Agmatine Mitigates Inflammation-Related Oxidative Stress in BV-2 Cells by Inducing a Pre-Adaptive Response.

Neuroinflammation and microglial activation, common components of most neurodegenerative diseases, can be imitated in vitro by challenging microglia cells with Lps. We here aimed to evaluate the effects of agmatine pretreatment on Lps-induced oxidative stress in a mouse microglial BV-2 cell line. Our findings show that agmatine suppresses nitrosative and oxidative burst in Lps-stimulated microglia by reducing iNOS and XO activity and decreasing O2- levels, arresting lipid peroxidation, increasing total glutathione content, and preserving GR and CAT activity. In accordance with these results, agmatine suppresses inflammatory NF-kB, and stimulates antioxidant Nrf2 pathway, resulting in decreased TNF, IL-1 beta, and IL-6 release, and reduced iNOS and COX-2 levels. Together with increased ARG1, CD206 and HO-1 levels, our results imply that, in inflammatory conditions, agmatine pushes microglia towards an anti-inflammatory phenotype. Interestingly, we also discovered that agmatine alone increases lipid peroxidation end product levels, induces Nrf2 activation, increases total glutathione content, and GPx activity. Thus, we hypothesize that some of the effects of agmatine, observed in activated microglia, may be mediated by induced oxidative stress and adaptive response, prior to Lps stimulation.

Understanding Nanomaterial Biotransformation: An Unmet Challenge to Achieving Predictive Nanotoxicology.

More than a decade has passed since the first concepts of predictive nanotoxicology were formulated. During this time, many advancements have been achieved in multiple disciplines, including the success stories of the fiber paradigm and the oxidative stress paradigm. However, important knowledge gaps are slowing down the development of predictive nanotoxicology and require a mutidisciplinary effort to be overcome. Among these gaps, understanding, reproducing, and modeling of nanomaterial biotransformation in biological environments is a central challenge, both in vitro and in silico. This dynamic and complex process is still a challenge for today's bioanalytics. This work explores and discusses selected approaches of the multidisciplinary efforts taken in the last decade and the challenges that remain unmet, in particular concerning nanomaterial biotransformation. It highlights some future advancements that, together, can help to understand such complex processes and accelerate the development of predictive nanotoxicology.

The sex-specific patterns of changes in hypothalamic-pituitary-gonadal axis during experimental autoimmune encephalomyelitis.

Multiple sclerosis develops during reproductive years in a sex-specific manner. Various neuroendocrine changes have been described in this inflammatory, demyelinating, and debilitating disease. We here aimed to determine the extent and sex specificity of alterations in the hypothalamic-pituitary-gonadal axis in the rat model of multiple sclerosis named experimental autoimmune encephalomyelitis. During the disease course, the hypothalamic tissue showed transient upregulation of inflammatory marker genes Gfap, Cd68, Ccl2, and Il1b in both sexes, but accompanied by sex-specific downregulation of Kiss1 (in females only) and Gnrh1 (in males only) expression. In females, the expression of gonadotrope-specific genes Lhb, Cga, and Gnrhr was also inhibited, accompanied by decreased basal but not stimulated serum luteinizing hormone levels and a transient arrest of the estrous cycle. In contrast, Fshb expression and serum progesterone levels were transiently elevated, findings consistent with the maintenance of the corpora lutea, and elevated immunohistochemical labeling of ovarian StAR, a rate limiting protein in steroidogenic pathway. In males, downregulation of Gnrhr expression and basal and stimulated serum luteinizing hormone and testosterone levels were accompanied by inhibited testicular StAR protein expression. We propose that inflammation of hypothalamic tissue downregulates Kiss1 and Gnrh1 expression in females and males, respectively, leading to sex-specific changes downstream the axis.

Beyond Global Charge: Role of Amine Bulkiness and Protein Fingerprint on Nanoparticle-Cell Interaction.

