Sirtuin 6 ameliorates arthritis through modulating cyclic AMP-responsive element binding protein/CCN1/cyclooxygenase 2 pathway in osteoblasts.

INTRODUCTION: CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MATERIALS AND METHODS: MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. RESULTS: SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at - 286 bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. CONCLUSION: Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.

The possible role of sirtuin 5 in the pathogenesis of apical periodontitis.

OBJECTIVES: We investigated the relation between expression of sirtuin 5 (SIRT5) in osteoblastic cells and progression of apical periodontitis. The role of SIRT5 in hypoxia-induced reactive oxygen species (ROS) formation and osteoblast apoptosis was also examined. MATERIALS AND METHODS: Progression of rat apical periodontitis was monitored by conventional radiography and microcomputed tomography. SIRT5 and oxidative stress biomarker 8-OHdG in bone-lining cells were assessed by immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was used to demonstrate apoptosis. In primary human osteoblasts cultured under hypoxia, Western blot was used to analyze SIRT5 expression and cleavage of pro-caspase 3 and poly(ADP-ribose) polymerase (PARP). SIRT5 was overexpressed through lentiviral technique. ROS formation and mitochondrial membrane potential changes were assessed by MitoSOX-Red and JC-1 fluorescence, respectively. Immunofluorescence microscope was used to evaluate mitochondrial release of cytochrome c. RESULTS: In rat apical periodontitis, disease progression was accompanied by decreased expression of SIRT5, increased oxidative stress, and enhanced apoptosis in bone-lining cells. SIRT5 was suppressed in cultured osteoblasts under hypoxia. SIRT5 overexpression ameliorated hypoxia-enhanced ROS formation, mitochondrial depolarization, cytochrome c leakage, activation of caspase-3, and PARP fragmentation. CONCLUSIONS: SIRT5 is able to alleviate hypoxia-enhanced osteoblast apoptosis. SIRT5 augmentation may have therapeutic potential for apical periodontitis.

CXCR4 receptor overexpression in mesenchymal stem cells facilitates treatment of acute lung injury in rats.

Novel therapeutic regimens for tissue renewal incorporate mesenchymal stem cells (MSCs) as they differentiate into a variety of cell types and are a stem cell type that is easy to harvest and to expand in vitro. However, surface chemokine receptors, such as CXCR4, which are involved in the mobilization of MSCs, are expressed only on the surface of a small proportion of MSCs, and the lack of CXCR4 expression may underlie the low efficiency of homing of MSCs toward tissue damage, which results in a poor curative effect. Here, a rat CXCR4 expressing lentiviral vector was constructed and introduced into MSCs freshly prepared from rat bone marrow. The influence of CXCR4 expression on migration, proliferation, differentiation, and paracrine effects of MSCs was examined in vitro. The in vivo properties of CXCR4-MSCs were also investigated in a model of acute lung injury in rats induced by lipopolysaccharide. Expression of CXCR4 in MSCs significantly enhanced the chemotactic and paracrine characteristics of the cells in vitro but did not affect self-renewal or differentiation into alveolar and vascular endothelial cells. In vivo, CXCR4 improved MSC homing and colonization of damaged lung tissue, and furthermore, the transplanted CXCR4-MSCs suppressed the development of acute lung injury in part by modulating levels of inflammatory molecules and the neutrophil count. These results indicated that efficient mobilization of MSCs to sites of tissue injury may be due to CXCR4, and therefore, increased expression of CXCR4 may improve their therapeutic potential in the treatment of diseases where tissue damage develops.

Autophagic degradation of CDK4 is responsible for G0/G1 cell cycle arrest in NVP-BEZ235-treated neuroblastoma.

