Palmitoleic acid protects microglia from palmitate-induced neurotoxicity in vitro.

Although palmitoleic acid (POA) is a lipokine with beneficial effects on obesity and is produced as a byproduct from the manufacture of prescription omega-3 fatty acids, its role in nervous system inflammation is still unknown. This study aims to examine the mechanisms and protective effects of POA against palmitic acid (PA)-induced microglial death. PA-induced microglial death was used as a model for POA intervention. Various inhibitors were employed to suppress potential routes of PA entry into the cell. Immunofluorescence staining and Western blotting were conducted to elucidate the protective pathways involved. The results suggest POA has the potential to eliminate PA-induced lactate dehydrogenase (LDH) release, which decreases the overall number of propidium iodide (PI)-positive cells compared with control. Moreover, POA has the potential to significantly increase lipid droplets (LDs) in the cytoplasm, without causing any lysosomal damage. POA inhibited both canonical and non-canonical gasdermin D (GSDMD)-mediated pyroptosis and gasdermin E (GSDME)-mediated pyroptosis, which PA typically induces. Additionally, POA inhibited the endoplasmic reticulum (ER) stress and apoptosis-related proteins induced by PA. Based on the findings, POA can exert a protective effect on microglial death induced by PA via pathways related to pyroptosis, apoptosis, ER stress, and LDs.

Palmitate lipotoxicity is closely associated with the fatty acid-albumin complexes in BV-2 microglia.

Palmitic acid (PA) is considered a major contributor to the inflammation in many metabolic diseases; however, this role has been questioned recently for the complicated procedures in preparing PA-bovine serum albumin (BSA) complex. This study is aimed to evaluate the effect of PA-BSA complexing methods on cell viability and inflammatory responses of BV-2 cells. Three commercially available BSA brands and two types of solvents were compared for their effects on the expression of inflammatory cytokines. Three commonly used proportions of PA-BSA were tested for cell viability and inflammatory responses. We found that all the three types of BSA were proinflammatory. Both ethanol and isopropanol dampened inflammation except that 1% isopropanol treatment increased the IL-1ß level by 26%. When reducing the BSA content in PA-BSA solutions from 3:1 to 5:1, a marked increase in cell viability (11%) was seen. To our surprise, reducing BSA content in PA-BSA solutions from 5:1 to 10:1 decreased cell viability by 11%. The 5:1 group exhibited the lowest inflammatory profile. Either PA-BSA or BSA alone increased the entry of LPS to the cytosol, which further caused pyroptosis. In summary, we found 5:1 (PA:BSA) to be the best binding ratio for studying inflammation in BV-2 microglia. The presence of LPS in the cytosol in the context of BSA might be the reason for confounding results from palmitate studies.

A single dose of lipopolysaccharide elicits autofluorescence in the mouse brain.

One hallmark of aging is autofluorescence (AF) in the brain. However, the underlying mechanism for inducing AF remains unknown. This study aims to determine the cause(s) of this phenomenon. The endogenous expression pattern of AF in mice was examined at differing ages. Intraperitoneal injection of a single dose of lipopolysaccharide (LPS) was performed to induce AF. Copper sulfate was applied to remove AF to allow for further immunofluorescence staining. AF appeared in the mouse brain as early as 3 months of age. In the cortex, AF occurs in the lysosomes of microglia, astrocytes, endothelial cells, and oligodendrocyte lineage cells and its prevalence increases with age. Interestingly, AF never occurs in the pericytes of young or aged brains. LPS administration resulted in a rapid and marked induction of brain AF, similar to the normal aging process. Finally, age-related and induced AF can be eliminated by low concentrations of copper sulfate solution. This pre-treatment is safe for aging and lineage tracing studies. These findings depict that AF in the brain could be associated with the innate immune response against Gram-negative bacteria infection.

Monkfish Peptides Mitigate High Fat Diet-Induced Hepatic Steatosis in Mice.