Amino groups presented on the surface of nanoparticles are well-known to be a predominant factor in the formation of the protein corona and subsequent cellular uptake. However, the molecular mechanism underpinning this relationship is poorly defined. This study investigates how amine type and density affect the protein corona and cellular association of gold nanoparticles with cells in vitro. Four specific poly(vinyl alcohol-co-N-vinylamine) copolymers are synthesized containing primary, secondary, or tertiary amines. Particle cellular association (i.e., cellular uptake and surface adsorption), as well as protein corona composition, are then investigated. It is found that the protein corona (as a consequence of "amine bulkiness") and amine density are both important in dictating cellular association. By evaluating the nanoparticle surface chemistry and the protein fingerprint, proteins that are significant in mediating particle-cell association are identified. In particular, primary amines, when exposed on the polymer side chain, are strongly correlated with the presence of alpha-2-HS-glycoprotein, and promote nanoparticle cellular association.

Assessing the Stability of Fluorescently Encoded Nanoparticles in Lysosomes by Using Complementary Methods.

Nanoparticles (NPs) are promising tools in biomedical research. In vitro testing is still the first method for initial evaluation; however, NP colloidal behavior and integrity, in particular inside cells (that is, in lysosomes), are largely unknown and difficult to evaluate because of the complexity of the environment. Furthermore, while the majority of NPs are usually labeled with fluorescent dyes for tracking purposes, the effect of the lysosomal environment on the fluorophore properties, as well as the ensuing effects on data interpretation, is often only sparsely addressed. In this work, we have employed several complementary analytical methods to better understand the fate of fluorescently encoded NPs and identify potential pitfalls that may arise from focusing primary analysis on a single attribute, for example, fluorophore detection. Our study shows that in a lysosomal environment NPs can undergo significant changes resulting in dye quenching and distorted fluorescence signals.

eGFP expression under UCHL1 promoter genetically labels corticospinal motor neurons and a subpopulation of degeneration-resistant spinal motor neurons in an ALS mouse model.

Understanding mechanisms that lead to selective motor neuron degeneration requires visualization and cellular identification of vulnerable neurons. Here we report generation and characterization of UCHL1-eGFP and hSOD1(G93A)-UeGFP mice, novel reporter lines for cortical and spinal motor neurons. Corticospinal motor neurons (CSMN) and a subset of spinal motor neurons (SMN) are genetically labeled in UCHL1-eGFP mice, which express eGFP under the UCHL1 promoter. eGFP expression is stable and continues through P800 in vivo. Retrograde labeling, molecular marker expression, electrophysiological analysis, and cortical circuit mapping confirmed CSMN identity of eGFP(+) neurons in the motor cortex. Anatomy, molecular marker expression, and electrophysiological analysis revealed that the eGFP expression is restricted to a subset of small-size SMN that are slow-twitch α and γ motor neurons. Crossbreeding of UCHL1-eGFP and hSOD1(G93A) lines generated hSOD1(G93A)-UeGFP mice, which displayed the disease phenotype observed in a hSOD1(G93A) mouse model of ALS. eGFP(+) SMN showed resistance to degeneration in hSOD1(G93A)-UeGFP mice, and their slow-twitch α and γ motor neuron identity was confirmed. In contrast, eGFP(+) neurons in the motor cortex of hSOD1(G93A)-UeGFP mice recapitulated previously reported progressive CSMN loss and apical dendrite degeneration. Our findings using these two novel reporter lines revealed accumulation of autophagosomes along the apical dendrites of vulnerable CSMN at P60, early symptomatic stage, suggesting autophagy as a potential intrinsic mechanism for CSMN apical dendrite degeneration.

Rat Ovarian Function Is Impaired during Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease affecting the CNS and occurring far more prevalently in women than in men. In both MS and its animal models, sex hormones play important immunomodulatory roles. We have previously shown that experimental autoimmune encephalomyelitis (EAE) affects the hypothalamic-pituitary-gonadal axis in rats of both sexes and induces an arrest in the estrous cycle in females. To investigate the gonadal status in female rats with EAE, we explored ovarian morphometric parameters, circulating and intraovarian sex steroid levels, and the expression of steroidogenic machinery components in the ovarian tissue. A prolonged state of diestrus was recorded during the peak of EAE, with maintenance of the corpora lutea, elevated intraovarian progesterone levels, and increased gene and protein expression of StAR, similar to the state of pseudopregnancy. The decrease in CYP17A1 protein expression was followed by a decrease in ovarian testosterone and estradiol levels. On the contrary, serum testosterone levels were slightly increased. With unchanged serum estradiol levels, these results point at extra-gonadal sites of sex steroid biosynthesis and catabolism as important regulators of their circulating levels. Our study suggests alterations in the function of the female reproductive system during central autoimmunity and highlights the bidirectional relationships between hormonal status and EAE.