BACKGROUND: CDK4 is highly expressed and associated with poor prognosis and decreased survival in advanced neuroblastoma (NB). Targeting CDK4 degradation presents a potentially promising therapeutic strategy compared to conventional CDK4 inhibitors. However, the autophagic degradation of the CDK4 protein and its anti-proliferation effect in NB cells has not been mentioned. RESULTS: We identified autophagy as a new pathway for the degradation of CDK4. Firstly, autophagic degradation of CDK4 is critical for NVP-BEZ235-induced G0/G1 arrest, as demonstrated by the overexpression of CDK4, autophagy inhibition, and blockade of autophagy-related genes. Secondly, we present the first evidence that p62 binds to CDK4 and then enters the autophagy-lysosome to degrade CDK4 in a CTSB-dependent manner in NVP-BEZ235 treated NB cells. Similar results regarding the interaction between p62 and CDK4 were observed in the NVP-BEZ235 treated NB xenograft mouse model. CONCLUSIONS: Autophagic degradation of CDK4 plays a pivotal role in G0/G1 cell cycle arrest in NB cells treated with NVP-BEZ235.

Optical and Electrical Properties of AlGaN-Based High Electron Mobility Transistors and Photodetectors with AlGaN/AlN/GaN Channel-Stacking Structure.

High channel current of the high electron mobility transistors (HEMTs) and high relative responsivity of the photodetectors (PDs) were demonstrated in the AlGaN/AlN/GaN channel-stacking epitaxial structures. The interference properties of the X-ray curves indicated high-quality interfaces of the conductive channels. The AlGaN/AlN/GaN interfaces were observed clearly in the transmission electron microscope micrograph. The saturation I ds currents of the HEMT structures were increased by adding a number of channels. The conductive properties of the channel-stacking structures corresponded to the peaks of the transconductance (g m) spectra in the HEMT structures. The depletion-mode one- and two-channel HEMT structures can be operated at the cutoff region by increasing the reverse V gs bias voltages. Higher I ds current in the active state and lower current in the cutoff state were observed in the two-channel HEMT structure compared with one- and three-channel HEMT structures. For the channel-stacking metal-semiconductor-metal photodetector structures, the peak responsivity was observed at almost 300 nm incident monochromic light, which was increased by adding a number of channel layers. The channel current of the HEMT devices and the photocurrent in the PD devices were increased by adding a number of two-dimensional electron gas (2DEG) channels. By using a flat gate metal layer, the two-channel AlGaN/AlN/GaN HEMT structures exhibited a high I ds current, a low cutoff current, and a high peak g m value and have the potential for GaN-based power devices, fast portable chargers, and ultraviolet PD applications.

The mechanisms of Porphyromonas gingivalis-derived outer membrane vesicles-induced neurotoxicity and microglia activation.

Background/purpose: Periodontitis is associated with various systemic diseases, potentially facilitated by the passage of Porphyromonas gingivalis outer membrane vesicles (Pg-OMVs). Several recent studies have suggested a connection between Pg-OMVs and neuroinflammation and neurodegeneration, but the precise causal relationship remains unclear. This study aimed to investigate the mechanisms underlying these associations using in vitro models. Materials and methods: Isolated Pg-OMVs were characterized by morphology, size, and gingipain activity. We exposed SH-SY5Y neuroblastoma cells and BV-2 microglial cells to various concentrations of Pg-OMVs. Cell morphology, a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, an enzyme-linked immunosorbent assay, and Western blot analysis were used to evaluate the cellular mechanism underlying Pg-OMV-induced neurotoxicity in neuronal cells and inflammatory responses in microglial cells. Results: Exposure to Pg-OMVs induced neurotoxicity in SH-SY5Y cells, as evidenced by cellular shrinkage, reduced viability, activation of apoptotic pathways, and diminished neuronal differentiation markers. Gingipain inhibition mitigated these effects, suggesting that gingipain mediates Pg-OMVs-induced neurotoxicity in SH-SY5Y cells. Our research on neuroinflammation suggests that upon endocytosis of Pg-OMVs by BV-2 cells, lipopolysaccharide (LPS) can modulate the production of inducible nitric oxide synthase and tumor necrosis factor-alpha by activating pathways that involve phosphorylated AKT and the phosphorylated JNK pathway. Conclusion: Our study demonstrated that following the endocytosis of Pg-OMVs, gingipain can induce neurotoxicity in SH-SY5Y cells. Furthermore, the Pg-OMVs-associated LPS can trigger neuroinflammation via AKT and JNK signaling pathways in BV-2 cells.

