Delineating Astrocytic Cytokine Responses in a Human Stem Cell Model of Neural Trauma

Neuroinflammation has been shown to mediate the pathophysiological response following traumatic brain injury (TBI). Accumulating evidence implicates astrocytes as key immune cells within the central nervous system (CNS), displaying both pro- and anti-inflammatory properties. The aim of this study was to investigate how in vitro human astrocyte cultures respond to cytokines across a concentration range that approximates the aftermath of human TBI. To this end, enriched cultures of human induced pluripotent stem cell (iPSC)-derived astrocytes were exposed to interleukin-1β (IL-1β) (1–10,000 pg/mL), IL-4 (1–10,000 pg/mL), IL-6 (100–1,000,000 pg/mL), IL-10 (1–10,000 pg/mL) and tumor necrosis factor (TNF)-α (1–10,000 pg/mL). After 1, 24, 48 and 72 h, cultures were fixed and immunolabeled, and the secretome/supernatant was analyzed at 24, 48, and 72 h using a human cytokine/chemokine 39-plex Luminex assay. Data were compared to previous in vitro studies of neuronal cultures and clinical TBI studies. The secretome revealed concentration-, time- and/or both concentration- and time-dependent production of downstream cytokines (29, 21, and 17 cytokines, respectively, p<0.05). IL-1β exposure generated the most profound downstream response (27 cytokines), IL-6 and TNF had intermediate responses (13 and 11 cytokines, respectively), whereas IL-4 and IL-10 only led to weak responses over time or in escalating concentration (8 and 8 cytokines, respectively). Notably, expression of IL-1β, IL-6, and TNF cytokine receptor mRNA was higher in astrocyte cultures than in neuronal cultures. Several secreted cytokines had temporal trajectories, which corresponded to those seen in the aftermath of human TBI. In summary, iPSC-derived astrocyte cultures exposed to cytokine concentrations reflecting those in TBI generated an increased downstream cytokine production, particularly IL-1β. Although more work is needed to better understand how different cells in the CNS respond to the neuroinflammatory milieu after TBI, our data shows that iPSC-derived astrocytes represent a tractable model to study cytokine stimulation in a cell type-specific manner

Multiplexed, High Density Electrophysiology with Nanofabricated Neural Probes

Extracellular electrode arrays can reveal the neuronal network correlates of behavior with single-cell, single-spike, and sub-millisecond resolution. However, implantable electrodes are inherently invasive, and efforts to scale up the number and density of recording sites must compromise on device size in order to connect the electrodes. Here, we report on silicon-based neural probes employing nanofabricated, high-density electrical leads. Furthermore, we address the challenge of reading out multichannel data with an application-specific integrated circuit (ASIC) performing signal amplification, band-pass filtering, and multiplexing functions. We demonstrate high spatial resolution extracellular measurements with a fully integrated, low noise 64-channel system weighing just 330 mg. The on-chip multiplexers make possible recordings with substantially fewer external wires than the number of input channels. By combining nanofabricated probes with ASICs we have implemented a system for performing large-scale, high-density electrophysiology in small, freely behaving animals that is both minimally invasive and highly scalable

Spatial encoding in spinal sensorimotor circuits differs in different wild type mice strains

<p>Abstract</p> <p>Background</p> <p>Previous studies in the rat have shown that the spatial organisation of the receptive fields of nociceptive withdrawal reflex (NWR) system are functionally adapted through experience dependent mechanisms, termed somatosensory imprinting, during postnatal development. Here we wanted to clarify 1) if mice exhibit a similar spatial encoding of sensory input to NWR as previously found in the rat and 2) if mice strains with a poor learning capacity in various behavioural tests, associated with deficient long term potention, also exhibit poor adaptation of NWR.</p> <p>The organisation of the NWR system in two adult wild type mouse strains with normal long term potentiation (LTP) in hippocampus and two adult wild type mouse strains exhibiting deficiencies in corresponding LTP were used and compared to previous results in the rat. Receptive fields of reflexes in single hindlimb muscles were mapped with CO₂laser heat pulses.</p> <p>Results</p> <p>While the spatial organisation of the nociceptive receptive fields in mice with normal LTP were very similar to those in rats, the LTP impaired strains exhibited receptive fields of NWRs with aberrant sensitivity distributions. However, no difference was found in NWR thresholds or onset C-fibre latencies suggesting that the mechanisms determining general reflex sensitivity and somatosensory imprinting are different.</p> <p>Conclusion</p> <p>Our results thus confirm that sensory encoding in mice and rat NWR is similar, provided that mice strains with a good learning capability are studied and raise the possibility that LTP like mechanisms are involved in somatosensory imprinting.</p

Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways

Background: Continuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS. Methods: Human bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting. Results: cLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2. Conclusions: Collectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS

Integrative Neuroinformatics for Precision Prognostication and Personalized Therapeutics in Moderate and Severe Traumatic Brain Injury.

