Social isolation initiated post-weaning augments ischemic brain injury by promoting pro-inflammatory responses.

Social isolation is associated with poor stroke outcome, but the underlying molecular mechanisms were largely unknown. In male Balb/C mice exposed to transient middle cerebral artery occlusion (MCAo), we examined the effects of social isolation initiated post-weaning on ischemic injury, cytokine/chemokine responses and cell signaling using a broad panel of techniques that involved immunocytochemistry, cytokine/chemokine array and Western blots. Social isolation initiated post-weaning elevated infarct size, brain edema and neuronal injury in the ischemic brain tissue 3 days after MCAo, and increased microglia/ macrophage and leukocyte accumulation. In line with the increased immune cell recruitment, levels of several proinflammatory cytokines (e.g., IL-1α, IL-1ß, IL-13, IL-17, TNF-α, IFN-γ), chemokines (e.g., CCL3, CCL4, CCL12, CXCL2, CXCL9, CXCL12) and adhesion molecules (i.e., ICAM-1) were increased in the ischemic brain tissue of socially isolated compared with paired housing mice, whereas levels of selected cytokines (IL-5, IL-6, IL-16) and colony-stimulating factors (G-CSF, GM-CSF) were reduced. The activity of the transcription factor nuclear factor-ĸB (NF-ĸB), which promotes cell injury via pro-inflammatory responses, was increased by social isolation, whereas that of nuclear factor erythroid related factor-2 (Nrf-2), which mediates anti-oxidative responses under oxidative stress conditions, was reduced. Our study shows that social isolation profoundly alters post-ischemic cell signaling in a way promoting pro-inflammatory responses. Our results highlight the importance of social support in preventing deleterious health effects of social isolation.

A highly bioavailable curcumin formulation ameliorates inflammation cytokines and neurotrophic factors in mice with traumatic brain injury.

A novel curcumin formulation increases relative absorption by 46 times (CurcuWIN®) of the total curcuminoids over the unformulated standard curcumin form. However, the exact mechanisms by which curcumin demonstrates its neuroprotective effects are not fully understood. This study aimed to investigate the impact of a novel formulation of curcumin on the expression of brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), a main component of the glial scar and growth-associated protein-43 (GAP-43), a signaling molecule in traumatic brain injury (TBI). Mice (adult, male, C57BL/6j) were randomly divided into three groups as follows: TBI group (TBI-induced mice); TBI + CUR group (TBI mice were injected i.p. curcumin just after TBI); TBI+ CurcuWIN® group (TBI mice were injected i.p. CurcuWIN® just after TBI). Brain injury was induced using a cold injury model. Injured brain tissue was stained with Cresyl violet to evaluate infarct volume and brain swelling, analyzed, and measured using ImageJ by Bethesda (MD, USA). Western blot analysis was performed to determine the protein levels related to injury. While standard curcumin significantly reduced brain injury, CurcuWIN® showed an even greater reduction associated with reductions in glial activation, NF-κB, and the inflammatory cytokines IL-1ß and IL-6. Additionally, both standard curcumin and CurcuWIN® led to increased BDNF, GAP-43, ICAM-1, and Nrf2 expression. Notably, CurcuWIN® enhanced their expression more than standard curcumin. This data suggests that highly bioavailable curcumin formulation has a beneficial effect on the traumatic brain in mice.

Knockdown of NEAT1 prevents post-stroke lipid droplet agglomeration in microglia by regulating autophagy.