Non-alcoholic fatty liver disease (NAFLD) is a hepatic metabolic syndrome usually accompanied by fatty degeneration and functional impairment. The aim of the study was to determine whether monkfish peptides (LPs) could ameliorate high-fat diet (HFD)-induced NAFLD and its underlying mechanisms. NAFLD was induced in mice by giving them an HFD for eight weeks, after which LPs were administered in various dosages. In comparison to the HFD control group: body weight in the LP-treated groups decreased by 23-28%; triacylglycerol levels in the blood decreased by 16-35%; and low-density lipoproteins levels in the blood decreased by 23-51%. Additionally, we found that LPs elevated the activity of hepatic antioxidant enzymes and reduced the inflammatory reactions within fatty liver tissue. Investigating the effect on metabolic pathways, we found that in LP-treated mice: the levels of phospho-AMP-activated protein kinase (p-AMPK), and phospho-acetyl CoA carboxylase (p-ACC) in the AMP-activated protein kinase (AMPK) pathway were up-regulated and the levels of downstream sterol regulatory element-binding transcription factor 1 (SREBP-1) were down-regulated; lipid oxidation increased and free fatty acid (FFA) accumulation decreased (revealed by the increased carnitine palmitoyltransferase-1 (CPT-1) and the decreased fatty acid synthase (FASN) expression, respectively); the nuclear factor erythroid-2-related factor 2 (Nrf2) antioxidant pathway was activated; and the levels of heme oxygenase-1 (HO-1) and nicotinamide quinone oxidoreductase 1 (NQO1) were increased. Overall, all these findings demonstrated that LPs can improve the antioxidant capacity of liver to alleviate NAFLD progression mainly through modulating the AMPK and Nrf2 pathways, and thus it could be considered as an effective candidate in the treatment of human NAFLD.

Protective mechanism of traditional Chinese medicine guizhi fuling pills against carbon tetrachloride-induced kidney damage is through inhibiting oxidative stress, inflammation and regulating the intestinal flora.

BACKGROUND: Chemical or drug-induced kidney damage has been recognized as a critical cause of kidney failure. The oxidative stress, inflammation, and imbalance of intestinal flora caused by carbon tetrachloride (CCl4) play a fundamental role in chronic kidney damage. Guizhi Fuling pills (GZFL) is a traditional formula consisting of five traditional Chinese medicinal herbs, which can promote blood circulation and improve kidney function. The underlying mechanisms of GZFL improving kidney damage are not fully understood yet. AIM: The current study aimed to explore the effects of GZFL on CCl4-induced kidney damage and intestinal microbiota in mice. METHODS: Male ICR mice were intraperitoneally administered with 20% CCl4 (mixed in a ratio of 1:4 in soybean oil) twice a week, for 4 weeks to induce kidney damage. Creatinine (CRE), urea nitrogen, antioxidant enzymes, and inflammatory cytokines were measured and the histology of the kidney, jejunum, and colon examination to assess kidney and intestinal damage. The expressions of nuclear factor-erythroid 2-related factor 2 (Nrf2) family members, nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome in kidney tissues, and the tight junction proteins in colonic tissues were detected by Western blot. The gut microbiota was analyzed through 16S rRNA gene sequencing. RESULTS: GZFL treatment decreased the serum CRE and urea nitrogen levels. Moreover, GZFL reduced the levels of pro-inflammatory cytokines and increased antioxidant enzyme activities in kidney and colonic tissues. GZFL improved the kidney, jejunum, and colon histology. Furthermore, GZFL inhibited the expressions of NLRP3, ASC, and cleaved-Caspase-1, while Nrf2, HO-1, NQO1, GCLM, and tight junction proteins were increased. The dysbiosis of intestinal microbiota improved after GZFL treatment. CONCLUSIONS: This study showed that GZFL could improve kidney damage, which might be mainly via the integrated regulations of the Nrf2 pathway, NLRP3 inflammasome, and composition of intestinal microbiota.

Fucoxanthin Attenuates Free Fatty Acid-Induced Nonalcoholic Fatty Liver Disease by Regulating Lipid Metabolism/Oxidative Stress/Inflammation via the AMPK/Nrf2/TLR4 Signaling Pathway.

