Targeted nanocarriers effectively remove cholesterol from macrophages.

TMA-DPH (1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene), a hydrophobic fluorescent membrane probe, interacts with living cells by instantaneous incorporation into the plasma membrane, where it becomes fluorescent. It then follows the intracellular constitutive membrane traffic and acts as a bulk membrane marker of the endocytic pathway (Illinger, D., P. Poindron, P. Fonteneau, M. Modolell, and J. G. Kuhry. 1990. Biochim. Biophys. Acta. 1030:73-81; Illinger, D., P. Poindron, and J. G. Kuhry. 1991. Biol. Cell. 73:131-138). As such, TMA-DPH displays particular properties mainly due to partition between membranes and aqueous media. From these properties, original arguments can be inferred in favor of the maturation model for the endocytic pathway, against that of pre-existing compartments, in L929 cultured mouse fibroblasts. (a) TMA-DPH labeling is seen to progress from the cell periphery to perinuclear regions during endocytosis without any noticeable loss in fluorescence intensity; with a vesicle shuttle model this evolution would be accompanied by probe dilution with a decrease in the overall intracellular fluorescence intensity, and the labeling of the inner (late) compartments could in no way become more intense than that of the peripheral (early) ones. (b) From TMA-DPH fluorescence anisotropy assays, it is concluded that membrane fluidity is the same in the successive endocytic compartments as in the plasma membrane, which probably denotes a similar phospholipidic membrane composition, as might be expected in the maturation model. (c) TMA-DPH internalization and release kinetics are more easily described with the maturation model.

<b>Abstract ID 17323</b> <b>Poster Board 339</b> <b>Aim:</b> We have previously reported that expression of multidrug resistance associated protein 5 (MRP5/ABCC5) in murine primary cortical neurons (PCNs) is much higher than other ABCC family transporters. However, its physiological function remains uncertain. We hypothesized that inhibition of MRP5 would promote neurite elongation because MRP5 efflux the second messengers such as cGMP and cAMP, which are important modulators of neurite elongation. The present study aimed to evaluate the effect of the knockdown of MRP5 on neurite outgrowth and to screen for MRP5 inhibitors among clinically used drugs acting in the central nervous system. <b>Methods:</b> Protein expression of MRP5 in PCNs was examined by immunocytochemical analysis. To evaluate the effect of MRP5 knockdown on neurite elongation, a neuronal cell line Neuro2A was transfected with siRNA for Mrp5 (siMrp5), followed by counting cells with neurites longer than the cell diameter. To check the efflux activity of MRP5 in Neuro2A, we used CMFDA, which is metabolized inside the cells to become a fluorescent substrate of MRP5; Neuro2A was incubated with CMFDA, and intracellular fluorescence intensity was measured. <b>Results and Discussion:</b> Immunocytochemical analysis showed that MRP5 was expressed in the PCNs and Neuro2A, which are positive for the neuronal marker βIII-tubulin. Transfection with siMrp5 in Neuro2A showed significantly lower mRNA expression of Mrp5 compared to negative siRNA-treated cells. The siMrp5 transfection significantly increased the number of cells with longer neurites and mRNA expression of neurite marker gap43 compared to negative siRNA transfection. The Mrp5 knockdown increased the expression of cGMP-dependent protein kinase (PKG), whereas a PKG inhibitor KT5823 significantly suppressed the neurite elongation induced by the knockdown with a concomitant decrease in the expression of PKG. These results suggest that inhibition of MRP5 promotes neurite elongation by activating PKG. Furthermore, we performed the screening for MRP5 inhibitors among clinical drugs. Neuro2A transfected with siMrp5 showed a higher intensity of intracellular fluorescence of CMFDA metabolite during incubation with CMFDA compared to negative siRNA. In addition, a typical inhibitor for MRP5 zaprinast also increased the fluorescent intensity, confirming that the assay is useful to assess the efflux activity of MRP5. Midazolam, perampanel, and nitrazepam remarkably increased the intracellular fluorescent intensity of the CMFDA metabolite. Exposure to midazolam or perampanel significantly promoted neurite elongation in PCNs. These drugs may thus potentially affect the neurite elongation by MRP5 inhibition and would be useful for the regulation of neurite outgrowth. Further studies are needed to elucidate the detailed mechanisms and to evaluate the possible involvement of clinically used MRP5 inhibitors in neurite elongation. <b>Conclusion:</b> Inhibition of MRP5 promotes neurite elongation by activating PKG. Midazolam and perampanel have been found to be novel MRP5 inhibitors, which promote neurite elongation in PCNs.