[Comparative study of the effects of intramedullary nail fixation and minimally invasive percutaneous plate internal fixation technique on platelet activation and serum transforming growth factor-ß1(TGF-ß) 1 and bone morphogenetic protein-2 (BMP-2) in patients with tibial and fibular fracture].

OBJECTIVE: To investigate the effect of intramedullary nail fixation (IMN) and minimally invasive percutaneous plate internal fixation (MIPPO) techniques on tibiofibular fractures and their effect on platelet activation and serum transforming growth factor-ß1 (TGF-ß1) and bone morphogenetic protein-2 (BMP-2). METHODS: Total of 105 patients with tibiofibular fractures from February 2019 to February 2020 were selected and divided into 53 cases in the MIPPO group and 52 cases in the IMN group. There were 29 males and 24 females with an average age of (41.74±6.05) years old in MIPPO group;in IMN group, 31 males and 21 females with an average age of (40.59±5.26) years old. The perioperative surgical indexes, postoperative complications, ankle function recovery at 12 months postoperatively, platelet activation indexes at 3 and 7 days preoperatively and postoperatively, and serum TGF-ß1 and BMP-2 levels at 4 and 8 weeks preoperatively and postoperatively were compared between the two groups. RESULTS: The operating time and fracture healing time in the MIPPO group were shorter than those in the IMN group(P<0.05); Compared with the preoperative period, the levels of GMP-140, PAC-1, CD63, and CD61 increased in both groups at 3 and 7 days after surgery, but were lower in the MIPPO group than in the IMN group(P<0.05);the levels of serum TGF-ß1 and BMP-2 increased in both groups at 4 and 8 weeks after surgery compared with the preoperative period, and the postoperative complication rate in the MIPPO group was lower than that in the IMN group(P<0.05);the difference was not statistically significant in the excellent rate of ankle function recovery at 12 months follow-up after surgery between two groups(P>0.05). CONCLUSION: Both intramedullary nail fixation and MIPO technique for treatment of tibia and fibula fractures can improve ankle joint function, but the latter has the advantages of short operation time, fast fracture healing, fewer complications, and light platelet activation. Serum TGF-ß1, BMP-2 level improves quickly.

Metformin Reduces Bone Resorption in Apical Periodontitis Through Regulation of Osteoblast and Osteoclast Differentiation.

INTRODUCTION: We have previously demonstrated that auxiliary metformin therapy promotes healing of apical periodontitis. Here we aimed to investigate the effects of metformin on osteoblast differentiation and osteoclast formation in cultured cells and rat apical periodontitis. METHODS: Murine pre-osteoblasts MC3T3-E1 and macrophages RAW264.7 were cultured under hypoxia (2% oxygen) or normoxia (21% oxygen) and stimulated with receptor activator of nuclear factor-κB ligand (RANKL) when indicated. Metformin was added to the cultures to evaluate its anti-hypoxic effects. Expressions of osteoblast differentiation regulator runt-related transcription factor 2 (RUNX2), RANKL, and osteoclast marker tartrate-resistant acid phosphatase (TRAP) were assessed by Western blot. Apical periodontitis was induced in mandibular first molars of 10 Sprague-Dawley rats. Root canal therapy with or without metformin supplement was performed. Periapical bone resorption was measured by micro-computed tomography. Immunohistochemistry was used to examine RUNX2, RANKL, and TRAP expressions. RESULTS: Hypoxia suppressed RUNX2 expression and enhanced RANKL synthesis in pre-osteoblasts. TRAP production increased in macrophages after hypoxia and/or RANKL stimulation. Metformin reversed hypoxia-induced RUNX2 suppression and RANKL synthesis in pre-osteoblasts. Metformin also inhibited hypoxia and RANKL-enhanced TRAP synthesis in macrophages. Intracanal metformin diminished bone loss in rat apical periodontitis. Comparing with vehicle control, cells lining bone surfaces in metformin-treated lesions had significantly stronger expression of RUNX2 and decreased synthesis of RANKL and TRAP. CONCLUSIONS: Alleviation of bone resorption by intracanal metformin was associated with enhanced osteoblast differentiation and diminished osteoclast formation in rat apical periodontitis. Our results endorsed the role of metformin as an effective medicament for inflammatory bone diseases.

