Anti-NeuN Antibody [1B7] (A85405)

Although depression-induced altered pain perception has been described in several laboratory and clinical studies, its neurobiological mechanism in the central nervous system (CNS), particularly in the spinal dorsal horn, remains unclear. Therefore, in this study, we aimed to clarify whether nociceptive sensitivity of neuropathic pain is altered in the olfactory bulbectomy (OB) model of depression and whether glucocorticoid receptor (GR), which is involved in the etio-pathologic mechanisms of both major depression and neuropathic pain, contributes to these processes in the spinal dorsal horn of male Sprague-Dawley rats. The results showed that mechanical allodynia and thermal hyperalgesia induced by spinal nerve ligation (SNL) were attenuated in OB-SNL rats with decreased spinal GR expression and nuclear translocation, whereas non-olfactory bulbectomy (NOB)-SNL rats showed increased spinal GR nuclear translocation. In addition, decreased GR nuclear translocation with normal mechanical nociception and hypoalgesia of thermal nociception were observed in OB-Sham rats. Intrathecal injection (i.t.) of GR agonist dexamethasone (Dex; 4 μg/rat/day for 1 week) eliminated the attenuating effect of depression on nociceptive hypersensitivity in OB-SNL rats and aggravated neuropathic pain in NOB-SNL rats, which was associated with the up-regulation of brain-derived neurotrophic factor (BDNF), TrkB and NR2B expression in the spinal dorsal horn. The present study shows that depression attenuates the mechanical allodynia and thermal hyperalgesia of neuropathic pain and suggests that altered spinal GR-BDNF-TrkB signaling may be one of the reasons for depression-induced hypoalgesia.

Spinal cord injury (SCI) is a devastating condition affecting hundreds of thousands of people worldwide annually. SCI results in activation of the inflammatory response and apoptosis, and generates oxidative stress, which has deleterious effects on the recovery of motor function. Apocynin, an inhibitor of NADPH oxidase, has been demonstrated to improve neuronal functional recovery in rat models of SCI. However, the efficacy of apocynin treatment post-SCI has not been investigated. The aim of this study was to observe the effects of apocynin on the repair of acute spinal cord damage in rats and to examine the potential beneficial effects. A rat model of SCI was established, and apocynin (50 mg/kg) was administered intraperitoneally at 30 min after SCI and then every 12 h for 3 days. In order to examine oxidative tissue injury, the levels of malondialdehyde and glutathione and activities of myeloperoxidase and superoxide dismutase in the spinal cord tissues were measured. Histological evaluations were also conducted. NeuN labeling, TUNEL staining and caspase 3 immunohistochemical staining were performed to analyze neuronal damage and apoptosis around the lesion. Immunohistochemical analysis was also carried out to observe the expression of CD11b and glial fibrillary acidic protein. The expression levels of bax, bcl-2, tumor necrosis-α, interleukin (IL)-1β and IL-6 in the spinal cord tissue were assayed by western blotting. Finally, locomotor function was evaluated using the inclined plane test and Basso, Beattie and Bresnahan scores. The results showed that treatment with apocynin decreased oxidative damage, alleviated neuronal apoptosis, inhibited the inflammatory response and resulted in the promotion of locomotor function. Therefore, this study confirmed the therapeutic efficacy of apocynin in the repair of SCI, which was probably mediated via the inhibition of apoptosis and the inflammatory response, thus promoting the restoration of nerve function.

The G protein-coupled receptor 17 (GPR17), a Gi-coupled GPCR, acts as an intrinsic timer of oligodendrocyte differentiation and myelination. The expression of GPR17 is upregulated during differentiation of oligodendrocyte precursor cells (OPCs) into premyelinating oligodendrocytes (preoligodendrocytes), whereas it is markedly downregulated during terminal maturation of myelinating oligodendrocytes. Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder caused by a loss-of-function mutation of either TYROBP (DAP12) or TREM2. Pathologically, the brains of NHD patients exhibit extensive demyelination designated leukoencephalopathy, astrogliosis, accumulation of axonal spheroids, and activation of microglia predominantly in the white matter of frontal and temporal lobes. Although GPR17 is a key regulator of oligodendrogenesis, a pathological role of GPR17 in NHD brains with relevance to development of leukoencephalopathy remains unknown. We studied the expression of GPR17 in five NHD brains and eight control brains by immunohistochemistry. We identified GPR17-immunoreactive preoligodendrocytes with a multipolar ramified morphology distributed in the white matter and the grey matter of all cases examined. However, we did not find statistically significant differences in the number of GPR17-expressing cells between NHD and control brains both in the white matter and the grey matter due to great variability from case to case. These observations do not support the view that GPR17-positive preoligodendrocytes play a central role in the development of leukoencephalopathy in NHD brains.

We previously reported transcriptional repression-induced atypical cell death of neuron (TRIAD), a new type of necrosis that is mainly regulated by Hippo pathway signaling and distinct from necroptosis regulated by RIP1/3 pathway. Here, we examined the ultrastructural and biochemical features of neuronal cell death in the brains of human HD patients in parallel with the similar analyses using mutant Htt-knock-in (Htt-KI) mice. LATS1 kinase, the critical regulator and marker of TRIAD, is actually activated in cortical neurons of postmortem human HD and of Htt-KI mouse brains, while apoptosis promoter kinase Plk1 was inactivated in human HD brains. Expression levels of YAP/YAPdeltaC were decreased in cortical neurons of human HD brains. Ultra-structural analyses revealed extreme enlargement of endoplasmic reticulum (ER), which characterizes TRIAD, in cortical neurons of human HD and those of Htt-KI mice. These biochemical and morphological results support that TRIAD occurs in human and mouse neurons under the HD pathology.

In the central nervous system, 24(S)-hydroxycholesterol (24(S)-HC) is an oxysterol synthesized from cholesterol by cholesterol 24-hydroxylase (CYP46A1) encoded by the cyp46a1 gene. In the present study using a rat ex vivo glaucoma model, we found that retinal 24(S)-HC synthesis is facilitated by pressure elevation. Moreover, we found that 24(S)-HC is neuroprotective against pressure mediated retinal degeneration. Quantitative real-time RT-PCR, ELISA, and immunohistochemistry revealed that elevated pressure facilitated the expression of cyp46a1 and CYP46A1. Immunohistochemically, the enhanced expression of CYP46A1 was mainly observed in retinal ganglion cells (RGC). LC-MS/MS revealed that 24(S)-HC levels increased in a pressure-dependent manner. Axonal injury and apoptotic RGC death induced by 75 mmHg high pressure was ameliorated by exogenously administered 1 μM 24(S)-HC. In contrast, voriconazole, a CYP46A1 inhibitor, was severely toxic even at normobaric pressure. Under normobaric conditions, 30 μM 24(S)-HC was required to prevent the voriconazole-mediated retinal damage. Taken together, our findings indicate that 24(S)-HC is facilitated by elevated pressure and plays a neuroprotective role under glaucomatous conditions, while voriconazole, an antifungal drug, is retinotoxic. 24(S)-HC and related compounds may serve as potential therapeutic targets for protecting glaucomatous eyes from pressure-induced injuries.