ZBTB7A regulates MDD-specific chromatin signatures and astrocyte-mediated stress vulnerability in orbitofrontal cortex.

Hyperexcitability in the orbitofrontal cortex (OFC) is a key clinical feature of anhedonic domains of Major Depressive Disorder (MDD). However, the cellular and molecular substrates underlying this dysfunction remain unknown. Here, cell-population-specific chromatin accessibility profiling in human OFC unexpectedly mapped genetic risk for MDD exclusively to non-neuronal cells, and transcriptomic analyses revealed significant glial dysregulation in this region. Characterization of MDD-specific cis-regulatory elements identified ZBTB7A - a transcriptional regulator of astrocyte reactivity - as an important mediator of MDD-specific chromatin accessibility and gene expression. Genetic manipulations in mouse OFC demonstrated that astrocytic Zbtb7a is both necessary and sufficient to promote behavioral deficits, cell-type-specific transcriptional and chromatin profiles, and OFC neuronal hyperexcitability induced by chronic stress - a major risk factor for MDD. These data thus highlight a critical role for OFC astrocytes in stress vulnerability and pinpoint ZBTB7A as a key dysregulated factor in MDD that mediates maladaptive astrocytic functions driving OFC hyperexcitability.

Testicular steroidogenesis is suppressed during experimental autoimmune encephalomyelitis in rats.

Multiple sclerosis (MS) is an autoimmune disease that usually occurs during the reproductive years in both sexes. Many male patients with MS show lower blood testosterone levels, which was also observed in male rats during experimental autoimmune encephalomyelitis (EAE), an animal model of MS. To better understand the causes of decreased testosterone production during EAE, we investigated the expression status of genes and proteins associated with steroidogenesis in the testes. No changes in the number of interstitial cells were observed in EAE animals, but the expression of the insulin-like 3 gene was reduced at the peak of the disease, implying that the Leydig cell functional capacity was affected. Consistent with this finding, the expression of most steroidogenic enzyme genes and proteins was reduced during EAE, including StAR, CYP11A1, CYP17A1 and HSD3B. No signs of testicular inflammation were observed. Recovery of steroidogenesis was observed after injection of hCG, the placental gonadotropin, or buserelin acetate, a gonadotropin-releasing hormone analogue, at the peak of EAE. Together, our results are consistent with the hypothesis that impaired testicular steroidogenesis originates upstream of the testes and that low serum LH is the main cause of decreased testosterone levels during EAE.

The Function of the Hypothalamic-Pituitary-Adrenal Axis During Experimental Autoimmune Encephalomyelitis: Involvement of Oxidative Stress Mediators.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease with an unknown origin. Previous studies showed the involvement of the hypothalamic-pituitary-adrenal (HPA) axis to susceptibility to autoimmune diseases, including MS, and its best-characterized animal model, experimental autoimmune encephalomyelitis (EAE). During MS/EAE, innate immune cells are activated and release cytokines and other inflammatory mediators, leading to a vicious cycle of inflammation. In response to inflammation, the activated HPA axis modulates immune responses via glucocorticoid activity. Because the mechanisms involving oxidative stress to the HPA axis are relatively unrevealed, in this study, we investigate the inflammatory and oxidative stress status of HPA axis during EAE. Our results reveal an upregulation of Pomc gene expression, followed by POMC and ACTH protein increase at the peak of the EAE in the pituitary. Also, prostaglandins are well-known contributors of HPA axis activation, which increases during EAE at the periphery. The upregulated Tnf expression in the pituitary during the peak of EAE occurred. This leads to the activation of oxidative pathways, followed by upregulation of inducible NO synthase expression. The reactive oxidant/nitrosative species (ROS/RNS), such as superoxide anion and NO, increase their levels at the onset and peak of the disease in the pituitary and adrenal glands, returning to control levels at the end of EAE. The corticotrophs in the pituitary increased in number and volume at the peak of EAE that coincides with high lipid peroxidation levels. The expression of MC2R in the adrenal glands increases at the peak of EAE, where strong induction of superoxide anion and malondialdehyde (MDA), reduced total glutathione (GSH) content, and catalase activity occurred at the peak and end of EAE compared with controls. The results obtained from this study may help in understanding the mechanisms and possible pharmacological modulation in MS and demonstrate an effect of oxidative stress exposure in the HPA activation during the course of EAE.