Label-free bond-selective imaging by listening to vibrationally excited molecules.

We report the realization of vibrational photoacoustic (VPA) microscopy using optical excitation of molecular overtone vibration and acoustic detection of the resultant pressure transients. Our approach eliminates the tissue scattering problem encountered in near-infrared spectroscopy and enables depth-resolved signal collection. The 2nd overtone of the CH bond stretch around 8300 cm(-1), where blood interference is minimal, is excited. We demonstrate 3D VPA imaging of lipid-rich atherosclerotic plaques by excitation from the artery lumen, and lipid storage in live Drosophila larvae, with millimeter scale penetration depth [corrected].

Size and PEG Length-Controlled PEGylated Monocrystalline Superparamagnetic Iron Oxide Nanocomposite for MRI Contrast Agent.

OBJECTIVE: PEGylated superparamagnetic iron oxide (SPIO) is the most promising alternatives to gadolinium-based contrast agents (GBCAs) in MRI. This paper is to explore the imaging effects of PEGylated SPIO, which is influenced by particle sizes and surface polyethylene glycol (PEG) coating, using as MRI contrast agents at different magnetic field intensities. METHODS: Firstly, nine PEGylated monocrystalline SPIO nanoparticles with different nanocrystal sizes and different molecular weights PEG coating were prepared, and then physical and biological properties were analyzed. Finally, MRI imaging in vivo was performed to observe the imaging performance. RESULTS: Nine PEGylated monocrystalline SPIO nanoparticles have good relaxivities, serum stability, and biosecurity. At the same time, they show different imaging characteristics at different magnetic field intensities. Eight-nanometer SPIO@PEG5k is an effective T 2 contrast agent at 3.0 T (r 2/r 1 = 14.0), is an ideal T 1-T 2 dual-mode contrast agent at 1.5 T (r 2/r 1 = 6.52), and is also an effective T 1 contrast agent at 0.5 T (r 2/r 1 = 2.49), while 4-nm SPIO@PEG5k is a T 1-T 2 dual-mode contrast agent at 3.0 T (r 2/r 1 = 5.24), and is a useful T 1 contrast agent at 0.5 T (r 2/r 1 = 1.74) and 1.5 T (r 2/r 1 = 2.85). MRI studies in vivo at 3.0 T further confirm that 4-nm SPIO@PEG5k displays excellent T 1-T 2 dual-mode contrast enhancement, whereas 8-nm SPIO@PEG5k only displays T 2 contrast enhancement. CONCLUSION: PEGylated SPIOs with different nanocrystal sizes and PEG coating can be used as T 1, T 2, or T 1-T 2 dual-mode contrast agents to meet the clinical demands of MRI at specific magnetic fields.

Imaging and quantitative analysis of atherosclerotic lesions by CARS-based multimodal nonlinear optical microscopy.

OBJECTIVE: The purpose of this study was to assess the ability of label-free multimodal nonlinear optical (NLO) microscopy to characterize, and thus enable quantitative in situ analyses of, different atherosclerotic lesion types, according to the original scheme suggested by the AHA Committee. METHODS AND RESULTS: Iliac arteries were taken from 24 male Ossabaw pigs divided into lean control and metabolic syndrome groups and were imaged by multimodal NLO microscopy where sum-frequency generation (SFG) and 2-photon excitation fluorescence (TPEF) were integrated on a coherent anti-Stokes Raman scattering (CARS) microscope platform. Foam cells, lipid deposits, matrices, and fibrous caps were visualized with submicron 3D resolution. Starting from the adaptive intimal thickening in the initial stage to the fibrous atheroma or mineralization in the advanced stages, lesions were visualized without labels. Histological staining of each lesion confirmed the lesion stages. Lipid and collagen contents were quantitatively analyzed based on the CARS and SFG signals. Lipid accumulation in thickened intima culminated in type IV whereas the highest collagen deposition was found in Type V lesions. Luminal CARS imaging showed the capability of viewing the location of superficial foam cells that indicate relatively active locus in a lesion artery. CONCLUSIONS: We have demonstrated the capability of CARS-based multimodal NLO microscopy to interrogate different stages of lesion development with subcellular detail to permit quantitative analysis of lipid and collagen contents.

