BACKGROUND AND PURPOSE—Blood-brain barrier (BBB) disruption is a critical pathological feature after stroke. MicroRNA-126 (miR-126) maintains BBB integrity by regulating endothelial cell function ...during development. However, the role of miR-126-3p and -5p in BBB integrity after stroke is unclear. Here, we investigated whether miR-126-3p and -5p overexpression regulates BBB integrity after cerebral ischemia.
METHODS—A lentivirus carrying genes encoding miR-126-3p or -5p was stereotactically injected into adult male Institute of Cancer Research mouse brains (n=36). Permanent middle cerebral artery occlusion was performed 2 weeks after virus injection. Brain infarct volume, edema volume, and modified neurological severity score were assessed at 1 and 3 days after ischemia. Immunostaining of ZO-1 (zonula occludens-1) and occludin was used to evaluate BBB integrity. IL-1β (interleukin-1β), TNF-α (tumor necrosis factor-α), VCAM-1 (vascular cell adhesion molecule-1), and E-selectin expression levels were determined by real-time polymerase chain reaction and Western blot analysis.
RESULTS—The expression of miR-126-3p and -5p decreased at 1 and 3 days after ischemia (P<0.05). Injection of lentiviral miR-126-3p or -5p reduced brain infarct volume and edema volume (P<0.05) and attenuated the decrease in ZO-1/occludin protein levels and IgG leakage at 3 days after stroke (P<0.05). Injection of lentiviral miR-126-5p improved behavioral outcomes at 3 days after stroke (P<0.05). miR-126-3p and -5p overexpression downregulated the expression of proinflammatory cytokines IL-1β and TNF-α and adhesion molecules VCAM-1 and E-selectin, as well as decreased MPO (myeloperoxidase positive) cell numbers at 3 days after ischemia (P<0.05).
CONCLUSIONS—miR-126-3p and -5p overexpression reduced the expression of proinflammatory cytokines and adhesion molecules, and attenuated BBB disruption after ischemic stroke, suggesting that miR-126-3p and -5p are new therapeutic targets in the acute stage of stroke.
Microalgae cultivation in wastewater has received increasing attention in recent years due to its many advantages. In this work, microalgae were cultured in seafood processing wastewater (SPW) for ...algal biomass and lipid production as well as nutrient removal. The biomass yield of Chlorella sp. achieved in the batch cultivation was 896 mg L−1, indicating that SPW contains a certain amount of nutrients which can be used for the growth of microalgae. However, the maximum specific growth rate of Chlorella sp. cultured in SPW throughout the whole cultivation period was only 0.040 d−1, suggesting that the growth of algal cells was inhibited during the culture process. High concentration of unionized ammonia in the SPW was found to be a factor inhibiting the growth of Chlorella sp. Aerated SPW (ASPW) and diluted SPW (DSPW) proved to be better culture media than SPW without pretreatment. The maximum specific growth rates of Chlorella sp. cultured in ASPW and DSPW during the culture interval were 0.156 and 0.091 d−1, respectively. Aeration pretreatment of SPW reduced the amount of toxic unionized ammonia, while most of the nutrients were retained in the wastewater. Therefore, higher biomass productivity (77.7 mg L−1 d−1) and higher lipid productivity (20.4 mg L−1 d−1) of microalgae were achieved in ASPW. Additionally, improved nutrient removal rates from ASPW were also achieved due to the faster growth of microalgae. The average nutrient removal rates in ASPW during the whole cultivation period were 4.98 and 1.91 mg L−1 d−1 for nitrogen and phosphorus, respectively.
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•Seafood processing wastewater (SPW) was used to produce algal biomass/lipid.•NH3-N concentration in SPW increased rapidly in the first few days of cultivation.•Aeration pretreatment could reduce the amount of toxic unionized ammonia in SPW.•Aerated SPW supported the largest algal biomass and lipid productivity.•Assimilation of microalgae removed only a small fraction of phosphorus in wastewater.
Ischemic stroke is a primary vascular disease of the central nervous system characterized by high morbidity, mortality, and healthcare costs. As conventional ischemic stroke models fail to predict ...therapeutic efficacy, in vitro neurovascular unit (NVU)/blood–brain barrier (BBB) models are utilized to model ischemic stroke through replicating the cell–cell interactions and mimicking the blood flow and anatomical features of the brain. Here, an overview of transwell, microfluidic, and hydrogel‐based NVU/BBB models is provided, including cell types, engineering approaches, and the simulation of physiological and pathological features of NVU/BBB after ischemic stroke. Collectively, recent advances in 3D‐printed NVU models are emphasized, which are anticipated to be a promising system for more reliable mechanistic studies and preclinical drug screenings that can eventually accelerate the drug development process for the ischemic stroke therapy.
In vitro neurovascular unit (NVU)/blood–brain barrier (BBB) models are utilized to model ischemic stroke through replicating the cell–cell interactions and mimicking the blood flow and anatomical features. An overview of transwell, brain‐on‐a‐chip, 3D hydrogel/bioprinted, and spheroid models is provided, including cell types, engineering approaches, and the simulation of physiological and pathological features of NVU/BBB after ischemic stroke.
Metformin, a widely used hypoglycemic drug, reduces stroke incidence and alleviates chronic inflammation in clinical trials. However, the effect of metformin in ischemic stroke is unclear. Here, we ...investigated the effect of metformin on ischemic stroke in mice and further explored the possible underlying mechanisms.
Ninety-eight adult male CD-1 mice underwent 90-minute transient middle cerebral artery occlusion (tMCAO). Metformin (200 mg/kg) was administrated for up to 14 days. Neurobehavioral outcomes, brain infarct volume, inflammatory factors, blood-brain barrier (BBB) permeability and AMPK signaling pathways were evaluated following tMCAO. Oxygen glucose deprivation was performed on bEND.3 cells to explore the mechanisms of metformin in inhibiting inflammatory signaling pathways.
Infarct volume was reduced in metformin-treated mice compared to the control group following tMCAO (P < 0.05). Neurobehavioral outcomes were greatly improved in metformin-treated mice (P < 0.05). MPO+ cells, Gr1+ cells, MPO activity and BBB permeability were decreased after metformin administration (P < 0.05). In addition, metformin activated AMPK phosphorylation, inhibited NF-κB activation, down-regulated cytokine (IL-1β, IL-6, TNF-α) and ICAM-1 expression following tMCAO (P < 0.05). Furthermore, metformin activated AMPK signaling pathway and alleviated oxygen-glucose deprivation-induced ICAM-1 expression in bEND.3 cells (P < 0.05). Compound C, a selective AMPK inhibitor, eliminated this promotional effect.
Metformin down-regulated ICAM-1 in an AMPK-dependent manner, which could effectively prevent ischemia-induced brain injury by alleviating neutrophil infiltration, suggesting that metformin is a promising therapeutic agent in stroke therapy.
Mesenchymal stem cell (MSC) transplantation has been shown to be beneficial in treating cerebral ischemia. However, such benefit is limited by the low survival of transplanted MSCs in an ischemic ...microenvironment. Previous studies showed that melatonin pretreatment can increase MSC survival in the ischemic kidney. However, whether it will improve MSC survival in cerebral ischemia is unknown. Our study examined the effect of melatonin pretreatment on MSCs under ischemia-related conditions in vitro and after transplantation into ischemic rat brain. Results showed that melatonin pretreatment greatly increased survival of MSCs in vitro and reduced their apoptosis after transplantation into ischemic brain. Melatonin-treated MSCs (MT-MSCs) further reduced brain infarction and improved neurobehavioral outcomes. Angiogenesis, neurogenesis, and the expression of vascular endothelial growth factor (VEGF) were greatly increased in the MT-MSC-treated rats. Melatonin treatment increased the level of p-ERK1/2 in MSCs, which can be blocked by the melatonin receptor antagonist luzindole. ERK phosphorylation inhibitor U0126 completely reversed the protective effects of melatonin, suggesting that melatonin improves MSC survival and function through activating the ERK1/2 signaling pathway. Thus, stem cells pretreated by melatonin may represent a feasible approach for improving the beneficial effects of stem cell therapy for cerebral ischemia.
Three‐dimensional (3D) bioprinting is driving significant innovations in biomedicine over recent years. Under certain scenarios such as in intraoperative bioprinting, the bioinks used should exhibit ...not only cyto/biocompatibility but also adhesiveness in wet conditions. Herein, an adhesive bioink composed of gelatin methacryloyl, gelatin, methacrylated hyaluronic acid, and skin secretion of Andrias davidianus is designed. The bioink exhibits favorable cohesion to allow faithful extrusion bioprinting in wet conditions, while simultaneously showing good adhesion to a variety of surfaces of different chemical properties, possibly achieved through the diverse bonds presented in the bioink formulation. As such, this bioink is able to fabricate sophisticated planar and volumetric constructs using extrusion bioprinting, where the dexterity is further enhanced using ergonomic handheld bioprinters to realize in situ bioprinting. In vitro experiments reveal that cells maintain high viability; further in vivo studies demonstrate good integration and immediate injury sealing. The characteristics of the bioink indicate its potential widespread utility in extrusion bioprinting and will likely broaden the applications of bioprinting toward situations such as in situ dressing and minimally invasive tissue regeneration.
A multi‐component bioink is designed, to exhibit favorable cohesion to allow faithful extrusion bioprinting under wet conditions while simultaneously showing good adhesion to a variety of surfaces. The characteristics of the bioink indicate its potential widespread utility in extrusion bioprinting and will likely broaden the applications of bioprinting toward situations such as in situ dressing and minimally invasive tissue regeneration.
Summary
Introduction
Dl‐3‐N‐butylphthalide (NBP), a small molecule drug used clinically in the acute phase of ischemic stroke, has been shown to improve functional recovery and promote angiogenesis ...and collateral vessel circulation after experimental cerebral ischemia. However, the underlying molecular mechanism is unknown.
Aims
To explore the potential molecular mechanism of angiogenesis induced by NBP after cerebral ischemia.
Results
NBP treatment attenuated body weight loss, reduced brain infarct volume, and improved neurobehavioral outcomes during focal ischemia compared to the control rats (P < 0.05). NBP increased the number of CD31+ microvessels, the number of CD31+/BrdU+ proliferating endothelial cells, and the functional vascular density (P < 0.05). Further study demonstrated that NBP also promoted the expression of vascular endothelial growth factor and angiopoietin‐1 (P < 0.05), which was accompanied by upregulated sonic hedgehog expression in astrocytes in vivo and in vitro.
Conclusion
NBP treatment promoted the expression of vascular endothelial growth factor and angiopoietin‐1, induced angiogenesis, and improved neurobehavioral recovery. These effects were associated with increased sonic hedgehog expression after NBP treatment. Our results broadened the clinical application of NBP to include the later phase of ischemia.
Inflammatory response plays a critical role in propagating tissue damage after focal cerebral ischemia. CXCL12 is a key chemokine for leukocyte recruitment. However, the role of CXCL12 and its ...receptor CXCR4 in ischemia-induced inflammatory response is unclear. Here we use the pharmacological antagonist of CXCR4, AMD3100, to investigate the function of CXCL12/CXCR4 in regulating inflammatory response during acute ischemia.
Adult male CD-1 mice (n=184) underwent permanent suture middle cerebral artery occlusion (MCAO). AMD3100 was injected for 3 days (1 mg/kg/day) after MCAO. Brain water content, infarct volume, neurological score, and myeloperoxidase (MPO) expression and activity were examined at 24, 48, and 72 hours after MCAO. Proinflammatory cytokine RNA and protein levels in brain tissue were measured by RT-PCR and enzyme linked immunosorbent assay.
Neurological score was greatly improved in AMD3100-treated mice compared with the control mice 3 days after MCAO (P<0.05). Brain edema-induced change of water content, IgG protein leakage, Evans blue extravasation, occludin, and ZO-1 expression in ipsilateral hemisphere were alleviated by acute treatment of AMD3100. MPO expression and activity revealed that AMD3100 profoundly reduced the number of MPO-positive cells in the ischemic region (P<0.05). It also attenuated proinflammatory cytokines including interleukin 6, tumor necrosis factor α, and interferon γ; their mRNA and protein levels changed accordingly compared with the controls (P<0.05).
CXCR4 antagonist AMD3100 significantly suppressed inflammatory response and reduced blood-brain barrier disruption after MCAO. AMD3100 attenuated ischemia-induced acute inflammation by suppressing leukocyte migration and infiltration, in addition to reducing proinflammatory cytokine expression in the ischemic region.
Matrix metalloproteinase 9 (MMP-9) plays a beneficial role in the delayed phase of middle cerebral artery occlusion (MCAO). However, the mechanism is obscure. Here, we constructed hypoxia response ...element (HRE)-regulated MMP-9 to explore its effect on glial scars and neurogenesis in delayed ischemic stroke. Adult male Institute of Cancer Research (ICR) mice underwent MCAO and received a stereotactic injection of lentivirus carrying HRE-MMP-9 or normal saline (NS)/lentivirus-GFP 7 days after ischemia. We found that HRE-MMP-9 improved neurological outcomes, reduced ischemia-induced brain atrophy, and degraded glial scars (p < 0.05). Furthermore, HRE-MMP-9 increased the number of microvessels in the peri-infarct area (p < 0.001), which may have been due to the accumulation of endogenous endothelial progenitor cells (EPCs) in the peri-infarct area after glial scar degradation. Finally, HRE-MMP-9 increased the number of bromodeoxyuridine-positive (BrdU+)/NeuN+ cells and the expression of PSD-95 in the peri-infarct area (p < 0.01). These changes could be blocked by vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor SU5416 and MMP-9 inhibitor 2-(4-phenoxyphenyl)sulfonylmethyl-thiirane (SB-3CT). Our results provided a novel mechanism by which glial scar degradation and vascular endothelial growth factor (VEGF)/VEGFR2-dependent angiogenesis may be key procedures for neurological recovery in delayed ischemic stroke after HRE-MMP-9 treatment. Therefore, HRE-MMP-9 overexpression in the delayed ischemic brain is a promising approach for neurological recovery.
Our findings challenged the traditional view that MMP-9 overexpression is unsuitable for stroke treatment because it induces hemorrhage. We provided the first evidence to link glial scar degradation and angiogenesis/neurogenesis-related downstream molecular events after brain ischemia and proposed that HRE-MMP-9 may be a promising target for ischemia therapy.
Glial scars present a major obstacle for neuronal regeneration after stroke. Thus, approaches to promote their degradation and inhibit their formation are beneficial for stroke recovery. The ...interaction of microglia and astrocytes is known to be involved in glial scar formation after stroke; however, how microglia affect glial scar formation remains unclear.
Mice were treated daily with M2 microglial small extracellular vesicles through tail intravenous injections from day 1 to day 7 after middle cerebral artery occlusion. Glial scar, infarct volume, neurological score were detected after ischemia. microRNA and related protein were examined in peri-infarct areas of the brain following ischemia.
M2 microglial small extracellular vesicles reduced glial scar formation and promoted recovery after stroke and were enriched in miR-124. Furthermore, M2 microglial small extracellular vesicle treatment decreased the expression of the astrocyte proliferation gene signal transducer and activator of transcription 3, one of the targets of miR-124, and glial fibrillary acidic protein and inhibited astrocyte proliferation both
and
. It also decreased Notch 1 expression and increased Sox2 expression in astrocytes, which suggested that astrocytes had transformed into neuronal progenitor cells. Finally, miR-124 knockdown in M2 microglial small extracellular vesicles blocked their effects on glial scars and stroke recovery.
Our results showed, for the first time, that microglia regulate glial scar formation
small extracellular vesicles, indicating that M2 microglial small extracellular vesicles could represent a new therapeutic approach for stroke.
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