure pulse which is transmitted to the intact dura mater of the animal via the tubing and produces the experimental TBI. Proper care and maintenance are essential for the LFPI device to function reliably, as the character and severity of the injury can vary greatly depending on the condition of the device. Here, we demonstrate how to properly clean, fill, and assemble the LFPI device, and ensure that it is adequately maintained for optimal results.

Blocking Cross-Species Secondary Binding When Performing Double Immunostaining With Mouse and Rat Primary Antibodies.

Immunostaining is a powerful technique and widely used to identify molecules in tissues and cells, although critical steps are necessary to block cross-reaction. Here we focused on an overlooked cross immunoreactivity issue where a secondary antibody (secondary) cross-reacts with a primary antibody (primary) from a different species. We first confirmed the previously reported cross-species binding of goat anti-mouse secondary to rat primary. This was accomplished by staining with a rat primary against glial fibrillary acidic protein (GFAP) and visualizing with goat (or donkey) anti-mouse secondary. We then further revealed the converse cross-species binding by staining with a mouse primary against neuronal nuclear protein (NeuN) and visualizing with anti-rat secondaries. We speculate that mouse and rat primaries share antigenicity, enabling either secondary to recognize either primary. To block this cross-species binding in double staining experiments, we compared three protocols using mouse anti-NeuN and rat anti-GFAP, two primaries whose antigens have non-overlapping distributions in brain tissues. Simultaneous staining resulted in cross-species astrocytic staining (anti-mouse secondary to rat anti-GFAP primary) but no cross-species neuronal staining (anti-rat secondary to mouse anti-NeuN primary). Cross-species astrocytic staining was missing after sequential same-species staining with mouse anti-NeuN primary, followed by rat anti-GFAP. However, cross-species astrocytic staining could not be diminished after sequential same-species staining with rat anti-GFAP primary, followed by mouse anti-NeuN. We thus hypothesize that a competition exists between anti-mouse and anti-rat secondaries in their binding to both primaries. Single staining for NeuN or GFAP visualized with dual secondaries at different dilution ratio supported this hypothesis.

A Comparison of Different Slicing Planes in Preservation of Major Hippocampal Pathway Fibers in the Mouse.

The hippocampus plays a critical role in learning and memory and higher cognitive functions, and its dysfunction has been implicated in various neuropathological disorders. Electrophysiological recording undertaken in live brain slices is one of the most powerful tools for investigating hippocampal cellular and network activities. The plane for cutting the slices determines which afferent and/or efferent connections are best preserved, and there are three commonly used slices: hippocampal-entorhinal cortex (HEC), coronal and transverse. All three slices have been widely used for studying the major afferent hippocampal pathways including the perforant path (PP), the mossy fibers (MFs) and the Schaffer collaterals (SCs). Surprisingly, there has never been a systematic investigation of the anatomical and functional consequences of slicing at a particular angle. In the present study, we focused on how well fiber pathways are preserved from the entorhinal cortex (EC) to the hippocampus, and within the hippocampus, in slices generated by sectioning at different angles. The postmortem neural tract tracer 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) was used to label afferent fibers to hippocampal principal neurons in fixed slices or whole brains. Laser scanning confocal microscopy was adopted for imaging DiI-labeled axons and terminals. We demonstrated that PP fibers were well preserved in HEC slices, MFs in both HEC and transverse slices and SCs in all three types of slices. Correspondingly, field excitatory postsynaptic potentials (fEPSPs) could be consistently evoked in HEC slices when stimulating PP fibers and recorded in stratum lacunosum-moleculare (sl-m) of area CA1, and when stimulating the dentate granule cell layer (gcl) and recording in stratum lucidum (sl) of area CA3. The MF evoked fEPSPs could not be recorded in CA3 from coronal slices. In contrast to our DiI-tracing data demonstrating severely truncated PP fibers in coronal slices, fEPSPs could still be recorded in CA1 sl-m in this plane, suggesting that an additional afferent fiber pathway other than PP might be involved. The present study increases our understanding of which hippocampal pathways are best preserved in the three most common brain slice preparations, and will help investigators determine the appropriate slices to use for physiological studies depending on the subregion of interest.

Concussion Induces Hippocampal Circuitry Disruption in Swine.

Hippocampal-dependent deficits in learning and memory formation are a prominent feature of traumatic brain injury (TBI); however, the role of the hippocampus in cognitive dysfunction after concussion (mild TBI) is unknown. We therefore investigated functional and structural changes in the swine hippocampus following TBI using a model of head rotational acceleration that closely replicates the biomechanics and neuropathology of closed-head TBI in humans. We examined neurophysiological changes using a novel ex vivo hippocampal slice paradigm with extracellular stimulation and recording in the dentate gyrus and CA1 occurring at 7 days following non-impact inertial TBI in swine. Hippocampal neurophysiology post-injury revealed reduced axonal function, synaptic dysfunction, and regional hyperexcitability at one week following even "mild" injury levels. Moreover, these neurophysiological changes occurred in the apparent absence of intra-hippocampal neuronal or axonal degeneration. Input-output curves demonstrated an elevated excitatory post-synaptic potential (EPSP) output for a given fiber volley input in injured versus sham animals, suggesting a form of homeostatic plasticity that manifested as a compensatory response to decreased axonal function in post-synaptic regions. These data indicate that closed-head rotational acceleration-induced TBI, the common cause of concussion in humans, may induce significant alterations in hippocampal circuitry function that have not resolved at 7 days post-injury. This circuitry dysfunction may underlie some of the post-concussion symptomatology associated with the hippocampus, such as post-traumatic amnesia and ongoing cognitive deficits.