Fucoxanthin, a xanthophyll carotenoid abundant in brown algae, is reported to have several biological functions, such as antioxidant, anti-inflammatory, and anti-tumor activities, in mice. We investigated the effects and mechanisms of fucoxanthin in the mixture oleate/palmitate = 2/1(FFA)-induced nonalcoholic fatty liver disease (NAFLD) cell model in this study. The results showed that the content of superoxide dismutase in the FFA group was 9.8 ± 1.0 U/mgprot, while that in the fucoxanthin high-dose (H-Fx) group (2 µg/mL) increased to 22.9 ± 0.6 U/mgprot. The content of interleukin-1ß in the FFA group was 89.3 ± 3.6 ng/mL, while that in the H-Fx group was reduced to 53.8 ± 2.8 ng/mL. The above results indicate that fucoxanthin could alleviate the FFA-induced oxidative stress and inflammatory levels in the liver cells. Oil red-O staining revealed visible protrusions and a significant decrease in the number of lipid droplets in the cytoplasm of cells in the fucoxanthin group. These findings on the mechanisms of action suggest that fucoxanthin can repair FFA-induced NAFLD via the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway and nuclear factor erythroid-2-related factor 2-mediated (Nrf2) signaling pathway, as well as by downregulating the expression of the Toll-like receptor 4-mediated (TLR4) signaling pathway. Fucoxanthin exhibited alleviating effects in the FFA-induced NAFLD model and could be explored as a potential anti-NAFLD substance.

Furazolidone and Nitrofurazone Metabolic Studies in Crucian Carp by Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry.

In this work, the detection of the furazolidone (FZD) and nitrofurazone (NFZ) metabolites residuals in crucian carp are focused. Crucian carps of identical size were exposed to the mixed nitrofuran antibiotics under optimized bath conditions at a concentration of 50 mg/L, 26 ± 0.5°C for 24 h. Then, liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (LC-ESI-MSMS) was performed after the drug exposure experiments when the nitrofuran metabolites were enriched in organisms. During the period of 0-144 h, residue levels of the 3-amino-2-oxazolidinone (AOZ) gradually decreased with a prolonged sampling time. The changing trend in semicarbazide (SEM) with the sample collection duration is divided into two stages, and its concentration showed a trend of rising first and then falling. The metabolite concentration-time curve demonstrates that 24 h was used as a sampling time, and fish muscle was selected as tissue samples in the further quantitative study. A novel crucian carp-enrichment procedure coupled to LC-ESI-MSMS quantitative method was further explored based on much metabolite data. According to the exponential curve of the SEM-to-AOZ concentration ratio at a precisely designed FZD-to-NFZ mass ratio, the final FZD content of the veterinary NFZ antibiotics was 0.069 ± 0.005% (in terms of mass).

Using Collagen Peptides From the Skin of Monkfish (Lophius litulon) to Ameliorate Kidney Damage in High-Fat Diet Fed Mice by Regulating the Nrf2 Pathway and NLRP3 Signaling.

BACKGROUND: Oxidative stress and inflammation play important roles in high-fat diet (HFD) induced kidney damage. Previous studies show that the collagen extracted from the skin of monkfish (Lophius litulon) with pepsin (pepsin-solubilized collagen, PSC) exhibits good biological activities. This study investigates the protective effect of PSCP against chronic kidney injury in HFD-fed mice. METHODS: Pepsin-solubilized collagen was further hydrolyzed into collagen peptides, and the compound with the best 2,2-diphenyl-1-picrylhydrazyl (DPPH) clearance rate was named pepsin-solubilized collagen peptide (PSCP). A group of mice were fed an HFD for 4 weeks, and then for another 6 weeks PSCP was added to their diet at the amount of either 100 or 200 mg/kg. RESULTS: Pepsin-solubilized collagen peptide treatment (200 mg/kg) reduced the mice's serum levels of uric acid (UA), creatinine (CRE), and blood urea nitrogen (BUN) by 27, 20, and 37%, respectively. This treatment also remarkably improved renal histopathology. Moreover, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were increased by 96, 52, and 74%, respectively, and decreased the malondialdehyde (MDA) level by 36%. Additionally, PSCP activated the Nrf2 pathway and inhibited NLRP3 signaling to significantly reduce the levels of inflammatory cytokines IL-1ß, IL-6, and TNF-α. CONCLUSIONS: Our results indicate that compound PSCP has the potential to prevent or control chronic kidney damage.

