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Implantable neuroelectronic interfaces have enabled advances in both fundamental research and treatment of neurological diseases but traditional intracranial depth electrodes require invasive surgery to place and can disrupt neural networks during implantation. We developed an ultrasmall and flexible endovascular neural probe that can be implanted into sub-100-micrometer-scale blood vessels in the brains of rodents without damaging the brain or vasculature. In vivo electrophysiology recording of local field potentials and single-unit spikes have been selectively achieved in the cortex and olfactory bulb. Histology analysis of the tissue interface showed minimal immune response and long-term stability. This platform technology can be readily extended as both research tools and medical devices for the detection and intervention of neurological diseases.

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We previously demonstrated that inhibition of miR-200c was protective against stroke in young adult male mice by augmenting sirtuin-1 (Sirt1). In the present study we assessed the role of miR-200c on injury, Sirt1, and bioenergetic and neuroinflammatory markers in aged male and female mice after experimental stroke. Mice were subjected to 1hr of transient middle cerebral artery occlusion (MCAO) and assessed for post-injury expression of miR-200c, Sirt1 protein and mRNA, N6-methyladenosine (m6A) methylated Sirt1 mRNA, ATP, cytochrome C oxidase activity, tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), infarct volume and motor function. MCAO induced a decrease in Sirt1 expression at 1d post-injury only in males. No differences in SIRT1 mRNA were observed between the sexes. Females had greater baseline miR-200c expression and a greater increase in miR-200c in response to stroke, while pre-MCAO levels of m6A SIRT1 was greater in females. Males had lower post-MCAO ATP levels and cytochrome C oxidase activity, and higher TNFα and IL-6. Post-injury intravenous treatment with anti-miR-200c reduced miR-200c expression in both sexes. In males, anti-miR-200c increased Sirt1 protein expression, reduced infarct volume, and improved neurological score. Conversely in females anti-miR-200c had no effect on Sirt1 levels and provided no protection against injury from MCAO. These results provide the first evidence of sexual dimorphism in the role of a microRNA in aged mice after experimental stroke and suggest sex-differences in epigenetic modulation of the transcriptome and downstream effects on miR biological activity may play a role in sexually dimorphic outcomes after stroke in aged brains.

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Implantable neuroelectronic interfaces have enabled significant advances in both fundamental research and treatment of neurological diseases, yet traditional intracranial depth electrodes require invasive surgery to place and can disrupt the neural networks during implantation. To address these limitations, we have developed an ultra-small and flexible endovascular neural probe that can be implanted into small 100-micron scale blood vessels in the brains of rodents without damaging the brain or vasculature. The structure and mechanical properties of the flexible probes were designed to meet the key constraints for implantation into tortuous blood vessels inaccessible with existing techniques. In vivo electrophysiology recording of local field potentials and single-unit spikes has been selectively achieved in the cortex and the olfactory bulb. Histology analysis of the tissue interface showed minimal immune response and long-term stability. This platform technology can be readily extended as both research tools and medical devices for the detection and intervention of neurological diseases.

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Perioperative organ injury is a frequent and major complication for the ∼240 million people undergoing surgery worldwide annually. Ischaemic preconditioning is a powerful technique that reduces organ injury in experimental models of heart, lung, gut, brain, and kidney ischaemia-reperfusion injury. However, ischaemic preconditioning has been a challenge to translate into clinical practice. We describe how utilising isolated pre-conditioned exosomes (secreted vesicles containing many cell-survival mediators), some of the translational hurdles of ischaemic preconditioning can be overcome. Delivery of exosomes in the perioperative period could become a promising new therapeutic strategy to prevent perioperative organ injury.

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Pain signals are relayed to the brain via a nociceptive system, and in rare cases, this nociceptive system contains genetic variants that can limit the pain response. Here, we questioned whether a human transient receptor potential vanilloid 1 (TRPV1) missense variant causes a resistance to noxious stimuli and, further, whether we could target this region with a cell-permeable peptide as a pain therapeutic. Initially using a computational approach, we identified a human K710N TRPV1 missense variant in an otherwise highly conserved region of mammalian TRPV1. After generating a TRPV1K710N-knockin mouse using CRISPR/Cas9, we discovered that the K710N variant reduced capsaicin-induced calcium influx in dorsal root ganglion neurons. The TRPV1K710N rodents also had less acute behavioral responses to noxious chemical stimuli and less hypersensitivity to nerve injury, while their response to noxious heat remained intact. Furthermore, blocking this K710 region in WT rodents using a cell-penetrating peptide limited acute behavioral responses to noxious stimuli and returned pain hypersensitivity induced by nerve injury to baseline levels. These findings identify K710 TRPV1 as a discrete site that is crucial for the control of nociception and provide insights into how to leverage rare genetic variants in humans to uncover fresh strategies for developing pain therapeutics.

