Acute ischemia induces spatially and transcriptionally distinct microglial subclusters.

BACKGROUND: Damage in the ischemic core and penumbra after stroke affects patient prognosis. Microglia immediately respond to ischemic insult and initiate immune inflammation, playing an important role in the cellular injury after stroke. However, the microglial heterogeneity and the mechanisms involved remain unclear. METHODS: We first performed single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics (ST) on middle cerebral artery occlusion (MCAO) mice from three time points to determine stroke-associated microglial subclusters and their spatial distributions. Furthermore, the expression of microglial subcluster-specific marker genes and the localization of different microglial subclusters were verified on MCAO mice through RNAscope and immunofluorescence. Gene set variation analysis (GSVA) was performed to reveal functional characteristics of microglia sub-clusters. Additionally, ingenuity pathway analysis (IPA) was used to explore upstream regulators of microglial subclusters, which was confirmed by immunofluorescence, RT-qPCR, shRNA-mediated knockdown, and targeted metabolomics. Finally, the infarct size, neurological deficits, and neuronal apoptosis were evaluated in MCAO mice after manipulation of specific microglial subcluster. RESULTS: We discovered stroke-associated microglial subclusters in the brains of MCAO mice. We also identified novel marker genes of these microglial subclusters and defined these cells as ischemic core-associated (ICAM) and ischemic penumbra-associated (IPAM) microglia, according to their spatial distribution. ICAM, induced by damage-associated molecular patterns, are probably fueled by glycolysis, and exhibit increased pro-inflammatory cytokines and chemokines production. BACH1 is a key transcription factor driving ICAM generation. In contrast, glucocorticoids, which are enriched in the penumbra, likely trigger IPAM formation, which are presumably powered by the citrate cycle and oxidative phosphorylation and are characterized by moderate pro-inflammatory responses, inflammation-alleviating metabolic features, and myelinotrophic properties. CONCLUSIONS: ICAM could induce excessive neuroinflammation, aggravating brain injury, whereas IPAM probably exhibit neuroprotective features, which could be essential for the homeostasis and survival of cells in the penumbra. Our findings provide a biological basis for targeting specific microglial subclusters as a potential therapeutic strategy for ischemic stroke.

Pharmacological Upregulation of Microglial Lipid Droplet Alleviates Neuroinflammation and Acute Ischemic Brain Injury.

Lipid droplets (LDs) were reported to play an important role in the modulation of inflammation and various cellular processes among multiple cell types. However, LDs accumulation, its function and mechanisms of its formation during ischemic stroke remained poorly-identified. In this study, we observed increased LDs accumulation in microglia at the acute stage of ischemic stroke by immunofluorescence and flow cytometry. Transcriptomic analysis indicated that microglia accumulated with LDs were associated with inflammation and phagocytosis. Both inflammatory activation and phagocytosis of tissue debris in microglia could contribute to LDs formation. Moreover, through specific LDs depletion and overload experiments by pharmacological approaches, we proposed that LDs was critical for the maintenance of anti-inflammatory properties of microglia. Furthermore, Atglistatin, a specific adipose triglyceride lipase (ATGL) inhibitor, was shown to prevent proinflammatory cytokines production in primary microglia through decreased LDs lipolysis. After Atglistatin treatment, middle cerebral artery occlusion (MCAO) mice showed decreased infarct volume and improved neurobehavioral performance at the acute stage of stroke. Our findings provided a biological basis for microglial LDs regulation as a potential therapeutic strategy for acute ischemic stroke and uncovered the neuroprotective role of Atglistatin in the treatment of MCAO mice.

Optogenetic Stimulation of mPFC Alleviates White Matter Injury-Related Cognitive Decline after Chronic Ischemia through Adaptive Myelination.