Rapid and sensitive quantification of cell-associated multi-walled carbon nanotubes.

Evaluating nanomaterial uptake and association by cells is relevant for in vitro studies related to safe-by-design approaches, nanomedicine or applications in photothermal therapy. However, standard analytical techniques are time-consuming, involve complex sample preparation or include labelling of the investigated sample system with e.g. fluorescent dyes. Here, we explore lock-in thermography to analyse and compare the association trends of epithelial cells, mesothelial cells, and macrophages exposed to gold nanoparticles and multi-walled carbon nanotubes over 24 h. The presence of nanomaterials in the cells was confirmed by dark field and transmission electron microscopy. The results obtained by lock-in thermography for gold nanoparticles were validated with inductively coupled plasma optical emission spectrometry; with data collected showing a good agreement between both techniques. Furthermore, we demonstrate the detection and quantification of carbon nanotube-cell association in a straightforward, non-destructive, and non-intrusive manner without the need to label the carbon nanotubes. Our results display the first approach in utilizing thermography to assess the carbon nanotube amount in cellular environments.

Progenitors from the postnatal forebrain subventricular zone differentiate into cerebellar-like interneurons and cerebellar-specific astrocytes upon transplantation.

Forebrain subventricular zone (SVZ) progenitor cells give rise to glia and olfactory bulb interneurons during early postnatal life in rats. We investigated the potential of SVZ cells to alter their fate by transplanting them into a heterotypic neurogenic and gliogenic environment-the cerebellum. Transplanted cells were examined 1 to 7 weeks and 6 months post transplantation. Forebrain progenitors populated the cerebellum and differentiated into oligodendrocytes, cerebellar-specific Bergmann glia and velate astrocytes, and neurons. The transplanted cells that differentiated into neurons maintained an interneuronal fate: they were GABA-positive, expressed interneuronal markers, such as calretinin, and exhibited membrane properties that are characteristic of interneurons. However, the transplanted interneurons lost the expression of the olfactory bulb transcription factors Tbr2 and Dlx1, and acquired a cerebellar-like morphology. Forebrain SVZ progenitors thus have the potential to adapt to a new environment and integrate into diverse regions, and may be a useful tool in transplantation strategies.

Induction of NTPDase1/CD39 by Reactive Microglia and Macrophages Is Associated With the Functional State During EAE.

Purinergic signaling is critically involved in neuroinflammation associated with multiple sclerosis (MS) and its major inflammatory animal model, experimental autoimmune encephalomyelitis (EAE). Herein, we explored the expression of ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1/CD39) in the spinal cord, at the onset (Eo), peak (Ep), and end (Ee) of EAE. Several-fold increase in mRNA and in NTPDase1 protein levels were observed at Eo and Ep. In situ hybridization combined with fluorescent immunohistochemistry showed that reactive microglia and infiltrated mononuclear cells mostly accounted for the observed increase. Colocalization analysis revealed that up to 80% of Iba1 immunoreactivity and ∼50% of CD68 immunoreactivity was colocalized with NTPDase1, while flow cytometric analysis revealed that ∼70% of mononuclear infiltrates were NTPDase1+ at Ep. Given the main role of NTPDase1 to degrade proinflammatory ATP, we hypothesized that the observed up-regulation of NTPDase1 may be associated with the transition between proinflammatory M1-like to neuroprotective M2-like phenotype of microglia/macrophages during EAE. Functional phenotype of reactive microglia/macrophages that overexpress NTPDase1 was assessed by multi-image colocalization analysis using iNOS and Arg1 as selective markers for M1 and M2 reactive states, respectively. At the peak of EAE NTPDase1 immunoreactivity showed much higher co-occurrence with Arg1 immunoreactivity in microglia and macrophages, compared to iNOS, implying its stronger association with M2-like reactive phenotype. Additionally, in ∼80% of CD68 positive cells NTPDase1 was coexpressed with Arg1 compared to negligible fraction coexpresing iNOS and ∼15% coexpresing both markers, additionally indicating prevalent association of NTPDase1 with M2-like microglial/macrophages phenotype at Ep. Together, our data suggest an association between NTPDase1 up-regulation by reactive microglia and infiltrated macrophages and their transition toward antiinflammatory phenotype in EAE.