BACKGROUND: Lipid droplets (LD), lipid-storing organelles containing neutral lipids like glycerolipids and cholesterol, are increasingly accepted as hallmarks of inflammation. The nuclear paraspeckle assembly transcript 1 (NEAT1), a long non-coding RNA with over 200 nucleotides, exerts an indispensable impact on regulating both LD agglomeration and autophagy in multiple neurological disorders. However, knowledge as to how NEAT1 modulates the formation of LD and associated signaling pathways is limited. METHODS: In this study, primary microglia were isolated from newborn mice and exposed to oxygen-glucose-deprivation/reoxygenation (OGD/R). To further explore NEAT1-dependent mechanisms, an antisense oligonucleotide (ASO) was adopted to silence NEAT1 under in vitro conditions. Studying NEAT1-dependent interactions with regard to autophagy and LD agglomeration under hypoxic conditions, the inhibitor and activator of autophagy 3-methyladenine (3-MA) and rapamycin (RAPA) were used, respectively. In a preclinical stroke model, mice received intraventricular injections of ASO NEAT1 or control vectors in order to yield NEAT1 knockdown. Analysis of readout parameters included qRT-PCR, immunofluorescence, western blot assays, and behavioral tests. RESULTS: Microglia exposed to OGD/R displayed a temporal pattern of NEAT1 expression, peaking at four hours of hypoxia followed by six hours of reoxygenation. After effectively silencing NEAT1, LD formation and autophagy-related proteins were significantly repressed in hypoxic microglia. Stimulating autophagy in ASO NEAT1 microglia under OGD/R conditions by means of RAPA reversed the downregulation of LD agglomeration and perilipin 2 (PLIN2) expression. On the contrary, application of 3-MA promoted repression of both LD agglomeration and expression of the LD-associated protein PLIN2. Under in vivo conditions, NEAT1 was significantly increased in mice at 24 h post-stroke. Knockdown of NEAT1 significantly alleviated LD agglomeration and inhibited autophagy, resulting in improved cerebral perfusion, reduced brain injury and increased neurological recovery. CONCLUSION: NEAT1 is a key player of LD agglomeration and autophagy stimulation, and NEAT1 knockdown provides a promising therapeutic value against stroke.

Preconditioned extracellular vesicles from hypoxic microglia reduce poststroke AQP4 depolarization, disturbed cerebrospinal fluid flow, astrogliosis, and neuroinflammation.

Background: Stroke stimulates reactive astrogliosis, aquaporin 4 (AQP4) depolarization and neuroinflammation. Preconditioned extracellular vesicles (EVs) from microglia exposed to hypoxia, in turn, reduce poststroke brain injury. Nevertheless, the underlying mechanisms of such effects are elusive, especially with regards to inflammation, AQP4 polarization, and cerebrospinal fluid (CSF) flow. Methods: Primary microglia and astrocytes were exposed to oxygen-glucose deprivation (OGD) injury. For analyzing the role of AQP4 expression patterns under hypoxic conditions, a co-culture model of astrocytes and microglia was established. Further studies applied a stroke model, where some mice also received an intracisternal tracer infusion of rhodamine B. As such, these in vivo studies involved the analysis of AQP4 polarization, CSF flow, astrogliosis, and neuroinflammation as well as ischemia-induced brain injury. Results: Preconditioned EVs decreased periinfarct AQP4 depolarization, brain edema, astrogliosis, and inflammation in stroke mice. Likewise, EVs promoted postischemic CSF flow and cerebral blood perfusion, and neurological recovery. Under in vitro conditions, hypoxia stimulated M2 microglia polarization, whereas EVs augmented M2 microglia polarization and repressed M1 microglia polarization even further. In line with this, astrocytes displayed upregulated AQP4 clustering and proinflammatory cytokine levels when exposed to OGD, which was reversed by preconditioned EVs. Reduced AQP4 depolarization due to EVs, however, was not a consequence of unspecific inflammatory regulation, since LPS-induced inflammation in co-culture models of astrocytes and microglia did not result in altered AQP4 expression patterns in astrocytes. Conclusions: These findings show that hypoxic microglia may participate in protecting against stroke-induced brain damage by regulating poststroke inflammation, astrogliosis, AQP4 depolarization, and CSF flow due to EV release.

Nanodrugs for the Treatment of Ischemic Stroke: A Systematic Review.