Efficacy, dosage, and duration of action of branched chain amino Acid therapy for traumatic brain injury.

Traumatic brain injury (TBI) results in long-lasting cognitive impairments for which there is currently no accepted treatment. A well-established mouse model of mild to moderate TBI, lateral fluid percussion injury (FPI), shows changes in network excitability in the hippocampus including a decrease in net synaptic efficacy in area CA1 and an increase in net synaptic efficacy in dentate gyrus. Previous studies identified a novel therapy consisting of branched chain amino acids (BCAAs), which restored normal mouse hippocampal responses and ameliorated cognitive impairment following FPI. However, the optimal BCAA dose and length of treatment needed to improve cognitive recovery is unknown. In the current study, mice underwent FPI then consumed 100 mM BCAA supplemented water ad libitum for 2, 3, 4, 5, and 10 days. BCAA therapy ameliorated cognitive impairment at 5 and 10 days duration. Neither BCAA supplementation at 50 mM nor BCAAs when dosed 5 days on then 5 days off was sufficient to ameliorate cognitive impairment. These results suggest that brain injury causes alterations in hippocampal function, which underlie and contribute to hippocampal cognitive impairment, which are reversible with at least 5 days of BCAA treatment, and that sustaining this effect is dependent on continuous treatment. Our findings have profound implications for the clinical investigation of TBI therapy.

A toolbox for spatiotemporal analysis of voltage-sensitive dye imaging data in brain slices.

Voltage-sensitive dye imaging (VSDI) can simultaneously monitor the spatiotemporal electrical dynamics of thousands of neurons and is often used to identify functional differences in models of neurological disease. While the chief advantage of VSDI is the ability to record spatiotemporal activity, there are no tools available to visualize and statistically compare activity across the full spatiotemporal range of the VSDI dataset. Investigators commonly analyze only a subset of the data, and a majority of the dataset is routinely excluded from analysis. We have developed a software toolbox that simplifies visual inspection of VSDI data, and permits unaided statistical comparison across spatial and temporal dimensions. First, the three-dimensional VSDI dataset (x,y,time) is geometrically transformed into a two-dimensional spatiotemporal map of activity. Second, statistical comparison between groups is performed using a non-parametric permutation test. The result is a 2D map of all significant differences in both space and time. Here, we used the toolbox to identify functional differences in activity in VSDI data from acute hippocampal slices obtained from epileptic Arx conditional knock-out and control mice. Maps of spatiotemporal activity were produced and analyzed to identify differences in the activity evoked by stimulation of each of two axonal inputs to the hippocampus: the perforant pathway and the temporoammonic pathway. In mutant hippocampal slices, the toolbox identified a widespread decrease in spatiotemporal activity evoked by the temporoammonic pathway. No significant differences were observed in the activity evoked by the perforant pathway. The VSDI toolbox permitted us to visualize and statistically compare activity across the spatiotemporal scope of the VSDI dataset. Sampling error was minimized because the representation of the data is standardized by the toolbox. Statistical comparisons were conducted quickly, across the spatiotemporal scope of the data, without a priori knowledge of the character of the responses or the likely differences between them.

Augmented Inhibition from Cannabinoid-Sensitive Interneurons Diminishes CA1 Output after Traumatic Brain Injury.

The neurological impairments associated with traumatic brain injury include learning and memory deficits and increased risk of seizures. The hippocampus is critically involved in both of these phenomena and highly susceptible to damage by traumatic brain injury. To examine network activity in the hippocampal CA1 region after lateral fluid percussion injury, we used a combination of voltage-sensitive dye, field potential, and patch clamp recording in mouse hippocampal brain slices. When the stratum radiatum (SR) was stimulated in slices from injured mice, we found decreased depolarization in SR and increased hyperpolarization in stratum oriens (SO), together with a decrease in the percentage of pyramidal neurons firing stimulus-evoked action potentials. Increased hyperpolarization in SO persisted when glutamatergic transmission was blocked. However, we found no changes in SO responses when the alveus was stimulated to directly activate SO. These results suggest that the increased SO hyperpolarization evoked by SR stimulation was mediated by interneurons that have cell bodies and/or axons in SR, and form synapses in stratum pyramidale and SO. A low concentration (100 nM) of the synthetic cannabinoid WIN55,212-2, restored CA1 output in slices from injured animals. These findings support the hypothesis that increased GABAergic signaling by cannabinoid-sensitive interneurons contributes to the reduced CA1 output following traumatic brain injury.

