Respiratory Syncytial Virus Infects Peripheral and Spinal Nerves and Induces Chemokine-Mediated Neuropathy.

Respiratory syncytial virus (RSV) primarily infects the respiratory epithelium, but growing evidence suggests that it may also be responsible for neurologic sequelae. In 3-dimensional microphysiologic peripheral nerve cultures, RSV infected neurons, macrophages, and dendritic cells along 2 distinct trajectories depending on the initial viral load. Low-level infection was transient, primarily involved macrophages, and induced moderate chemokine release with transient neural hypersensitivity. Infection with higher viral loads was persistent, infected neuronal cells in addition to monocytes, and induced robust chemokine release followed by progressive neurotoxicity. In spinal cord cultures, RSV infected microglia and dendritic cells but not neurons, producing a moderate chemokine expression pattern. The persistence of infection was variable but could be identified in dendritic cells as long as 30 days postinoculation. This study suggests that RSV can disrupt neuronal function directly through infection of peripheral neurons and indirectly through infection of resident monocytes and that inflammatory chemokines likely mediate both mechanisms.

Fully Integrated 3D Microelectrode Arrays with Polydopamine-Mediated Silicon Dioxide Insulation for Electrophysiological Interrogation of a Novel 3D Human, Neural Microphysiological Construct.

Advances within in vitro biological system complexity have enabled new possibilities for the "Organs-on-a-Chip" field. Microphysiological systems (MPS) as such incorporate sophisticated biological constructs with custom biological sensors. For microelectromechanical systems (MEMS) sensors, the dielectric layer is critical for device performance, where silicon dioxide (SiO2) represents an excellent candidate due to its biocompatibility and wide utility in MEMS devices. Yet, high temperatures traditionally preclude SiO2 from incorporation in polymer-based BioMEMS. Electron-beam deposition of SiO2 may provide a low-temperature, dielectric serving as a nanoporous MPS growth substrate. Herein, we enable improved adherence of nanoporous SiO2 to polycarbonate (PC) and 316L stainless steel (SS) via polydopamine (PDA)-mediated chemistry. The resulting stability of the combinatorial PDA-SiO2 film was interrogated, along with the nature of the intrafilm interactions. A custom polymer-metal three-dimensional (3D) microelectrode array (3D MEA) is then reported utilizing PDA-SiO2 insulation, for definition of novel dorsal root ganglion (DRG)/nociceptor and dorsal horn (DH) 3D neural constructs in excess of 6 months for the first time. Spontaneous/evoked compound action potentials (CAPs) are successfully reported. Finally, inhibitory drugs treatments showcase pharmacological responsiveness of the reported multipart biological activity. These results represent the initiation of a novel 3D MEA-integrated, 3D neural MPS for the long-term electrophysiological study.

Immunoblot Detection of the Phosphorylation of the Estrogen Receptor α as an Outcome of GPR30 /GPER1 Activation.

Phosphorylation of the serine residues in estrogen receptor (ER) α is important in transcriptional activation. Hence, methods to detect such posttranslational modification events are valuable. We describe, in detail, the analysis of the phosphorylated ERα by electrophoretic separation of proteins and subsequent immunoblotting techniques. In particular, phosphorylation of the ERα is one possible outcome of activation of the putative membrane estrogen receptor (mER), GPR30 or GPER1. Hence, phosphorylation represents a crosstalk event between GPR30 and ERα and may be important in estrogen-regulated physiology.

Enhancing the use of exposure science across EU chemical policies as part of the European Exposure Science Strategy 2020-2030.

BACKGROUND: A scientific framework on exposure science will boost the multiuse of exposure knowledge across EU chemicals-related policies and improve risk assessment, risk management and communication across EU safety, security and sustainability domains. OBJECTIVE: To stimulate public and private actors to align and strengthen the cross-policy adoption of exposure assessment data, methods and tools across EU legislation. METHODS: By mapping and analysing the EU regulatory landscape making use of exposure information, policy and research challenges and key areas of action are identified and translated into opportunities enhancing policy and scientific efficiency. RESULTS: Identified key areas of actions are to develop a common scientific exposure assessment framework, supported by baseline acceptance criteria and a shared knowledge base enhancing exchangeability and acceptability of exposure knowledge within and across EU chemicals-related policies. Furthermore, such framework will improve communication and management across EU chemical safety, security and sustainability policies comprising sourcing, manufacturing and global trade of goods and waste management. In support of building such a common framework and its effective use in policy and industry, exposure science innovation needs to be better embedded along the whole policymaking cycle, and be integrated into companies' safety and sustainability management systems. This will help to systemically improve regulatory risk management practices. SIGNIFICANCE: This paper constitutes an important step towards the implementation of the EU Green Deal and its underlying policy strategies, such as the Chemicals Strategy for Sustainability.

