Lysophosphatidic Acid Receptor 1 Plays a Pathogenic Role in Permanent Brain Ischemic Stroke by Modulating Neuroinflammatory Responses.

Lysophosphatidic acid receptor 1 (LPA1) plays a critical role in brain injury following a transient brain ischemic stroke. However, its role in permanent brain ischemic stroke remains unknown. To address this, we investigated whether LPA1 could contribute to brain injury of mice challenged by permanent middle cerebral artery occlusion (pMCAO). A selective LPA1 antagonist (AM152) was used as a pharmacological tool for this investigation. When AM152 was given to pMCAO-challenged mice one hour after occlusion, pMCAO-induced brain damage such as brain infarction, functional neurological deficits, apoptosis, and blood-brain barrier disruption was significantly attenuated. Histological analyses demonstrated that AM152 administration attenuated microglial activation and proliferation in injured brain after pMCAO challenge. AM152 administration also attenuated abnormal neuroinflammatory responses by decreasing expression levels of pro-inflammatory cytokines while increasing expression levels of anti-inflammatory cytokines in the injured brain. As underlying effector pathways, NF-κB, MAPKs (ERK1/2, p38, and JNKs), and PI3K/Akt were found to be involved in LPA1-dependent pathogenesis. Collectively, these results demonstrate that LPA1 can contribute to brain injury by permanent ischemic stroke, along with relevant pathogenic events in an injured brain.

Neuroprotective effects of oxytocin against ischemic stroke in rats by blocking glutamate release and CREB-mediated DNA hypermethylation.

Glutamate plays a crucial role in cognitive impairments after ischemic stroke. There is a scarcity of information about how glutamate-induced activation of cAMP-response element binding (CREB) signaling pathway regulates both the negative and positive regulators of synaptic plasticity. Recent studies have demonstrated the involvement of prominent epigenetic repressors, such as MeCP2 and DNMTs, in stroke. Neuroprotective effects of oxytocin against ischemia have been previously reported, while the underlying mechanism is still elusive. In this research, the possible role of CREB-mediated DNA hypermethylation and the potential mechanism of oxytocin in a rat model of permanent middle cerebral artery occlusion (pMCAO) were assessed. Adult male Sprague-Dawley rats were pretreated with intraperitoneal injection of oxytocin at the onset of pMCAO. The effects of oxytocin on spines and the expression levels of synaptic genes were determined. The regulatory effects of oxytocin on glutamate level, N-methyl-D-aspartate receptors (NMDARs), its downstream CREB pathway, and global or gene-specific DNA methylation status were evaluated by immunofluorescence, co-immunoprecipitation, and chromatin immunoprecipitation, respectively. We found that CREB could act as a common transcription factor for MeCP2 and DNMT3B after ischemic stroke. Oxytocin dose-dependently deactivated NR2B-related CaM-CREB pathway and inhibited DNA hypermethylation at the CpG islands of Ngf gene in pMCAO-operated rats. Moreover, oxytocin prevented pMCAO-induced reduction in the number of spines and neural cells. DNA hypermethylation in Ngf gene contributed to the cognitive deficits post-stroke. The neuroprotective effects of oxytocin against ischemia could be attributed to inhibiting glutamate release, providing additional evidence on the mechanism of oxytocin against ischemic stroke.

Lauric acid provides neuroprotection against oxidative stress in mouse model of hyperglycaemic stroke.

During ischemic stroke, higher glucose level linked worse outcomes were reported even in patients without pre-existing diabetes. Evidence suggest that such worse stroke outcomes were mainly due to production of reactive, toxic glucose metabolites that expands oxidative damage inside the brain. As a consequence of high oxidative stress, microvasculature structures and tight junctions compromised their functionally, infarct volume expands and brain edema exacerbates. In a mouse model of ischemic stroke with induced acute hyperglycaemia, Lauric acid (LA) as a natural saturated fatty acid demonstrated neuroprotection by attenuating infarct volume and brain edema. In addition, in the ipsilateral hyperglycaemic brain, the LA significantly increased the expression of tight junction representative protein (occludin) as well as anti-oxidative markers; Manganese superoxide dismutase (Mn) SOD, Extracellular superoxide dismutase (Ec-SOD) and nuclear factor-erythroid factor 2-related factor 2 (Nrf2) in the ipsilateral region against hyperglycemic ischemic stroke. LA treated animals showed a significant reduction in the production of lipid peroxidation products (4-HNE) in the microvascular structures, maintained the blood brain barrier (BBB) integrity. LA linked neuroprotective outcomes were further confirmed by behavioral tests, where functional outcomes and motor coordination were improved significantly. Furthermore, LA treatment enhanced food intake, decreased mortality rate, and net body weight loss. Conclusively, LA modulated ischemic insult exacerbated by hyperglycemia and provided neuroprotection.