Monkfish (Lophius litulon) Peptides Ameliorate High-Fat-Diet-Induced Nephrotoxicity by Reducing Oxidative Stress and Inflammation via Regulation of Intestinal Flora.

BACKGROUND: Renal damage and intestinal flora imbalance due to lipotoxicity are particularly significant in terms of oxidative stress and inflammation, which can be alleviated with bioactive peptides. The monkfish (Lophius litulon) is rich in proteins, which can be used as a source of quality bioactive peptides. This study aimed to examine the protective effect of monkfish peptides on renal injury and their potential role in regulating gut microbiota. METHODS: Monkfish meat was hydrolyzed using neutral protease and filtered, and the component with the highest elimination rate of 2,2-diphenyl-1-picrylhydrazyl was named lophius litulon peptides (LPs). Lipid nephrotoxicity was induced via high-fat diet (HFD) feeding for 8 weeks and then treated with LPs. Oxidative stress, inflammatory factors, and intestinal flora were evaluated. RESULTS: LP (200 mg/kg) therapy reduced serum creatinine, uric acid, and blood urea nitrogen levels by 49.5%, 31.6%, and 31.6%, respectively. Renal vesicles and tubules were considerably improved with this treatment. Moreover, the activities of superoxide dismutase, glutathione peroxidase, and total antioxidant capacity increased significantly by 198.7%, 167.9%, 61.5%, and 89.4%, respectively. LPs attenuated the upregulation of HFD-induced Toll-like receptor 4 and phospho-nuclear factor-kappa B and increased the protein levels of heme oxygenase 1, nicotinamide quinone oxidoreductase 1, and nuclear factor erythroid 2-related factor 2. The dysbiosis of intestinal microbiota improved after LP treatment. CONCLUSIONS: LPs significantly improve antioxidant activity, reduce inflammatory cytokine levels, and regulate intestinal dysbiosis. Thus, LPs are potential compounds that can alleviate HFD-induced renal lipotoxicity.

Astaxanthin attenuated hyperuricemia and kidney inflammation by inhibiting uric acid synthesis and the NF-κ B/NLRP3 signaling pathways in potassium oxonate and hypoxanthine-induced hyperuricemia mice.

Inflammation is an important pathological feature of hyperuricemia, which in turn aggravates hyperuricemia. Astaxanthin is a carotenoid with strong antioxidant capacity and possesses many biological activities. This study was aimed to evaluate the effect of astaxanthin (ASX) on hyperuricemia and kidney inflammation in potassium oxonate (PO) and hypoxanthine (HX)-induced hyperuricemic mice. Male ICR mice were administered intragastrically with PO and HX (250 mg/kg, respectively) for 14 days. ASX was given by gavage one hour after PO and HX administration. ASX treatment significantly reversed PO and HX-induced hyperuricemia and kidney inflammation in mice as evidenced by decreased serum levels of uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN), and inflammatory factors (IL-1ß, IL-6, and TNF-α) and increased activities of antioxidant enzymes (CAT, SOD and GSH-Px). Furthermore, ASX administration effectively inhibited the activities of key enzymes related to UA synthesis (xanthine oxidase (XOD) and adenosine deaminase (ADA)) and modulated the protein expressions of NF-κ B p65, p-NF-κ B p65, Iκ Bα, p-Iκ Bα, NLRP3, ASC, Caspase-1, and cleavedCaspase-1 involved in inflammation pathways. Our results suggested that ASX improved hyperuricemia and kidney inflammation induced by PO and HX, probably by reducing UA synthesis and suppressing the NF-κ B and NLRP3 pathways simultaneously.