OBJECTIVE5-ALA guided resection of glioma in adults enables better delineation between tumor and normal brain, allowing improved resection and improved patients’ outcome. Recently, several reports were published regarding 5-ALA for resection of pediatric brain tumors. The aim of the study was to determine the intracellular fluorescence of PPIX in pediatric brain tumors by hyperspectral imaging and to compare it with visually observed intraoperative fluorescence.METHODS5-ALA was administered orally four hours prior to surgery. During tumor resection the surgeon assessed the fluorescence signal to be strong, weak or absent. Subsequently, fluorescence intensity of samples was measured via spectroscopy. In addition, clinical data, imaging and laboratory data were analyzed.RESULTSEleven children (1–16 years) were operated. Tumor entities included: three medulloblastomas, two pilocytic astrocytomas (PA), two anaplastic ependymomas and one diffuse astrocytoma, anaplastic astrocytoma, pilomyxoid astrocytoma and anaplastic pleomorphic xanthoastrocytoma. Strong fluorescence was visible in all anaplastic tumors and one PA; one PA demonstrated weak fluorescence. Visible fluorescence was strongly associated with intracellular fluorescence intensity and PPIX concentration (P<0.05). Within all tumors with visible fluorescence the intracellular PPIX concentration was greater than 4 µg/ml. Except for moderate and transient elevation of liver enzymes, no 5-ALA related adverse events were reported.CONCLUSIONWe demonstrate a strong association between intraoperative observations and spectrometric measurements of PPIX fluorescence in tumor tissue. As in former studies, fluorescence signal was more commonly observed in malignant glial tumors. Further prospective controlled trials should be conducted to investigate the feasibility of 5-ALA guided resection of pediatric brain tumors.

5-Aminolevulinic acid (5-ALA)-guided resection of gliomas in adults enables better delineation between tumor and normal brain, allowing improved resection and improved patients' outcome. Recently, several reports were published regarding 5-ALA for resection of pediatric brain tumors. The aim of the study was to determine the intracellular fluorescence of protoporphyrin IX (PPIX) in pediatric brain tumors by hyperspectral imaging and to compare it with visually observed intraoperative fluorescence. 5-ALA was administered orally 4h prior to surgery. During tumor resection, the surgeon assessed the fluorescence signal to be strong, weak, or absent. Subsequently, fluorescence intensity of tumor samples was measured via spectroscopy. In addition, clinical data, imaging, and laboratory data were analyzed. Eleven children (1-16years) were operated. Tumor entities included three (n = 3) medulloblastomas, two (n = 2) pilocytic astrocytomas (PA), two (n = 2) anaplastic ependymomas and one (n = 1) diffuse astrocytoma, anaplastic astrocytoma (n = 1), pilomyxoid astrocytoma (n = 1) and anaplastic pleomorphic xanthoastrocytoma (n = 1). Strong fluorescence was visible in all anaplastic tumors and one PA; one PA demonstrated weak fluorescence. Visible fluorescence was strongly associated with intracellular fluorescence intensity and PPIX concentration (P < 0.05). Within all tumors with visible fluorescence, the intracellular PPIX concentration was greater than 4μg/ml. Except for moderate and transient elevation of liver enzymes, no 5-ALA related adverse events were reported. We demonstrate a strong association between intraoperative observations and spectrometric measurements of PPIX fluorescence in tumor tissue. As in former studies, fluorescence signal was more commonly observed in malignant glial tumors. Further prospective controlled trials should be conducted to investigate the feasibility of 5-ALA-guided resection of pediatric brain tumors.