A Comparative Study on the Neuroprotective Effect of Geopung-Chunghyuldan on In Vitro Oxygen-Glucose Deprivation and In Vivo Permanent Middle Cerebral Artery Occlusion Models.

Geopung-Chunghyuldan (GCD), which is a mixture of Chunghyuldan (CD), Radix Salviae Miltiorrhizae, Radix Notoginseng, and Borneolum Syntheticum, is used to treat ischemic stroke in traditional Korean medicine. This study aimed to investigate the effects of GCD and CD on ischemic brain damage using in vitro and in vivo stroke models, as well as to elucidate the synergistic effects of GCD against ischemic insult. To study the effect of GCD in an in vitro ischemia model, SH-SY5Y cells were exposed to oxygen-glucose deprivation (OGD). Cell death after 16 h of OGD exposure was measured using the MTT assay and live/dead cell counting methods. An in vivo ischemia mice model was established through permanent middle cerebral artery occlusion (pMCAO). To determine the neuroprotective effect of GCD, it was orally administered immediately and 2 h after pMCAO. The infarct volume was measured through 2,3,5-triphenyltetrazolium chloride staining at 24 h after pMCAO. Compared with the control group, GCD treatment significantly reduced OGD-induced cell death in SH-SY5Y cells; however, CD treatment did not show a significant protective effect. In the pMCAO model, compared with the control group, treatment with GCD and CD significantly and mildly reduced the infarct volume, respectively. Our findings indicate that compared with CD, GCD may allow a more enhanced neuroprotective effect in acute ischemic stroke, indicating a potential synergistic neuroprotective effect. The possibility of GCD as a novel alternative choice for the prevention and treatment of ischemic stroke is suggested.

Neuroprotective Effects of Geopung-Chunghyuldan Based on Its Salvianolic Acid B Content Using an In Vivo Stroke Model.

BACKGROUND: Geopung-Chunghyuldan (GCD) has neuroprotective properties. Salviae miltiorrhizae Radix plays an essential role in GCD's effect. The Salviae miltiorrhizae Radix marker compound is salvianolic acid B; however, its content is not uniform among samples. This study aimed to evaluate the neuroprotective effects of GCD based on salvianolic acid B content. METHODS: The neuroprotective effects of GCD based on the salvianolic acid B content were evaluated by measuring infarct volume 24 h after permanent middle cerebral artery occlusion in an in vivo stroke model. For the experimental group, each GCD was administered immediately before surgery. The control groups were administered distilled water and aspirin (30 mg/kg) in the same way. The salvianolic acid B content in five types of Salviae Miltiorrhizae Radix (two Chinese and three Korean regions) based on different cultivation regions was analyzed by high-performance liquid chromatography. RESULTS: Three samples met the Korean and Chinese Pharmacopeia standards for salvianolic acid B. However, two samples did not. GCDs with high salvianolic acid B showed marked neuroprotective effects compared to the control groups, whereas GCDs with low salvianolic acid B did not. CONCLUSIONS: The salvianolic acid B content of Salviae miltiorrhizae Radix affects the neuroprotection effect of GCD. Stable, raw Salviae miltiorrhizae Radix is essential for GCD homogenization.

Loganin attenuates neuroinflammation after ischemic stroke and fracture by regulating α7nAChR-mediated microglial polarization.