Ischemic stroke, a significant neurovascular disorder, currently lacks effective restorative medication. However, recently developed nanomedicines bring renewed promise for alleviating ischemia's effects and facilitating the healing of neurological and physical functions. The aim of this systematic review was to evaluate the efficacy of nanotherapies in animal models of stroke and their potential impact on future stroke therapies. We also assessed the scientific quality of current research focused on nanoparticle-based treatments for ischemic stroke in animal models. We summarized the effectiveness of nanotherapies in these models, considering multiple factors such as their anti-inflammatory, antioxidant, and angiogenetic properties, as well as their safety and biodistribution. We conclude that the application of nanomedicines may reduce infarct size and improve neurological function post-stroke without causing significant organ toxicity.

Reduced folate carrier 1 is present in retinal microvessels and crucial for the inner blood retinal barrier integrity.

BACKGROUND: Reduced folate carrier 1 (RFC1; SLC19a1) is the main responsible transporter for the B9 family of vitamins named folates, which are essential for normal tissue growth and development. While folate deficiency resulted in retinal vasculopathy, the expression and the role of RFC1 in blood-retinal barrier (BRB) are not well known. METHODS: We used whole mount retinas and trypsin digested microvessel samples of adult mice. To knockdown RFC1, we delivered RFC1-targeted short interfering RNA (RFC1-siRNA) intravitreally; while, to upregulate RFC1 we delivered lentiviral vector overexpressing RFC1. Retinal ischemia was induced 1-h by applying FeCl3 to central retinal artery. We used RT-qPCR and Western blotting to determine RFC1. Endothelium (CD31), pericytes (PDGFR-beta, CD13, NG2), tight-junctions (Occludin, Claudin-5 and ZO-1), main basal membrane protein (Collagen-4), endogenous IgG and RFC1 were determined immunohistochemically. RESULTS: Our analyses on whole mount retinas and trypsin digested microvessel samples of adult mice revealed the presence of RFC1 in the inner BRB and colocalization with endothelial cells and pericytes. Knocking down RFC1 expression via siRNA delivery resulted in the disintegration of tight junction proteins and collagen-4 in twenty-four hours, which was accompanied by significant endogenous IgG extravasation. This indicated the impairment of BRB integrity after an abrupt RFC1 decrease. Furthermore, lentiviral vector-mediated RFC1 overexpression resulted in increased tight junction proteins and collagen-4, confirming the structural role of RFC1 in the inner BRB. Acute retinal ischemia decreased collagen-4 and occludin levels and led to an increase in RFC1. Besides, the pre-ischemic overexpression of RFC1 partially rescued collagen-4 and occludin levels which would be decreased after ischemia. CONCLUSION: In conclusion, our study clarifies the presence of RFC1 protein in the inner BRB, which has recently been defined as hypoxia-immune-related gene in other tissues and offers a novel perspective of retinal RFC1. Hence, other than being a folate carrier, RFC1 is an acute regulator of the inner BRB in healthy and ischemic retinas.

Age-Associated Resilience Against Ischemic Injury in Mice Exposed to Transient Middle Cerebral Artery Occlusion.

Ischemic stroke is the leading cause of death and disability. Although stroke mainly affects aged individuals, animal research is mostly one on young rodents. Here, we examined the development of ischemic injury in young (9-12-week-old) and adult (72-week-old) C57BL/6 and BALB/c mice exposed to 30 min of intraluminal middle cerebral artery occlusion (MCAo). Post-ischemic reperfusion did not differ between young and adult mice. Ischemic injury assessed by infarct area and blood-brain barrier (BBB) integrity assessed by IgG extravasation analysis was smaller in adult compared with young mice. Microvascular viability and neuronal survival assessed by CD31 and NeuN immunohistochemistry were higher in adult than young mice. Tissue protection was associated with stronger activation of cell survival pathways in adult than young mice. Microglial/macrophage accumulation and activation assessed by F4/80 immunohistochemistry were more restricted in adult than young mice, and pro- and anti-inflammatory cytokine and chemokine responses were reduced by aging. By means of liquid chromatography-mass spectrometry, we identified a hitherto unknown proteome profile comprising the upregulation of glycogen degradation-related pathways and the downregulation of mitochondrial dysfunction-related pathways, which distinguished post-ischemic responses of the aged compared with the young brain. Our study suggests that aging increases the brain's resilience against ischemic injury.