Intracanal Metformin Promotes Healing of Apical Periodontitis via Suppressing Inducible Nitric Oxide Synthase Expression and Monocyte Recruitment.

INTRODUCTION: We have previously shown that intracanal metformin ameliorates apical periodontitis, partially by modulation of osteoblast apoptosis. The action of metformin on other cell types pertinent to the development of apical periodontitis needs to be examined. In the present study, we aimed to analyze whether its effects on the expression of inducible nitric oxide synthase (iNOS) and monocyte recruitment contribute to the therapeutic effect on apical periodontitis. METHODS: Lipopolysaccharide (LPS)-induced expression of iNOS in a human monocytic cell line, Mono-Mac-6, was assessed by Western blot. The amount of nitrite in culture medium was assessed to quantify nitric oxide (NO) production. C-C motif chemokine ligand-2 (CCL-2) synthesis was measured by enzyme-linked immunosorbent assay. Experimental apical periodontitis in rats was treated with root canal debridement with or without intracanal metformin medication. Lesion progression was assessed by conventional radiography and micro-computed tomographic imaging. Cellular expression of iNOS and the number of monocytes/macrophages were assessed by immunohistochemistry. RESULTS: Metformin suppressed LPS-induced iNOS and NO production by monocytes. More importantly, metformin inhibited LPS-enhanced CCL-2 synthesis through modulation of the iNOS/NO pathway. Intracanal metformin reduced bone resorption associated with apical periodontitis and suppressed iNOS expression and monocyte recruitment. CONCLUSIONS: Our results confirmed the therapeutic efficacy of intracanal metformin for apical periodontitis. Suppression of monocyte recruitment through modulation of iNOS expression and NO production is an important mechanism underlying the beneficial effect of metformin.

Effects of Surgery Combined with Chemoradiotherapy on Short- and Long-Term Outcomes of Early-Stage Nasopharyngeal Carcinoma.

OBJECTIVE: The efficacy of surgery as the primary treatment modality for nasopharyngeal carcinoma (NPC) is yet to be clarified. Therefore, we aimed to explore the short- and long-term efficacy of surgery for early-stage NPC. METHODS: We retrospectively evaluated 341 patients diagnosed with early-stage NPC between September 2010 and December 2015. Among them, 58 patients underwent endoscopic nasopharyngectomy combined with chemoradiotherapy, whereas 283 patients underwent conventional chemoradiotherapy. The patients who underwent concurrent chemoradiotherapy or radiotherapy alone were matched to patients who underwent surgery in a 1:2 ratio using propensity score matching to analyze the clinical efficacy of each therapeutic modality. The primary endpoint was survival, and the secondary endpoints were tumor regression rate and reduction in Epstein-Barr virus (EBV)-DNA levels. RESULTS: After matching, 156 patients were enrolled (58 patients in the surgery group; 98 patients in the non-surgery group). The baseline data of the matched patients had good inter-group comparability (All P>0.05). The surgery group had significantly higher 5-year overall survival (98.30% vs. 91.70%), disease-free survival (98.30% vs. 81.40%), and recurrence-free survival (100.00% vs. 90.10%) rates than did the non-surgery group (All P<0.05). In total, 0 and 14 patients in the surgery and non-surgery groups, respectively, had residual cancer at the end of treatment (P=0.001). All patients in the surgery group tested negative for EBV-DNA, whereas two patients in the non-surgery group tested positive. The incidence of hematologic toxicity during treatment was similar between the two groups (All P>0.05). Still, the incidence of severe oral mucositis was lower in the surgery group than in the non-surgery group (37.9% vs. 54.08%, P=0.051). CONCLUSION: Surgery can improve the clearance rate of EB virus and reduce tumor residue. Surgery may be a safe and effective treatment for early NPC.