Fracture in acute stage of ischemic stroke can increase inflammatory response and enhance stroke injury. Loganin alleviates the symptoms of many inflammatory diseases through its anti-inflammatory effect, but its role in ischemic stroke and fracture remains to be explored. Here, mice were handled with permanent middle cerebral artery occlusion (pMCAO) followed by tibial fracture 1 day later to establish a pMCAO+fracture model. Loganin or Methyllycaconitine (MLA, a specific a7nAchR inhibitor) were intragastrically administered 2 or 0.5 h before pMCAO, respectively. And mouse motor function and infarct volume were evaluated 3 days after pMCAO. We found that loganin alleviated the neurological deficit, cerebral infarction volume, and neuronal apoptosis (NeuN+ TUNEL+ ) in mice with pMCAO+fracture. And loganin suppressed pMCAO+fracture-induced neuroinflammation by promoting M2 microglia polarization (Iba1+ CD206+ ) and inhibiting M1 microglia polarization (Iba1+ CD11b+ ). While administration with MLA reversed the protective effect of loganin on pMCAO+fracture-induced neurological deficit and neuroinflammation. Next, LPS was used to stimulate BV2 microglia to simulate pMCAO+fracture-induced inflammatory microenvironment in vitro. Loganin facilitated the transformation of LPS-stimulated BV2 cells from M1 pro-inflammatory state (CD11b+ ) to M2 anti-inflammatory state (CD206+ ), which was antagonized by treatment with MLA. And loganin induced autophagy activation in LPS-stimulated BV2 cells by activating a7nAchR. Moreover, treatment with rapamycin (an autophagy activator) neutralized the inhibitory effect of MLA on loganin induced transformation of BV2 cells to M2 phenotype. Furthermore, BV2 cells were treated with LPS, LPS + loganin, LPS + loganin+MLA, or LPS + loganin+MLA+ rapamycin to obtain conditioned medium (CM) for stimulating primary neurons. Loganin reduced the damage of primary neurons caused by LPS-stimulated BV2 microglia through activating a7nAchR and inducing autophagy activation. In conclusion, loganin played anti-inflammatory and neuroprotective roles in pMCAO + fracture mice by activating a7nAchR, enhancing autophagy and promoting M2 polarization of microglia.

Unilateral cerebral ischemia induces morphological changes in the layer V projection neurons of the contralateral hemisphere.

Decreased blood flow to the brain causes stroke and damage to neuronal networks. Neuronal damage occurs not only in the infarct core but also in areas away from the infarcts. This study was aimed to assess alterations of the cortical projection neurons that were distantly connected with the infarcts. Unilateral cortical ischemia was generated by middle cerebral artery occlusion in the right somatosensory cortex. Pre-labeled thalamocortical neurons disappeared, whereas contralateral callosal projection neurons survived 48 h post-ischemia. The unilateral ischemia increased the total length, segment length and the spine volume of dendrites from layer V callosal neurons in the homotopic cortex of the contralateral hemisphere. The morphological remolding of the contralateral cortical neurons cannot be reproduced by the spinal cord hemisection that cuts axons of corticospinal projection neurons of layer V. The data suggest that the retrograde degeneration of axons may not account for the early morphological changes in the contralateral cortex. We hypothesize that the loss of innervations from the ischemic cortex may bring in adaptive changes to the connected neurons, and adult cortical neurons can adjust their morphology to meet the reduction of synaptic inputs. This study may improve our understanding of the re-organization of cortical circuits following focal cerebral ischemia and help the development of new treatments designed to minimize the disability associated with stroke.

Resolution of inflammation is disturbed in acute ischemic stroke with diabetes mellitus and rescued by resolvin D2 treatment.

BACKGROUND: Inflammation plays an important role in diabetes mellitus (DM)-related acute ischemic stroke (AIS). The mechanisms of un-resolved inflammation in DM-related AIS are not fully understood. Specialized pro-resolving mediators (SPMs) are key regulators that promote resolution of inflammation. We aimed to examine resolution function in patients with AIS complicated with DM, and explore potential treatment effects of one of the SPMs, resolvin D2 (RvD2) ex vivo and in vivo. METHODS: Cultured human macrophages, which were derived from peripheral blood mononuclear cells of AIS and none-AIS patients with or without DM, were stimulated with oxidized-low density lipoprotein (ox-LDL). Levels of SPMs and inflammatory markers were analysed, and RvD2 treatment effects were evaluated in these cells. For experiments in vivo, challenges with high fat diet and low-dose streptozotocin (STZ) were used to induce DM in C57BL/6J mice. AIS model was established by permanent middle cerebral artery occlusion (pMCAO) followed by intra-cerebroventricular injection of RvD2. RESULTS: Compared with macrophages of AIS patients without DM, the ratios of SPMs to leukotriene B4 (LTB4) were decreased in AIS patients with DM, accompanied by reduced expression of SPM synthesis enzyme, 15-lipoxygenase-1. Moreover, the levels of pro-inflammatory pathway markers were increased, and the macrophages were skewed to M1 polarization in AIS patients with DM. In mice, treatment with RvD2 ameliorated pMCAO-induced brain injury, neurological dysfunction, and inflammatory response. Furthermore, RvD2 rescued resolution of inflammation by promoting macrophage/microglia polarization to pro-resolving M2 phenotype ex vivo and in vivo. CONCLUSIONS: Our data demonstrate resolution of inflammation is impaired by DM in AIS patients, implicating a novel mechanism of un-resolved inflammation in DM-related AIS. Furthermore, RvD2 promotes inflammation resolution in macrophages/microglia and protects DM-related AIS, and may thus serve as a novel therapeutic target.