White matter injury (WMI), which reflects myelin loss, contributes to cognitive decline or dementia caused by cerebral vascular diseases. However, because pharmacological agents specifically for WMI are lacking, novel therapeutic strategies need to be explored. It is recently found that adaptive myelination is required for homeostatic control of brain functions. In this study, adaptive myelination-related strategies are applied to explore the treatment for ischemic WMI-related cognitive dysfunction. Here, bilateral carotid artery stenosis (BCAS) is used to model ischemic WMI-related cognitive impairment and uncover that optogenetic and chemogenetic activation of glutamatergic neurons in the medial prefrontal cortex (mPFC) promote the differentiation of oligodendrocyte precursor cells (OPCs) in the corpus callosum, leading to improvements in myelin repair and working memory. Mechanistically, these neuromodulatory techniques exert a therapeutic effect by inducing the secretion of Wnt2 from activated neuronal axons, which acts on oligodendrocyte precursor cells and drives oligodendrogenesis and myelination. Thus, this study suggests that neuromodulation is a promising strategy for directing myelin repair and cognitive recovery through adaptive myelination in the context of ischemic WMI.

Ultrasensitive detection of butyrylcholinesterase activity based on self-polymerization modulated fluorescence of sulfur quantum dots.

Butyrylcholinesterase (BChE) is an important clinical diagnosing index for liver dysfunction and organophosphate toxicity. However, the current assays for BChE activity are suffering from the relative poor detection sensitivity. In this work, an ultrasensitive fluorescence assay for BChE activity was developed based on the self-polymerization modulated fluorescence of sulfur quantum dots (S-dots). The luminescence of S-dots can be quenched by the self-polymerized dopamine. The hydrolysate of substrates, thiocholine, under the catalysis of BChE can reduce dopamine, which results in the inhibition of self-polymerization and the fluorescence recovery of S-dots. BChE can be quantitatively detected by recording the recovered fluorescence of S-dots, and a linear relationship is observed between the ratio of fluorescence and the concentration of BChE in the range from 0.01 to 10 U/L. A limit of detection as low as 0.0069 U/L calculated, which is the lowest number so far. The assay also shows excellent selectivity towards various interference species and acetylcholinesterase. These features allowed the direct detection of BChE activity in human serum, demonstrating the great practical applications of our assay.

Adropin-based dual treatment enhances the therapeutic potential of mesenchymal stem cells in rat myocardial infarction.

Both weak survival ability of stem cells and hostile microenvironment are dual dilemma for cell therapy. Adropin, a bioactive substance, has been demonstrated to be cytoprotective. We therefore hypothesized that adropin may produce dual protective effects on the therapeutic potential of stem cells in myocardial infarction by employing an adropin-based dual treatment of promoting stem cell survival in vitro and modifying microenvironment in vivo. In the current study, adropin (25 ng/ml) in vitro reduced hydrogen peroxide-induced apoptosis in rat bone marrow mesenchymal stem cells (MSCs) and improved MSCs survival with increased phosphorylation of Akt and extracellular regulated protein kinases (ERK) l/2. Adropin-induced cytoprotection was blocked by the inhibitors of Akt and ERK1/2. The left main coronary artery of rats was ligated for 3 or 28 days to induce myocardial infarction. Bromodeoxyuridine (BrdU)-labeled MSCs, which were in vitro pretreated with adropin, were in vivo intramyocardially injected after ischemia, following an intravenous injection of 0.2 mg/kg adropin (dual treatment). Compared with MSCs transplantation alone, the dual treatment with adropin reported a higher level of interleukin-10, a lower level of tumor necrosis factor-α and interleukin-1ß in plasma at day 3, and higher left ventricular ejection fraction and expression of paracrine factors at day 28, with less myocardial fibrosis and higher capillary density, and produced more surviving BrdU-positive cells at day 3 and 28. In conclusion, our data evidence that adropin-based dual treatment may enhance the therapeutic potential of MSCs to repair myocardium through paracrine mechanism via the pro-survival pathways.

Xingnaojing ameliorates synaptic plasticity and memory deficits in an Aß1-42 induced mouse model of Alzheimer's disease.