Investigations on alterations of hippocampal circuit function following mild traumatic brain injury.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation. Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI. These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

Flexible casting of modular self-aligning microfluidic assembly blocks.

The recent shift among developers of microfluidic technologies toward modularized "plug and play" construction reflects the steadily increasing realization that, for many would-be users of microfluidic tools, traditional clean-room microfabrication is prohibitively complex and/or expensive. In this work, we present an advanced modular microfluidic construction scheme in which pre-fabricated microfluidic assembly blocks (MABs) can be quickly fashioned, without expertise or specialized facilities, into sophisticated microfluidic devices for a wide range of applications. Specifically, we describe three major advances to the MAB concept: (1) rapid production and extraction of MABs using flexible casting trays, (2) use of pre-coated substrates for simultaneous assembly and bonding, and (3) modification of block design to include automatic alignment and sealing structures. Finally, several exemplary applications of these MABs are demonstrated in chemical gradient synthesis, droplet generation, and total internal reflection fluorescence microscopy.

Somatostatin signaling in neuronal cilia is critical for object recognition memory.

Most neurons possess a single, nonmotile cilium that projects out from the cell surface. These microtubule-based organelles are important in brain development and neurogenesis; however, their function in mature neurons is unknown. Cilia express a complement of proteins distinct from other neuronal compartments, one of which is the somatostatin receptor subtype SST(3). We show here that SST(3) is critical for object recognition memory in mice. sst3 knock-out mice are severely impaired in discriminating novel objects, whereas they retain normal memory for object location. Further, systemic injection of an SST(3) antagonist (ACQ090) disrupts recall of familiar objects in wild-type mice. To examine mechanisms of SST(3), we tested synaptic plasticity in CA1 hippocampus. Electrically evoked long-term potentiation (LTP) was normal in sst3 knock-out mice, while adenylyl cyclase/cAMP-mediated LTP was impaired. The SST(3) antagonist also disrupted cAMP-mediated LTP. Basal cAMP levels in hippocampal lysate were reduced in sst3 knock-out mice compared with wild-type mice, while the forskolin-induced increase in cAMP levels was normal. The SST(3) antagonist inhibited forskolin-stimulated cAMP increases, whereas the SST(3) agonist L-796,778 increased basal cAMP levels in hippocampal slices but not hippocampal lysate. Our results show that somatostatin signaling in neuronal cilia is critical for recognition memory and suggest that the cAMP pathway is a conserved signaling motif in cilia. Neuronal cilia therefore represent a novel nonsynaptic compartment crucial for signaling involved in a specific form of synaptic plasticity and in novelty detection.

K+ M-current regulates the transition to seizures in immature and adult hippocampus.

PURPOSE: Loss-of-function mutations in Kv7.2 or Kv7.3 K(+) channel subunits underlies the neonatal epilepsy benign familial neonatal convulsions (BFNC). These two subunits interact to form a functional K(+) channel that underlies the M-current (I(M)), a voltage-dependent noninactivating K(+) current. In BFNC, seizures begin shortly after birth, and spontaneously remit in the first few months of life. The nature of this window of vulnerability is unclear. We address this issue using a hippocampal slice model, to study the effects of I(M) blockade or augmentation on epileptiform activity. METHODS: We used the Mg(+)(+)-free seizure model in adult and immature (P8-P15) acute rat hippocampal slices. We recorded from both CA1 and CA3 regions using extracellular and intracellular methods. RESULTS: When M-channels are blocked pharmacologically, the transition from interictal to ictal bursting becomes much more likely, especially in immature brain. We also show augmentation of I(M) is effective in stopping ictal events in immature brain, at the developmental age that approximates a human newborn in cortical development. I(M) appears to counter the sustained N-methyl-D-aspartate (NMDA) receptor-mediated depolarizations needed to trigger an ictal event. The increased likelihood of ictal bursting by I(M) blockade is not shared by other selective K(+) channel blockers that increase hippocampal excitability. CONCLUSIONS: Voltage-dependent M-channels are activated during interictal bursts and contribute to burst termination. When these channels are compromised, interictal burst duration becomes sufficient to trigger the sustained depolarizations that underlie ictal bursts. This transition to ictal bursts upon I(M) blockade is especially likely to occur in immature hippocampus. This selective function of M-channels likely contributes to the transient window of vulnerability to seizures that occurs with BFNC.

Phase change microvalve for integrated devices.

An active microvalve that uses a meltable piston in place of a conventional solid material to obstruct fluid flow in a microfluidic channel has been developed. This phase change valve is simple to operate and requires no additional fabrication steps. The valve is inherently latched, reusable, and leak-proof (to at least 250 psi) and can be electronically addressed using resistive heaters. The valve has been characterized for a range of operational parameters that will serve as a design guide. For the designs tested, piston displacements of 5 mm or more in 1 s have been achieved. Valves 1.4 mm in length in a 50 microm x 200 microm channel have been integrated on a biochemical reaction device, and successful DNA amplification using PCR has been achieved. The phase change valve can be easily implemented in an array format that can be used to realize complex microfluidic circuits.