Imeglimin Is Neuroprotective Against Ischemic Brain Injury in Rats-a Study Evaluating Neuroinflammation and Mitochondrial Functions.

Imeglimin is a novel oral antidiabetic drug modulating mitochondrial functions. However, neuroprotective effects of this drug have not been investigated. The aim of this study was to investigate effects of imeglimin against ischemia-induced brain damage and neurological deficits and whether it acted via inhibition of mitochondrial permeability transition pore (mPTP) and suppression of microglial activation. Ischemia in rats was induced by permanent middle cerebral artery occlusion (pMCAO) for 48 h. Imeglimin (135 µg/kg/day) was injected intraperitoneally immediately after pMCAO and repeated after 24 h. Immunohistochemical staining was used to evaluate total numbers of neurons, astrocytes, and microglia as well as interleukin-10 (IL-10) producing cells in brain slices. Respiration of isolated brain mitochondria was assessed using high-resolution respirometry. Assessment of ionomycin-induced mPTP opening in intact cultured primary rat neuronal, astrocytic, and microglial cells was performed using fluorescence microscopy. Treatment with imeglimin significantly decreased infarct size, brain edema, and neurological deficits after pMCAO. Moreover, imeglimin protected against pMCAO-induced neuronal loss as well as microglial proliferation and activation, and increased the number of astrocytes and the number of cells producing anti-inflammatory cytokine IL-10 in the ischemic hemisphere. Imeglimin in vitro acutely prevented mPTP opening in cultured neurons and astrocytes but not in microglial cells; however, treatment with imeglimin did not prevent ischemia-induced mitochondrial respiratory dysfunction after pMCAO. This study demonstrates that post-stroke treatment with imeglimin exerts neuroprotective effects by reducing infarct size and neuronal loss possibly via the resolution of neuroinflammation and partly via inhibition of mPTP opening in neurons and astrocytes.

Neurovascular protection by adropin in experimental ischemic stroke through an endothelial nitric oxide synthase-dependent mechanism.

Adropin is a highly-conserved peptide that has been shown to preserve endothelial barrier function. Blood-brain barrier (BBB) disruption is a key pathological event in cerebral ischemia. However, the effects of adropin on ischemic stroke outcomes remain unexplored. Hypothesizing that adropin exerts neuroprotective effects by maintaining BBB integrity, we investigated the role of adropin in stroke pathology utilizing loss- and gain-of-function genetic approaches combined with pharmacological treatment with synthetic adropin peptide. Long-term anatomical and functional outcomes were evaluated using histology, MRI, and a battery of sensorimotor and cognitive tests in mice subjected to ischemic stroke. Brain ischemia decreased endogenous adropin levels in the brain and plasma. Adropin treatment or transgenic adropin overexpression robustly reduced brain injury and improved long-term sensorimotor and cognitive function in young and aged mice subjected to ischemic stroke. In contrast, genetic deletion of adropin exacerbated ischemic brain injury, irrespective of sex. Mechanistically, adropin treatment reduced BBB damage, degradation of tight junction proteins, matrix metalloproteinase-9 activity, oxidative stress, and infiltration of neutrophils into the ischemic brain. Adropin significantly increased phosphorylation of endothelial nitric oxide synthase (eNOS), Akt, and ERK1/2. While adropin therapy was remarkably protective in wild-type mice, it failed to reduce brain injury in eNOS-deficient animals, suggesting that eNOS is required for the protective effects of adropin in stroke. These data provide the first causal evidence that adropin exerts neurovascular protection in stroke through an eNOS-dependent mechanism. We identify adropin as a novel neuroprotective peptide with the potential to improve stroke outcomes.

Evaluation of the Effectiveness of Post-Stroke Metformin Treatment Using Permanent Middle Cerebral Artery Occlusion in Rats.