Hyperthermia alters neurobehavior by affecting cell proliferation and neuronal survival in young male rats.

Maintenance of body temperature within physiological range is critical for the fetal and neonatal development. Hyperthermia is one of the most frequently encountered pediatric complaints and may cause neurological disorders due to neuronal injury. In this study, we aimed to investigate the effects of hyperthermia on behavioral alterations, neuronal survival, apoptosis, and cell proliferation in young male Sprague-Dawley rats. Twenty-one 13-day-old rats were randomly divided into three groups (n = 7 per group). Body temperature was increased to 39°C and 41°C in a hyperthermia induction chamber for 30 min, whereas the animals in control group were maintained at 36°C. Twenty-four hours after hyperthermia, animals were subjected to the open field test, elevated-O-maze test, and grip strength test to assess the locomotor activity, anxiety, and motor function. Neuronal survival, apoptosis, and cell proliferation were investigated in cortex, hippocampal dentate gyrus (DG) and CA1 regions, and corpus callosum (CC). Decreased locomotor activity and motor function and increased anxiety were observed in the hyperthermia groups, and these were more pronounced in the 41°C group. Neuronal survival was significantly decreased in DG, CA1, and CC in the hyperthermia groups (**p < 0.01). Apoptosis was significantly induced in cortex, DG, and CC of the animals exposed to heat (*p < 0.05). In addition, cell proliferation positivity decreased significantly only in DG and CC of the animals exposed to heat (*p < 0.05). Our results suggest that neurobehavioral deficits caused by hyperthermia may be due to the increased apoptosis and neuronal cell death and decreased cell proliferation in the brain of postnatal developing rats.

The Unconventional Growth Factors Cerebral Dopamine Neurotrophic Factor and Mesencephalic Astrocyte-Derived Neurotrophic Factor Promote Post-ischemic Neurological Recovery, Perilesional Brain Remodeling, and Lesion-Remote Axonal Plasticity.

Considerable efforts are currently made to develop strategies that boost endogenous recovery once a stroke has occurred. Owing to their restorative properties, neurotrophic factors are attractive candidates that capitalize on endogenous response mechanisms. Non-conventional growth factors cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) promote neuronal survival and reduce neurological deficits in the acute phase of ischemic stroke in mice. Their effects on endogenous repair and recovery mechanisms in the stroke recovery phase were so far unknown. By intracerebroventricular delivery of CDNF or MANF starting 3 days post-stroke (1 µg/day for 28 days via miniosmotic pumps), we show that delayed CDNF and MANF administration promoted functional neurological recovery assessed by a battery of behavioral tests, increased long-term neuronal survival, reduced delayed brain atrophy, glial scar formation, and, in case of CDNF but not MANF, increased endogenous neurogenesis in the perilesional brain tissue. Besides, CDNF and MANF administration increased long-distance outgrowth of terminal axons emanating from the contralesional pyramidal tract, which crossed the midline to innervate ipsilesional facial nucleus. This plasticity promoting effect was accompanied by downregulation of the axonal growth inhibitor versican and the guidance molecules ephrin B1 and B2 in the previously ischemic hemisphere at 14 dpi, which represents a sensitive time-point for axonal growth. CDNF and MANF reduced the expression of the proinflammatory cytokines IL1ß and TNFα in both hemispheres. The effects of non-conventional growth factors in the ischemic brain should further be examined since they might help to identify targets for restorative stroke therapy.

Lithium promotes long-term neurological recovery after spinal cord injury in mice by enhancing neuronal survival, gray and white matter remodeling, and long-distance axonal regeneration.

Spinal cord injury (SCI) induces neurological deficits associated with long-term functional impairments. Since the current treatments remain ineffective, novel therapeutic options are needed. Besides its effect on bipolar mood disorder, lithium was reported to have neuroprotective activity in different neurodegenerative conditions, including SCI. In SCI, the effects of lithium on long-term neurological recovery and neuroplasticity have not been assessed. We herein investigated the effects of intraperitoneally administered lithium chloride (LiCl) on motor coordination recovery, electromyography (EMG) responses, histopathological injury and remodeling, and axonal plasticity in mice exposed to spinal cord transection. At a dose of 0.2, but not 2.0 mmol/kg, LiCl enhanced motor coordination and locomotor activity starting at 28 days post-injury (dpi), as assessed by a set of behavioral tests. Following electrical stimulation proximal to the hemitransection, LiCl at 0.2 mmol/kg decreased the latency and increased the amplitude of EMG responses in the denervated hindlimb at 56 dpi. Functional recovery was associated with reduced gray and white matter atrophy rostral and caudal to the hemitransection, increased neuronal survival and reduced astrogliosis in the dorsal and ventral horns caudal to the hemitransection, and increased regeneration of long-distance axons proximal and distal to the lesion site in mice receiving 0.2 mmol/kg, but not 2 mmol/kg LiCl, as assessed by histochemical and immunohistochemical studies combined with anterograde tract tracing. Our results indicate that LiCl induces long-term neurological recovery and neuroplasticity following SCI.

Phosphodiesterase 10A deactivation induces long-term neurological recovery, Peri-infarct remodeling and pyramidal tract plasticity after transient focal cerebral ischemia in mice.

The phosphodiesterase (PDE) superfamily comprises enzymes responsible for the cAMP and cGMP degradation to AMP and GMP. PDEs are abundant in the brain, where they are involved in several neuronal functions. High PDE10A abundance was previously observed in the striatum; however its consequences for stroke recovery were unknown. Herein, we evaluated the effects of PDE10A deactivation by TAK-063 (0.3 or 3 mg/kg, initiated 72 h post-stroke) in mice exposed to intraluminal middle cerebral artery occlusion. We found that PDE10A deactivation over up to eight weeks dose-dependently increased long-term neuronal survival, angiogenesis, and neurogenesis in the peri-infarct striatum, which represents the core of the middle cerebral artery territory, and reduced astroglial scar formation, whole brain atrophy and, more specifically, striatal atrophy. Functional motor-coordination recovery and the long-distance plasticity of pyramidal tract axons, which originate from the contralesional motor cortex and descend through the contralesional striatum to innervate the ipsilesional facial nucleus, were enhanced by PDE10A deactivation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed a set of dopamine receptor-related and neuronal plasticity-related PDE10A targets, which were elevated (e.g., protein phosphatase-1 regulatory subunit 1B) or reduced (e.g., serine/threonine protein phosphatase 1α, ß-synuclein, proteasome subunit α2) by PDE10A deactivation. Our results identify PDE10A as a therapeutic target that critically controls post-ischemic brain tissue remodeling and plasticity.

ACKR3 regulates platelet activation and ischemia-reperfusion tissue injury.

Platelet activation plays a critical role in thrombosis. Inhibition of platelet activation is a cornerstone in treatment of acute organ ischemia. Platelet ACKR3 surface expression is independently associated with all-cause mortality in CAD patients. In a novel genetic mouse strain, we show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo. Further, we performed ischemia/reperfusion experiments (transient LAD-ligation and tMCAO) in mice to assess the impact of genetic ACKR3 deficiency in platelets on tissue injury in ischemic myocardium and brain. Loss of platelet ACKR3 enhances tissue injury in ischemic myocardium and brain and aggravates tissue inflammation. Activation of platelet-ACKR3 via specific ACKR3 agonists inhibits platelet activation and thrombus formation and attenuates tissue injury in ischemic myocardium and brain. Here we demonstrate that ACKR3 is a critical regulator of platelet activation, thrombus formation and organ injury following ischemia/reperfusion.

A Novel Theanine Complex, Mg-L-Theanine Improves Sleep Quality via Regulating Brain Electrochemical Activity.

L-Theanine is commonly used to improve sleep quality through inhibitory neurotransmitters. On the other hand, Mg2+, a natural NMDA antagonist and GABA agonist, has a critical role in sleep regulation. Using the caffeine-induced brain electrical activity model, here we investigated the potency of L-theanine and two novel Mg-L-theanine compounds with different magnesium concentrations on electrocorticography (ECoG) patterns, GABAergic and serotonergic receptor expressions, dopamine, serotonin, and melatonin levels. Furthermore, we evaluated the sleep latency and duration in the pentobarbital induced sleep model. We herein showed that L-theanine, particularly its various complexes with magnesium increases the expression of GABAergic, serotonergic, and glutamatergic receptors, which were associated with decreased ECoG frequency, increased amplitude, and enhanced delta wave powers. Besides increased dopamine, serotonin, and melatonin; decreased MDA and increased antioxidant enzyme levels were also observed particularly with Mg-complexes. Protein expression analyses also showed that Mg-L-theanine complexes decrease inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) levels significantly. In accordance with these results, Mg complexes improved the sleep latency and duration even after caffeine administration. As a result, our data indicate that Mg-L-theanine compounds potentiate the effect of L-theanine on sleep by boosting slow-brain waves, regulating brain electrical activity, and increasing neurotransmitter and GABA receptor levels.

Delayed Therapeutic Administration of Melatonin Enhances Neuronal Survival Through AKT and MAPK Signaling Pathways Following Focal Brain Ischemia in Mice.

Melatonin has a role in the cell survival signaling pathways as a candidate for secondary stroke prevention. Therefore, in the present study, the coordination of ipsilateral and contralateral hemispheres to evaluate delayed post-acute effect of melatonin was examined on recovery of the cell survival and apoptosis after stroke. Melatonin was administered (4 mg/kg/day) intraperitoneally for 45 days, starting 3 days after 30 min of middle cerebral artery occlusion. The genes and proteins related to the cell survival and apoptosis were investigated by immunofluorescence, western blotting, and RT-PCR techniques after behavioral experiments. Melatonin produced delayed neurological recovery by improving motor coordination on grip strength and rotarod tests. This neurological recovery was also reflected by high level of NeuN positive cells and low level of TUNEL-positive cells suggesting enhanced neuronal survival and reduced apoptosis at the fifty-fifth day of stroke. The increase of NGF, Nrp1, c-jun; activation of AKT; and dephosphorylation of ERK and JNK at the fifty-fifth day showed that cell survival and apoptosis signaling molecules compete to contribute to the remodeling of brain. Furthermore, an increase in the CREB and Atf-1 expressions suggested the melatonin's strong reformative effect on neuronal regeneration. The contralateral hemisphere was more active at the latter stages of the molecular and functional regeneration which provides a further proof of principle about melatonin's action on the promotion of brain plasticity and recovery after stroke.

Stroke induces disease-specific myeloid cells in the brain parenchyma and pia.

Inflammation triggers secondary brain damage after stroke. The meninges and other CNS border compartments serve as invasion sites for leukocyte influx into the brain thus promoting tissue damage after stroke. However, the post-ischemic immune response of border compartments compared to brain parenchyma remains poorly characterized. Here, we deeply characterize tissue-resident leukocytes in meninges and brain parenchyma and discover that leukocytes respond differently to stroke depending on their site of residence. We thereby discover a unique phenotype of myeloid cells exclusive to the brain after stroke. These stroke-associated myeloid cells partially resemble neurodegenerative disease-associated microglia. They are mainly of resident microglial origin, partially conserved in humans and exhibit a lipid-phagocytosing phenotype. Blocking markers specific for these cells partially ameliorates stroke outcome thus providing a potential therapeutic target. The injury-response of myeloid cells in the CNS is thus compartmentalized, adjusted to the type of injury and may represent a therapeutic target.

Phosphorylation of PI3K/Akt at Thr308, but not phosphorylation of MAPK kinase, mediates lithium-induced neuroprotection against cerebral ischemia in mice.

Lithium, in addition to its effect on acute and long-term bipolar disorder, is involved in neuroprotection after ischemic stroke. Yet, its mechanism of action is still poorly understood, which was only limited to its modulatory effect on GSK pathway. Therefore, we initially analyzed the dose-dependent effects of lithium on neurological deficits, infarct volume, brain edema and blood-brain barrier integrity, along with neuronal injury and survival in mice subjected to focal cerebral ischemia. Thereafter, we investigated the involvement of the PI3K/Akt and MEK signal transduction pathways and their components. Our observations revealed that 2 mmol/kg lithium significantly improved post-ischemic brain tissue survival. Although, 2 mmol/kg lithium had no negative effect on brain microcirculation, 5 and 20 mmol/kg lithium reduced brain perfusion. Furthermore, supratherapeutic dose of lithium in 20 mmol/kg lead to animal death. In addition, improvement of brain perfusion with L-arginine, did not change the effect of 5 mmol/kg lithium on brain injury. Additionally, post-stroke blood-brain barrier leakage, hemodynamic impairment and apoptosis have been reversed by lithium treatment. Interestingly, lithium-induced neuroprotection was associated with increased phosphorylation of Akt at Thr308 and suppressed GSK-3ß phosphorylation at Ser9 residue. Lithium upregulated Erk-2 and downregulated JNK-2 phosphorylation. To distinguish whether neuroprotective effects of lithium are modulated by PI3K/Akt or MEK, we sequentially blocked these pathways and demonstrated that the neuroprotective activity of lithium persisted during MEK/ERK inhibition, whereas PI3K/Akt inhibition abolished neuroprotection. Collectively, we demonstrated lithium exerts its post-stroke neuroprotective activity via the PI3K/Akt pathway, specifically via Akt phosphorylation at Thr308, but not via MEK/ERK.

Right ventricle early inflow-outflow index may inform about the severity of pneumonia in patients with COVID-19.

BACKGROUND: Echocardiography is generally used in our daily practice to detect cardiovascular complications in COVID-19 patients and for etiological research in the case of worsened clinical status. Many echocardiographic parameters have been the subject of investigation in previous studies on COVID-19. Recently, the right ventricle early inflow-outflow (RVEIO) index has been identified as a possible and indirect marker of the severity of tricuspid regurgitation and right ventricular dysfunction in pulmonary embolism. In this study, we aimed to investigate the relationship between the severity of pneumonia in COVID-19 patients and the RVEIO index. METHODS: A total of 54 patients diagnosed with COVID-19 pneumonia were enrolled in this study. Our study population was separated into two groups as severe pneumonia and nonsevere pneumonia based on computed tomography imaging. RESULTS: Saturation O2 , C-reactive protein, D-dimer, deceleration time, tricuspid annular plane systolic excursion, tricuspid lateral annular systolic velocity, and RVEIO index values were found to be significantly different between severe and nonsevere pneumonia groups. The result of the multivariate logistic regression test revealed that saturation O2, D-dimer, Sm, and RVEIO index were the independent predictive parameters for severe pneumonia. Receiver operating characteristic curve analysis demonstrated that RVEIO index >4.2 predicted severe pneumonia with 77% sensitivity and 79% specificity. CONCLUSION: The RVEIO index can be used as a bedside, noninvasive, easily accessible, and useful marker to identify the COVID-19 patient group with widespread pneumonia and, therefore high risk of complications, morbidity, and mortality.

Phosphodiesterase 10A Is a Critical Target for Neuroprotection in a Mouse Model of Ischemic Stroke.

Phosphodiesterase 10A (PDE10A) hydrolyzes adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). It is highly expressed in the striatum. Recent evidence implied that PDE10A may be involved in the inflammatory processes following injury, such as ischemic stroke. Its role in ischemic injury was unknown. Herein, we exposed mice to 90 or 30-min middle cerebral artery occlusion, followed by the delivery of the highly selective PDE10A inhibitor TAK-063 (0.3 mg/kg or 3 mg/kg) immediately after reperfusion. Animals were sacrificed after 24 or 72 h, respectively. Both TAK-063 doses enhanced neurological function, reduced infarct volume, increased neuronal survival, reduced brain edema, and increased blood-brain barrier integrity, alongside cerebral microcirculation improvements. Post-ischemic neuroprotection was associated with increased phosphorylation (i.e., activation) of pro-survival Akt, Erk-1/2, GSK-3α/ß and anti-apoptotic Bcl-xL abundance, decreased phosphorylation of pro-survival mTOR, and HIF-1α, MMP-9 and pro-apoptotic Bax abundance. Interestingly, PDE10A inhibition reduced inflammatory cytokines/chemokines, including IFN-γ and TNF-α, analyzed by planar surface immunoassay. In addition, liquid chromatography-tandem mass spectrometry revealed 40 proteins were significantly altered by TAK-063. Our study established PDE10A as a target for ischemic stroke therapy.

