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Hypoxic-ischaemic encephalopathy (HIE) is an important cause of morbidity and mortality globally. Although mild therapeutic hypothermia (TH) may improve outcomes in selected babies, the mechanism of action is not fully understood. A proteomics discovery study was carried out to analyse proteins in the plasma of newborns with HIE. Proteomic analysis of plasma from 22 newborns with moderate-severe HIE that had initially undergone TH, and relative controls including 10 newborns with mild HIE who did not warrant TH and also cord blood from 10 normal births (non-HIE) were carried out using the isobaric Tandem Mass Tag (TMT®) 10plexTM labelling with tandem mass spectrometry. A total of 7818 unique peptides were identified in all TMT10plexTM samples, translating to 3457 peptides representing 405 proteins, after applying stringent filter criteria. Apart from the unique protein signature from normal cord blood, unsupervised analysis revealed several significantly regulated proteins in the TH-treated moderate-severe HIE group. GO annotation and functional clustering revealed various proteins associated with glucose metabolism: the enzymes fructose-bisphosphate aldolase A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase 1, phosphoglycerate kinase 1, and pyruvate kinase PKM were upregulated in newborns with favourable (sHIE+) outcomes compared to newborns with unfavourable (sHIE−) outcomes. Those with favourable outcomes had normal MR imaging or mild abnormalities not predictive of adverse outcomes. However, in comparison to mild HIE and the sHIE− groups, the sHIE+ group had the additional glucose metabolism-related enzymes upregulated, including triosephosphate isomerase, α-enolase, 6-phosphogluconate dehydrogenase, transaldolase, and mitochondrial glutathione reductase. In conclusion, our plasma proteomic study demonstrates that TH-treated newborns with favourable outcomes have an upregulation in glucose metabolism. These findings may open new avenues for more effective neuroprotective therapy. [ABSTRACT FROM AUTHOR]

• Serotonin and serotonin transporter expression are similar in the brain of zebra finches. • Different serotonin expression at different song nuclei in zebra finches. • Different serotonin expression at different peri-song nuclei in zebra finches. • Various forms of interactivity between serotoninergic and cholinergic neurones in the brain stem of zebra finches. • Different co-expression of serotonin in dopaminergic neurones in zebra finches. Serotonin is an important neurotransmitter of the brain, but its role in song control remains to be fully demonstrated. Using male zebra finches (Taeniopygia guttata) that have song learning and production capabilities, we analysed the serotonin expression levels in the song nuclei and adjacent areas (peri-song nuclei) using immunohistochemistry. Key song nuclei were identified using combinations of Hoechst, choline acetyltransferase, and a neurofilament (NN18) marker in reference to the ZEBrA atlas. Mean serotonin expression was highest in interfacial nucleus (Nif) and lower in the other song nuclei in the following order (in order of highest first): interfacial nucleus (Nif) > Area X > dorsomedial part of the intercollicular nucelus (DM) > robust nucleus of the archistriatum (RA) > lateral magnocellular nucleus of the anterior neostriatum (LMAN) > ventral respiratory group (VRG) > dorsolateral nucleus of the medial thalamus (DLM) > the nucleus HVC (proper name) > tracheosyringeal motor nucleus (nXIIts). However, the mean serotonin expression (in order of highest first) in the peri-song nuclei regions was: peri-DM > peri-nXIIts > supra-peri-HVC > peri-RA > peri-DLM > peri-Area X > infra-peri-HVC > peri-VRG > peri-LMAN > peri-Nif. Interestingly, serotoninergic fibers immunostained for serotonin or the serotonin transporter can be found as a basket-like peri-neuronal structure surrounding cholinergic cell bodies, and appear to form contacts onto dopaminergic neurones. In summary, serotonin fibers are present at discrete song nuclei, and peri-song nuclei regions, which suggest serotonin may have a direct and/or modulatory role in song control. [ABSTRACT FROM AUTHOR]

• Automated behaviour systems allow long-term assessment of complex CNS injury models. • Home cage grouped-housed animal are relevant for subtle spontaneous behaviour studies. • Spinal cord injury affects locomotion and the grooming /rearing ability of animals. • Group behaviour analysis facilitates the study of aggression in brain injury models. • Home cage non-invasive behaviour systems provides a unique 3Rs advantage. Neurotrauma patients face major neurological sequelae. The failure in the preclinical-to-clinical translation of candidate therapies could be due to poor evaluation of rodent behaviours after neurotrauma. A home cage automated system was used to study the long term behaviour of individual rats with traumatic brain injury (TBI), spinal cord injury (SCI) and non-CNS injured controls, whilst group-housed in their home cages. Naïve rats were used as baseline controls. Automated locomotor activity and body temperature recordings were carried out 24 h /day for 3 days/week during 12 weeks post-injury. Behavioural patterns, including aggression, rearing, grooming, feeding and drinking were analysed from automated video recordings during week 1, 6 and 12. SCI animals showed a lower locomotor activity compared to TBI or control animals during light and dark phases. TBI animals showed a higher aggression during the dark phase in the first week post-injury compared to SCI or control animals. Individual grooming and rearing were reduced in SCI animals compared to TBI and control animals in the first week post-injury during the dark phase. No differences in drinking or feeding were detected between groups. Locomotor activity did not differ between naïve male and female rats, but body temperature differ between light and dark phases for both. Injury severity was compared to standard SCI and TBI behaviour scores (BBB and mNSS, respectively) and histological analysis. This study demonstrates the practical benefits of using a non-intrusive automated home cage recording system to observe long term individual behaviour of group-housed SCI and TBI rats. [ABSTRACT FROM AUTHOR]