Optimization of Extraction of Bioactive Peptides from Monkfish (Lophius litulon) and Characterization of Their Role in H2O2-Induced Lesion.

BACKGROUND: Marine fish meat has been widely used for the extraction of bioactive peptides. This study was aimed to optimize the preparation of monkfish muscle peptides (LPs) using response surface methodology (RSM) and explore the antioxidant activities of <1 kDa LPs. METHODS: Peptides were prepared from the muscles of monkfish (Lophius litulon), and five proteases were tested to hydrolyze muscle proteins. The hydrolysate that was treated using neutrase showed the highest degree of hydrolysis (DH) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activities. RESULTS: The optimized conditions were as follows: water/material ratio of 5.4:1, a time span of 5 h, pH of 7.0, enzyme concentration of 2000 U/g, and temperature of 45 °C; the maximum DPPH scavenging activity and DH were 92.861% and 19.302%, respectively. LPs exhibited appreciable antioxidant activities, including DPPH radical, hydroxyl radical, 2,2'-azinobis-3-ethylbenzthiazoline-6-sulphonate (ABTS) radical, and superoxide anion scavenging activities. LPs attenuated H2O2-related oxidative injury in RAW264.7 cells, reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and increased the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) levels. CONCLUSION: We concluded that LPs could be an ideal source of bioactive peptides from monkfish and also have pharmaceutical potential.

Collagen Peptides from Swim Bladders of Giant Croaker (Nibea japonica) and Their Protective Effects against H2O2-Induced Oxidative Damage toward Human Umbilical Vein Endothelial Cells.

Five different proteases were used to hydrolyze the swim bladders of Nibea japonica and the hydrolysate treated by neutrase (collagen peptide named SNNHs) showed the highest DPPH radical scavenging activity. The extraction process of SNNHs was optimized by response surface methodology, and the optimal conditions were as follows: a temperature of 47.2 °C, a pH of 7.3 and an enzyme concentration of 1100 U/g, which resulted in the maximum DPPH clearance rate of 95.44%. Peptides with a Mw of less than 1 kDa (SNNH-1) were obtained by ultrafiltration, and exhibited good scavenging activity for hydroxyl radicals, ABTS radicals and superoxide anion radicals. Furthermore, SNNH-1 significantly promoted the proliferation of HUVECs, and the protective effect of SNNH-1 against oxidative damage of H2O2-induced HUVECs was investigated. The results indicated that all groups receiving SNNH-1 pretreatment showed an increase in GSH-Px, SOD, and CAT activities compared with the model group. In addition, SNNH-1 pretreatment reduced the levels of ROS and MDA in HUVECs with H2O2-induced oxidative damage. These results indicate that collagen peptides from swim bladders of Nibea japonica can significantly reduce the oxidative stress damage caused by H2O2 in HUVECs and provides a basis for the application of collagen peptides in the food industry, pharmaceuticals, and cosmetics.

Netrin-1 attenuates brain injury after middle cerebral artery occlusion via downregulation of astrocyte activation in mice.

BACKGROUND: Netrin-1 functions largely via combined receptors and downstream effectors. Evidence has shown that astrocytes express netrin-1 receptors, including DCC and UNC5H2. However, whether netrin-1 influences the function of astrocytes was previously unknown. METHODS: Lipopolysaccharide was used to stimulate the primary cultured astrocytes; interleukin release was used to track astrocyte activation. In vivo, shRNA and netrin-1 protein were injected in the mouse brain. Infarct volume, astrocyte activation, and interleukin release were used to observe the function of netrin-1 in neuroinflammation and brain injury after middle cerebral artery occlusion. RESULTS: Our results demonstrated that netrin-1 reduced lipopolysaccharide-induced interleukin-1ß and interleukin-12ß release in cultured astrocytes, and blockade of the UNC5H2 receptor with an antibody reversed this effect. Additionally, netrin-1 increased p-AKT and PPAR-γ expression in primary cultured astrocytes. In vivo studies showed that knockdown of netrin-1 increased astrocyte activation in the mouse brain after middle cerebral artery occlusion (p < 0.05). Moreover, injection of netrin-1 attenuated GFAP expression (netrin-1 0.27 ± 0.06 vs. BSA 0.62 ± 0.04, p < 0.001) and the release of interleukins and reduced infarct volume after brain ischemia (netrin-1 0.27 ± 0.06 vs. BSA 0.62 ± 0.04 mm3, p < 0.05). CONCLUSION: Our results indicate that netrin-1 is an important molecule in regulating astrocyte activation and neuroinflammation in cerebral ischemia and provides a potential target for ischemic stroke therapy.