Polymer-Coated Gold Nanospheres Do Not Impair the Innate Immune Function of Human B Lymphocytes in Vitro.

Gold nanoparticles (GNPs) are intended for use within a variety of biomedical applications due to their physicochemical properties. Although, in general, biocompatibility of GNPs with immune cells such as macrophages and dendritic cells is well established, the impact of GNPs on B lymphocyte immune function remains to be determined. Since B lymphocytes play an important role in health and disease, the suitability of GNPs as a B cell-targeting tool is of high relevance. Thus, we provide information on the interactions of GNPs with B lymphocytes. Herein, we exposed freshly isolated human B lymphocytes to a set of well-characterized and biomedically relevant GNPs with distinct surface (polyethylene glycol (PEG), PEG/poly(vinyl alcohol) (PEG/PVA)) and shape (spheres, rods) characteristics. Polymer-coated GNPs poorly interacted with B lymphocytes, in contrast to uncoated GNPs. Importantly, none of the GNPs significantly affected cell viability, even at the highest concentration of 20 µg/mL over a 24 h suspension exposure period. Furthermore, none of the nanosphere formulations affected the expression of activation markers (CD69, CD86, MHC II) of the naive B lymphocytes, nor did they cause an increase in the secretion of pro-inflammatory cytokines ( i.e. , IL-6, IL-1ß). However, the absence of polymer coating on the sphere GNPs and the rod shape caused a decrease in IL-6 cytokine production by activated B lymphocytes, suggesting a functional impairment. With these findings, the present study contributes imperative knowledge toward the safe-by-design approaches being conducted to benefit the development of nanomaterials, specifically those as theranostic tools.

Nanoparticle administration method in cell culture alters particle-cell interaction.

As a highly interdisciplinary field, working with nanoparticles in a biomedical context requires a robust understanding of soft matter physics, colloidal behaviors, nano-characterization methods, biology, and bio-nano interactions. When reporting results, it can be easy to overlook simple, seemingly trivial experimental details. In this context, we set out to understand how in vitro technique, specifically the way we administer particles in 2D culture, can influence experimental outcomes. Gold nanoparticles coated with poly(vinylpyrrolidone) were added to J774A.1 mouse monocyte/macrophage cultures as either a concentrated bolus, a bolus then mixed via aspiration, or pre-mixed in cell culture media. Particle-cell interaction was monitored via inductively coupled plasma-optical emission spectroscopy and we found that particles administered in a concentrated dose interacted more with cells compared to the pre-mixed administration method. Spectroscopy studies reveal that the initial formation of the protein corona upon introduction to cell culture media may be responsible for the differences in particle-cell interaction. Modeling of particle deposition using the in vitro sedimentation, diffusion and dosimetry model helped to clarify what particle phenomena may be occurring at the cellular interface. We found that particle administration method in vitro has an effect on particle-cell interactions (i.e. cellular adsorption and uptake). Initial introduction of particles in to complex biological media has a lasting effect on the formation of the protein corona, which in turn mediates particle-cell interaction. It is of note that a minor detail, the way in which we administer particles in cell culture, can have a significant effect on what we observe regarding particle interactions in vitro.

Biodistribution, Clearance, and Long-Term Fate of Clinically Relevant Nanomaterials.

Realization of the immense potential of nanomaterials for biomedical applications will require a thorough understanding of how they interact with cells, tissues, and organs. There is evidence that, depending on their physicochemical properties and subsequent interactions, nanomaterials are indeed taken up by cells. However, the subsequent release and/or intracellular degradation of the materials, transfer to other cells, and/or translocation across tissue barriers are still poorly understood. The involvement of these cellular clearance mechanisms strongly influences the long-term fate of used nanomaterials, especially if one also considers repeated exposure. Several nanomaterials, such as liposomes and iron oxide, gold, or silica nanoparticles, are already approved by the American Food and Drug Administration for clinical trials; however, there is still a huge gap of knowledge concerning their fate in the body. Herein, clinically relevant nanomaterials, their possible modes of exposure, as well as the biological barriers they must overcome to be effective are reviewed. Furthermore, the biodistribution and kinetics of nanomaterials and their modes of clearance are discussed, knowledge of the long-term fates of a selection of nanomaterials is summarized, and the critical points that must be considered for future research are addressed.