Abstract Neonates can develop hypoxic-ischaemic encephalopathy (HIE) due to lack of blood supply or oxygen, resulting in a major cause of death and disability among term newborns. However, current definitive treatment of therapeutic hypothermia, will only benefit one out of nine babies. Furthermore, the mechanisms of HIE and therapeutic hypothermia are not fully understood. Recently, microRNAs (miRNAs) have become of interest to many researchers due to their important role in post-transcriptional control and deep evolutionary history. Despite this, role of miRNAs in newborns with HIE remains largely unknown due to limited research in this field. Therefore, this review aims to understand the role of miRNAs in normal brain development and HIE pathophysiology with reliance on extrapolated data from other diseases, ages and species due to current limited data. This will provide us with an overview of how miRNAs in normal brain development changes after HIE. Furthermore, it will indicate how miRNAs are affected specifically or globally by the various pathophysiological events. In addition, we discuss about how drugs and commercially available agents can specifically target certain miRNAs as a mechanism of action and potential safety issue with off-target effects. Improving our understanding of the role of miRNAs on the cellular response after HIE would enhance the success of effective diagnosis, prognosis, and treatment of newborns with HIE. Highlights • MicroRNAs are important in normal newborn brain development and during brain injury, especially cell death and inflammation. • The changes involving microRNAs in newborn brain injury are complex due to the additional ongoing newborn brain development. • MicroRNAs are potential targets for treatment of brain injury in newborns. [ABSTRACT FROM AUTHOR]

Task-specific rehabilitation has been shown to promote functional recovery after acute spinal cord injury (SCI). Recently, the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), has been shown to promote neuroplasticity after SCI. Here, we investigated whether the combination of a single bolus of DHA with rehabilitation can enhance the effect of DHA or rehabilitation therapy in adult injured spinal cord. We found enhanced functional improvement with DHA in combination with rehabilitation compared with either treatment alone in a rat cervical lateral hemisection SCI model. This behavioral improvement correlated with a significant sprouting of uninjured corticospinal and serotonergic fibers. We also observed that the greatest increase in the synaptic vesicle protein, synaptophysin, and the synaptic active zone protein, Bassoon, occurred in animals that received both DHA and rehabilitation. In summary, the functional, anatomical, and synaptic plasticity induced by task-specific rehabilitation can be further enhanced by DHA treatment. This study shows the potential beneficial effects of DHA combined with rehabilitation for the treatment of patients with SCI. [ABSTRACT FROM AUTHOR]

Docosahexaenoic acid (DHA) is an ω-3 polyunsaturated fatty acid that is essential in brain development and has structural and signaling roles. Acute DHA administration is neuroprotective and promotes functional recovery in animal models of adult spinal cord injury (SCI). However, the mechanisms underlying this recovery have not been fully characterized. Here we investigated the effects of an acute intravenous bolus of DHA delivered after SCI and characterized DHA-induced neuroplasticity within the adult injured spinal cord. We found robust sprouting of uninjured corticospinal and serotonergic fibers in a rat cervical hemisection SCI model. A mouse pyramidotomy model was used to confirm that this robust sprouting was not species or injury model specific. Furthermore, we demonstrated that corticospinal fibers sprouting to the denervated side of the cord following pyramidotomy contact V2a interneurons. We also demonstrated increased serotonin fibers and synaptophysin in direct contact with motor neurons. DHA also increased synaptophysin in rat cortical cell cultures. A reduction in phosphatase and tensin homolog (PTEN) has been shown to be involved in axonal regeneration and synaptic plasticity. We showed that DHA significantly upregulates miR-21 and downregulates PTEN in corticospinal neurons. Downregulation of PTEN and upregulation of phosphorylated AKT by DHA were also seen in primary cortical neuron cultures and were accompanied by increased neurite outgrowth. In summary, acute DHA induces anatomical and synaptic plasticity in adult injured spinal cord. This study shows that DHA has therapeutic potential in cervical SCI and provides evidence that DHA could exert its beneficial effects in SCI via enhancement of neuroplasticity. [ABSTRACT FROM AUTHOR]

Spinal cord injury can have a range of debilitating effects, permanently impacting a patient's quality of life. Initially thought to be an immune privileged site, the spinal cord is able to mount a timely and well organized inflammatory response to injury. Intricate immune cell interactions are triggered, typically consisting of a staggered multiphasic immune cell response, which can become deregulated if left unchecked. Although several immunomodulatory compounds have yielded success in experimental rodent spinal cord injury models, their translation to human clinical studies needs further consideration. Because temporal differences between rodent and human inflammatory responses to spinal cord injury do exist, drug delivery timing will be a crucial component in recovery from spinal cord injury. Given too early, immunomodulatory therapies may impede beneficial inflammatory reactions to the injured spinal cord or even miss the opportunity to dampen delayed harmful autoimmune processes. Therefore, this review aims to summarize the temporal inflammatory response to spinal cord injury, as well as detailing specific immune cell functions. By clearly defining the chronological order of inflammatory events after trauma, immunomodulatory drug delivery timing can be better optimized. Further, we compare spinal cord injury-induced inflammatory responses in rodent and human studies, enabling clinicians to consider these differences when initiating clinical trials. Improved understanding of the cellular immune response after spinal cord injury would enhance the efficacy of immunomodulatory agents, enabling combined therapies to be considered. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease characterised by loss of motor neurons that currently has no cure. Omega-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have many health benefits including neuroprotective and myoprotective potential. We tested the hypothesis that a high level of dietary EPA could exert beneficial effects in ALS. The dietary exposure to EPA (300 mg/kg/day) in a well-established mouse model of ALS expressing the G93A superoxide dismutase 1 (SOD1) mutation was initiated at a pre-symptomatic or symptomatic stage, and the disease progression was monitored until the end stage. Daily dietary EPA exposure initiated at the disease onset did not significantly alter disease presentation and progression. In contrast, EPA treatment initiated at the pre-symptomatic stage induced a significantly shorter lifespan. In a separate group of animals sacrificed before the end stage, the tissue analysis showed that the vacuolisation detected in G93A-SOD1 mice was significantly increased by exposure to EPA. Although EPA did not alter motor neurone loss, EPA reversed the significant increase in activated microglia and the astrocytic activation seen in G93A-SOD1 mice. The microglia in the spinal cord of G93A-SOD1 mice treated with EPA showed a significant increase in 4-hydroxy-2-hexenal, a highly toxic aldehydic oxidation product of omega-3 fatty acids. These data show that dietary EPA supplementation in ALS has the potential to worsen the condition and accelerate the disease progression. This suggests that great caution should be exerted when considering dietary omega-3 fatty acid supplements in ALS patients. [ABSTRACT FROM AUTHOR]

Stroke is the dominant cause of sensorimotor disability that primarily affects the elderly. We now show that neuroplasticity and functional recovery after stroke is constrained by inhibitory chondroitin sulphates. In two blinded, randomized preclinical trials, degradation of chondroitin sulphate using chondroitinase ABC reactivated neuroplasticity and promoted sensorimotor recovery after stroke in elderly rats. Three days after stroke, chondroitinase ABC was microinjected into the cervical spinal cord to induce localized plasticity of forelimb sensorimotor spinal circuitry. Chondroitinase ABC effectively removed chondroitin sulphate from the extracellular matrix and perineuronal nets. Three different tests of sensorimotor function showed that chondroitinase ABC promoted recovery of forelimb function. Anterograde and retrograde tracing showed that chondroitinase ABC also induced sprouting of the contralesional corticospinal tract in the aged treated hemicord. Chondroitinase ABC did not neuroprotect the peri-infarct region. We show for the first time delayed chondroitinase ABC treatment promotes neuroanatomical and functional recovery after focal ischaemic stroke in an elderly nervous system. [ABSTRACT FROM AUTHOR]

Pain remains an area of considerable unmet clinical need, and this is particularly true of pain associated with bone metastases, in part because existing analgesic drugs show only limited efficacy in many patients and in part because of the adverse side effects associated with these agents. An important issue is that the nature and roles of the algogens produced in bone that drive pain-signalling systems remain unknown. Here, we tested the hypothesis that adenosine triphosphate is one such key mediator through actions on P2X3 and P2X2/3 receptors, which are expressed selectively on primary afferent nocioceptors, including those innervating the bone. Using a well-established rat model of bone cancer pain, AF-353, a recently described potent and selective P2X3 and P2X2/3 receptor antagonist, was administered orally to rats and found to produce highly significant prevention and reversal of bone cancer pain behaviour. This attenuation occurred without apparent modification of the disease, since bone destruction induced by rat MRMT-1 carcinoma cells was not significantly altered by AF-353. Using in vivo electrophysiology, evidence for a central site of action was provided by dose-dependent reductions in electrical, mechanical and thermal stimuli-evoked dorsal horn neuronal hyperexcitability following direct AF-353 administration onto the spinal cord of bone cancer animals. A peripheral site of action was also suggested by studies on the extracellular release of adenosine triphosphate from MRMT-1 carcinoma cells. Moreover, elevated phosphorylated-extracellular signal-regulated kinase expression in dorsal root ganglion neurons, induced by co-cultured MRMT-1 carcinoma cells, was significantly reduced in the presence of AF-353. These data suggest that blockade of P2X3 and P2X2/3 receptors on both the peripheral and central terminals of nocioceptors contributes to analgesic efficacy in a model of bone cancer pain. Thus, systemic P2X3 and P2X2/3 receptor antagonists with central nervous system penetration may offer a promising therapeutic tool in treating bone cancer pain. [ABSTRACT FROM PUBLISHER]

Following trauma of the adult brain or spinal cord the injured axons of central neurons fail to regenerate or if intact display only limited anatomical plasticity through sprouting. Adult cortical neurons forming the corticospinal tract (CST) normally have low levels of the neuronal calcium sensor-1 (NCS1) protein. In primary cultured adult cortical neurons, the lentivectorinduced overexpression of NCS1 induces neurite sprouting associated with increased phospho-Akt levels. When the PI3K/ Akt signalling pathway was pharmacologically inhibited the NCS1-induced neurite sprouting was abolished. The overexpression of NCS1 in uninjured corticospinal neurons exhibited axonal sprouting across the midline into the CSTdenervated side of the spinal cord following unilateral pyramidotomy. Improved forelimb function was demonstrated behaviourally and electrophysiologically. In injured corticospinal neurons, overexpression of NCS1 induced axonal sprouting and regeneration and also neuroprotection. These findings demonstrate that increasing the levels of intracellular NCS1 in injured and uninjured central neurons enhances their intrinsic anatomical plasticity within the injured adult central nervous system. [ABSTRACT FROM AUTHOR]

P2X3 and P2X2/3 receptors are localized on sensory afferents both peripherally and centrally and have been implicated in various sensory functions. However, the physiological role of these receptors expressed presynaptically in the spinal cord in regulating sensory transmission remains to be elucidated. Here, a novel selective P2X3 and P2X2/3 antagonist, AF-792 [5-(5-ethynyl-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine, previously known as RO-5], in addition to less selective purinoceptor ligands, was applied intrathecally in vivo. Cystometry recordings were made to assess changes in the micturition reflex contractions after drug treatments. We found that AF-792 inhibited micturition reflex activity significantly (300nmol; from baseline contraction intervals of 1.18±0.07 to 9.33#x00B1;2.50min).Furthermore,inhibition of P2X3 and P2X2/3 receptors in the spinal cord significantly attenuated spinal activation of extracellular-signal regulated kinases induced by acute peripheral stimulation of the bladder with 1% acetic acid by 46.4 ± 12.0% on average. Hence, the data suggest that afferent signals originating from the bladder are regulated by spinal P2X3 and P2X2/3 receptors and establish directly an endogenous central presynaptic purinergic mechanism to regulate visceral sensory transmission. Identification of this spinal purinergic control in visceral activities may help the development of P2X3 and P2X2/3 antagonist to treat urological dysfunction, such as overactive bladder, and possibly other debilitating sensory disorders, including chronic pain states. [ABSTRACT FROM AUTHOR]

Background: Traumatic injuries can undermine neurological functions and act as risk factors for the development of irreversible and fatal neurodegenerative disorders like amyotrophic lateral sclerosis (ALS). In this study, we have investigated how a mutation of the superoxide dismutase 1 (SOD1) gene, linked to the development of ALS, modifies the acute response to a gentle mechanical compression of the spinal cord. In a 7-day post-injury time period, we have performed a comparative ontological analysis of the gene expression profiles of injured spinal cords obtained from pre-symptomatic rats over-expressing the G93A-SOD1 gene mutation and from wild type (WT) littermates. Results: The steady post-injury functional recovery observed in WT rats was accompanied by the early activation at the epicenter of injury of several growth-promoting signals and by the down-regulation of intermediate neurofilaments and of genes involved in the regulation of ion currents at the 7 day post-injury time point. The poor functional recovery observed in G93A-SOD1 transgenic animals was accompanied by the induction of fewer pro-survival signals, by an early activation of inflammatory markers, of several pro-apoptotic genes involved in cytochrome-C release and by the persistent up-regulation of the heavy neurofilament subunits and of genes involved in membrane excitability. These molecular changes occurred along with a pronounced atrophy of spinal cord motor neurones in the G93A-SOD1 rats compared to WT littermates after compression injury. Conclusions: In an experimental paradigm of mild mechanical trauma which causes no major tissue damage, the G93A-SOD1 gene mutation alters the balance between pro-apoptotic and pro-survival molecular signals in the spinal cord tissue from the pre-symptomatic rat, leading to a premature activation of molecular pathways implicated in the natural development of ALS. [ABSTRACT FROM AUTHOR]

Abstract: Microglia are important in homeostasis and widely considered to have roles in the pathogenesis of conditions such as neuropathic pain and multiple sclerosis. The need to study microglia from the adult spinal cord is essential to further understand the role of these cells in disease pathology. Primary microglia are often prepared from brain tissues obtained from embryonic or perinatal age rodents and the process can take over a week to complete. The protocol in this study provides rapid isolation of microglia from adult spinal cord, allowing immediate availability for experimentation of both ex vivo and in vitro within a few hours. A purity of 99% with little or no neuronal or astrocytic contamination can be achieved. Between 70% and 85% of these adult microglia were in a relatively non-activated state. Functionally, these microglia respond to lipopolysaccharide incubation with increases in both phospho-p38 MAPK and OX42 immunostaining, as well as release of ATP, as compared to un-stimulated microglia. This technique provides a protocol to achieve rapid and efficient extraction of high purity, quiescent and functionally active microglia from adult mouse spinal cord, allowing greater study of adult spinal microglia in physiological and pathophysiological states. [Copyright &y& Elsevier]

Understanding of the sequence and nature of the events that govern neuron-microglia communication is critical for the discovery of new mechanisms and targets for chronic pain treatment. The neuronal chemokine fractalkine (FKN) and its microglial receptor CX3CR1 may mediate such a function in the dorsal horn of the spinal cord after cleavage of the extracellular domain of this transmembrane chemokine by a protease. Here we report that in neuropathic rat dorsal horn, with dorsal root-attached preparations, soluble FKN (sFKN) contents are increased in the superfusates collected after noxious-like electrical stimulation of ipsilateral primary afferent fibers. The increase of sFKN is prevented by morpholinurea-leucine-homophenylalanine-vinyl sulfone-phenyl (LHVS), an irreversible inhibitor of cathepsin S (CatS) whose proteolytic activity is also increased in the superfusates. The source of CatS activity is microglial cells activated by the peripheral nerve injury and secreting the enzyme, as a result of primary afferent fiber stimulation. Indeed, the acute activation of dorsal horn microglia by lipopolysaccharide results in increased CatS activity in the superfusates, followed by increased sFKN contents. Consistent with these observations ex vivo, the levels of both sFKN and CatS activity in CSF samples increased significantly after peripheral nerve injury, associated with spinal microglial activation. Finally, because we found that both FKN immunoreactivity and mRNA are confined to dorsal horn neurons, we suggest that under neuropathic conditions, noxious stimulation of primary afferent fibers induces release of CatS from microglia, which liberates FKN from dorsal horn neurons, thereby contributing to the amplification and maintenance of chronic pain. [ABSTRACT FROM AUTHOR]

A recent major conceptual advance has been the recognition of the importance of immune system-neuronal interactions in the modulation of brain function, one example of which is spinal pain processing in neuropathic states. Here, we report that in peripheral nerve-injured rats, the lysosomal cysteine protease cathepsin S (CatS) is critical for the maintenance of neuropathic pain and spinal microglia activation. After injury, CatS was exclusively expressed by activated microglia in the ipsilateral dorsal horn, where expression peaked at day 7, remaining high on day 14. Intrathecal delivery of an irreversible Cats inhibitor, morpholinurea-leucinehomophenylalanineovinyl phenyl sulfone (LHVS), was antihyperalgesic and antiallodynic in neuropathic rats and attenuated spinal microglia activation. Consistent with a pronociceptive role of endogenous CatS, spinal intrathecal delivery of rat recombinant Cats (rrCatS) induced hyperalgesia and allodynia in naYve rats and activated p38 mitogen-activated protein kinase (MAPK) in spinal cord microglia. A bioinformatics approach revealed that the transmembrane chemokine fractalkine (FKN) is a potential substrate for Cats cleavage. We show that rrCatS incubation reduced the levels of cell-associated FKN in cultured sensory neurons and that a neutralizing antibody against FKN prevented both FKN- and CatS- induced allodynia, hyperalgesia, and p38 MAPK activation. Furthermore, rrCatS induced allodynia in wild-type but not CX3CR1-knockout mice. We suggest that under conditions of increased nociception, microglial CatS is responsible for the liberation of neuronal FKN, which stimulates p38 MAPK phosphorylation in microglia, thereby activating neurons via the release of pronociceptive mediators. [ABSTRACT FROM AUTHOR]

Abstract: Many studies have shown a role of retinoid signalling in neurite outgrowth in vitro, and that the retinoic acid receptor (RAR) β2 is critical for this process. We show here that RARβ2 is expressed predominantly in dorsal root ganglia (DRG) neuronal subtypes that express neurofilament (NF) 200 and calcitonin gene-related peptide (CGRP), and that these neurons extend neurites in response to RA. We demonstrate that retinoid signalling has a role in neurite outgrowth in vivo, by showing that in a peripheral nerve crush model there is less neurite outgrowth from RARβ null DRG compared to wild-type. We identify sonic hedgehog (Shh) as a downstream target of the RARβ2 signalling pathway as it is expressed in the injured DRG of wild-type but not RARβ null mice. This regulation is direct as when RARβ2 is overexpressed in adult motoneurons Shh is induced in them. Finally we show that Shh alone cannot induce neurite outgrowth but potentiates RARβ2 signalling in this process. [Copyright &y& Elsevier]

The embryonic CNS readily undergoes regeneration, unlike the adult CNS, which has limited axonal repair after injury. Here we tested the hypothesis that retinoic acid receptor β2 (RARβ2), critical in development for neuronal growth, may enable adult neurons to grow in an inhibitory environment. Overexpression of RARβ2 in adult rat dorsal root ganglion cultures increased intracellular levels of cyclic AMP and stimulated neurite outgrowth. Stable RARβ2 expression in DRG neurons in vitro and in vivo enabled their axons to regenerate across the inhibitory dorsal root entry zone and project into the gray matter of the spinal cord. The regenerated neurons enhanced second-order neuronal activity in the spinal cord, and RARβ2-treated rats showed highly significant improvement in sensorimotor tasks. These findings show that RARβ2 induces axonal regeneration programs within injured neurons and may thus offer new therapeutic opportunities for CNS regeneration. [ABSTRACT FROM AUTHOR]

In this study, the plausible role of trimethylamine N-oxide (TMAO), a microbiota metabolite, was investigated as a link between peripheral inflammation and the inflammation of the central nervous system using different cell lines. TMAO treatment favored the differentiation of adipocytes from preadipocytes (3T3-L1 cell line). In macrophages (RAW 264.7 cell line), which infiltrate adipose tissue in obesity, TMAO increased the expression of pro-inflammatory cytokines. The treatment with 200 μM of TMAO seemed to disrupt the blood–brain barrier as it induced a significant decrease in the expression of occludin in hCMECs. TMAO also increased the expression of pro-inflammatory cytokines in primary neuronal cultures, induced a pro-inflammatory state in primary microglial cultures, and promoted phagocytosis. Data obtained from this project suggest that microbial dysbiosis and increased TMAO secretion could be a key link between peripheral and central inflammation. Thus, TMAO-decreasing compounds may be a promising therapeutic strategy for neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

An abstract of the article "Lentiviral Vector Expressing Retinoic Acid Receptor â2 Promotes Regeneration in a Rat Spinal Cord Injury Model," by Liang-Fong Wong and colleagues is presented.

Neuroinflammation has recently been identified as a fundamentally important pathological process in most, if not all, CNS diseases. The main contributor to neuroinflammation is the microglia, which constitute the innate immune response system. Accurate identification of microglia and their reactivity state is therefore essential to further our understanding of CNS pathophysiology. Many staining techniques have been used to visualise microglia in rodent and human tissue, and immunostaining is currently the most frequently used. Historically, identification of microglia was predominantly based on morphological structure, however, recently there has been a reliance on selective antigen expression, and microglia-specific markers have been identified providing increased certainty that the cells observed are in fact microglia, rather than the similar yet distinct macrophages. To date, the most microglia-specific markers are P2Y12 and TMEM119. However, other microglia-related markers can also be useful for demonstrating activation state, phagocytic state, and for neuroimaging purposes in longitudinal studies. Overall, it is important to be aware of the microglia-selectivity issues of the various stains and immunomarkers used by researchers to distinguish microglia in CNS tissue to avoid misinterpretation. [ABSTRACT FROM AUTHOR]