Transient receptor potential channel 1/4 (TRPC1/4) are considered to be related to subarachnoid hemorrhage (SAH)-induced cerebral vasospasm. In this study, a SAH rat model was employed to study the roles of TRPC1/4 in the early brain injury (EBI) after SAH. Primary cultured hippocampal neurons were exposed to oxyhemoglobin to mimic SAH in vitro. The protein levels of TRPC1/4 increased and peaked at 5 days after SAH in rats. Inhibition of TRPC1/4 by SKF96365 aggravated SAH-induced EBI, such as cortical cell death (by TUNEL staining) and degenerating (by FJB staining). In addition, TRPC1/4 overexpression could increase calcineurin activity, while increased calcineurin activity could promote the dephosphorylation of N-methyl-D-aspartate receptor (NMDAR). Calcineurin antagonist FK506 could weaken the neuroprotection and the dephosphorylation of NMDAR induced by TRPC1/4 overexpression. Contrarily, calcineurin agonist chlorogenic acid inhibited SAH-induced EBI, even when siRNA intervention of TRPC1/4 was performed. Moreover, calcineurin also could lead to the nuclear transfer of nuclear factor of activated T cells (NFAT), which is a transcription factor promoting the expressions of TRPC1/4. TRPC1/4 could inhibit SAH-induced EBI by supressing the phosphorylation of NMDAR via calcineurin. TRPC1/4-induced calcineurin activation also could promote the nuclear transfer of NFAT, suggesting a positive feedback regulation of TRPC1/4 expressions.

We have reported that nuclear translocation of Receptor-interacting protein 3 (RIP3) involves in neuronal programmed necrosis after 20-min global cerebral ischemia/reperfusion (I/R) injury. Herein, the underlying mechanisms and the nuclear role of RIP3 were investigated further. The necroptosis inhibitor necrostatin-1 (Nec-1), the autophagy inhibitor 3-methyladenine (3-MA), and the caspase-3 inhibitor acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-al (Ac-DMQD-CHO) were administered intracerebroventricularly 1 h before ischemia. Protein expression, location and interaction was determined by western blot, immunofluorescence or immunoprecipitation. Most CA1 neuronal death induced by 20-min global cerebral I/R injury was TUNEL-positive. Neuronal death and rat mortality rates were greatly inhibited by Nec-1 and 3-MA pre-treatment, but not by Ac-DMQD-CHO. And no activation of caspase-3 was detected after I/R injury. Caspase-8 was expressed richly in GFAP-positive astrocytes and Iba-1-positive microglia, but was not detected in Neun-positive neurons. The nuclear translocation and co-localization of RIP3 and AIF, and their interaction were detected after I/R injury. These processes were inhibited by Nec-1 and 3-MA pre-treatment, but not by Ac-DMQD-CHO. The formation of an RIP3-AIF complex and its nuclear translocation are critical to ischemic neuronal DNA degradation and programmed necrosis. Neurons are more likely to enter the programmed necrosis signal pathway for the loss of caspase-8 suppression.

Testosterone plays a central role in the facilitation of male-type social behaviors, such as sexual and aggressive behaviors, and the development of their neural bases in male mice. The action of testosterone via estrogen receptor (ER) α, after being aromatized to estradiol, has been suggested to be crucial for the full expression of these behaviors. We previously reported that silencing of ERα in adult male mice with the use of a virally mediated RNAi method in the medial preoptic area (MPOA) greatly reduced sexual behaviors without affecting aggressive behaviors whereas that in the medial amygdala (MeA) had no effect on either behavior. It is well accepted that testosterone stimulation during the pubertal period is necessary for the full expression of male-type social behaviors. However, it is still not known whether, and in which brain region, ERα is involved in this developmental effect of testosterone. In this study, we knocked down ERα in the MeA or MPOA in gonadally intact male mice at the age of 21 d and examined its effects on the sexual and aggressive behaviors later in adulthood. We found that the prepubertal knockdown of ERα in the MeA reduced both sexual and aggressive behaviors whereas that in the MPOA reduced only sexual, but not aggressive, behavior. Furthermore, the number of MeA neurons was reduced by prepubertal knockdown of ERα. These results indicate that ERα activation in the MeA during the pubertal period is crucial for male mice to fully express their male-type social behaviors in adulthood.

Patients with tumors that metastasize to bone frequently suffer from debilitating pain, and effective therapies for treating bone cancer are lacking. This study employed a novel strategy in which herpes simplex virus (HSV) carrying a small interfering RNA (siRNA) targeting platelet-derived growth factor (PDGF) was used to alleviate bone cancer pain. HSV carrying PDGF siRNA was established and intrathecally injected into the cavum subarachnoidale of animals suffering from bone cancer pain and animals in the negative group. Sensory function was assessed by measuring thermal and mechanical hyperalgesia. The mechanism by which PDGF regulates pain was also investigated by comparing the differential expression of pPDGFRα/β and phosphorylated ERK and AKT. Thermal and mechanical hyperalgesia developed in the rats with bone cancer pain, and these effects were accompanied by bone destruction in the tibia. Intrathecal injection of PDGF siRNA and morphine reversed thermal and mechanical hyperalgesia in rats with bone cancer pain. In addition, we observed attenuated astrocyte hypertrophy, down-regulated pPDGFRα/β levels, reduced levels of the neurochemical SP, a reduction in CGRP fibers and changes in pERK/ERK and pAKT/AKT ratios. These results demonstrate that PDGF siRNA can effectively treat pain induced by bone cancer by blocking the AKT-ERK signaling pathway.