Striatal dopaminergic neurons as a potential target for GDNF based ischemic stroke therapy.

BACKGROUND: Glial cell-line-derived neurotrophic factor (GDNF) is a well-known regulatory neurotrophic factor on dopaminergic neurons. Several pathologies have been documented so far in case of any impairment in the dopaminergic system. This study aimed to investigate the potential protective role of lentiviral GNDF delivery on the small population of tyrosine hydroxylase (TH) positive dopamine producing striatal neurons after ischemic stroke. METHODS: Fourteen C57BL/6J male mice (8-10 weeks) were intracerebrally treated with lentiviral GDNF (Lv-GDNF) or vehicle. Ten days after injections, cerebral ischemia was induced by blockage of the middle cerebral artery. Animals were terminated 72 h after ischemia, and their brains were taken for histological and molecular investigations. Following confirmation of GDNF overexpression, TH immunostaining and immunoblotting were used to evaluate the role of GDNF on dopaminergic neurons. Next, Fluro Jade C staining was implemented to examine the degree of neuronal degeneration at the damaged parenchyma. RESULTS: Neither the amount of TH positive dopaminergic neurons nor the expression of TH changed in the Lv-GDNF treated animals comparing to the vehicle group. On the other hand, GDNF exposure caused a significant increase in the expression of Nurr1, an essential transcription factor for dopaminergic neurons and Gap43, growth and plasticity promoting protein, in the ischemic striatum. Treatment with Lv-GDNF gave rise to a significant reduction in the number of degenerated neurons. Finally, enhanced GDNF expression also induced expression of an important stress-related transcription factor NF-κB as well as the nitric oxide synthase enzymes iNOS and nNOS in the contralesional hemisphere.

Extracellular vesicles from hypoxia-preconditioned microglia promote angiogenesis and repress apoptosis in stroke mice via the TGF-ß/Smad2/3 pathway.

Systemic transplantation of oxygen-glucose deprivation (OGD)-preconditioned primary microglia enhances neurological recovery in rodent stroke models, albeit the underlying mechanisms have not been sufficiently addressed. Herein, we analyzed whether or not extracellular vesicles (EVs) derived from such microglia are the biological mediators of these observations and which signaling pathways are involved in the process. Exposing bEnd.3 endothelial cells (ECs) and primary cortical neurons to OGD, the impact of EVs from OGD-preconditioned microglia on angiogenesis and neuronal apoptosis by the tube formation assay and TUNEL staining was assessed. Under these conditions, EV treatment stimulated both angiogenesis and tube formation in ECs and repressed neuronal cell injury. Characterizing microglia EVs by means of Western blot analysis and other techniques revealed these EVs to be rich in TGF-ß1. The latter turned out to be a key compound for the therapeutic potential of microglia EVs, affecting the Smad2/3 pathway in both ECs and neurons. EV infusion in stroke mice confirmed the aforementioned in vitro results, demonstrating an activation of the TGF-ß/Smad2/3 signaling pathway within the ischemic brain. Furthermore, enriched TGF-ß1 in EVs secreted from OGD-preconditioned microglia stimulated M2 polarization of residing microglia within the ischemic cerebral environment, which may contribute to a regulation of an early inflammatory response in postischemic hemispheres. These observations are not only interesting from the mechanistic point of view but have an immediate therapeutic implication as well, since stroke mice treated with such EVs displayed a better functional recovery in the behavioral test analyses. Hence, the present findings suggest a new way of action of EVs derived from OGD-preconditioned microglia by regulating the TGF-ß/Smad2/3 pathway in order to promote tissue regeneration and neurological recovery in stroke mice.