The accumulation of insoluble amyloid ß (Aß) peptides is one of the pathological changes in Alzheimer's disease (AD), which induced synaptic plasticity impairment and excitatory amino acid toxicity associated with decreased memory function. Xingnaojing (XNJ), a well-known prescription in traditional Chinese medicine, has been used for the treatment of stroke for many years in China. In this study, we aim to investigate the therapeutic effects of XNJ in a hippocampus of Aß1-42 induced mouse model of AD which showed significant memory loss and impaired synaptic morphology and function. Treatment of XNJ could attenuate spatial and working memory dysfunction, increase dendritic spine density and improve long-term potential (LTP) induction. In addition, XNJ treatment significantly increased the level of N-methyl-d-aspartate receptors (NMDARs) and inhibit the NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) ratio in AD mice. XNJ treatment also activated the AKT/mechanistic target of rapamycin (mTOR) pathway, while inhibition of the mTOR pathway by rapamycin could reverse the protective effects of XNJ treatment. In conclusion, XNJ protected against synaptic plasticity and memory impairment in AD mice via the activation of AKT/mTOR signaling pathway, suggesting XNJ as an alternative treatment for AD.

Ultrasensitive detection of butyrylcholinesterase activity based on the inner filter effect of MnO2 nanosheets on sulfur nanodots.

Butyrylcholinesterase (BChE) activity is an important index for a variety of diseases. In this work, a "turn-on" assay is proposed based on controlling the inner filter effect (IFE) of MnO2 nanosheets (NSs) on sulfur nanodots (S-dots). The fluorescence of S-dots is effectively quenched by the MnO2 NSs, due to the wide overlap of the emission spectrum of S-dots and absorption spectrum of MnO2 NSs, together with the superior light absorption capability of MnO2 NSs. BChE can catalyze acetylthiocholine and produce thiocholine, which effectively decomposes the MnO2 NSs into Mn2+, resulting in the disappearance of the IFE and recovery of fluorescence of S-dots. Two-stage linear relationships between the ratio of fluorescence intensity and concentration of BChE are observed from 0.05 to 10 and from 10 to 500 U L-1. A limit of detection of 0.035 U L-1 is achieved, which is the best performance so far. The as-proposed assay is robust enough for practical detection in human serum, and it can avoid interference from its sister enzyme (acetylcholinesterase) and glutathione at the micromolar level. The presented results provide a clue for the functionalization of S-dots, and offer a powerful tool as an analytic technique for nanomedicine and environmental science.

Microstructural disruption of the right inferior fronto-occipital and inferior longitudinal fasciculus contributes to WMH-related cognitive impairment.

AIMS: White matter hyperintensity (WMH) is the most common neuroimaging manifestation of cerebral small vessel disease and is related to cognitive dysfunction or dementia. This study aimed to investigate the mechanism and effective indicators to predict WMH-related cognitive impairment. METHODS: We recruited 22 healthy controls (HC), 25 cases of WMH with normal cognition (WMH-NC), and 23 cases of WMH with mild cognitive impairment (WMH-MCI). All individuals underwent diffusion tensor imaging (DTI) and a standardized neuropsychological assessment. Automated Fiber Quantification was used to extract altered DTI metrics between groups, and partial correlation was performed to assess the associations between WM integrity and cognitive performance. Furthermore, machine learning analyses were performed to determine underlying imaging markers of WMH-related cognitive impairment. RESULTS: Our study found that mean diffusivity (MD) values of several fiber bundles including the bilateral anterior thalamic radiation (ATR), the left inferior fronto-occipital fasciculus (IFOF), the right inferior longitudinal fasciculus (ILF), and the right superior longitudinal fasciculus (SLF) were negatively correlated with memory function, while that of the anterior component of the right IFOF and the posterior and intermediate component of the right ILF showed significant negative correlation with MMSE and episodic memory, respectively. Furthermore, machine learning analyses showed that the accuracy of recognizing WMH-MCI patients from the WMH populations was up to 80.5% and the intermediate and posterior components of the right ILF and the anterior component of the right IFOF contribute the most. CONCLUSIONS: Changes in the properties of DTI may be the potential mechanism of WMH-related MCI, especially the right IFOF and the right ILF, which may become imaging markers for predicting WMH-related cognitive dysfunction.