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Epiretinal membranes (ERMs) are the result of fibro-cellular proliferation that cause distortion and impairment of central vision. We hypothesized that select microRNAs (miRs) regulate retinal fibro-proliferation and ERM formation. Following IRB approval, a pilot study was performed in patients presenting for retina surgery with and without clinical ERMs. Total RNA was isolated from ERM tissue and controls from non-ERM vitreous and subjected to miR profiling via microarray analysis. MiR-494 was identified as the only miR selectively expressed at significantly greater levels, and in silico analysis identified p27 as a putative fibroproliferative gene target of miR-494. In vitro testing of miR-494 and p27 in fibrotic transformation was assessed in spontaneously immortalized human retinal pigment epithelial (RPE) and human Müller cell lines, stimulated to transform into a fibroproliferative state via transforming growth factor beta (TGFß). Fibroproliferative transformation was characterized by de novo cellular expression of alpha smooth muscle actin (αSMA). In both RPE and Müller cells, both TGFß and miR-494 mimic decreased p27 expression. In parallel experiments, transfection with p27 siRNA augmented TGFß-induced αSMA expression, while only in RPE cells did co-transfection with miR-494 inhibitor decrease αSMA levels. These results demonstrate that miR-494 augments fibrotic transformation in both Müller cells and RPEs, however only in RPEs does miR-494 mediate fibrotic transformation via p27. As p27 is known to regulate cellular proliferation and differentiation, future studies should extend clinical testing of miR-494 and/or p27 as a potential novel non-surgical therapy for ERMs, as well as identify relevant miR-494 targets in Müller cells.

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Memory impairment remains a leading disability in survivors of global cerebral ischemia, occurring secondary to delayed neurodegeneration of hippocampal cornu ammonis-1 (CA1) neurons. MicroRNA-200c (miR-200c) is induced following ischemic stress and we have previously demonstrated that pre-treatment with anti-miR-200c is protective against embolic stroke in mice. In the present study we assessed the role of miR-200c on CA1 neurodegeneration, sirtuin-1 (SIRT1), and mitochondrial dynamic protein expression in a mouse model of transient global cerebral ischemia and in vitro in primary mouse astrocyte cultures after simulated ischemia. Mice were subjected to 10 min bilateral common carotid artery occlusion plus hypotension with 5% isoflurane. After 2 h recovery mice were treated with intravenous injection of either anti-miR-200c or mismatch control. Memory function was assessed by Barnes maze at post-injury days 3 and 7. Mice were sacrificed at post-injury day 7 for assessment of brain cell-type specific expression of miR-200c, SIRT1, and the mitochondrial fusion proteins mitofusin-2 (MFN2) and OPA1 via complexed fluorescent in situ hybridization and fluorescent immunohistochemistry. Global cerebral ischemia induced significant loss of CA1 neurons, impaired memory performance and decreased expression of CA1 SIRT1, MFN2, and OPA1. Post-injury treatment with anti-miR-200c significantly improved survival, prevented CA1 neuronal loss, improved post-injury performance in Barnes maze, and was associated with increased post-injury expression of CA1 SIRT1 and MFN2 in astrocytes. In vitro, primary mouse astrocyte cultures pre-treated with miR-200c inhibitor prior to oxygen/glucose deprivation preserved expression of SIRT1 and MFN2, and decreased reactive oxygen species generation, whereas pre-treatment with miR-200c mimic had opposite effects that could be reversed by co-treatment with SIRT1 activator. These results suggest that miR-200c regulates astrocyte mitochondrial homeostasis via targeting SIRT1, and that CA1 astrocyte mitochondria and SIRT1 represent potential post-injury therapeutic targets to preserve cognitive function in survivors of global cerebral ischemia.