Stroke is the second leading cause of death worldwide. Treatment options for ischemic stroke are limited, and the development of new therapeutic agents or combined therapies is imperative. Growing evidence suggests that metformin treatment, due to its anti-inflammatory action, exerts a neuroprotective effect against ischemia/reperfusion-induced brain damage. Experimental assessment has typically been performed in models of cerebral transient ischemia followed by long-term reperfusion. The aim of this study was to evaluate the neuroprotective effect of metformin treatment after permanent middle cerebral artery occlusion (pMCAO) without reperfusion in rats. Neurological deficits were assessed using the Longa scale, which offers a graded scale on body movement following pMCAO. Both infarct size and brain oedema area were measured by staining with 2,3,5-triphenyltetrazolium chloride. The number of neurons and total and activated microglia, as well as interleukin 10 (IL-10) production, in brain sections were evaluated by immunohistochemical staining. Our results show that metformin treatment improves the neurological state and reduces infarct size after 120 h of pMCAO. Metformin also prevents neuronal loss in the ischemic cortex but not in the striatum after 48 h of pMCAO. Moreover, post-stroke treatment with metformin significantly decreases the number of total and activated microglia at 48 h. The anti-inflammatory effect of metformin is associated with increased IL-10 production at 48 h after pMCAO. The results of the present study suggest that post-stroke treatment with metformin exerts anti-inflammatory and neuroprotective effects in a pMCAO model.

Benzoinum from Styrax tonkinensis (Pierre) Craib ex Hart exerts a NVU protective effect by inhibiting cell apoptosis in cerebral ischaemia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Benzoinum (Styraceae) is a traditional Chinese medicine used to treat stroke and other cardio-cerebrovascular diseases for thousands of years. Benzoinum has also proven to have diverse pharmacological activity, but the neuroprotection mechanism of apoptosis in ischaemic stroke was not determined. AIM OF THIS STUDY: To investigate the protective effect of a neurovascular unit (NVU) and the mechanisms of benzoinum on cerebral ischaemic rats. MATERIALS AND METHODS: The neuroprotective activity of benzoinum against middle cerebral artery occlusion (MCAO)-induced cerebral ischaemic injury. Neurological scores, 2,3,5-Triphenyltetrazolium chloride (TTC) staining, and hematoxylin-eosin staining (HE) staining were conducted to evaluate the neurological damage. Infarction rate and denatured cell index (DCI) were also calculated. The ultrastructure of neuron and blood-brain-barrier (BBB) was observed by transmission electron microscopy (TEM). Immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect Bax, Bcl-2 and Caspase 3 expression. Furthermore, Claudin 5 also was detected through immunohistochemistry. RESULTS: Benzoinum could significantly improve neurological function score and reduce cerebral infarction rate and DCI. In addition, benzoinum alleviated pathomorphological change and apoptosis in the brain tissue of MCAO rats. The results of TEM and claudin 5 expression of immunohistochemistry showed that benzoinum could play a neuroprotective effect in NVU. Also, benzoinum-enhanced Bcl2, and reduced Bax and Bax/Bcl-2 and Caspase 3, suggest that benzoinum provided a neuroprotective effect by inhibited cell apoptosis. CONCLUSION: Benzoinum could play a neuroprotective role and regulate apoptosis for repair and stabilisation of NVU. This anti-apoptosis activity might be associated with the downregulation of Bax and Caspase 3, and the upregulation of Bcl2. Our present findings provide a promising medication for the treatment of ischaemic stroke.

Early Reperfusion Following Ischemic Stroke Provides Beneficial Effects, Even After Lethal Ischemia with Mature Neural Cell Death.

Ischemic stroke is a critical disease caused by cerebral artery occlusion in the central nervous system (CNS). Recent therapeutic advances, such as neuroendovascular intervention and thrombolytic therapy, have allowed recanalization of occluded brain arteries in an increasing number of stroke patients. Although previous studies have focused on rescuing neural cells that still survive despite decreased blood flow, expanding the therapeutic time window may allow more patients to undergo reperfusion in the near future, even after lethal ischemia, which is characterized by death of mature neural cells, such as neurons and glia. However, it remains unclear whether early reperfusion following lethal ischemia results in positive outcomes. The present study used two ischemic mouse models-90-min transient middle cerebral artery occlusion (t-MCAO) paired with reperfusion to induce lethal ischemia and permanent middle cerebral artery occlusion (p-MCAO)-to investigate the effect of early reperfusion up to 8 w following MCAO. Although early reperfusion following 90-min t-MCAO did not rescue mature neural cells, it preserved the vascular cells within the ischemic areas at 1 d following 90-min t-MCAO compared to that following p-MCAO. In addition, early reperfusion facilitated the healing processes, including not only vascular but also neural repair, during acute and chronic periods and improved recovery. Furthermore, compared with p-MCAO, early reperfusion after t-MCAO prevented behavioral symptoms of neurological deficits without increasing negative complications, including hemorrhagic transformation and mortality. These results indicate that early reperfusion provides beneficial effects presumably via cytoprotective and regenerative mechanisms in the CNS, suggesting that it may be useful for stroke patients that experienced lethal ischemia.