Chasing lipids in health and diseases by coherent anti-Stokes Raman scattering microscopy.

The integration of near IR picosecond pulse excitation, collinear beam geometry, epi-detection, and laser-scanning has produced a coherent anti-Stokes Raman scattering (CARS) microscope with a detection sensitivity of 10(5) vibrational oscillators, sub-micron 3D resolution, and video-rate acquisition speed. The incorporation of spectral detection and other imaging modalities has added versatility to the CARS microscope. These advances allowed sensitive interrogation of biological samples, particularly lipids that have a high density of CH(2) groups. With initial applications to membrane domains, lipid bodies, demyelinating diseases, obesity, and cardiovascular diseases, CARS microscopy is poised to become a powerful bio-imaging tool with the availability of a multifunctional, affordable, easy-to-operate CARS microscope, and the development of CARS endoscopy for in vivo diagnosis.

Rapid, non-invasive fluorescence margin assessment: Optical specimen mapping in oral squamous cell carcinoma.

OBJECTIVE: Surgical resection remains the primary treatment for the majority of solid tumors. Despite efforts to obtain wide margins, close or positive surgical margins (<5 mm) are found in 15-30% of head and neck cancer patients. Obtaining negative margins requires immediate, intraoperative feedback of margin status. To this end, we propose optical specimen mapping of resected tumor specimens immediately after removal. MATERIALS AND METHODS: A first-in-human pilot study was performed in patients (n = 8) after infusion of fluorescently labeled antibody, panitumumab-IRDye800 to allow surgical mapping of the tumor specimen. Patients underwent standard of care surgical resection for head and neck squamous cell carcinoma (HNSCC). Optical specimen mapping was performed on the primary tumor specimen and correlated with pathological findings after tissue processing. RESULTS: Optical mapping of the specimen had a 95% sensitivity and 89% specificity to detect cancer within 5 mm (n = 160) of the cut surface. To detect tumor within 2 mm of the specimen surface, the sensitivity of optical specimen mapping was 100%. The maximal observed penetration depth of panitumumab-IRDye800 through human tissue in our study was 6.3 mm. CONCLUSION: Optical specimen mapping is a highly sensitive and specific method for evaluation of margins within <5 mm of the tumor mass in HNSCC specimens. This technology has potentially broad applications for ensuring adequate tumor resection and negative margins in head and neck cancers.

Microfluidic CARS cytometry.

Coherent anti-stokes Raman scattering (CARS) flow cytometry was demonstrated by combining a laser-scanning CARS microscope with a polydimethylsiloxane (PDMS) based microfluidic device. Line-scanning across the hydrodynamically focused core stream was performed for detection of flowing objects. Parameters were optimized by utilizing polystyrene beads as flowing particles. Population measurements of adipocytes isolated from mouse fat tissues demonstrated the viability of microfluidic CARS cytometry for quantitation of adipocyte size distribution. CARS cytometry could be a new modality for quantitative analysis with vibrational selectivity.

Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy.

Coherent anti-Stokes Raman scattering (CARS) microscopy was applied to image myelinated fibers in different regions of a mouse brain. The CARS signal from the CH2 symmetric stretching vibration allows label-free imaging of myelin sheath with 3D sub-micron resolution. Compared with two-photon excited fluorescence imaging with lipophilic dye labeling, CARS microscopy provides sharper contrast and avoids photobleaching. The CARS signal exhibits excitation polarization dependence which can be eliminated by reconstruction of two complementary images with perpendicular excitation polarizations. The capability of imaging myelinated fibers without exogenous labeling was used to map the whole brain white matter in brain slices and to analyze the microstructural anatomy of brain axons. Quantitative information about fiber volume%, myelin density, and fiber orientations was derived. Combining CARS with two-photon excited fluorescence allowed multimodal imaging of myelinated axons and other cells. Furthermore, in vivo CARS imaging on an upright microscope clearly identified fiber bundles in brain subcortex white matter. These advances open up new opportunities for the study of brain connectivity and neurological disorders.