Polydopamine/Transferrin Hybrid Nanoparticles for Targeted Cell-Killing.

Polydopamine can form biocompatible particles that convert light into heat. Recently, a protocol has been optimized to synthesize polydopamine/protein hybrid nanoparticles that retain the biological function of proteins, and combine it with the stimuli-induced heat generation of polydopamine. We have utilized this novel system to form polydopamine particles, containing transferrin (PDA/Tf). Mouse melanoma cells, which strongly express the transferrin receptor, were exposed to PDA/Tf nanoparticles (NPs) and, subsequently, were irradiated with a UV laser. The cell death rate was monitored in real-time. When irradiated, the melanoma cells exposed to PDA/Tf NPs underwent apoptosis, faster than the control cells, pointing towards the ability of PDA/Tf to mediate UV-light-induced cell death. The system was also validated in an organotypic, 3D-printed tumor spheroid model, comprising mouse melanoma cells, and the exposure and subsequent irradiation with UV-light, yielded similar results to the 2D cell culture. The process of apoptosis was found to be targeted and mediated by the lysosomal membrane permeabilization. Therefore, the herein presented polydopamine/protein NPs constitute a versatile and stable system for cancer cell-targeting and photothermal apoptosis induction.

A rational and iterative process for targeted nanoparticle design and validation.

The lack of understanding of fundamental nano-bio interactions, and difficulties in designing particles stable in complex biological environments are major limitations to their translation into biomedical clinical applications. Here we present a multi-parametric approach to fully characterize targeted nanoparticles, and emphasizes the significant effect that each detail in the synthetic process can have on downstream in vitro results. Through an iterative process, particles were designed, synthesized and tested for physico-chemical and bio-interactive properties which allowed the optimization of nanoparticle functionality. Taken together all interative steps demonstrate that we have synthesized a multifunctional gold nanoparticles that can detect ERBB2-positive breast cancer cells while showing stealth-like behavior toward ERBB2-negative cells and excellent physicochemical stability.

A rapid screening method to evaluate the impact of nanoparticles on macrophages.

Nanotechnology is an emerging and highly promising field to develop new approaches for biomedical applications. There is however at present an unmet need for a rapid and universal method to screen nanoparticles (NP) for immunocompatibility at early stages of their development. Indeed, although many types of highly diverse NP are currently under investigation, their interaction with immune cells remains fairly unpredictable. Macrophages which are professional phagocytic cells are believed to be among the first cell types that take up NP, mediating inflammation and thus immunological responses. The present work describes a highly reproducible screening method to study the NP interaction with macrophages. Three essential questions are answered in parallel, in a single multiwell plate: Are the NP taken up by macrophages? Do the NP cause macrophage cell death? Do the NP induce inflammatory reactions? This assay is proposed as a standardized screening protocol to obtain a rapid overview of the impact of different types of NP on macrophages. Due to high reproducibility, this method also allows quality control assessment for such aspects as immune-activating contaminants and batch-to-batch variability.

Cell- and region-specific expression of depression-related protein p11 (S100a10) in the brain.

P11 (S100a10), a member of the S100 family of proteins, has widespread distribution in the vertebrate body, including in the brain, where it has a key role in membrane trafficking, vesicle secretion, and endocytosis. Recently, our laboratory has shown that a constitutive knockout of p11 (p11-KO) in mice results in a depressive-like phenotype. Furthermore, p11 has been implicated in major depressive disorder (MDD) and in the actions of antidepressants. Since depression affects multiple brain regions, and the role of p11 has only been determined in a few of these areas, a detailed analysis of p11 expression in the brain is warranted. Here we demonstrate that, although widespread in the brain, p11 expression is restricted to distinct regions, and specific neuronal and nonneuronal cell types. Furthermore, we provide comprehensive mapping of p11 expression using in situ hybridization, immunocytochemistry, and whole-tissue volume imaging. Overall, expression spans multiple brain regions, structures, and cell types, suggesting a complex role of p11 in depression. J. Comp. Neurol. 525:955-975, 2017. © 2016 Wiley Periodicals, Inc.