An enriched environment increases the expression of fibronectin type III domain-containing protein 5 and brain-derived neurotrophic factor in the cerebral cortex of the ischemic mouse brain.

Many studies have shown that fibronectin type III domain-containing protein 5 (FDNC5) and brain-derived neurotrophic factor (BDNF) play vital roles in plasticity after brain injury. An enriched environment refers to an environment that provides animals with multi-sensory stimulation and movement opportunities. An enriched environment has been shown to promote the regeneration of nerve cells, synapses, and blood vessels in the animal brain after cerebral ischemia; however, the exact mechanisms have not been clarified. This study aimed to determine whether an enriched environment could improve neurobehavioral functions after the experimental inducement of cerebral ischemia and whether neurobehavioral outcomes were associated with the expression of FDNC5 and BDNF. This study established ischemic mouse models using permanent middle cerebral artery occlusion (pMCAO) on the left side. On postoperative day 1, the mice were randomly assigned to either enriched environment or standard housing condition groups. Mice in the standard housing condition group were housed and fed under standard conditions. Mice in the enriched environment group were housed in a large cage, containing various toys, and fed with a standard diet. Sham-operated mice received the same procedure, but without artery occlusion, and were housed and fed under standard conditions. On postoperative days 7 and 14, a beam-walking test was used to assess coordination, balance, and spatial learning. On postoperative days 16-20, a Morris water maze test was used to assess spatial learning and memory. On postoperative day 15, the expression levels of FDNC5 and BDNF proteins in the ipsilateral cerebral cortex were analyzed by western blot assay. The results showed that compared with the standard housing condition group, the motor balance and coordination functions (based on beam-walking test scores 7 and 14 days after operation), spatial learning abilities (based on the spatial learning scores from the Morris water maze test 16-19 days after operation), and memory abilities (based on the memory scores of the Morris water maze test 20 days after operation) of the enriched environment group improved significantly. In addition, the expression levels of FDNC5 and BDNF proteins in the ipsilateral cerebral cortex increased in the enriched environment group compared with those in the standard housing condition group. Furthermore, the Pearson correlation coefficient showed that neurobehavioral functions were positively associated with the expression levels of FDNC5 and BDNF (r = 0.587 and r = 0.840, respectively). These findings suggest that an enriched environment upregulates FDNC5 protein expression in the ipsilateral cerebral cortex after cerebral ischemia, which then activates BDNF protein expression, improving neurological function. BDNF protein expression was positively correlated with improved neurological function. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Fudan University, China (approval Nos. 20160858A232, 20160860A234) on February 24, 2016.

CCR2 deficiency in monocytes impairs angiogenesis and functional recovery after ischemic stroke in mice.

Inflammatory Ly6ChiCCR2+ monocytes infiltrate the brain after stroke but their functions are not entirely clear. We report that CCR2+ monocytes and CCR2+ lymphocytes infiltrate the brain after permanent ischemia. To underscore the role of CCR2+ monocytes, we generated mice with selective CCR2 deletion in monocytes. One day post-ischemia, these mice showed less infiltrating monocytes and reduced expression of pro-inflammatory cytokines, markers of alternatively macrophage activation, and angiogenesis. Accordingly, Ly6Chi monocytes sorted from the brain of wild type mice 24 h post-ischemia expressed pro-inflammatory genes, M2 genes, and pro-angiogenic genes. Flow cytometry showed heterogeneous phenotypes within the infiltrating Ly6ChiCCR2+ monocytes, including a subgroup of Arginase-1+ cells. Mice with CCR2-deficient monocytes displayed a delayed inflammatory rebound 15 days post-ischemia that was not found in wild type mice. Furthermore, they showed reduced angiogenesis and worse behavioral performance. Administration of CCR2+/+ bone-marrow monocytes to mice with CCR2-deficient monocytes did not improve the behavioral performance suggesting that immature bone-marrow monocytes lack pro-reparative functions. The results show that CCR2+ monocytes contribute to acute post-ischemic inflammation and participate in functional recovery. The study unravels heterogeneity in the population of CCR2+ monocytes infiltrating the ischemic brain and suggests that pro-reparative monocyte subsets promote functional recovery after ischemic stroke.

Effect of Experimental Ischemic Stroke and PGE2 EP1 Selective Antagonism in Alzheimer's Disease Mouse Models.

BACKGROUND: Neuroinflammation has been recognized as an important factor in the pathogenesis of Alzheimer's disease (AD). One of the most recognized pathways in mediating neuroinflammation is the prostaglandin E2-EP1 receptor pathway. OBJECTIVE: Here, we examined the efficacy of the selective EP1 antagonist ONO-8713 in limiting amyloid-ß (Aß), lesion volumes, and behavioral indexes in AD mouse models after ischemic stroke. METHODS: Transgenic APP/PS1, 3xTgAD, and wildtype (WT) mice were subjected to permanent distal middle cerebral artery occlusion (pdMCAO) and sham surgeries. Functional outcomes, memory, anatomical outcomes, and Aß concentrations were assessed 14 days after surgery. RESULTS: pdMCAO resulted in significant deterioration in functional and anatomical outcomes in the transgenic mice compared with the WT mice. No relevant differences were observed in the behavioral tests when comparing the ONO-8713 and vehicle-treated groups. Significantly lower cavitation (p = 0.0373) and percent tissue loss (p = 0.0247) were observed in APP/PS1 + ONO-8713 mice compared with the WT + ONO-8713 mice. However, the percent tissue injury was significantly higher in APP/PS1 + ONO-8713 mice compared with the WT + ONO-8713 group (p = 0.0373). Percent tissue loss was also significantly lower in the 3xTgAD + ONO-8713 mice than in the WT + ONO-8713 mice (p = 0.0185). ONO-8713 treatment also attenuated cortical microgliosis in APP/PS1 mice as compared with the vehicle (p = 0.0079); however, no differences were observed in astrogliosis across the groups. Finally, APP/PS1 mice presented with characteristic Aß load in the cortex while 3xTgAD mice exhibited very low Aß levels. CONCLUSION: In conclusion, under the experimental conditions, EP1 receptor antagonist ONO-8713 showed modest benefits in anatomical outcomes after stroke, mainly in APP/PS1 mice.

Semi-Automated Cell and Tissue Analyses Reveal Regionally Specific Morphological Alterations of Immune and Neural Cells in a Porcine Middle Cerebral Artery Occlusion Model of Stroke.

Histopathological analysis of cellular changes in the stroked brain provides critical information pertaining to inflammation, cell death, glial scarring, and other dynamic injury and recovery responses. However, commonly used manual approaches are hindered by limitations in speed, accuracy, bias, and the breadth of morphological information that can be obtained. Here, a semi-automated high-content imaging (HCI) and CellProfiler histological analysis method was developed and used in a Yucatan miniature pig permanent middle cerebral artery occlusion (pMCAO) model of ischemic stroke to overcome these limitations. Evaluation of 19 morphological parameters in IBA1+ microglia/macrophages, GFAP+ astrocytes, NeuN+ neuronal, FactorVIII+ vascular endothelial, and DCX+ neuroblast cell areas was conducted on porcine brain tissue 4 weeks post pMCAO. Out of 19 morphological parameters assessed in the stroke perilesional and ipsilateral hemisphere regions (38 parameters), a significant change in 38 38 measured IBA1+ parameters, 34 38   GFAP+ parameters, 32 38 NeuN+ parameters, 31 38 FactorVIII+ parameters, and 28 38 DCX+ parameters were observed in stroked vs. non-stroked animals. Principal component analysis (PCA) and correlation analyses demonstrated that stroke-induced significant and predictable morphological changes that demonstrated strong relationships between IBA1+, GFAP+, and NeuN+ areas. Ultimately, this unbiased, semi-automated HCI and CellProfiler histopathological analysis approach revealed regional and cell specific morphological signatures of immune and neural cells after stroke in a highly translational porcine model. These identified features can provide information of disease pathogenesis and evolution with high resolution, as well as be used in therapeutic screening applications.

Role of rno-miR-124-3p in regulating MCT1 expression in rat brain after permanent focal cerebral ischemia.