Long-term effects of prenatal hypoxia on embryonic days E14 or E18 on the number, type and localization of cortical neurons, density of labile synaptopodin-positive dendritic spines, and parietal cortex-dependent behavioral tasks were examined in the postnatal ontogenesis of rats. An injection of 5'ethynyl-2'deoxyuridine to pregnant rats was used to label neurons generated on E14 or E18 in the fetuses. In control rat pups a majority of cells labeled on E14 were localized in the lower cortical layers V-VI while the cells labeled on E18 were mainly found in the superficial cortical layers II-III. It was shown that hypoxia both on E14 and E18 results in disruption of neuroblast generation and migration but affects different cell populations. In rat pups subjected to hypoxia on E14, the total number of labeled cells in the parietal cortex was decreased while the number of labeled neurons scattered within the superficial cortical layers was increased. In rat pups subjected to hypoxia on E18, the total number of labeled cells in the parietal cortex was also decreased but the number of scattered labeled neurons was higher in the lower cortical layers. It can be suggested that prenatal hypoxia both on E14 and E18 causes a disruption in neuroblast migration but with a different outcome. Only in rats subjected to hypoxia on E14 did we observe a reduction in the total number of pyramidal cortical neurons and the density of labile synaptopodin-positive dendritic spines in the molecular cortical layer during the first month after birth which affected development of the cortical functions. As a result, rats subjected to hypoxia on E14, but not on E18, had impaired development of the whisker-placing reaction and reduced ability to learn reaching by a forepaw. The data obtained suggest that hypoxia on E14 in the period of generation of the cells, which later differentiate into the pyramidal cortical neurons of the V-VI layers and form cortical minicolumns, affects formation of cortical cytoarchitecture, neuronal plasticity and behavior in postnatal ontogenesis which testify to cortical dysfunction. Hypoxia on E18 does not significantly affect cortical structure and parietal cortex-dependent behavioral tasks.

The cerebellum plays an essential role in balance and motor coordination. Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex and are critical for the execution of its functions, including motor coordination. Toll-like receptor (TLR) 4 is involved in the innate immune response and is abundantly expressed in the central nervous system; however, little is known about its role in cerebellum-related motor functions. To address this question, we evaluated motor behavior in TLR4 deficient mice. We found that TLR4(-∕-) mice showed impaired motor coordination. Morphological analyses revealed that TLR4 deficiency was associated with a reduction in the thickness of the molecular layer of the cerebellum. TLR4 was highly expressed in PCs but not in Bergmann glia or cerebellar granule cells; however, loss of TLR4 decreased the number of PCs. These findings suggest a novel role for TLR4 in cerebellum-related motor coordination through maintenance of the PC population.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping neurodegenerative disorders whose pathogenesis remains largely unknown. Using TDP-43(A315T) mice, an ALS and FTD model with marked cortical pathology, we found that hyperactive somatostatin interneurons disinhibited layer 5 pyramidal neurons (L5-PNs) and contributed to their excitotoxicity. Focal ablation of somatostatin interneurons efficiently restored normal excitability of L5-PNs and alleviated neurodegeneration, suggesting a new therapeutic target for ALS and FTD.

Human pluripotent stem cells (hPSC) have promise for regenerative medicine due to their auto-renovation and differentiation capacities. Nevertheless, there are several ethical and methodological issues about these cells that have not been resolved. Human amniotic epithelial cells (hAEC) have been proposed as source of pluripotent stem cells. Several groups have studied hAEC but have reported inconsistencies about their pluripotency properties. The aim of the present study was the in vitro characterization of hAEC collected from a Mexican population in order to identify transcription factors involved in the pluripotency circuitry and to determine their epigenetic state. Finally, we evaluated if these cells differentiate to cortical progenitors. We analyzed qualitatively and quantitatively the expression of the transcription factors of pluripotency (OCT4, SOX2, NANOG, KLF4 and REX1) by RT-PCR and RT-qPCR in hAEC. Also, we determined the presence of OCT4, SOX2, NANOG, SSEA3, SSEA4, TRA-1-60, E-cadherin, KLF4, TFE3 as well as the proliferation and epigenetic state by immunocytochemistry of the cells. Finally, hAEC were differentiated towards cortical progenitors using a protocol of two stages. Here we show that hAEC, obtained from a Mexican population and cultured in vitro (P0-P3), maintained the expression of several markers strongly involved in pluripotency maintenance (OCT4, SOX2, NANOG, TFE3, KLF4, SSEA3, SSEA4, TRA-1-60 and E-cadherin). Finally, when hAEC were treated with growth factors and small molecules, they expressed markers characteristic of cortical progenitors (TBR2, OTX2, NeuN and β-III-tubulin). Our results demonstrated that hAEC express naïve pluripotent markers (KLF4, REX1 and TFE3) as well as the cortical neuron phenotype after differentiation. This highlights the need for further investigation of hAEC as a possible source of hPSC.

X-ALD is an inherited neurodegenerative disorder where mutations in the ABCD1 gene result in clinically diverse phenotypes: the fatal disorder of cerebral childhood ALD (cALD) or a milder disorder of adrenomyeloneuropathy (AMN). The various models used to study the pathobiology of X-ALD disease lack the appropriate presentation for different phenotypes of cALD vs AMN. This study demonstrates that induced pluripotent stem cells (IPSC) derived brain cells astrocytes (Ast), neurons and oligodendrocytes (OLs) express morphological and functional activities of the respective brain cell types. The excessive accumulation of saturated VLCFA, a "hallmark" of X-ALD, was observed in both AMN OLs and cALD OLs with higher levels observed in cALD OLs than AMN OLs. The levels of ELOVL1 (ELOVL Fatty Acid Elongase 1) mRNA parallel the VLCFA load in AMN and cALD OLs. Furthermore, cALD Ast expressed higher levels of proinflammatory cytokines than AMN Ast and control Ast with or without stimulation with lipopolysaccharide. These results document that IPSC-derived Ast and OLs from cALD and AMN fibroblasts mimic the respective biochemical disease phenotypes and thus provide an ideal platform to investigate the mechanism of VLCFA load in cALD OLs and VLCFA-induced inflammatory disease mechanisms of cALD Ast and thus for testing of new therapeutics for AMN and cALD disease of X-ALD.

Central nervous system (CNS)-infiltrating effector T cells play critical roles in the development and progression of multiple sclerosis (MS). However, current drugs for MS are very limited due to the difficulty of delivering drugs into the CNS. Here we identify a cell-permeable peptide, dNP2, which efficiently delivers proteins into mouse and human T cells, as well as various tissues. Moreover, it enters the brain tissue and resident cells through blood vessels by penetrating the tightly organized blood-brain barrier. The dNP2-conjugated cytoplasmic domain of cytotoxic T-lymphocyte antigen 4 (dNP2-ctCTLA-4) negatively regulates activated T cells and shows inhibitory effects on experimental autoimmune encephalomyelitis in both preventive and therapeutic mouse models, resulting in the reduction of demyelination and CNS-infiltrating T helper 1 and T helper 17 cells. Thus, this study demonstrates that dNP2 is a blood-brain barrier-permeable peptide and dNP2-ctCTLA-4 could be an effective agent for treating CNS inflammatory diseases such as MS.