Hemichannel-mediated volume regulation contributes to IPC-induced cardiomyocyte protection.

Cx43 has been documented to be involved in ischemic preconditioning (IPC). However, the participation of Cx43-formed hemichannels in IPC and the potential underlying mechanisms remain unclear. The present study focused on cardiomyocytes' volume regulation during IPC to investigate the role of hemichannels in the IPC-induced cardioprotection. In the study, mice cardiomyocytes were respectively treated with a hemichannel blocker, octanol or 18a-Glycyrrhizic acid (18a-GA), and a Cx43-silenced lentivirus. They were subsequently cultured in hypotonic solution to simulate ischemic reperfusion (SIR) and systemic ischemic preconditioning (SIP). Cell morphology and volumetric (area) change were detected by inverted microscopy at 30 min following the addition of hypotonic solution. Cardiomyocyte mortality was assessed by trypan blue stain assay. The analyses revealed that regardless of the treatments, hypotonic solution aggravated cell edema: Compared with the initial condition (the moment before the solution addition, 0 min), the volumetric area increased significantly 30 min later (for hypotonic+DMSO, 5,050±1,511 vs. 3,464±723 µm2; for hypotonic+scramble lentiviral vector, 5,517±1,128 vs. 2,331±536 µm2; P<0.05, respectively). Either treatment alleviated the edematous condition when a comparison was made between 30 min after the hypotonic addition and 0 min (for hypotonic+octanol, 2,990±765 vs. 2,821±773 µm2; for hypotonic+18a-GA, 4,817±1,306 vs. 4,762±1,271 µm2; for hypotonic+Cx43-silenced, 3,627±688 vs. 3,419±814 µm2; P>0.05 for all). Notably, results indicated that the SIP group had lower mortality rates compared with its SIR counterpart; the hypotonic+octanol, hypotonic+18a-GA, and hypotonic+Cx43-silenced group showed markedly-declined mortality when compared with their respective control groups (respectively, 35.70±1.02, 30.76±2.20 vs. 53.58±2.14%; 30.89±2.37 vs. 54.12±2.55%; P<0.05 for all). The results suggest that ischemic preconditioning may provide cardioprotection by blocking the opening of the hemichannels and further mediating the volume regulation of cardiomyocytes.

Endowing Polymeric Assemblies with Unique Properties and Behaviors by Incorporating Versatile Nanogels in the Shell.

Herein, we report an example of self-driven morphological transition and the unique dissociation behavior of polymeric assemblies. Polymeric nanogels were introduced into the shell of polymeric assemblies and used as a powerful platform to endow the assemblies with unique properties and behaviors. It is exhibited that the nanogel can host an intrananogel cross-linking reaction and thus contracts automatically to change the geometrical packing parameters of the building blocks, driving morphological transitions of the assemblies; the transitions are self-driven without any external stimuli. Additionally, when the nanogels in the shell expand, the assemblies dissociate into small fragments even when the core is in a frozen state; in existing studies, polymeric assemblies with a frozen core cannot dissociate, except the core becomes dynamic under the stimuli. Both the self-driven morphological transition and the dissociation behavior are unique and have never been reported before.

Impacts of aquaculture wastewater irrigation on soil microbial functional diversity and community structure in arid regions.