Label-free Imaging of Arterial Cells and Extracellular Matrix Using a Multimodal CARS Microscope.

A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH(2)-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH(2) bonds. The extracellular matrix organization were further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.

Metformin Ameliorates Periapical Lesions through Suppression of Hypoxia-induced Apoptosis of Osteoblasts.

INTRODUCTION: Intramuscular injection of metformin has been shown to inhibit the progression of periapical lesions in rats by decreasing the number of receptor activator of nuclear factor-κß ligand- and tartrate-resistant acid phosphatase-positive cells. In this study, we investigated the effect of metformin on hypoxia-induced apoptosis of osteoblasts and the therapeutic activity of intracanal metformin in induced periapical lesions in rats. METHODS: The influence of metformin on hypoxia-induced mitochondrial superoxide production in human osteoblasts was examined by using MitoSOX (Invitrogen, Carlsbad, CA) fluorescence dye signaling. The release of cytochrome c from mitochondria and the cleavage of procaspase-9 and poly(adenosine diphosphate-ribose) polymerase were evaluated by Western blot analysis. Apoptotic cell fraction was assessed by DNA content flow cytometry. In a rat model of induced periapical lesions, the effect of intracanal metformin on disease progression was appraised by 2-dimensional radiography and micro-computed tomographic imaging. Oxidative lesions and apoptotic activity of osteoblasts in vivo were estimated, respectively, by 8-hydroxy-2'-deoxyguanosine staining and terminal deoxynucleotidyl transferase dUTP nick end labeling. RESULTS: Metformin inhibited hypoxia-enhanced mitochondrial superoxide production in osteoblasts. Metformin suppressed hypoxia-induced cytochrome c release from mitochondria and the cleavage of procaspase-9 and poly(adenosine diphosphate-ribose) polymerase. Metformin repressed hypoxia-augmented apoptotic cell fraction. In a rat model, intracanal metformin diminished the size of periapical lesions and the oxidative damage and apoptotic activity in osteoblasts. CONCLUSIONS: Hypoxia increased oxidative stress in osteoblasts and enhanced cell death through activation of the mitochondrial pathway of apoptosis. Metformin attenuated the oxidative and cytotoxic action of hypoxia. The therapeutic effect of metformin on periapical lesions is partially caused by its antioxidative activity.

Increased Expression of Glutaminase in Osteoblasts Promotes Macrophage Recruitment in Periapical Lesions.

INTRODUCTION: Recently, we have shown that tissue hypoxia stimulates the progression of periapical lesions by up-regulating glycolysis-dependent apoptosis of osteoblasts. Other facets of hypoxia-induced metabolic reprogramming in disease pathogenesis require further investigation. In this study, we examined the connection between hypoxia-augmented glutamine catabolism in osteoblasts and the development of periapical lesions. METHODS: Primary human osteoblasts were cultured under hypoxia. The expression of glutaminase 1 (GLS1) was examined using Western blot analysis. The production of glutamate was measured by colorimetric assay. Knockdown of GLS1 was performed with small interfering RNA technology. C-C motif chemokine ligand 2 (CCL2) secretion and chemotaxis of J774 macrophages were examined by enzyme-linked immunosorbent assay and transwell migration assay, respectively. In a rat model of induced periapical lesions, the relations between disease progression and osteoblastic expression of GLS1 or macrophage recruitment were studied. RESULTS: Hypoxia enhanced GLS1 expression and subsequent glutamate production in osteoblasts. Glutamate induced chemoattraction of macrophages by osteoblasts through up-regulation of CCL2 synthesis. Hypoxia promoted CCL2 secretion and macrophage recruitment through augmentation of glutaminolysis. Knockdown of GLS1 abolished hypoxia-induced effects. In rat periapical lesions, progressive bone resorption was significantly related to elevated GLS1 expression in osteoblasts and increased macrophage recruitment. CONCLUSIONS: In addition to the rise in glycolytic activity, the progression of periapical lesions is also associated with enhanced glutamine catabolism in osteoblasts. GLS1 may be a potential therapeutic target in the management of periapical lesions.