Cav1.2 and Cav1.3 are the major L-type voltage-gated Ca(2+) channels in the CNS. Yet, their individual in vivo functions are largely unknown. Both channel subunits are expressed in the auditory brainstem, where Cav1.3 is essential for proper maturation. Here, we investigated the role of Cav1.2 by targeted deletion in the mouse embryonic auditory brainstem. Similar to Cav1.3, loss of Cav1.2 resulted in a significant decrease in the volume and cell number of auditory nuclei. Contrary to the deletion of Cav1.3, the action potentials of lateral superior olive (LSO) neurons were narrower compared with controls, whereas the firing behavior and neurotransmission appeared unchanged. Furthermore, auditory brainstem responses were nearly normal in mice lacking Cav1.2. Perineuronal nets were also unaffected. The medial nucleus of the trapezoid body underwent a rapid cell loss between postnatal days P0 and P4, shortly after circuit formation. Phosphorylated cAMP response element-binding protein (CREB), nuclear NFATc4, and the expression levels of p75NTR, Fas, and FasL did not correlate with cell death. These data demonstrate for the first time that both Cav1.2 and Cav1.3 are necessary for neuronal survival but are differentially required for the biophysical properties of neurons. Thus, they perform common as well as distinct functions in the same tissue.

Neuronal development is a pro-survival process that involves neurite growth, synaptogenesis, synaptic and neuronal pruning. During development, these processes can be controlled by temporal gene expression that is orchestrated by both long non-coding RNAs and microRNAs. To examine the interplay between these different components of the transcriptome during neuronal differentiation, we carried out mRNA, long non-coding RNA and microRNA expression profiling on maturing primary neurons. Subsequent gene ontology analysis revealed regulation of axonogenesis and dendritogenesis processes by these differentially expressed mRNAs and non-coding RNAs. Temporally regulated mRNAs and their associated long non-coding RNAs were significantly over-represented in proliferation and differentiation associated signalling, cell adhesion and neurotrophin signalling pathways. Verification of expression of the Axin2, Prkcb, Cntn1, Ncam1, Negr1, Nrxn1 and Sh2b3 mRNAs and their respective long non-coding RNAs in an in vitro model of ischemic-reperfusion injury showed an inverse expression profile to the maturation process, thus suggesting their role(s) in maintaining neuronal structure and function. Furthermore, we propose that expression of the cell adhesion molecules, Ncam1 and Negr1 might be tightly regulated by both long non-coding RNAs and microRNAs.

A strict regulation of protein expression during developmental stages and in response to environmental signals is essential to every cell and organism. Recent research has shown that the mammalian brain is particularly sensitive to alterations in expression patterns of specific proteins and cognitive deficits as well as autistic behaviours have been linked to dysregulated protein expression. An intellectual disability characterised by changes in the expression of a variety of proteins is the fragile X syndrome. Due to the loss of a single mRNA binding protein, the Fragile X Mental Retardation Protein FMRP, vast misregulation of the mRNA metabolism is taking place in the disease. Here, we present the identification and characterisation of a novel protein named Simiate, whose mRNA contains several FMRP recognition motifs and associates with FMRP upon co-precipitation. Sequence analysis revealed that the protein evolved app. 1.7 billion years ago when eukaryotes developed. Applying antibodies generated against Simiate, the protein is detected in a variety of tissues, including the mammalian brain. On the subcellular level, Simiate localises to somata and nuclear speckles. We show that Simiate and nuclear speckles experience specific alterations in FMR1(-/-) mice. An antibody-based block of endogenous Simiate revealed that the protein is essential for cell survival. These findings suggest not only an important role for Simiate in gene transcription and/or RNA splicing, but also provide evidence for a function of nuclear speckles in the fragile X syndrome. Indeed, transcription and splicing are two fundamental mechanisms to control protein expression, that underlie not only synaptic plasticity and memory formation, but are also affected in several diseases associated with mental disabilities.

Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.

BACKGROUND:

Temporomandibular disorders (TMDs) have the highest prevalence in women of reproductive age. The role of estrogen in TMDs and especially in TMDs related pain is not fully elucidated. Voltage-gated sodium channel 1.7 (Nav1.7) plays a prominent role in pain perception and Nav1.7 in trigeminal ganglion (TG) is involved in the hyperalgesia of inflamed Temporomandibular joint (TMJ). Whether estrogen could upregulate trigeminal ganglionic Nav1.7 expression to enhance hyperalgesia of inflamed TMJ remains to be explored.

METHODS:

Estrous cycle and plasma levels of 17β-estradiol in female rats were evaluated with vaginal smear and enzyme linked immunosorbent assay, respectively. Female rats were ovariectomized and treated with 17β-estradiol at 0 μg, 20 μg and 80 μg, respectively, for 10 days. TMJ inflammation was induced using complete Freund's adjuvant. Head withdrawal thresholds and food intake were measured to evaluate the TMJ nociceptive responses. The expression of Nav1.7 in TG was examined using real-time PCR and western blot. The activity of Nav1.7 promoter was examined using luciferase reporter assay. The locations of estrogen receptors (ERα and ERβ), the G protein coupled estrogen receptor (GPR30), and Nav1.7 in TG were examined using immunohistofluorescence.

RESULTS:

Upregulation of Nav1.7 in TG and decrease in head withdrawal threshold were observed with the highest plasma 17β-estradiol in the proestrus of female rats. Ovariectomized rats treated with 80 μg 17β-estradiol showed upregulation of Nav1.7 in TG and decrease in head withdrawal threshold as compared with that of the control or ovariectomized rats treated with 0 μg or 20 μg. Moreover, 17β-estradiol dose-dependently potentiated TMJ inflammation-induced upregulation of Nav1.7 in TG and also enhanced TMJ inflammation-induced decrease of head withdrawal threshold in ovariectomized rats. In addition, the estrogen receptor antagonist, ICI 182,780, partially blocked the 17β-estradiol effect on Nav1.7 expression and head withdrawal threshold in ovariectomized rats. ERα and ERβ, but not GPR30, were mostly co-localized with Nav1.7 in neurons in TG. In the nerve growth factor-induced and ERα-transfected PC12 cells, 17β-estradiol dose-dependently enhanced Nav1.7 promoter activity, whereas mutations of the estrogen response element at -1269/-1282 and -1214/-1227 in the promoter completely abolished its effect on the promoter activity.

CONCLUSION:

Estradiol could upregulate trigeminal ganglionic Nav1.7 expression to contribute to hyperalgesia of inflamed TMJ.

INTRODUCTION:

Many patients suffering from migraine gain little relief from existing treatments partly because many existing acute and preventive therapies used in migraine have been adopted from other neurologic conditions such as depression or epilepsy. Here, we present data supporting a new migraine-specific target, the mGlu5 receptor.

METHODS:

We studied the effect of mGlu5 blockade using ADX10059, on neuronal firing in the trigeminocervical complex (TCC) and durovascular effects of nociceptive trigeminovascular activation in the anesthetized rat. The clinical potential of the mGlu5 mechanism was tested with ADX10059 orally in a double-blind placebo-controlled, parallel group, clinical trial.

RESULTS:

The negative allosteric mGlu5 modulator ADX10059 attenuated dural vasodilator responses to meningeal stimulation in a dose-dependent manner, comparable to naratriptan, while the N-methyl-d-aspartate receptor blocker MK-801 had no effect. ADX10059 reduced responses of trigeminocervical neurons to dural stimulation, most strikingly affecting their spontaneous firing rate. Immunostaining identified mGlu5 and not mGlu1a receptors in the TCC. The primary efficacy endpoint for the clinical trial, 2 h pain free, demonstrated a significant effect of ADX10059 375 mg, 17%, versus placebo, 5%. No serious adverse events were reported at the primary dose, with transient dizziness being the most common treatment-emergent event at 48%.

INTERPRETATION:

Our findings provide preclinical and clinical proof of concept establishing mGlu5 as a novel therapeutic target in the treatment of migraine. Although ADX10059 is unsuitable as a therapeutic candidate, because of hepatoxicity detected in a subsequent study, the data open a new direction for migraine research and therapy.

BACKGROUND:

Chorioamnionitis is associated with an increased risk of brain injury in preterm neonates. Inflammatory changes in brain could underlie this injury. Here, we evaluated whether neuroinflammation is induced by chorioamnionitis in a clinically relevant model.

METHODS:

Rhesus macaque fetuses were exposed to either intra-amniotic (IA) saline, or IA lipopolysaccharide (LPS) (1 mg) 16 or 48 h prior to delivery at 130 days (85 % of gestation) (n = 4-5 animals/group). We measured cytokines in the cerebrospinal fluid (CSF), froze samples from the left brain for molecular analysis, and immersion fixed the right brain hemisphere for immunohistology. We analyzed the messenger RNA (mRNA) levels of the pro-inflammatory cytokines IL-1β, CCL2, TNF-α, IL-6, IL-8, IL-10, and COX-2 in the periventricular white matter (PVWM), cortex, thalamus, hippocampus, and cerebellum by RT-qPCR. Brain injury was assessed by immunohistology for myelin basic protein (MBP), IBA1 (microglial marker), GFAP (astrocyte marker), OLIG2 (oligodendrocyte marker), NeuN (neuronal marker), CD3 (T cells), and CD14 (monocytes). Microglial proliferation was assessed by co-immunostaining for IBA1 and Ki67. Data were analyzed by ANOVA with Tukey's post-test.

RESULTS:

IA LPS increased mRNA expression of pro-inflammatory cytokines in the PVWM, thalamus, and cerebellum, increased IL-6 concentration in the CSF, and increased apoptosis in the periventricular area after 16 h. Microglial proliferation in the white matter was increased 48 h after IA LPS.

CONCLUSIONS:

LPS-induced chorioamnionitis caused neuroinflammation, microglial proliferation, and periventricular apoptosis in a clinically relevant model of chorioamnionitis in fetal rhesus macaques. These findings identify specific responses in the fetal brain and support the hypothesis that neuroinflammatory changes may mediate the adverse neurodevelopmental outcomes associated with chorioamnionitis.

BACKGROUND:

This study was designed to determine the role of the A1 adenosine receptors in intracerebral hemorrhage (ICH)-induced secondary brain injury and the underlying mechanisms.

METHODS:

A collagenase-induced ICH model was established in Sprague-Dawley rats, and cultured primary rat cortical neurons were exposed to oxyhemoglobin at a concentration of 10 μM to mimic ICH in vitro. The A1 adenosine receptor agonist N(6)-cyclohexyladenosine and antagonist 8-phenyl-1,3-dipropylxanthine were used to study the role of A1 adenosine receptor in ICH-induced secondary brain injury, and antagonists of P38 and Hsp27 were used to study the underlying mechanisms of A1 adenosine receptor actions.

RESULTS:

The protein level of A1 adenosine receptor was significantly increased by ICH, while there was no significant change in protein levels of the other 3 adenosine receptors. In addition, the A1 adenosine receptor expression could be increased by N(6)-cyclohexyladenosine and decreased by 8-phenyl-1,3-dipropylxanthine under ICH conditions. Activation of the A1 adenosine receptor attenuated neuronal apoptosis in the subcortex, which was associated with increased phosphorylation of P38, MAPK, MAPKAP2, and Hsp27. Inhibition of the A1 adenosine receptor resulted in opposite effects. Finally, the neuroprotective effect of the A1 adenosine receptor agonist N(6)-cyclohexyladenosine was inhibited by antagonists of P38 and Hsp27.

CONCLUSIONS:

This study demonstrates that activation of the A1 adenosine receptor by N(6)-cyclohexyladenosine could prevent ICH-induced secondary brain injury via the P38-MAPKAP2-Hsp27 pathway.

BACKGROUND:

Renewed attention has been directed to the functions of the Y chromosome in the central nervous system during early human male development, due to the recent proposed involvement in neurodevelopmental diseases. PCDH11Y and NLGN4Y are of special interest because they belong to gene families involved in cell fate determination and formation of dendrites and axon.

METHODS:

We used RNA sequencing, immunocytochemistry and a padlock probing and rolling circle amplification strategy, to distinguish the expression of X and Y homologs in situ in the human brain for the first time. To minimize influence of androgens on the sex differences in the brain, we focused our investigation to human embryos at 8-11 weeks post-gestation.

RESULTS:

We found that the X- and Y-encoded genes are expressed in specific and heterogeneous cellular sub-populations of both glial and neuronal origins. More importantly, we found differential distribution patterns of X and Y homologs in the male developing central nervous system.

CONCLUSIONS:

This study has visualized the spatial distribution of PCDH11X/Y and NLGN4X/Y in human developing nervous tissue. The observed spatial distribution patterns suggest the existence of an additional layer of complexity in the development of the male CNS.

BACKGROUND:

Recent evidence suggests that persistent pain and recurrent pain are due to the pain memory which is related to the phosphorylation of cAMP response element-binding protein (p-CREB) in anterior cingulate cortex (ACC). Eletroacupuncture (EA), as a complementary Chinese medical procedure, has a significant impact on the treatment of pain and is now considered as a mind-body therapy.

METHODS:

The rat model of pain memory was induced by two injections of carrageenan into the paws, which was administered separately by a 14-day interval, and treated with EA therapy. The paw withdrawal thresholds (PWTs) of animals were measured and p-CREB expressions in ACC were detected by using immunofluorescence (IF) and electrophoretic mobility shift assay (EMSA). Statistical comparisons among different groups were made by one-way, repeated-measures analysis of variance (ANOVA).

RESULTS:

The second injection of carrageenan caused the decrease of PWTs in the non-injected hind paw. EA stimulation applied prior to the second injection, increased the values of PWTs. In ACC, the numbers of p-CREB positive cells were significantly increased in pain memory model rats, which were significantly reduced by EA. EMSA results showed EA also down-regulated the combining capacity of p-CREB with its DNA. Furthermore, the co-expression of p-CREB with GFAP, OX-42, or NeuN in ACC was strengthened in the pain memory model rats. EA inhibited the co-expression of p-CREB with GFAP or OX-42, but not NeuN in ACC.

CONCLUSIONS:

The present results suggest the retrieval of pain memory could be alleviated by the pre-treatment of EA, which is at least partially attributed to the down-regulated expression and combining capacity of p-CREB and the decreased expression of p-CREB in astrocytes and microglia cells.

BACKGROUND & PURPOSE:

Toll-like receptor 4 (TLR4) signaling induces tissue pro-inflammatory cytokine release and endoplasmic reticulum (ER) stress. We examined the role of TLR4 in autonomic dysfunction and the contribution of ER stress.

EXPERIMENTAL APPROACH:

Our study included animals divided in 6 experimental groups: rats treated with saline (i.v., 0.9%), LPS (i.v., 10mg/kg), VIPER (i.v., 0.1 mg/kg), or 4-PBA (i.p., 10 mg/kg). Two other groups were pretreated either with VIPER (TLR4 viral inhibitory peptide) LPS + VIPER (i.v., 0.1 mg/kg) or 4-Phenyl butyric acid (4-PBA) LPS + PBA (i.p., 10 mg/kg). Arterial pressure (AP) and heart rate (HR) were measured in conscious Sprague-Dawley rats. AP, HR variability, as well as baroreflex sensitivity (BrS), was determined after LPS or saline treatment for 2 hours. Immunofluorescence staining for NeuN, Ib1a, TLR4 and GRP78 in the hypothalamic paraventricular nucleus (PVN) was performed. TNF-α, TLR4 and GRP78 protein expression in the PVN were evaluated by western blot. Plasma norepinephrine levels were determined by ELISA.

KEY RESULTS:

Acute LPS treatment increased HR and plasma norepinephrine concentration. It also decreased HR variability and high frequency (HF) components of HR variability, as well BrS. Acute LPS treatment increased TLR4 and TNF-α protein expression in the PVN. These hemodynamic and molecular effects were partially abrogated with TLR4 blocker or ER stress inhibitor pretreatment. In addition, immunofluorescence study showed that TLR4 is co-localized with GRP78in the neurons. Further inhibition of TLR4 or ER stress was able to attenuate the LPS-induced microglia activation.

CONCLUSIONS & IMPLICATIONS:

TLR4 signaling promotes autonomic dysfunction, inflammation and microglia activation, through neuronal ER stress, in the PVN.

BACKGROUND:

Microglia are the primary immune cells of the spinal cord that are activated in response to ischemia/reperfusion (IR) injury and release various neurotrophic and/or neurotoxic factors to determine neuronal survival. Among them, matrix metalloproteinase-9 (MMP-9), which cleaves various components of the extracellular matrix in the basal lamina and functions as part of the blood spinal cord barrier (BSCB), is considered important for regulating inflammatory responses and microenvironmental homeostasis of the BSCB in the pathology of ischemia. Sevoflurane has been reported to protect against neuronal apoptosis during cerebral IR. However, the effects of sevoflurane preconditioning on spinal cord IR injury remain unclear. In this study, we investigated the role of sevoflurane on potential genetic roles of microglial MMP-9 in tight junction protein breakdown, opening of the BSCB, and subsequent recruitment of microglia to apoptotic spinal cord neurons.

RESULTS:

The results showed significant upregulation of MMP-9 in rats with IR-induced inflammation of the BSCB compared to that of the sham group, manifested as dysfunctional BSCB with increased Evans blue extravasation and reduced expression of occludin protein. Increased MMP-9 expression was also observed to facilitate invasion and migration of activated microglia, imaging as high Iba-1 expression, clustered to neurons in the injured spinal cord, as shown by double immunofluorescence, and increased proinflammatory chemokine production (CXCL10, CCL2). Further, sevoflurane preconditioning markedly improved motor function by ameliorating neuronal apoptosis, as shown by reduced TUNEL-positive cell counts and expression of cleaved caspase-3. These protective effects were probably responsible for downregulation of MMP-9 and maintenance of normal expression of occludin protein indicating BSCB integrity from inflammatory damage, which was confirmed by decreased protein levels of Iba-1 and MMP-9, as well as reduced production of proinflammatory chemokines (CXCL10, CCL2) and proinflammatory cytokines (IL-1β). Intrathecal injection of specific siRNAs targeting MMP-9 had similar protective effects to those of sevoflurane preconditioning.

CONCLUSIONS:

Preconditioning with 2.4% sevoflurane attenuated spinal cord IR injury by inhibiting recruitment of microglia and secretion of MMP-9; thus inhibiting downstream effects on inflammatory damage to BSCB integrity and neuronal apoptosis.

BACKGROUND:

Receptor-interacting protein 3 (RIP3), a member of RIP family proteins, has been shown to participate in programmed necrosis or necroptosis in cell biology studies. Evidence suggests that necroptosis may be a mode of neuronal death in the retina.

RESULTS:

In the present study we determined the expression of RIP3 in normal rat retina and its changes following acute high intraocular pressure (aHIOP). RIP3 immunoreactivity (IR) was largely present in the inner retinal layers, localized to subsets of cells expressing neuron-specific nuclear antigen (NeuN), parvalbumin and calbindin in the ganglion cell layer (GCL) and inner nuclear layer (INL). No double labeling was detected for RIP3 with PKC-α or rhodopsin. RIP3 immunoreactivity was increased in the GCL at 6 hr and 12 hr, but reduced at 24 hr in the retina, without apparent alteration in laminar or cellular distribution pattern. Western blot analysis confirmed the above time-dependent alteration in RIP3 protein expression. RIP3 expressing cells frequently co-localized with propidium iodide (PI). A few co-localized cells were observed between RIP3 and Bax or cleaved caspase-3 in the GCL in 12 hr following aHIOP.

CONCLUSIONS:

The results indicate that RIP3 is expressed differentially in retinal neurons in adult rats, including subsets of ganglion cells, amacrine and horizontal cells. RIP3 protein levels are elevated rapidly following aHIOP. RIP3 labeling co-localized with PI, Bax or cleaved caspase-3 among cells in the ganglion cell layer following aHIOP, which suggest its involvement of RIP3 in neuronal responses to acute ischemic insults.

Congenital diaphragmatic hernia (CDH) can induce lung hypoplasia and pulmonary hypertension and is associated with high mortality. The purpose of this study is to examine the efficacy and safety of antenatal Saireito (TJ-114), a traditional Japanese herbal medicine, in a rat CDH model. Sprague-Dawley rats were exposed to an herbicide (nitrofen, 100 mg) on embryonic day 9 (E9) to induce CDH, and antenatal Saireito (2000 mg/kg/day) was orally administered from E10 to E20. On E21, fetuses were delivered. Antenatal Saireito significantly decreased the incidence of CDH (p < 0.01), increased lung volume (p < 0.01), improved alveolarization and pulmonary artery remodeling using histological analysis, and improved respiratory function using gasometric analysis (pH; p < 0.05, and PCO2 ; p < 0.01). In addition, antenatal Saireito significantly decreased endothelin-1 and endothelin receptor A expression in the pulmonary arteries. Taken together, our results demonstrated that antenatal Saireito can improve fetal pulmonary hypoplasia and pulmonary vascular remodeling and, as a result, can improve respiratory function in a rat CDH model.

Copyright © 2016 John Wiley & Sons, Ltd.

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling.

The circadian system has endowed animals with the ability to anticipate recurring food availability at particular times of day. As daily food anticipation (FA) is independent of the suprachiasmatic nuclei, the central pacemaker of the circadian system, questions arise of where FA signals originate and what role components of the circadian clock might play. Here we show that liver-specific deletion of Per2 in mice abolishes FA, an effect that is rescued by viral overexpression of Per2 in the liver. RNA sequencing indicates that Per2 regulates β-hydroxybutyrate (βOHB) production to induce FA leading to the conclusion that liver Per2 is important for this process. Unexpectedly, we show that FA originates in the liver and not in the brain. However, manifestation of FA involves processing of the liver-derived βOHB signal in the brain, indicating that the food-entrainable oscillator is not located in a single tissue but is of systemic nature.

Among the primary brain tumors, glioblastoma multiforme (GBM) has a radical proliferation ability that complicates the therapeutic modulation of cancer progression. The majority of GBM patients have a low survival rate (<1 year) due to radical tumor growth and late cancer diagnosis. Previous reports have shown that astrocytes have a specific metabolic organization that includes the production of lactate, the storage of glycogen, and use of lactate to support neurons which possess higher capacity of metabolism compared to neurons. We hypothesized that these characteristics of astrocytes could contribute to enhanced proliferation of GBM compared to neuroblastoma (NB). Here, we show that U87MG cells (a model of GBM) proliferate more rapidly than SH-SY5Y cells (a model of NB). A higher extracellular acidification rate and maximal mitochondrial oxygen consumption rate were observed in U87MG cells compared to SH-SY5Y cells. The expression levels of lactate dehydrogenase (LDH)-A and LDH-B were higher in U87MG cells and primary cultured astrocytes than in SH-SY5Y cells and neurons. Furthermore, the mRNA levels of succinate dehydrogenase and peroxisome proliferator-activated receptor-γ were high in U87MG cells, suggesting that these cells have high capacity for mitochondrial metabolism and uptake of fatty acids related to synthesis of the cell membrane, respectively. Taken together, we demonstrate that GBM cells are characterized by activation of the LDH-expression-related glycolytic pathway and mitochondrial metabolic capacity, suggesting two innate properties of astrocytes that could provide a driving force for the growth ability of GBM. Based on these findings, we propose that therapeutic approaches aimed at treating GBM could target LDH for modulating the metabolic properties of GBM cells.

The ability to change strategies in different contexts is a form of behavioral flexibility that is crucial for adaptive behavior. The striatum has been shown to contribute to certain forms of behavioral flexibility such as reversal learning. Here we report on the contribution of striatal cholinergic interneurons-a key element in the striatal neuronal circuit-to strategy set-shifting in which an attentional shift from one stimulus dimension to another is required. We made lesions of rat cholinergic interneurons in dorsomedial or ventral striatum using a specific immunotoxin and investigated the effects on set-shifting paradigms and on reversal learning. In shifting to a set that required attention to a previously irrelevant cue, lesions of dorsomedial striatum significantly increased the number of perseverative errors. In this condition, the number of never-reinforced errors was significantly decreased in both types of lesions. When shifting to a set that required attention to a novel cue, rats with ventral striatum lesions made more perseverative errors. Neither lesion impaired learning of the initial response strategy nor a subsequent switch to a new strategy when response choice was indicated by a previously relevant cue. Reversal learning was not affected. These results suggest that in set-shifting the striatal cholinergic interneurons play a fundamental role, which is dissociable between dorsomedial and ventral striatum depending on behavioral context. We propose a common mechanism in which cholinergic interneurons inhibit neurons representing the old strategy and enhance plasticity underlying exploration of a new rule.

Although multipotent cell types have enlarged nuclei with decondensed chromatin, this property has not been exploited to enhance the characterization of neural progenitor cell (NPC) populations in the brain. We found that mouse brain cell nuclei that expressed exceptionally high levels of the pan neuronal marker NeuN/FOX3 (NeuN-High) had decondensed chromatin relative to most NeuN-Low or NeuN-Neg (negative) nuclei. Purified NeuN-High nuclei expressed significantly higher levels of transcripts encoding markers of neurogenesis, neuroplasticity, and learning and memory (ARC, BDNF, ERG1, HOMER1, NFL/NEF1, SYT1), subunits of chromatin modifying machinery (SIRT1, HDAC1, HDAC2, HDAC11, KAT2B, KAT3A, KAT3B, KAT5, DMNT1, DNMT3A, Gadd45a, Gadd45b) and markers of NPC and cell cycle activity (BRN2, FOXG1, KLF4, c-MYC, OCT4, PCNA, SHH, SOX2) relative to neuronal NeuN-Low or to mostly non-neuronal NeuN-Neg nuclei. NeuN-High nuclei expressed higher levels of HDAC1, 2, 4, and 5 proteins. The cortex, hippocampus, hypothalamus, thalamus, and nucleus accumbens contained high percentages of large decondensed NeuN-High nuclei, while the cerebellum, and pons contained very few. NeuN-High nuclei have the properties consistent with their being derived from extremely active neurons with elevated rates of chromatin modification and/or NPC-like cells with multilineage developmental potential. The further analysis of decondensed neural cell nuclei should provide novel insights into neurobiology and neurodegenerative disease.

The blood-brain barrier (BBB) prevents the access of therapeutic antibodies to central nervous system (CNS) targets. The engineering of bispecific antibodies in which a therapeutic "arm" is combined with a BBB-transcytosing arm can significantly enhance their brain delivery. The BBB-permeable single-domain antibody FC5 was previously isolated by phenotypic panning of a naive llama single-domain antibody phage display library. In this study, FC5 was engineered as a mono- and bivalent fusion with the human Fc domain to optimize it as a modular brain delivery platform. In vitro studies demonstrated that the bivalent fusion of FC5 with Fc increased the rate of transcytosis (Papp) across brain endothelial monolayer by 25% compared with monovalent fusion. Up to a 30-fold enhanced apparent brain exposure (derived from serum and cerebrospinal fluid pharmacokinetic profiles) of FC5- compared with control domain antibody-Fc fusions after systemic dosing in rats was observed. Systemic pharmacological potency was evaluated in the Hargreaves model of inflammatory pain using the BBB-impermeable neuropeptides dalargin and neuropeptide Y chemically conjugated with FC5-Fc fusion proteins. Improved serum pharmacokinetics of Fc-fused FC5 contributed to a 60-fold increase in pharmacological potency compared with the single-domain version of FC5; bivalent and monovalent FC5 fusions with Fc exhibited similar systemic pharmacological potency. The study demonstrates that modular incorporation of FC5 as the BBB-carrier arm in bispecific antibodies or antibody-drug conjugates offers an avenue to develop pharmacologically active biotherapeutics for CNS indications.

Alterations in the activity of neural circuits are a common consequence of traumatic brain injury (TBI), but the relationship between single-neuron properties and the aggregate network behavior is not well understood. We recently reported that the GluN2B-containing NMDA receptors (NMDARs) are key in mediating mechanical forces during TBI, and that TBI produces a complex change in the functional connectivity of neuronal networks. Here, we evaluated whether cell-to-cell heterogeneity in the connectivity and aggregate contribution of GluN2B receptors to [Ca(2+)]i before injury influenced the functional rewiring, spontaneous activity, and network plasticity following injury using primary rat cortical dissociated neurons. We found that the functional connectivity of a neuron to its neighbors, combined with the relative influx of calcium through distinct NMDAR subtypes, together contributed to the individual neuronal response to trauma. Specifically, individual neurons whose [Ca(2+)]i oscillations were largely due to GluN2B NMDAR activation lost many of their functional targets 1 h following injury. In comparison, neurons with large GluN2A contribution or neurons with high functional connectivity both independently protected against injury-induced loss in connectivity. Mechanistically, we found that traumatic injury resulted in increased uncorrelated network activity, an effect linked to reduction of the voltage-sensitive Mg(2+) block of GluN2B-containing NMDARs. This uncorrelated activation of GluN2B subtypes after injury significantly limited the potential for network remodeling in response to a plasticity stimulus. Together, our data suggest that two single-cell characteristics, the aggregate contribution of NMDAR subtypes and the number of functional connections, influence network structure following traumatic injury.

BACKGROUND:

Neuroinflammation is the leading cause of neurological sequelae after traumatic brain injury (TBI). The aim of the present study was to investigate whether the neuroprotective effects of electroacupuncture (EA) are mediated by anti-neuroinflammatory effects in a rat model of TBI.

METHODS:

Male Sprague-Dawley rats were randomly divided into three groups: sham-operated, TBI control, and EA-treated. The animals in the sham-operated group underwent a sham operation, those in the TBI control group were subjected to TBI, but not EA, and those in the EA group were treated with EA for 60 min immediately after TBI, daily for 3 consecutive days. EA was applied at the acupuncture points GV20, GV26, LI4, and KI1, using a dense-dispersed wave, at frequencies of 0.2 and 1 Hz, and an amplitude of 1 mA. Cell infarction volume (TTC stain), neuronal apoptosis (markers: TUNEL and Caspase-3), activation of microglia (marker: Iba1) and astrocytes (marker: GFAP), and tumor necrosis factor (TNF)-α expression in the microglia and astrocytes were evaluated by immunofluorescence. Functional outcomes were assessed using the inclined plane test. All tests were performed 72 h after TBI.

RESULTS:

We found that TBI-induced loss of grasp strength, infarction volume, neuronal apoptosis, microglial and astrocyte activation, and TNF-α expression in activated microglia and astrocytes were significantly attenuated by EA treatment.

CONCLUSIONS:

Treatment of TBI in the acute stage with EA for 60 min daily for 3 days could ameliorate neuroinflammation. This may thus represent a mechanism by which functional recovery can occur after TBI.

BACKGROUND:

Adverse maternal lifestyle resulting in adverse early life environment (AELE) increases risks for neuropsychiatric disorders in offspring. Neuropsychiatric disorders are associated with impaired neurogenesis and neuro-inflammation in the hippocampus (HP). Microglia are neuro-inflammatory cells in the brain that regulate neurogenesis via toll-like receptors (TLR). TLR-9 is implicated in neurogenesis inhibition and is responsible for stress-related inflammatory responses. We hypothesized that AELE would increase microglia cell count and increase TLR-9 expression in juvenile mouse HP. These increases in microglia cell count and TLR-9 expression would be associated with decrease neural stem cell count and neuronal cell count.

METHODS:

We developed a mouse model of AELE combining Western diet and a stress environment. Stress environment consisted of random change from embryonic day 13 (E13) to E17 as well as static change in maternal environment from E13 to postnatal day 21(P21). At P21, we measured hippocampal cell numbers of microglia, neural stem cell and neuron, as well as hippocampal TLR-9 expression.

RESULTS:

AELE significantly increased total microglia number and TLR-9 expression in the hippocampus. Concurrently, AELE significantly decreased neural stem cell and neuronal numbers.

CONCLUSIONS:

AELE increased the neuro-inflammatory cellular response in the juvenile HP. We speculate that increased neuro-inflammatory responses may contribute to impaired neurogenesis seen in this model.

Copyright © 2016 ISDN. Published by Elsevier Ltd. All rights reserved.

BACKGROUND:

Tumor necrosis factor-alpha (TNF-α) is elevated early in injured brain after traumatic brain injury (TBI), in humans and in animals. Etanercept (a TNF-α antagonist with anti-inflammatory effects) attenuates TBI in rats by reducing both microglial and astrocytic activation and increased serum levels of TNF-α. However, it is not known whether etanercept improves outcomes of TBI by attenuating microglia-associated, astrocytes-associated, and/or neurons-associated TNF-α expression in ischemic brain. A well clinically relevant rat model, where a lateral fluid percussion is combined with systemic administration of etanercept immediately after TBI, was used. The neurological severity score and motor function was measured on all rats preinjury and on day 3 after etanercept administration. At the same time, the neuronal and glial production of TNF-α was measured by Immunofluorescence staining. In addition, TNFα contents of ischemic cerebral homogenates was measured using commercial enzyme-linked immunosorbent assay kits.

RESULTS:

In addition to inducing brain ischemia as well as neurological and motor deficits, TBI caused significantly higher numbers of microglia-TNF-α double positive cells, but not neurons-TNF-α or astrocytes-TNF-α double positive cells in the injured brain areas than did the sham operated controls, when evaluated 3 days after TBI. The TBI-induced cerebral ischemia, neurological motor deficits, and increased numbers of microglia-TNF-α double positive cells and increased TNF-α levels in the injured brain were all significantly attenuated by etanercept therapy.

CONCLUSION:

This finding indicates that early microglia overproduction of TNF-α in the injured brain region after TBI contributes to cerebral ischemia and neurological motor deficits, which can be attenuated by etanercept therapy. Studies in this model could provide insight into the mechanisms underlying neurological motor disturbance in brain-injured patients.