Contribution of Vascular Cells to Neointimal Formation.

The de-differentiation and proliferation of smooth muscle cells (SMCs) are widely accepted as the major contributor to vascular remodeling. However, recent studies indicate that vascular stem cells (VSCs) also play an important role, but their relative contribution remains to be elucidated. In this study, we used genetic lineage tracing approach to further investigate the contribution of SMCs and VSCs to neointimal thickening in response to endothelium denudation injury or artery ligation. In vitro and in vivo analysis of MYH11-cre/Rosa-loxP-RFP mouse artery showed that SMCs proliferated at a much slower rate than non-SMCs. Upon denudation or ligation injury, two distinct types of neointima were identified: Type-I neointimal cells mainly involved SMCs, while Type II mainly involved non-SMCs. Using Sox10-cre/Rosa-loxP-LacZ mice, we found that Sox10+ cells were one of the cell sources in neointima. In addition, lineage tracing using Tie2-cre/Rosa-LoxP-RFP showed that endothelial cells also contributed to the neointimal formation, but rarely transdifferentiated into mesenchymal lineages. These results provide a novel insight into the contribution of vascular cells to neointima formation, and have significant impact on the development of more effective therapies that target specific vascular cell types.

Effect of ischaemic brain injury on sexual function in adult mice.

OBJECTIVE: Priapism refers to a condition with persistent abnormal erection of the penis, which is usually caused by disease or injury in the brain or spinal cord, or obstruction to the outflow of blood through the dorsal vein at the root of the penis, without sexual desires. The effect of cerebral ischaemia on sexual function is unknown. The aim of this study is to explore whether priapism occurs in adult mice. Furthermore, we examined the relationship between priapism and the region of infarct in the brain. DESIGN: Adult male CD-1 mice who underwent permanent middle cerebral artery occlusion (pMCAO) were closely examined from 2 hours to 14 days postoperation. RESULTS: We found that priapism occurs in ∼80% of the mice with pMCAO, which could persist up to 14 days. Further study has demonstrated that the occurrence of priapism is related to the infarct region: priapism is found only in mice with ischaemic injury extending to the hypothalamus and the hippocampus regions. CONCLUSION: Our result suggested priapism may be used as a deep brain injury marker for evaluating brain injury in mice after pMCAO.

High-Resolution and Quantitative X-Ray Phase-Contrast Tomography for Mouse Brain Research.

Imaging techniques for visualizing cerebral vasculature and distinguishing functional areas are essential and critical to the study of various brain diseases. In this paper, with the X-ray phase-contrast imaging technique, we proposed an experiment scheme for the ex vivo mouse brain study, achieving both high spatial resolution and improved soft-tissue contrast. This scheme includes two steps: sample preparation and volume reconstruction. In the first step, we use heparinized saline to displace the blood inside cerebral vessels and then replace it with air making air-filled mouse brain. After sample preparation, X-ray phase-contrast tomography is performed to collect the data for volume reconstruction. Here, we adopt a phase-retrieval combined filtered backprojection method to reconstruct its three-dimensional structure and redesigned the reconstruction kernel. To evaluate its performance, we carried out experiments at Shanghai Synchrotron Radiation Facility. The results show that the air-tissue structured cerebral vasculatures are highly visible with propagation-based phase-contrast imaging and can be clearly resolved in reconstructed cross-images. Besides, functional areas, such as the corpus callosum, corpus striatum, and nuclei, are also clearly resolved. The proposed method is comparable with hematoxylin and eosin staining method but represents the studied mouse brain in three dimensions, offering a potential powerful tool for the research of brain disorders.