Aquaculture wastewater is one of the most important alternative water resources in arid regions where scarcity of fresh water is common. Irrigation with this kind of water may affect soil microbial functional diversity and community structure as changes of soil environment would be significant. Here, we conducted a field sampling to investigate these effects using Biolog and metagenomic methods. The results demonstrated that irrigation with aquaculture wastewater could dramatically reduce soil microbial functional diversity. The values of diversity indices and sole carbon source utilization were all significantly decreased. Increased soil salinity, especially Cl concentration, appeared primarily associated with the decreases. Differently, higher bacterial community diversity was obtained in aquaculture wastewater irrigated soils. More abundant phyla Actinobacteria, Chloroflexi, Acidobacteria, Gemmatimonadetes and fewer members of Proteobacteria, Bacteroidetes and Planctomycetes were found in this kind of soils. Changes in the concentration of soil Cl mainly accounted for the shifts of bacterial community composition. This research can improve our understanding of how aquaculture wastewater irrigation changes soil microbial process and as a result, be useful to manage soil and wastewater resources in arid regions.

Shifts in soil microbial metabolic activities and community structures along a salinity gradient of irrigation water in a typical arid region of China.

Saline water irrigation can change soil environment, which thereby influence soil microbial process. Based on a field experiment, the shifts in soil microbial metabolic activities and community structures under five irrigation salinities were studied using Biolog and metagenomic methods in this study. The results demonstrated that microbial metabolic activities were greatly restrained in saline water irrigated soils, as average well color development (AWCD) reduced under all saline water irrigation treatments. Although no significant difference in carbon substrate utilization of all six categories was observed among Mild, Medium, High and Severe treatments, the consumption of sole carbon source was significantly varied. Especially, asparagine, galacturonic, putrescine and 4-benzoic acid played a decisive role in dominating the differences. Soil bacterial richness and diversity increased with irrigation salinity while the number of bacterial phyla decreased. Three significantly increased (Proteobacteria, Actinobacteria and Chloroflexi), two decreased (Planctomycetes, Bacteroidetes) and two irresponsive (Gemmatimonadetes and Acidobacteria) phyla were observed as the dominant groups in saline water irrigated soils. The results presented here could improve the understanding of the soil biological process under saline circumstance.

Development and evaluation of a SYBR Green real-time RT-PCR assay for detection of avian hepatitis E virus.

BACKGROUND: Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease, as well as hepatitis-splenomegaly syndrome in chickens. To date, conventional reverse transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR methods have been used for the diagnosis of avian HEV infection in chickens. However, these assays are time consuming, inconvenient, and cannot detect the virus quantitatively. In this study, a rapid and sensitive SYBR Green real-time RT-PCR assay was developed to detect avian HEV RNA quantitatively in serum, liver, spleen, and fecal samples from chickens. RESULTS: Based on the sequence of the most conserved HEV gene, ORF3, the primers for the assay were designed, and the standard plasmid was constructed. The detection limit of the assay was shown to be 10 copies/µl of standard plasmid/reaction, with a corresponding cycle-threshold value of 29.3. The standard curve exhibited a dynamic linear range across at least 7 log units of DNA copy number. The specificity and reproducibility of this assay was high, showing that the assay detected avian HEV RNA specifically and with little variability. Compared to conventional RT-PCR, the current assay is more sensitive for detecting avian HEV in serum, liver, spleen, and fecal samples from chickens. CONCLUSIONS: A rapid, specific, and reproducible SYBR Green real-time RT-PCR assay was developed for the diagnosis of avian HEV infection in chickens. This assay can accurately detect avian HEV RNA in serum, liver, spleen, and fecal samples with more sensitivity than conventional RT-PCR.

Efficient fuzzy C-means architecture for image segmentation.

This paper presents a novel VLSI architecture for image segmentation. The architecture is based on the fuzzy c-means algorithm with spatial constraint for reducing the misclassification rate. In the architecture, the usual iterative operations for updating the membership matrix and cluster centroid are merged into one single updating process to evade the large storage requirement. In addition, an efficient pipelined circuit is used for the updating process for accelerating the computational speed. Experimental results show that the the proposed circuit is an effective alternative for real-time image segmentation with low area cost and low misclassification rate.