Morphine-sensitive synaptic transmission emerges in embryonic rat microphysiological model of lower afferent nociceptive signaling.

Debilitating chronic pain resulting from genetic predisposition, injury, or acquired neuropathy is becoming increasingly pervasive. Opioid analgesics remain the gold standard for intractable pain, but overprescription of increasingly powerful and addictive opioids has contributed to the current prescription drug abuse epidemic. There is a pressing need to screen experimental compounds more efficiently for analgesic potential that remains unmet by conventional research models. The spinal cord dorsal horn is a common target for analgesic intervention, where peripheral nociceptive signals are relayed to the central nervous system through synaptic transmission. Here, we demonstrate that coculturing peripheral and dorsal spinal cord nerve cells in a novel bioengineered microphysiological system facilitates self-directed emergence of native nerve tissue macrostructure and concerted synaptic function. The mechanistically distinct analgesics-morphine, lidocaine, and clonidine-differentially and predictably modulate this microphysiological synaptic transmission. Screening drug candidates for similar microphysiological profiles will efficiently identify therapeutics with analgesic potential.

Comparative Analysis of Chemotherapy-Induced Peripheral Neuropathy in Bioengineered Sensory Nerve Tissue Distinguishes Mechanistic Differences in Early-Stage Vincristine-, Cisplatin-, and Paclitaxel-Induced Nerve Damage.

Chemotherapy-induced peripheral neuropathy (CIPN) is a well-known, potentially permanent side effect of widely used antineoplastic agents. The mechanisms of neuropathic progression are poorly understood, and the need to test efficacy of novel interventions to treat CIPN continues to grow. Bioengineered microphysiological nerve tissue ("nerve on a chip") has been suggested as an in vitro platform for modeling the structure and physiology of in situ peripheral nerve tissue. Here, we find that length-dependent nerve conduction and histopathologic changes induced by cisplatin, paclitaxel, or vincristine in rat dorsal root ganglion-derived microphysiological sensory nerve tissue recapitulate published descriptions of clinical electrophysiological changes and neuropathologic biopsy findings in test animals and human patients with CIPN. We additionally confirm the postulated link between vincristine-induced axoplasmic transport failure and functional impairment of nerve conduction, the postulated paclitaxel-induced somal toxicity, and identify a potential central role of gliotoxicity in cisplatin-induced sensory neuropathy. Microphysiological CIPN combines the tight experimental control afforded by in vitro experimentation with clinically relevant functional and structural outputs that conventionally require in vivo models. Microphysiological nerve tissue provides a low-cost, high-throughput alternative to conventional nonclinical models for efficiently and effectively investigating lesions, mechanisms, and treatments of CIPN. Neural microphysiological systems are capable of modeling complex neurological disease at the tissue level offering unique advantages over conventional methodology for both testing and generating hypotheses in neurological disease modeling. Impact Statement Recapitulation of distinct hallmarks of clinical CIPN in microphysiological sensory nerve validates a novel peripheral neurotoxicity model with unique advantages over conventional model systems.

Systems Pharmacology Modeling Identifies a Novel Treatment Strategy for Bortezomib-Induced Neuropathic Pain.

Chemotherapy-induced peripheral neurotoxicity is a common dose-limiting side effect of several cancer chemotherapeutic agents, and no effective therapies exist. Here we constructed a systems pharmacology model of intracellular signaling in peripheral neurons to identify novel drug targets for preventing peripheral neuropathy associated with proteasome inhibitors. Model predictions suggested the combinatorial inhibition of TNFα, NMDA receptors, and reactive oxygen species should prevent proteasome inhibitor-induced neuronal apoptosis. Dexanabinol, an inhibitor of all three targets, partially restored bortezomib-induced reduction of proximal action potential amplitude and distal nerve conduction velocity in vitro and prevented bortezomib-induced mechanical allodynia and thermal hyperalgesia in rats, including a partial recovery of intraepidermal nerve fiber density. Dexanabinol failed to restore bortezomib-induced decreases in electrophysiological endpoints in rats, and it did not compromise bortezomib anti-cancer effects in U266 multiple myeloma cells and a murine xenograft model. Owing to its favorable safety profile in humans and preclinical efficacy, dexanabinol might represent a treatment option for bortezomib-induced neuropathic pain.

Neuroestrogen-Dependent Transcriptional Activity in the Brains of ERE-Luciferase Reporter Mice following Short- and Long-Term Ovariectomy.

Previous work has demonstrated that estrogen receptors are transcriptionally active in the absence of ovarian estrogens. The current work aims to determine whether brain-derived estrogens influence estrogen receptor-dependent transcription after short- or long-term loss of ovarian function. Experiments were conducted using estrogen response element (ERE)-Luciferase reporter mice, which express the gene for luciferase driven by consensus ERE, allowing for the quantification of ERE-dependent transcription. Brain regions examined were hippocampus, cortex, and hypothalamus. In Experiment 1, short-term (10 d) ovariectomy had no impact on ERE-dependent transcription across brain regions compared with sham surgery. In Experiment 2, chronic intracerebroventricular administration of the aromatase inhibitor letrozole significantly decreased transcriptional activity in 10-d-old ovariectomized mice across brain regions, indicating that the sustained transcription in short-term ovariectomized mice is mediated at least in part via actions of neuroestrogens. Additionally, intracerebroventricular administration of estrogen receptor antagonist ICI-182,780 blocked transcription in 10-d-old ovariectomized mice across brain regions, providing evidence that sustained transcription in ovariectomized mice is estrogen receptor dependent. In Experiment 3, long-term (70 d) ovariectomy significantly decreased ERE-dependent transcription across brain regions, though some residual activity remained. In Experiment 4, chronic intracerebroventricular letrozole administration had no impact on transcription in 70 d ovariectomized mice across brain regions, indicating that the residual ERE-dependent transcription in long-term ovariectomized mice is not mediated by neuroestrogens. Overall, the results indicate that ERE-dependent transcription in the brain continues after ovariectomy and that the actions of neuroestrogens contribute to the maintenance of ERE-dependent transcription in the brain following short-term, but not long-term, loss of ovarian function.

Nuclear estrogen receptor activation by insulin-like growth factor-1 in Neuro-2A neuroblastoma cells requires endogenous estrogen synthesis and is mediated by mutually repressive MAPK and PI3K cascades.

Non-canonical mechanisms of estrogen receptor activation may continue to support women's cognitive health long after cessation of ovarian function. These mechanisms of estrogen receptor activation may include ligand-dependent actions via locally synthesized neuroestrogens and ligand-independent actions via growth factor-dependent activation of intracellular kinase cascades. We tested the hypothesis that ligand-dependent and ligand-independent mechanisms interact to activate nuclear estrogen receptors in the Neuro-2A neuroblastoma cell line in the absence of exogenous estrogens. Transcriptional output of estrogen receptors was measured following treatment with insulin-like growth factor-1 (IGF-1) in the presence of specific inhibitors for mitogen-activated protein kinase (MAPK), phosphoinositde-3 kinase (PI3K), and neuroestrogen synthesis. Results indicate that IGF-1-dependent activation of nuclear estrogen receptors is mediated by MAPK, is opposed PI3K, and requires concomitant endogenous neuroestrogen synthesis. We conclude that both cellular signaling context and endogenous ligand availability are important modulators of ligand-independent nuclear estrogen receptor activation.

Semantic Segmentation of Microengineered Neural Tissues.

In this paper, we present a novel strategy for automatic segmentation of biomedical images acquired from bio-engineered nerve tissues exhibiting variable morphological characteristics. Automatic image segmentation is one step towards the end goal of automatic analysis of the impact of various neurotoxic drug treatments on these artificial nerve tissues. We propose a deep learning architecture to perform this task. Our proposed architecture can be seen as a variation of U-Net that helps deal with a small manually annotated training data set. We present promising preliminary results and our human expert analysis shows that in some cases the model is even more precise in detecting the relevant morphological characteristics of the tissue compared to the manually annotated data. In the future, our model can be adapted for end-to-end automatic analysis of treated tissues. Moreover, based on a very small set of annotated data, it provides a reasonable segmentation to be used by human annotators. This will reduce the time of manual annotation significantly and streamline the process of generating a larger manually annotated data set for training our final ideal segmentation model.

Detection of the Phosphorylation of the Estrogen Receptor α as an Outcome of GPR30 Activation.

Phosphorylation of the serine residues in estrogen receptor (ER) α is important in transcriptional activation. Hence, methods to detect such posttranslational modification events are valuable. We describe, in detail, the analysis of the phosphorylated ERα by electrophoretic separation of proteins and subsequent immuno-blotting techniques. In particular, phosphorylation of the ERα is one possible outcome of activation of the putative membrane estrogen receptor (mER), GPR30. Hence, phosphorylation represents a cross talk event between GPR30 and ERα and may be important in estrogen-regulated physiology.

Membrane-initiated non-genomic signaling by estrogens in the hypothalamus: cross-talk with glucocorticoids with implications for behavior.

The estrogen receptor and glucocorticoid receptor are members of the nuclear receptor superfamily that can signal using both non-genomic and genomic transcriptional modes. Though genomic modes of signaling have been well characterized and several behaviors attributed to this signaling mechanism, the physiological significance of non-genomic modes of signaling has not been well understood. This has partly been due to the controversy regarding the identity of the membrane ER (mER) or membrane GR (mGR) that may mediate rapid, non-genomic signaling and the downstream signaling cascades that may result as a consequence of steroid ligands binding the mER or the mGR. Both estrogens and glucocorticoids exert a number of actions on the hypothalamus, including feedback. This review focuses on the various candidates for the mER or mGR in the hypothalamus and the contribution of non-genomic signaling to classical hypothalamically driven behaviors and changes in neuronal morphology. It also attempts to categorize some of the possible functions of non-genomic signaling at both the cellular level and at the organismal level that are relevant for behavior, including some behaviors that are regulated by both estrogens and glucocorticoids in a potentially synergistic manner. Lastly, it attempts to show that steroid signaling via non-genomic modes may provide the organism with rapid behavioral responses to stimuli.

GPR30 activation decreases anxiety in the open field test but not in the elevated plus maze test in female mice.

The GPR30 is a novel estrogen receptor (ER) that is a candidate membrane ER based on its binding to 17ß estradiol and its rapid signaling properties such as activation of the extracellular-regulated kinase (ERK) pathway. Its distribution in the mouse limbic system predicts a role for this receptor in the estrogenic modulation of anxiety behaviors in the mouse. A previous study showed that chronic administration of a selective agonist to the GPR30 receptor, G-1, in the female rat can improve spatial memory, suggesting that GPR30 plays a role in hippocampal-dependent cognition. In this study, we investigated the effect of a similar chronic administration of G-1 on behaviors that denote anxiety in adult ovariectomized female mice, using the elevated plus maze (EPM) and the open field test as well as the activation of the ERK pathway in the hippocampus. Although estradiol benzoate had no effect on behaviors in the EPM or the open field, G-1 had an anxiolytic effect solely in the open field that was independent of ERK signaling in either the ventral or dorsal hippocampus. Such an anxiolytic effect may underlie the ability of G-1 to increase spatial memory, by acting on the hippocampus.

Activation of the G-protein coupled receptor 30 (GPR30) has different effects on anxiety in male and female mice.

The GPR30, a former orphan GPCR, is a putative membrane estrogen receptor that can activate rapid signaling pathways such as extracellular regulated kinase (ERK) in a variety of cells and may contribute to estrogen's effects in the central nervous system. The distribution of GPR30 in the limbic system predicts a role for this receptor in the regulation of learning and memory and anxiety by estrogens. Though acute G-1 treatment is reported to be anxiogenic in ovariectomised female mice and in gonadally intact male mice, the effect of GPR30 activation is unknown in gonadectomised male mice. In this study, we show that an acute administration of G-1 to gonadectomised male mice, but not female mice, was anxiolytic on an elevated plus maze task, without affecting locomotor activity. In addition, though G-1 treatment did not regulate ERK, it was associated with increased estrogen receptor (ER)α phosphorylation in the ventral, but not dorsal, hippocampus of males. In the female, G-1 increased the ERK activation solely in the dorsal hippocampus, independent of state anxiety. This is the first study to report an anxiolytic effect of GPR30 activation in male mice, in a rapid time frame that is commensurate with non-genomic signaling by estrogen.