This study aimed to assess the role of microRNAs (miRNAs) in regulating monocarboxylate transporter-1 (MCT1) expression in rat brain after permanent focal cerebral ischemia to identify a new target for early treatment of cerebral ischemia. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO) in rats. Morphology and protein expression levels of MCT1 were assessed by immunofluorescence and Western blotting. Using bioinformatics and double luciferase reporter assays, rno-miR-124-3p was selected as a direct target for rat MCT1. Expression of rno-miR-124-3p after pMCAO was detected. Then, rats were treated with rno-miR-124-3p agomir via lateral ventricle injection, and after 6 h or 24 h ischemia, rno-miR-124-3p expression and gene and protein expression of MCT-1 were detected by qRT-PCR and Western blotting. Brain infarction was identified by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Results showed that pMCAO induced brain infarction and increased the expression of MCT1. The levels of rno-miR-124-3p after pMCAO were in contrast to those of MCT1 protein in ischemic region, while declined after 3, 6 and 12 h of pMCAO in ischemic penumbra. After administration of rno-miR-124-3p agomir, MCT1 mRNA and protein levels were increased after 6 h of pMCAO, while decreased after 24 h of pMCAO. Meanwhile, rno-miR-124-3p levels increased after both times. TTC staining showed treatment with rno-miR-124-3p agomir reduced brain infarction. The role of rno-miR-124-3p in regulating MCT1 was as a positive regulator after 6 h of pMCAO, while a negative regulator after 24 h of pMCAO, however, both activities had protective effects against cerebral ischemia.

Catalpol Enhances Neurogenesis And Inhibits Apoptosis Of New Neurons Via BDNF, But Not The BDNF/Trkb Pathway.

BACKGROUND: The role of catalpol in brain neurogenesis and newborn neuron survival has not been previously determined in permanent middle cerebral artery occlusion (pMCAO). METHODS: Fifty-four rats were divided into 6 groups: pMCAO (model, n=9); sham operation (NS, n=9); catalpol treatment (5 mg/kg and 10 mg/kg subgroups, n=9 each); K252a (n=9); and K252a+catalpol 5 mg/kg (n=9) with stroke. The effects of catalpol on behavior, neurogenesis surrounding the infarction ipsilateral to pMCAO, and the expression of brain-derived neurotrophic factor (BDNF) and its receptor (TrkB) were evaluated. Vehicle or, K252a (i.p.), an inhibitor of TrkB phosphorylase. RESULTS: Repeated administration of catalpol reduced neurological deficits and significantly improved neurogenesis. Catalpol increased the number of newborn immature neurons, as determined by BrdU+-Nestin+ and BrdU+-Tuj-1+ staining, and downregulated cleaved caspase 3 in Tuj-1+ cells at day 7 following stroke. Moreover, catalpol increased the protein expression of Tuj-1, MAP2, and the Bcl-2/Bax ratio, as determined using Western blot. Catalpol also significantly increased brain levels of BDNF, but not TrkB, resulting in enhanced survival of newborn neurons via inhibition of apoptosis. CONCLUSION: Catalpol may contribute to neurogenesis in infarcted brain regions and help promote the survival of newborn neurons by activating BDNF, but not BDNF/TrkB signaling.

Piperine as a neuroprotective functional component in rats with cerebral ischemic injury.

Long pepper (Piper longum L.) and black pepper (Piper nigrum L.) plants are commonly used as spices around the world and have also been postulated to have medicinal effects. Piperine, as the major alkaloid of P. nigrum and P. longum, has gained wide attention of the medical community and culinary enthusiasts. This study seeks to determine the effects of piperine on neuronal apoptosis in peri-infarcted cerebral cortices of rats with permanent middle cerebral artery occlusion (pMCAO) injury. Evaluation of the different behavioral components was conducted after pMCAO. 2, 3, 5-Triphenyltetrazolium chloride (TTC) was used to evaluate the area of cortical ischemia. Gross histopathological changes, as well as microscopic neuronal changes, were observed in brain tissue samples. The protein expression of Caspase-3, Caspase-9, Bax, Bcl-2, and Cytochrome C (Cyt-c) was analyzed using western blotting. The findings reveal that rats that received piperine treatment show markedly decreased neurological deficit, less ischemia-induced cellular damage, as well as smaller areas of cerebral infarction, with less severe macro and microcellular cerebral structural changes. Western blotting analysis reveals that piperine administration inhibits Bax, while enhancing Bcl-2 expression. The protein expression of Caspase-3, Caspase-9, and Cyt-c was also found to be significantly inhibited. We conclude that piperine may provide several beneficial neuroprotective effects that warrant further investigation.