Despite changes in guideline-based management of moderate/severe traumatic brain injury (TBI) over the preceding decades, little impact on mortality and morbidity have been seen. This argues against the "one-treatment fits all" approach to such management strategies. With this, some preliminary advances in the area of personalized medicine in TBI care have displayed promising results. However, to continue transitioning toward individually-tailored care, we require integration of complex "-omics" data sets. The past few decades have seen dramatic increases in the volume of complex multi-modal data in moderate and severe TBI care. Such data includes serial high-fidelity multi-modal characterization of the cerebral physiome, serum/cerebrospinal fluid proteomics, admission genetic profiles, and serial advanced neuroimaging modalities. Integrating these complex and serially obtained data sets, with patient baseline demographics, treatment information and clinical outcomes over time, can be a daunting task for the treating clinician. Within this review, we highlight the current status of such multi-modal omics data sets in moderate/severe TBI, current limitations to the utilization of such data, and a potential path forward through employing integrative neuroinformatic approaches, which are applied in other neuropathologies. Such advances are positioned to facilitate the transition to precision prognostication and inform a top-down approach to the development of personalized therapeutics in moderate/severe TBI

Development of transparent ceramic Ce-doped gadolinium garnet gamma spectrometers

Abstract not provide

Nuclear expression of FLT1 and its ligand PGF in FUS-DDIT3 carrying myxoid liposarcomas suggests the existence of an intracrine signaling loop

<p>Abstract</p> <p>Background</p> <p>The FUS-DDIT3 fusion oncogene encodes an abnormal transcription factor that has a causative role in the development of myxoid/round-cell liposarcomas (MLS/RCLS). We have previously identified <it>FLT1 </it>(<it>VEGFR1</it>) as a candidate downstream target gene of FUS-DDIT3. The aim of this study was to investigate expression of FLT1 and its ligands in MLS cells.</p> <p>Methods</p> <p>HT1080 human fibrosarcoma cells were transiently transfected with <it>FUS-DDIT3</it>-GFP variant constructs and FLT1 expression was measured by quantitative real-time PCR. In addition, <it>FLT1</it>, <it>PGF, VEGFA and VEGFB </it>expression was measured in MLS/RCLS cell lines, MLS/RCLS tumors and in normal adiopocytes. We analyzed nine cases of MLS/RCLS and one cell line xenografted in mice for FLT1 protein expression using immunohistochemistry. MLS/RCLS cell lines were also analyzed for FLT1 by immunofluorescence and western blot. MLS/RCLS cell lines were additionally treated with FLT1 tyrosine kinase inhibitors and assayed for alterations in proliferation rate.</p> <p>Results</p> <p><it>FLT1 </it>expression was dramatically increased in transfected cells stably expressing FUS-DDIT3 and present at high levels in cell lines derived from MLS. The FLT1 protein showed a strong nuclear expression in cells of MLS tissue as well as in cultured MLS cells, which was confirmed by cellular fractionation. Tissue array analysis showed a nuclear expression of the FLT1 protein also in several other tumor and normal cell types including normal adipocytes. The FLT1 ligand coding gene <it>PGF </it>was highly expressed in cultured MLS cells compared to normal adipocytes while the other ligand genes <it>VEGFA </it>and <it>VEGFB </it>were expressed to lower levels. A more heterogeneous expression pattern of these genes were observed in tumor samples. No changes in proliferation rate of MLS cells were detected at concentrations for which the kinase inhibitors have shown specific inhibition of FLT1.</p> <p>Conclusions</p> <p>Our results imply that <it>FLT1 </it>is induced as an indirect downstream effect of FUS-DDIT3 expression in MLS. This could be a consequence of the ability of FUS-DDIT3 to hijack parts of normal adipose tissue development and reprogram primary cells to a liposarcoma-like phenotype. The findings of nuclear FLT1 protein and expression of corresponding ligands in MLS and normal tissues may have implications for tissue homeostasis and tumor development through auto- or intracrine signaling.</p

Short-pulse Laser Capability on the Mercury Laser System

Applications using high energy ''petawatt-class'' laser drivers operating at repetition rates beyond 0.01 Hz are only now being envisioned. The Mercury laser system is designed to operate at 100 J/pulse at 10 Hz. We investigate the potential of configuring the Mercury laser to produce a rep-rated, ''petawatt-class'' source. The Mercury laser is a prototype of a high energy, high repetition rate source (100 J, 10 Hz). The design of the Mercury laser is based on the ability to scale in energy through scaling in aperture. Mercury is one of several 100 J, high repetition rate (10 Hz) lasers sources currently under development (HALNA, LUCIA, POLARIS). We examine the possibility of using Mercury as a pump source for a high irradiance ''petawatt-class'' source: either as a pump laser for an average power Ti:Sapphire laser, or as a pump laser for OPCPA based on YCa{sub 4}O(BO{sub 3}){sub 3} (YCOB), ideally producing a source approaching 30 J /30 fs /10 Hz--a high repetition rate petawatt. A comparison of the two systems with nominal configurations and efficiencies is shown in Table 1