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Ischemic stroke remains a devastating cerebrovascular disease that accounts for a high proportion of mortality and disability worldwide. MicroRNAs (miRNAs) are a class of small non-coding RNAs that are responsible for regulation of post-transcriptional gene expression, and growing evidence supports a role for miRNAs in stroke injury and recovery. The current study examined the role of miR-182 in experimental stroke using both in vitro and in vivo models of ischemic injury. Brain levels of miR-182 significantly increased after transient middle cerebral artery occlusion (MCAO) in mice and in primary astrocyte cultures subjected to combined oxygen-glucose deprivation/reperfusion (OGD/R) injury. In vivo, stroke volume and neurological score were significantly improved by pre-treatment with miR-182 antagomir. Astrocyte cultures stressed with OGD/R resulted in mitochondrial fragmentation and downregulation of cortactin, an actin-binding protein. Inhibition of miR-182 significantly preserved cortactin expression, reduced mitochondrial fragmentation and improved astrocyte survival after OGD/R. In parallel, lipopolysaccharide (LPS)-induced nitric-oxide release in astrocyte cultures was significantly reduced by miR-182 inhibition, translating to reduced injury in primary neuronal cultures subjected to conditioned medium from LPS-treated astrocytes. These findings identify miR-182 and/or cortactin as potential clinical targets to preserve mitochondrial structure and mitigate neuroinflammation and cell death after ischemic stroke.

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Intracerebral hemorrhage (ICH) is devastating among stroke types with high mortality. To date, not a single therapeutic intervention has been successful. Cofilin plays a critical role in inflammation and cell death. In the current study, we embarked on designing and synthesizing a first-in-class small-molecule inhibitor of cofilin to target secondary complications of ICH, mainly neuroinflammation. A series of compounds were synthesized, and two lead compounds SZ-3 and SK-1-32 were selected for further studies. Neuronal and microglial viabilities were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay using neuroblastoma (SHSY-5Y) and human microglial (HMC-3) cell lines, respectively. Lipopolysaccharide (LPS)-induced inflammation in HMC-3 cells was used for neurotoxicity assay. Other assays include nitric oxide (NO) by Griess reagent, cofilin inhibition by F-actin depolymerization, migration by scratch wound assay, tumor necrosis factor (TNF-α) by enzyme-linked immunosorbent assay (ELISA), protease-activated receptor-1 (PAR-1) by immunocytochemistry and Western blotting (WB), and protein expression levels of several proteins by WB. SK-1-32 increased neuronal/microglial survival, reduced NO, and prevented neurotoxicity. However, SZ-3 showed no effect on neuronal/microglial survival but prevented microglia from LPS-induced inflammation by decreasing NO and preventing neurotoxicity. Therefore, we selected SZ-3 for further molecular studies, as it showed potent anti-inflammatory activities. SZ-3 decreased cofilin severing activity, and its treatment of LPS-activated HMC-3 cells attenuated microglial activation and suppressed migration and proliferation. HMC-3 cells subjected to thrombin, as an in vitro model for hemorrhagic stroke, and treated with SZ-3 after 3 h showed significantly decreased NO and TNF-α, significantly increased protein expression of phosphocofilin, and decreased PAR-1. In addition, SZ-3-treated SHSY-5Y showed a significant increase in cell viability by significantly reducing nuclear factor-κ B (NF-κB), caspase-3, and high-temperature requirement (HtrA2). Together, our results support the novel idea of targeting cofilin to counter neuroinflammation during secondary injury following ICH.

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Embolic stroke results in a necrotic core of cells destined to die, but also a peri-ischemic, watershed penumbral region of potentially salvageable brain tissue. Approaches to effectively differentiate between the ischemic and peri-ischemic zones is critical for novel therapeutic discovery to improve outcomes in survivors of stroke. MicroRNAs are a class of small non-coding RNAs regulating gene translation that have region- and cell-specific expression and responses to ischemia. We have previously reported that global inhibition of cerebral microRNA-200c after experimental stroke in mice is protective, however delineating the post-stroke sub-regional and cell-type specific patterns of post-stroke miR-200c expression are necessary to minimize off-target effects and advance translational application. Here, we detail a novel protocol to visualize regional miR-200c expression after experimental stroke, complexed with visualization of regional ischemia and markers of oxidative stress in an experimental stroke model in mice. In the present study we demonstrate that the fluorescent hypoxia indicator pimonidazole hydrochloride, the reactive-oxygen-species marker 8-hydroxy-deoxyguanosine, neuronal marker MAP2 and NeuN, and the reactive astrocyte marker GFAP can be effectively complexed to determine regional differences in ischemic injury as early as 30 min post-reperfusion after experimental stroke, and can be effectively used to distinguish ischemic core from surrounding penumbral and unaffected regions for targeted therapy. This multi-dimensional post-stroke immunofluorescent imaging protocol enables a greater degree of sub-regional mechanistic investigation, with the ultimate goal of developing more effective post-stroke pharmaceutical therapy.

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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.

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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.

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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.

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The details of adult neurogenesis, including environmental triggers, region specificity, and species homology remain an area of intense investigation. Slowing or halting age-related cognitive dysfunction, or restoring neurons lost to disease or injury represent just a fraction of potential therapeutic applications. New neurons can derive from stem cells, pluripotent neural progenitor cells, or non-neuronal glial cells, such as astrocytes. Astrocytes must be epigenetically "reprogrammed" to become neurons, which can occur both naturally in vivo, and via artificial exogenous treatments. While neural progenitor cells are localized to a few neurogenic zones in the adult brain, astrocytes populate almost every brain structure. In this review, we will summarize recent research into neurogenesis that arises from conversion of post-mitotic astrocytes, detail the genetic and epigenetic pathways that regulate this process, and discuss the possible clinical relevance in supplementing stem-cell neurogenic therapies.

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Drug addiction remains a prevalent and fatal disease worldwide that carries significant social and economic impacts. Recent reports suggest illicit pregabalin (Lyrica) use may be increasing among youth, however the addictive potential of pregabalin has not been well established. Drug seeking behavior and chronic drug use are associated with deficits in glutamate clearance and activation of postsynaptic glutamatergic receptors. In the current study, we investigated the abuse potential of pregabalin using conditioned place preference (CPP) paradigm. Different doses of pregabalin (30, 60, 90, and 120 mg/kg) were used to assess the seeking behavior in mice. Glutamate homeostasis is maintained by glutamate transporter type-1 (GLT-1), which plays a vital role in clearing the released glutamate from synapses and drug seeking behavior. Therefore, we investigated the role of glutamate in pregabalin-seeking behavior with ceftriaxone (CEF), a potent GLT-1 upregulator. Mice treated with pregabalin 60 and 90 mg/kg doses demonstrated drug seeking-like behavior, which was significantly blocked by CEF pretreatment. These results suggest that pregabalin-induced CPP was successfully modulated by CEF which could serve as a lead compound for developing treatment for pregabalin abuse.

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In the present study, we assessed efficacy of exosomes harvested from human and mouse stem cell cultures in protection of mouse primary astrocyte and neuronal cell cultures following in vitro ischemia, and against ischemic stroke in vivo. Cell media was collected from primary mouse neural stem cell (NSC) cultures or from human induced pluripotent stem cell-derived cardiomyocyte (iCM) cultures. Exosomes were extracted and purified by polyethylene glycol complexing and centrifugation, and exosome size and concentration were determined with a NanoSiteTM particle analyzer. Exosomes were applied to primary mouse cortical astrocyte or neuronal cultures prior to, and/or during, combined oxygen-glucose deprivation (OGD) injury. Cell death was assessed via lactate dehydrogenase (LHD) and propidium iodide staining 24 h after injury. NSC-derived exosomes afforded marked protection to astrocytes following OGD. A more modest (but significant) level of protection was observed with human iCM-derived exosomes applied to astrocytes, and with NSC-derived exosomes applied to primary neuronal cultures. In subsequent experiments, NSC-derived exosomes were injected intravenously into adult male mice 2 h after transient (1 h) middle cerebral artery occlusion (MCAO). Gross motor function was assessed 1 day after reperfusion and infarct volume was assessed 4 days after reperfusion. Mice treated post-stroke with intravenous NSC-derived exosomes exhibited significantly reduced infarct volumes. Together, these results suggest that exosomes isolated from mouse NSCs provide neuroprotection against experimental stroke possibly via preservation of astrocyte function. Intravenous NSC-derived exosome treatment may therefore provide a novel clinical adjuvant for stroke in the immediate post-injury period.

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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 exhibited de novo DCX expression in response to injury. In vitro experiments 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.

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Recent studies demonstrate significant neuroimmune changes in postpartum females, a period that also carries an increased risk of stroke. Oxytocin, a major hormone upregulated in the brains of nursing mothers, has been shown to both modulate neuroinflammation and protect against stroke. In the present study we assessed whether and how nursing modulates the neuroimmune response and injury after stroke. We observed that postpartum nursing mice were markedly protected from 1 h of transient middle cerebral artery occlusion (MCAO) relative to either non-pregnant/non-postpartum or non-nursing (pups removed) postpartum females. Nursing mice also expressed reduced levels of pro-inflammatory cytokines, had decreased migration of blood leukocytes into the brain following MCAO, and displayed peripheral neuroimmune changes characterized by increased spleen weight and increased fraction of spleen monocytes. Intranasal oxytocin treatment in non-pregnant females in part recapitulated the protective and anti-inflammatory effects associated with nursing. In summary, the results of the present study demonstrate that nursing in the postpartum period provides relative protection against transient ischemic stroke associated with decreased brain leukocytes and increased splenic monocytes. These effects appear to be regulated, at least in part, by oxytocin.

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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.

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