Electrospun bilayer fibrous scaffolds for enhanced cell infiltration and vascularization in vivo.

Bilayer poly(L-lactic acid) fibrous scaffolds consisting of a thin aligned-fiber layer (AFL) and a relatively thick random-fiber layer (RFL) were fabricated by an electrospinning technique, which uses two slowly rotating parallel disks as the collector. The morphology and structure of the bilayer scaffolds were examined by high-magnification scanning electron microscopy and confocal microscopy. The bilayer scaffolds demonstrated a gradual variation in through-thickness porosity and fiber alignment and an average porosity much higher than that of conventionally electrospun scaffolds (controls) with randomly distributed fibers. The biocompatibility and biological performance of the bilayer fibrous scaffolds were evaluated by in vivo experiments involving subcutaneous scaffold implantation in Sprague-Dawley rats, followed by histology and immunohistochemistry studies. The results illustrate the potential of the bilayer scaffolds to overcome major limitations of conventionally electrospun scaffolds associated with intrinsically small pores, low porosity and, consequently, poor cell infiltration. The significantly higher porosity and larger pore size of RFL enhances cell motility through the scaffold thickness, whereas the relatively dense structure of AFL provides the scaffold with the necessary mechanical strength. The bilayer scaffolds show more than two times higher cell infiltration than controls during implantation in vivo. The unique structure of the bilayer scaffolds promotes collagen fiber deposition, cell proliferation, and ingrowth of smooth muscle cells and endothelial cells in vivo. The results of this study illustrate the high prospect of the fabricated bilayer fibrous scaffolds in tissue engineering and regeneration.

Collateral circulation prevents masticatory muscle impairment in rat middle cerebral artery occlusion model.

The rat suture middle cerebral artery occlusion (MCAO) is a frequently used animal model for investigating the mechanisms of ischemic brain injury. During suture MCAO, transection of the external carotid artery (ECA) potentially restrains blood flow and impairs masticatory muscle and other ECA-supported territories, consequently influencing post-operation animal survival. This study was aimed at investigating the effect of ECA transection on the hemodynamic alterations using a novel synchrotron radiation (SR) angiography technique and magnetic resonance imaging in live animals. Fifteen male adult Sprague-Dawley rats were used in this study. Animals underwent MCAO, in which the ECA was transected. SR angiography was performed before and after MCAO. Rats then underwent magnetic resonance imaging (MRI) to detect the tissue lesion both intra- and extra-cranially. Animals with SR angiography without other manipulations were used as control. High-resolution cerebrovascular morphology was analyzed using a novel technique of SR angiography. The masticatory muscle lesion was further examined by hematoxylin and eosin staining. MRI and histological results showed that there was no masticatory muscle lesion at 1, 7 and 28 days following MCAO with ECA transection. In normal condition, the ECA and its branch external maxillary artery were clearly detected. Following ECA transection, the external maxillary artery was still observed and the blood supply appeared from the anastomotic branch from the pterygopalatine artery. SR angiography further revealed the inter-relationship of hemisphere extra- and intra-cranial vasculature in the rat following MCAO. Transection of the ECA did not impair masticatory muscles in rat suture MCAO. Interrupted blood flow could be compensated by the collateral circulation from the pterygopalatine artery.

Melatonin Pretreatment Improves the Survival and Function of Transplanted Mesenchymal Stem Cells after Focal Cerebral Ischemia.

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.

Biophysical regulation of epigenetic state and cell reprogramming.

Biochemical factors can help reprogram somatic cells into pluripotent stem cells, yet the role of biophysical factors during reprogramming is unknown. Here, we show that biophysical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells' epigenetic state. Specifically, decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)--a subunit of H3 methyltranferase--by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications.