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Brain-enriched microRNA-338 (miR-338) is known to play a central role in brain mitochondrial function, however the role of miR-338 in stroke injury remains unknown. This study investigated the role of miR-338 in injury from transient focal cerebral ischemia in mice, and in cell survival and mitochondrial function after in vitro ischemia in astrocyte and neuronal cultures. Pre-treatment of mice with intracerebroventricular injection of miR-338 antagomir 24 h prior to 1 h of middle cerebral artery occlusion (MCAO) significantly reduced infarct size and improved neurological score at both 24 h and 7d after injury. Levels of the miR-338 target cytochrome-c oxidase subunit 4I1 (COX4I1), which plays an essential role in maintaining brain mitochondrial ATP production, were increased in miR-338 antagomir-treated mice. Mouse primary astrocyte cell cultures subjected to glucose deprivation exhibited increased cell survival when pre-treated with miR-338 inhibitor, and greater cell death with miR-338 mimic. Decreased miR-338 levels were associated with increased ATP production, augmented cytochrome c oxidative (CcO) activity and preservation of COX4I1. In vitro protection with miR-338 inhibitor was blocked by concurrent knockdown of COX4I1 with small interfering RNA. Parallel studies in mouse neuronal N2a cultures resulted in preserved ATP content and CcO activity with miR-338 inhibition, indicating a shared miR-338-dependent response to ischemic stress between brain cell types. These results suggest that miR-338 inhibition and/or COX4I1-targeted therapies may be novel clinical strategies to protect against stroke injury via preservation of mitochondrial function in multiple cell types.

Stroke induces a robust inflammatory response. However, it still lacks a systematic view of the various immune cell types due to the limited numbers of fluorophore used in the traditional FACS technique. In our current study, we utilized the novel technique mass cytometry (CyTOF) to analyze multiple immune cell types. We detected these immune cells from the ischemic brain, peripheral blood, spleen, and bone marrow at different time courses after stroke. Our data showed (1) dynamic changes in the immune cell numbers in the ischemic brain and peripheral organs. (2) The expression levels of cell surface markers indicate the inflammation response status after stroke. Interestingly, CD62L, a key adhesion molecule, regulates the migration of leukocytes from blood vessels into secondary lymphoid tissues and peripheral tissues. (3) A strong leukocyte network across the brain and peripheral immune organs was identified using the R program at day 1 after ischemia, suggesting that the peripheral immune cells dramatically migrated into the ischemic areas after stroke. This study provides a systematic, wide view of the immune components in the brain and peripheral organs for a deep understanding of the immune response after ischemic stroke.

The gut-brain-microbiota axis (GBMAx) coordinates bidirectional communication between the gut and brain, and is increasingly recognized as playing a central role in physiology and disease. MicroRNAs are important intracellular components secreted by extracellular vesicles (EVs), which act as vital mediators of intercellular and interspecies communication. This review will present current advances in EV-derived microRNAs and their potential functional link with GBMAx. We propose that EV-derived microRNAs comprise a novel regulatory system for GBMAx, and a potential novel therapeutic target for modifying GBMAx in clinical therapy.

Serum prealbumin is a recognized marker of malnutrition, but its prognostic role in patients with hemorrhagic stroke remains unclear. In this study, we retrospectively reviewed the records of 105 patients with hemorrhagic stroke admitted to Renmin Hospital of Wuhan University, China, from January to December 2015. We collected demographic and radiological data, and recorded serum prealbumin levels at admission and on days 1, 3, 6, 9, and 14-21. The existence of infections and gastrointestinal hemorrhage, and clinical condition at discharge were also recorded. Serum prealbumin levels during hospitalization were significantly lower in patients with infections compared with those without infections, and also significantly lower in patients with gastrointestinal hemorrhage compared with those without. Serum prealbumin levels at discharge were significantly higher in patients with good recovery than in those with poor recovery. We conclude that regular serum prealbumin measurements in patients with hemorrhagic stroke may be a useful indicator for determining clinical status and prognosis, which may therefore help to guide clinical decision-making.

Background: Stroke is a sexually dimorphic disease with age-dependent sex differences in incidence, prevalence, and outcome. Delineating the molecular mechanisms that determine sexual dimorphism in stroke is necessary to overcome translational barriers and advance the development of novel stroke treatments. Non-coding RNAs, including microRNAs ( miRs ), are upstream regulators of genes that regulate cell survival. In young adult male animals, miR-181a and miR-200c have been established as central regulators in the cellular response to stroke, however their role in females or aged animal cohorts represents a critical knowledge gap. Therefore, in the present study we investigated the response of miR-181a and miR-200c in the brain after stroke in aged male and female mice. Methods: Aged (20 month old) male and female mice were subjected to 1h middle cerebral artery occlusion (MCAO) followed by 24h reperfusion. RT-qPCR, fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) were used to quantify and localize post-MCAO cellular expression of miR-181a and miR-200c. Results: There was no baseline difference in miR-181a between sexes, however miR-200c expression was ~3-fold higher at baseline in female animals versus males. MCAO induced ~2-fold increase in miR-181a in male animals but post-MCAO expression remained unchanged in females. In contrast, miR-200c expression increased by ~16-fold in males and ~4-fold in females. At baseline, miR-181a and miR-200c primarily localized to cortical neurons (NeuN+). In response to MCAO astrocyte reactivity (GFAP+) coincided with robust expression of miR-200c in both males and females. Conclusions: MiR-181a and miR-200c exhibit sexual dimorphism in response to stroke. Augmented expression of miR-200c appears secondary to astrocyte reactivity. Future experiments will determine if inhibition of miR-181a and miR-200c are protective against ischemic stroke in aged animals.

Transient forebrain ischemia, as occurs with cardiac arrest and resuscitation, results in impaired cognitive function secondary to delayed neuronal cell death in hippocampal cornu ammonis-1 (CA1). Comparatively, hippocampal neurons in the adjacent dentate gyrus (DG) survive, suggesting that elucidating the molecular mechanisms underpinning hippocampal sub-regional differences in ischemic tolerance could be central in the development of novel interventions to improve outcome in survivors of forebrain ischemia. MicroRNAs (miRNAs) are non-coding RNAs that modulate the translation of target genes and have been established as an effective therapeutic target for other models of injury. The objective of the present study was to assess and compare post-injury miRNA profiles between CA1 and DG using a rat model of forebrain ischemia. CA1 and DG sub-regions were dissected from rat hippocampi following 10 min of forebrain ischemia at three time points (3 h, 24 h, and 72 h) and at baseline. Pronounced differences between CA1 and DG were observed for several select miRNAs, including miR-181a-5p, a known regulator of cerebral ischemic injury. We complexed fluorescent in situ hybridization with immunohistochemistry to observe cell-type specific and temporal differences in mir-181a-5p expression between CA1 and DG in response to injury. Using established miRNA-mRNA prediction algorithms, we extended our observations in CA1 miRNA dysregulation to identify key functional pathways as potential modulators of CA1 ischemic vulnerability. In summary, our observations support a central role for miRNAs in selective CA1 ischemic vulnerability and suggest that cell-specific miRNA targeting could be a viable clinical approach to preserve CA1 neurons and improve cognitive outcomes for survivors of transient forebrain ischemia.

Mild traumatic brain injury (mTBI) can result in permanent impairment in memory and learning and may be a precursor to other neurological sequelae. Clinical treatments to ameliorate the effects of mTBI are lacking. Inhibition of microRNA-181a (miR-181a) is protective in several models of cerebral injury, but its role in mTBI has not been investigated. In the present study, miR-181a-5p antagomir was injected intracerebroventricularly 24 h prior to closed-skull cortical impact in young adult male mice. Paw withdrawal, open field, zero maze, Y maze, object location and novel object recognition tests were performed to assess neurocognitive dysfunction. Brains were assessed immunohistologically for the neuronal marker NeuN, the perineuronal net marker wisteria floribunda lectin (WFA), cFos, and the interneuron marker parvalbumin. Protein quantification was performed with immunoblots for synaptophysin and postsynaptic density 95 (PSD95). Fluorescent in situ hybridization was utilized to localize hippocampal miR-181a expression. MiR-181a antagomir treatment reduced neuronal miR-181a expression after mTBI, restored deficits in novel object recognition and increased hippocampal parvalbumin expression in the dentate gyrus. These changes were associated with decreased dentate gyrus hyperactivity indicated by a relative reduction in PSD95 and cFos expression. These results suggest that miR-181a inhibition may be a therapeutic approach to reduce hippocampal excitotoxicity and prevent cognitive dysfunction following mTBI.