Abstract: We have developed a pyramidotomy model in mice to lesion the corticospinal tract at the level of the brainstem pyramidal tract, and evaluated the resultant impairments in motor function in a series of behavioural tests. Adult C57BL/6 mice received a unilateral pyramidotomy and a control group of mice underwent sham surgery. We studied the effects of this lesion on forepaw function using five behavioural paradigms, some of which have been widely used in rat studies but have not been fully explored in mice. The tests used were: a rearing test, which assesses forepaw use for weight support during spontaneous vertical exploration of a cylinder; a grid walking test, which assesses the ability to accurately place the forepaws during exploration of an elevated grid; a tape-removal test, which measures both sensory and motor function of the forepaw; a CatWalk automated gait analysis, which provides a number of quantitative measures including stride length and stride width during locomotion; and a staircase reaching task, which assesses skilled independent forepaw use. All tests revealed lesion effects on forepaw function with the tape removal, grid walking, rearing and CatWalk tests demonstrating robust effects throughout the testing period. The development of a pyramidotomy lesion model in mice, together with behavioural tests which can reliably measure functional impairments, will provide a valuable tool for assessing therapeutic strategies to promote regeneration and plasticity. [Copyright &y& Elsevier]

Despite increasing knowledge on microRNAs, their role in the pathogenesis of neonatal encephalopathy remains to be elucidated. Herein, we identify let-7b-5p as a significant microRNA in neonates with moderate to severe encephalopathy from dried blood spots using next generation sequencing. Validation studies using Reverse Transcription and quantitative Polymerase Chain Reaction on 45 neonates showed that let-7b-5p expression was increased on day 1 in neonates with moderate to severe encephalopathy with unfavourable outcome when compared to those with mild encephalopathy. Mechanistic studies performed on glucose deprived cell cultures and the cerebral cortex of two animal models of perinatal brain injury, namely hypoxic-ischaemic and intrauterine inflammation models confirm that let-7b-5p is associated with the apoptotic Hippo pathway. Significant reduction in neuronal let-7b-5p expression corresponded with activated Hippo pathway, with increased neuronal/nuclear ratio of Yes Associated Protein (YAP) and increased neuronal cleaved caspase-3 expression in both animal models. Similar results were noted for let-7b-5p and YAP expression in glucose-deprived cell cultures. Reduced nuclear YAP with decreased intracellular let-7b-5p correlated with neuronal apoptosis in conditions of metabolic stress. This finding of the Hippo-YAP association with let-7b needs validation in larger cohorts to further our knowledge on let-7b-5p as a biomarker for neonatal encephalopathy. Using next generation sequencing of dried blood spots and subsequent validation, Ponnusamy et al identify let-7b-5p as an elevated microRNA in neonates with moderate to severe encephalopathy. Using cell culture and murine models of perinatal brain injury they demonstrate that the effects of let-7b-5p are elicited via the Hippo-YAP pathway, which should be validated in large neonate cohorts to expand our understanding of let-7b-5p as a biomarker for neonatal encephalopathy. [ABSTRACT FROM AUTHOR]

The impact of antithrombotic agents on patients with primary intracerebral hemorrhage (ICH) remains controversial, especially patients who require emergent craniotomy. This study was undertaken to evaluate clinical outcomes in operated patients with ICH with and without previous antithrombotic agents. This is a retrospective cohort study. Between January 2001 and December 2013, all patients with ICH who received emergent craniotomy and who were present in Taiwan's National Health Insurance Research Database were screened and divided into those with previous antiplatelet therapy, anticoagulant therapy, and nonantithrombotic therapy according to their health care claims data within 3 months of index admission. The primary end points included in-hospital mortality and complications and short-term outcome. Of 18,872 eligible patients, 16,251 (87.1%) did not receive any antithrombotic therapy, 2267 patients had antiplatelet therapy, and 354 patients had anticoagulation therapy. After propensity score matching, significantly more blood transfusions and craniectomies were identified in the patients with previous antithrombotic treatment compared with those undergoing nonantithrombotic therapy. Compared with the nonantithrombotic treatment cohort, patients under previous anticoagulant treatment had significantly higher in-hospital mortality (odds ratio, 2.12; 95% confidence interval, 1.45–3.10). Furthermore, during the 6-month follow-up period, previous anticoagulant therapy was independently associated with a greater risk of all-cause mortality (P = 0.001). The in-hospital and 6-month all-cause mortality of patients with previous antiplatelet treatment was not significantly different from patients with nonantithrombotic treatment. These findings suggested an increased risk of in-hospital mortality and poor short-term outcome among operated patients with ICH with previous antithrombotic therapy, particularly anticoagulant therapy, but not with antiplatelet therapy. [ABSTRACT FROM AUTHOR]

Objective: The objective of the present study was to determine whether selective inflammatory cytokine concentrations within cerebrospinal fluid are useful markers for the differential diagnosis of aseptic and bacterial meningitis within neurosurgical patients.Design: Prospective, open-label, observational, cohort study.Setting: Neurosurgical ICU, Chang Gung Memorial Hospital.Patients: Thirty-two consecutive neurosurgical patients who had postoperative fever following external ventricular drain insertion for the treatment of brain injury underwent serial cerebrospinal fluid cytokine analysis pre and post fever to determine the value of such markers in ascertaining the differential diagnosis of meningitis.Intervention: Cerebrospinal fluid samples were collected on the day of fever onset, as well as on day 2 and 4 pre and post fever development. Tumor necrosis factor-α, interleukin-1β, interleukin-6, interleukin-8, transforming growth factor-β, and procalcitonin were subsequently analyzed using enzyme-linked immunosorbent assay analysis techniques.Measurement and Main Results: Inflammatory marker levels were compared among febrile aseptic, bacterial, and nonmeningitis patients to determine cerebrospinal fluid inflammatory changes over time. Significant increases in cerebrospinal fluid tumor necrosis factor -α, interleukin-1β, interleukin-6, and interleukin-8 levels were observed within patients with bacterial meningitis at fever onset, which was not evident in aseptic or nonmeningitis patients. Furthermore, significant increases in cerebrospinal fluid tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8 levels were detected as early as 4 days prior to fever onset within patients with bacterial meningitis when compared with both aseptic and nonmeningitis groups. Interestingly, procalcitonin was only significantly increased in patients with bacterial meningitis on the fourth day post fever.Conclusion: The present study suggests that raised cerebrospinal fluid tumor necrosis factor -α, interleukin-1β, and interleukin-8 in a temporal manner may indicate early bacterial meningitis development in neurosurgical patients, enabling earlier diagnostic certainty and improved patient outcomes. [ABSTRACT FROM AUTHOR]

Chronic visceral pain affects millions of individuals worldwide and remains poorly understood, with current therapeutic options constrained by gastrointestinal adverse effects. Visceral pain is strongly associated with inflammation and distension of the gut. Here we report that the voltage-gated sodium channel subtype NaV1.9 is expressed in half of gut-projecting rodent dorsal root ganglia sensory neurons. We show that NaV1.9 is required for normal mechanosensation, for direct excitation and for sensitization of mouse colonic afferents by mediators from inflammatory bowel disease tissues, and by noxious inflammatory mediators individually. Excitatory responses to ATP or PGE2 were substantially reduced in NaV1.9(-/-) mice. Deletion of NaV1.9 substantially attenuates excitation and subsequent mechanical hypersensitivity after application of inflammatory soup (IS) (bradykinin, ATP, histamine, PGE2, and 5HT) to visceral nociceptors located in the serosa and mesentery. Responses to mechanical stimulation of mesenteric afferents were also reduced by loss of NaV1.9, and there was a rightward shift in stimulus-response function to ramp colonic distension. By contrast, responses to rapid, high-intensity phasic distension of the colon are initially unaffected; however, run-down of responses to repeat phasic distension were exacerbated in NaV1.9(-/-) afferents. Finally colonic afferent activation by supernatants derived from inflamed human tissue was greatly reduced in NaV1.9(-/-) mice. These results demonstrate that NaV1.9 is required for persistence of responses to intense mechanical stimulation, contributes to inflammatory mechanical hypersensitivity, and is essential for activation by noxious inflammatory mediators, including those from diseased human bowel. These observations indicate that NaV1.9 represents a high-value target for development of visceral analgesics. [ABSTRACT FROM AUTHOR]

Chronic visceral pain affects millions of individuals worldwide and remains poorly understood, with current therapeutic options constrained by gastrointestinal adverse effects. Visceral pain is strongly associated with inflammation and distension of the gut. Here we report that the voltage-gated sodium channel subtype Na V 1.9 is expressed in half of gut-projecting rodent dorsal root ganglia sensory neurons. We show that Na V 1.9 is required for normal mechanosensation, for direct excitation and for sensitization of mouse colonic afferents by mediators from inflammatory bowel disease tissues, and by noxious inflammatory mediators individually. Excitatory responses to ATP or PGE 2 were substantially reduced in Na V 1.9 −/− mice. Deletion of Na V 1.9 substantially attenuates excitation and subsequent mechanical hypersensitivity after application of inflammatory soup (IS) (bradykinin, ATP, histamine, PGE 2 , and 5HT) to visceral nociceptors located in the serosa and mesentery. Responses to mechanical stimulation of mesenteric afferents were also reduced by loss of Na V 1.9, and there was a rightward shift in stimulus–response function to ramp colonic distension. By contrast, responses to rapid, high-intensity phasic distension of the colon are initially unaffected; however, run-down of responses to repeat phasic distension were exacerbated in Na V 1.9 −/− afferents. Finally colonic afferent activation by supernatants derived from inflamed human tissue was greatly reduced in Na V 1.9 −/− mice. These results demonstrate that Na V 1.9 is required for persistence of responses to intense mechanical stimulation, contributes to inflammatory mechanical hypersensitivity, and is essential for activation by noxious inflammatory mediators, including those from diseased human bowel. These observations indicate that Na V 1.9 represents a high-value target for development of visceral analgesics. [ABSTRACT FROM AUTHOR]

To restore lost functions while repairing the neuronal structure after spinal cord injury (SCI), pharmacological interventions with multiple therapeutic agents will be a more effective modality given the complex pathophysiology of acute SCI. Toward this end, we prepared electrospun membranes containing aligned core–shell fibers with a polylactic acid (PLA) shell, and docosahexaenoic acid (DHA) or a brain-derived neurotropic factor (BDNF) in the core. The controlled release of both pro-regenerative agents is expected to provide combinatory treatment efficacy for effective neurogenesis, while aligned fiber topography is expected to guide directional neurite extension. The in vitro release study indicates that both DHA and BDNF could be released continuously from the electrospun membrane for up to 50 days, while aligned microfibers guide the neurite extension of primary cortical neurons along the fiber axis. Furthermore, the PLA/DHA/BDNF core–shell fibrous membrane (CSFM) provides a significantly higher neurite outgrowth length from the neuron cells than the PLA/DHA CSFM. This is supported by the upregulation of genes associated with neuroprotection and neuroplasticity from RT-PCR analysis. From an in vivo study by implanting a drug-loaded CSFM into the injury site of a rat suffering from SCI with a cervical hemisection, the co-delivery of DHA and BDNF from a PLA/DHA/BDNF CSFM could significantly improve neurological function recovery from behavioral assessment, as well as provide neuroprotection and promote neuroplasticity changes in recovered neuronal tissue from histological analysis. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) is associated with disability and a drastic decrease in quality of life for affected individuals. Previous studies support the idea that docosahexaenoic acid (DHA)-based pharmacological approach is a promising therapeutic strategy for the management of acute SCI. We postulated that a nanostructured material for controlled delivery of DHA at the lesion site may be well suited for this purpose. Toward this end, we prepare drug-loaded fibrous mats made of core-shell nanofibers by electrospinning, which contained a polylactic acid (PLA) shell for encapsulation of DHA within the core, for delivery of DHA in situ. In vitro study confirmed sustained DHA release from PLA/DHA core-shell nanofiber membrane (CSNM) for up to 36 days, which could significantly increase neurite outgrowth from primary cortical neurons in 3 days. This is supported by the upregulation of brain-derived neurotropic factor (BDNF) and neurotrophin-3 (NT-3) neural marker genes from qRT-PCR analysis. Most importantly, the sustained release of DHA could significantly increase the neurite outgrowth length from cortical neuron cells in 7 days when co-cultured with PLA/DHA CSNM, compared with cells cultured with 3 μM DHA. From in vivo study with a SCI model created in rats, implantation of PLA/DHA CSNM could significantly improve neurological functions revealed by behavior assessment in comparison with the control (no treatment) and the PLA CSNM groups. According to histological analysis, PLA/DHA CSNM also effectively reduced neuron loss and increased serotonergic nerve sprouting. Taken together, the PLA/DHA CSNM may provide a nanostructured drug delivery system for DHA and contribute to neuroprotection and promoting neuroplasticity change following SCI. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) could result in edema and cause an increase in intracranial pressure of the brain resulting in mortality and morbidity. Although there is hyperosmolarity therapy available for this pathophysiological event, it remains controversial. Recently, several groups have shown docosahexaenoic acid (DHA) to improve functional and histological outcomes following brain injury based on reduction of neuroinflammation and apoptosis. However, the effect of DHA on blood–brain barrier (BBB) dysfunction after brain injury has not been fully studied. Here, a controlled cortical impact rat model was used to test the effect of a single dose of DHA administered 30 min post injury. Modified neurological severity score (mNSS) and forelimb asymmetry were used to determine the functional outcomes. Neuroimaging and histology were used to characterize the edema and BBB dysfunction. The study showed that DHA-treated TBI rats had better mNSS and forelimb asymmetry score than vehicle-treated TBI rats. Temporal analysis of edema using MRI revealed a significant reduction in edema level with DHA treatment compared to vehicle in TBI rats. Histological analysis using immunoglobulin G (IgG) extravasation showed that there was less extravasation, which corresponded with a reduction in aquaporin 4 and astrocytic metalloprotease 9 expression, and greater endothelial occludin expression in the peri-contusional site of the TBI rat brain treated with DHA in comparison to vehicle treatment. In conclusion, the study shows that DHA can exert its functional improvement by prevention of the edema formation via prevention of BBB dysfunction after TBI. [ABSTRACT FROM AUTHOR]

This report was produced by an Expert Working Group (EWG) consisting of UK-based researchers, veterinarians and regulators of animal experiments with specialist knowledge of the use of animal models of spinal cord injury (SCI). It aims to facilitate the implementation of the Three Rs (Replacement, Reduction and Refinement), with an emphasis on refinement. Specific animal welfare issues were identified and discussed, and practical measures proposed, with the aim of reducing animal use and suffering, reducing experimental variability, and increasing translatability within this critically important research field. • Pre-clinical research into SCI is currently dominated by the use of animal models. • These models can involve significant injury and have the potential to cause high levels of suffering. • A UK-based expert working group has identified animal welfare issues associated with modelling SCI in animals. • This article sets out practical refinements to improve animal welfare. [ABSTRACT FROM AUTHOR]

Songbirds communicate through learned vocalizations, using a forebrain circuit with convergent similarity to vocal-control circuitry in humans. This circuit is incomplete in female zebra finches, hence only males sing. We show that the UTS2B gene, encoding Urotensin-Related Peptide (URP), is uniquely expressed in a key pre-motor vocal nucleus (HVC), and specifically marks the neurons that form a male-specific projection that encodes timing features of learned song. UTS2B-expressing cells appear early in males, prior to projection formation, but are not observed in the female nucleus. We find no expression evidence for canonical receptors within the vocal circuit, suggesting either signalling to other brain regions via diffusion or transduction through other receptor systems. Urotensins have not previously been implicated in vocal control, but we find an annotation in Allen Human Brain Atlas of increased UTS2B expression within portions of human inferior frontal cortex implicated in human speech and singing. Thus UTS2B (URP) is a novel neural marker that may have conserved functions for vocal communication. [ABSTRACT FROM AUTHOR]

Abstract Damage to peripheral nerves is associated with changes in excitability and/or phenotype of primary afferent neurons as well as increased neuronal excitability (central sensitization) and reduced inhibitory tone in the dorsal horn. For instance, in dorsal root ganglia (DRG) brain derived neurotrophic factor (BDNF) is down-regulated in small cells whilst de novo expressed in large diameter cells. In the dorsal horn, GABA content is decreased. In this study, in a dorsal horn, ‘with dorsal roots attached’ preparation obtained from spinal nerve lesioned Wistar rats, stimulation of ipsilateral dorsal roots at either A fibre or A + C fibre strength did not evoke release of BDNF. In separate experiments, activity-induced release of GABA in the isolated dorsal horn of neuropathic rats was significantly reduced compared to release in sham operated rats. GABA release could be significantly restored following topical application of BDNF through the dorsal horn preparation. Finally, neuropathic rats developed thermal and mechanical hypersensitivity and thermal hyperalgesia was reduced by intrathecal injection of BDNF. We concluded that BDNF-induced release of GABA could be a mechanism to explain the antinociceptive action of intrathecal BDNF in neuropathic animals. Furthermore, reduced availability of sensory neuron-derived BDNF might contribute to the reduced GABAergic tone in the dorsal horn of neuropathic rats. [ABSTRACT FROM AUTHOR]