Introduction Perineuronal nets (PNNs) are extracellular matrices that encompass parvalbumin‐expressing parvalbumin positive (PVALB+) fast‐spiking inhibitory interneurons where they protect and stabilize afferent synapses. Recent observations that gonadal hormones influence PVALB+ neuron development suggest that PNN regulation may be sexually dimorphic. Sex differences in PNN abundance and complexity have been reported in sexually dimorphic nuclei in zebra finch brains; however, corresponding differences in mammalian brains have not been investigated. Methods In this study we assessed the number of cortical and hippocampal PNNs in juvenile and young adult male and female rats using fluorescent immunohistochemistry for PVALB and the PNN marker Wisteria Floribunda Lectin. Results We report here that PNNs are numerous and well developed in hippocampal cornu ammonis‐1 of adult males but are lower in juvenile and possibly adult females. No significant differences were observed between sexes in cornu ammonis‐3 or adjacent neocortex. There was an observed developmental difference in the neocortex as juveniles had more PVALB+ cells, but fewer PNN+ cells, than adults. Conclusions Because PNNs are integral for several hippocampal‐mediated learning and memory tasks, these observations have potential sex‐dependent translational implications for clinical strategies targeting cognitive dysfunction.

Astrocytes and microglia play crucial roles in the response to cerebral ischemia and are effective targets for stroke therapy in animal models. MicroRNAs (miRs) are important posttranscriptional regulators of gene expression that function by inhibiting the translation of select target genes. In astrocytes, miR expression patterns regulate mitochondrial function in response to oxidative stressviatargeting of Bcl2 and heat shock protein 70 family members. Mitochondria play an active role in microglial activation, and miRs regulate the microglial neuroinflammatory response. As endogenous miR expression patterns can be altered with exogenous mimics and inhibitors, miR-targeted therapies represent a viable intervention to optimize glial mitochondrial function and improve clinical outcome following cerebral ischemia. In the present article, we review the role that astrocytes and microglia play in neuronal function and fate following ischemic stress, discuss the relevance of mitochondria in the glial response to injury, and present current evidence implicating miRs as critical regulators in the glial mitochondrial response to cerebral ischemia.

Cerebral ischemia remains a major cause of death and disability worldwide, yet therapeutic options remain limited. Differences in sex and age play an important role in the final outcome in response to cerebral ischemia in both experimental and clinical studies: males have a higher risk and worse outcome than females at younger ages and this trend reverses in older ages. Although the molecular mechanisms underlying sex dimorphism are complex and are still not well understood, studies suggest steroid hormones, sex chromosomes, differential cell death and immune pathways, and sex-specific microRNAs may contribute to the outcome following cerebral ischemia. This review focuses on differential effects between males and females on cell death and immunological pathways in response to cerebral ischemia, the central role of innate sex differences in steroid hormone signaling, and upstreamregulation of sexually dimorphic gene expression by microRNAs.

Background and Purpose— MicroRNA (miR)-200c increases rapidly in the brain after transient cerebral ischemia but its role in poststroke brain injury is unclear. Reelin, a regulator of neuronal migration and synaptogenesis, is a predicted target of miR-200c. We hypothesized that miR-200c contributes to injury from transient cerebral ischemia by targeting reelin. Methods— Brain infarct volume, neurological score and levels of miR-200c, reelin mRNA, and reelin protein were assessed in mice subjected to 1 hour of middle cerebral artery occlusion with or without intracerebroventricular infusion of miR-200c antagomir, mimic, or mismatch control. Direct targeting of reelin by miR-200c was assessed in vitro by dual luciferase assay and immunoblot. Results— Pretreatment with miR-200c antagomir decreased post–middle cerebral artery occlusion brain levels of miR-200c, resulting in a significant reduction in infarct volume and neurological deficit. Changes in brain levels of miR-200c inversely correlated with reelin protein expression. Direct targeting of the Reln 3′ untranslated region by miR-200c was verified with dual luciferase assay. Inhibition of miR-200c resulted in an increase in cell survival subsequent to in vitro oxidative injury. This effect was blocked by knockdown of reelin mRNA, whereas application of reelin protein afforded protection. Conclusions— These findings suggest that the poststroke increase in miR-200c contributes to brain cell death by inhibiting reelin expression, and that reducing poststroke miR-200c is a potential target to mitigate stroke-induced brain injury.

Significance: Cerebral ischemia is a major cause of death and disability throughout the world, yet therapeutic options remain limited. The interplay between the cellular redox state and the immune response plays a critical role in determining the extent of neural cell injury after ischemia and reperfusion. Excessive amounts of reactive oxygen species (ROS) generated by mitochondria and other sources act both as triggers and effectors of inflammation. This review will focus on the interplay between these two mechanisms. Recent Advances: MicroRNAs (miRNAs) are important post-transcriptional regulators that interact with multiple target messenger RNAs coordinately regulating target genes, including those involved in controlling mitochondrial function, redox state, and inflammatory pathways. This review will focus on the regulation of mitochondria, ROS, and inflammation by miRNAs in the chain of deleterious intra- and intercellular events that lead to brain cell death after cerebral ischemia. Critical Issues: Although pretreatment using miRNAs was effective in cerebral ischemia in rodents, testing treatment after the onset of ischemia is an essential next step in the development of acute stroke treatment. In addition, miRNA formulation and delivery into the CNS remain a challenge in the clinical translation of miRNA therapy. Future Directions: Future research should focus on post-treatment and potential clinical use of miRNAs. Antioxid. Redox Signal. 22, 187–202.

Circuit remodeling driven by pathological forms of synaptic plasticity underlies several psychiatric diseases including addiction. Deep brain stimulation (DBS) has been applied to treat a number of neurological and psychiatric conditions although its effects are transient and mediated by largely unknown mechanisms. Recently optogenetic protocols that restore normal transmission at identified synapses in mice have provided proof of the idea that cocaine adaptive behavior can be reversed in vivo. The most efficient protocol relies on the activation of metabotropic glutamate receptors mGluRs which depotentiates excitatory synaptic inputs onto dopamine D1 receptor medium sized spiny neurons and normalizes drug adaptive behavior. We discovered that acute low frequency DBS refined by selective blockade of dopamine D1 receptors mimics optogenetic mGluR dependent normalization of synaptic transmission. Consequently there was a long lasting abolishment of behavioral sensitization.

Ischemic stroke remains a leading cause of death and disability with few treatment options. MicroRNAs (miRs) are short, non-coding RNAs that regulate gene expression. They have important potential applications as biomarkers for stroke severity and outcome, as well as presenting unique possibilities for interventions to minimize injury and improve recovery and outcome following stroke. MiRs function by binding messenger RNAs (mRNA) and silencing translation of target genes. Endogenous miR expression levels change in response to stress, and they can be altered by application of exogenous nucleotides—miR mimics—to increase or inhibitors to decrease levels of specific miRs. By virtue of their relatively short binding sequences, a single miR can simultaneously modulate numerous related gene targets. As miR expression can be cell-type specific, miRs can also be used to target specific brain cell types, such as microglia and astrocytes, which helps determine neuronal cell fate following stress. MiR-based therapeutics may therefore provide a novel approach to the development of effective therapeutics for ischemic stroke.

We report the first description of oculocardiac reflex elicited with injection of local anesthetic in an empty orbit, and highlight clinical indicators for patients that may be at risk for an exaggerated oculocardiac reflex. We describe a patient with prior head and eye trauma treated for anophthalmic socket reconstruction at an outpatient eye surgery center. Injection of local anesthetic into the empty orbit induced an extended sinus arrest. This exaggerated response was avoided in a subsequent surgery by pretreatment with high-dose anticholinergics.

Whether the effect of miR-181a is sexually dimorphic in stroke is unknown. Prior work showed protection of male mice with miR-181a inhibition. Estrogen receptor-α (ERα) is an identified target of miR181 in endometrium. Therefore we investigated the separate and joint effects of miR-181a inhibition and 17β-estradiol (E2) replacement after ovariectomy. Adult female mice were ovariectomized and implanted with an E2- or vehicle-containing capsule for 14d prior to 1h middle cerebral artery occlusion (MCAO). Each group received either miR-181a antagomir or mismatch control by intracerebroventricular injection 24h before MCAO. After MCAO neurologic deficit and infarct volume were assessed. Primary male and female astrocyte cultures were subjected to glucose deprivation with miR-181a inhibitor or transfection control, and E2 or vehicle control, with/without ESRα knockdown with small interfering RNA. Cell death was assessed by propidium iodide staining, and lactate dehydrogenase assay. A miR-181a/ERα target site blocker (TSB), with/without miR-181a mimic, was used to confirm targeting of ERα by miR-181a in astrocytes. Individually, miR-181a inhibition or E2 decreased infarct volume and improved neurologic score in female mice, and protected male and female astrocyte cultures. Combined miR-181a inhibition plus E2 afforded greater protection of female mice and female astrocyte cultures, but not in male astrocyte cultures. MiR-181a inhibition only increased ERα levels in vivo and in female cultures, while ERα knockdown with siRNA increased cell death in both sexes. Treatment with ERα TSB was strongly protective in both sexes. In conclusion, the results of the present study suggest miR-181a inhibition enhances E2-mediated stroke protection in females in part by augmenting ERα production, a mechanism detected in female mice and female astrocytes. Sex differences were observed with combined miR-181a inhibition/E2 treatment, and miR-181a targeting of ERα.

The cellular and molecular mechanisms regulating postinjury neurogenesis in the adult hippocampus remain undefined. We have previously demonstrated that preinjury treatment with anti-microRNA (miR)-181a preserved neurons and prevented astrocyte dysfunction in the hippocampal cornu ammonis-1 (CA1) following transient forebrain ischemia. In the present study, we assessed postinjury treatment with anti-miR-181a on recovery of CA1 neurons following transient forebrain ischemia in rats. Stereotactic CA1 injection of miR-181a antagomir at either 2 h or 7 d postinjury resulted in improved restoration of CA1 measured at 28 d postinjury. Treatment with antagomir was associated with overexpression of the mir-181a target cell adhesion-associated, oncogene-related protein and enhanced expression of the neuroprogenitor cell marker doublecortin (DCX) in the CA1. Assessment of GFAP+cell fate by Cre/Lox-mediated deletion demonstrated that some GFAP+cells in CA1 exhibitedde novoDCX expression in response to injury.In vitroexperiments using primary neuronal stem cells confirmed that miR-181a inhibition augmented the expression of DCX and directed cellular differentiation toward a neuronal fate. These results suggest that miR-181a inhibition plays a central role in the restoration of CA1 neurons via augmentation of early latent neurogenic gene activation in neural progenitor cells, including some reactive astrocytes. Therapeutic interventions targeting this restorative process may represent a novel postinjury approach to improve clinical outcomes in survivors of forebrain ischemia.

One reason for the translational failure of antiinflammatory approaches for stroke therapy is that strategies targeting neuronal survival alone may be insufficient to improve outcome. There has been an increased interest in the roles of other cell types in the inflammatory response in the brain following thrombotic stroke. Within minutes of ischemia, local neuroinflammatory events occur mediated by the activation of microglia and astrocytes, specialized glial cells that represent the largest proportion of cells in the mammalian brain. This chapter will discuss the critical role that microglia and astrocytes play in the induction of poststroke neuroinflammation, and their potential as targets in the next generation of therapeutic strategies for thrombotic stroke.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults. Patients with GBM have poor outcomes, even with the current gold-standard first-line treatment: maximal safe resection combined with radiotherapy and temozolomide chemotherapy. Accumulating evidence suggests that advances in antigen-specific cancer vaccines and immune checkpoint blockade in other advanced tumors may provide an appealing promise for immunotherapy in glioma. The future of therapy for GBM will likely incorporate a combinatorial, personalized approach, including current conventional treatments, active immunotherapeutics, plus agents targeting immunosuppressive checkpoints.

Stroke is one of the leading causes of death and disability worldwide. Because stroke is a multifactorial disease with a short therapeutic window many clinical stroke trials have failed and the only currently approved therapy is thrombolysis. MicroRNAs (miRNA) are endogenously expressed noncoding short single-stranded RNAs that play a role in the regulation of gene expression at the post-transcriptional level, via degradation or translational inhibition of their target mRNAs. The study of miRNAs is rapidly growing and recent studies have revealed a significant role of miRNAs in ischemic disease. miRNAs are especially important candidates for stroke therapeutics because of their ability to simultaneously regulate many target genes and since to date targeting single genes for therapeutic intervention has not yet succeeded in the clinic. Although there are already quite a few review articles about miRNA in ischemic heart disease, much less is currently known about miRNAs in cerebral ischemia. This review summarizes current knowledge about miRNAs and cerebral ischemia, focusing on the role of miRNAs in ischemia, both changes in expression and identification of potential targets, as well as the potential of miRNAs as biomarkers and therapeutic targets in cerebral ischemia.

The interleukin-1 family of cytokines are potent inducers of inflammation and pain. Proteolytic activation of this family of cytokines is under the control of several innate immune receptors that coordinate to form large multiprotein signalling platforms, termed inflammasomes. Recent evidence suggests that a wide range of inflammatory diseases, cancers, and metabolic and autoimmune disorders, in which pain is a common complaint, may be coordinated by inflammasomes. Activation of inflammasomes results in cleavage of caspase-1, which subsequently induces downstream initiation of several potent pro-inflammatory cascades. Therefore, it has been proposed that targeting inflammasome activity may be a novel and effective therapeutic strategy for these pain-related diseases. The purpose of this narrative review article is to provide the reader with an overview of the activation and regulation of inflammasomes and to investigate the potential therapeutic role of inflammasome inhibition in the treatment of diseases characterized by pain, including the following: complex regional pain syndrome, gout, rheumatoid arthritis, inflammatory pain, neuropathic pain, chronic prostatitis, chronic pelvic pain syndrome, and fibromyalgia. We conclude that the role of the inflammasome in pain-associated diseases is likely to be inflammasome subtype and disease specific. The currently available evidence suggests that disease-specific targeting of the assembly and activity of the inflammasome complex may be a novel therapeutic opportunity for the treatment of refractory pain in many settings.