Real-Time Recording of the Cellular Effects of the Anion Transporter Prodigiosin

In vitro study of H +-sensitive neurons in the ventral medullary surface of neonate rats

Poly(amido)amine dendrimers generation 4.0 (PAMAM G4) reduce blood hyperglycaemia and restore impaired blood–brain barrier permeability in streptozotocin diabetes in rats

Persistent activation of macrophages (MP)s into a proinflammatory M1 or anti-inflammatory M2 phenotype plays a role in several pathological conditions, including autoimmune diseases, fibrosis, infections, atherosclerosis and tumor development. The mannose receptor (MR, CD206), expressed at low levels on resting MPs and absent on M1 MPs, is highly expressed on M2 MPs, making it a potential target and drug delivery portal. Recently, we developed a novel, highly selective MR targeting ligand (MRTL), consisting of two mannose molecules separated by a monodisperse 12 unit poly(ethylene glycol) linker, to enhance the cellular uptake of polymeric nanocarriers. The feasibility of using the MRTL ligand for selectively targeting M2 MPs for intracellular delivery of nanoparticles (NPs) was investigated. Rat peritoneal MPs were differentiated into an M1 or M2 phenotype using IFN-γ and IL-4/IL-13, respectively. Expression of the M1 marker, inducible nitric oxide synthase (iNOS), and the M2 markers arginase (Arg)-1 and MR (at both the mRNA and protein levels) confirmed MP phenotypic activation. Resting, M1 and M2 MPs were treated with fluorescein isothiocyanate (FITC)-labeled MRTL or NPs displaying FITC-labeled MRTL at two surface densities (1 and 10%) and examined by confocal microscopy. Intracellular fluorescence was also quantified. Uptake of the MRTL was 2.4- and 11.8-fold higher in M2 MPs when compared to resting or M1 MPs, respectively, consistent with marker expression levels. Mannan, a competitive inhibitor of the MR, abrogated MRTL uptake. MRTL also co-localized with a fluid-phase endocytosis marker, further suggesting that uptake was mediated by MR-mediated endocytosis. Intracellular NP fluorescence was confirmed by flow cytometry and by confocal microscopy. MRTL-NPs accumulated intracellularly with no significant cell surface binding, suggesting efficient translocation. NPs displaying a low surface density (1%) of the MRTL exhibited significantly higher (2.3-fold) uptake into M2 MPs, relative to resting and M1 MPs. The 10% MRTL-NPs displayed greater uptake by M2 MPs when compared to resting and M1 MPs, but less uptake than 1% MRTL-NPs into M2 MPs. Control FITC-labeled plain NPs did not exhibit selective MP uptake. These studies demonstrate that M2 MPs are selectively targeted by NPs displaying a novel bivalent ligand that utilizes the MR as a target/portal for cell entry. This study also establishes the feasibility of the approach allowing for further investigation in vivo.

Near-infrared absorption and emission probes with optimal connection bridges for live monitoring of NAD(P)H dynamics in living systems

To investigate the effect of peroxynitrite on the formation of diabetic cataract and its reversal by puerarin. in rat. Normal Sprague-Dawley (SD) rats were equally divided into control group, streptozotocin group (STZ) and treatment group. STZ and treatment group were treated with STZ to establish animal model of diabetic rat cataract and in treatment group puerarin was given by peritoneal injection. General condition and lens shape of rats were examined at 20th, 40th, 60th day respectively. Lens epithelial cells (LEC) were observed with optical microscope. percentage of apoptotic cells, and fluorescent intensity of positive cells for nitrotyrosine (NT) which is the symbol of peroxynitrite were tested by flow cytometry. The expression of NT in the lens was analyzed by Western blot analysis. On 20th, 40th, 60th day of the experiment, clear, mild and severe lens opacity were found in control group, treatment group, and STZ group respectively and the lens opacity was gradually increased in all of rats included in the study. Under optical microscope, no changes were presented in control group, but mild changes were showed in treatment group and remarkable changes were found in STZ group. On all the specified days, distinguishable differences were found in the amount and percentage of apoptotic cells compared with that of control group in treatment group and STZ groups as well as between treatment and STZ groups (P < 0.05). There was significant increase in the fluorescent intensity and amount of NT in treatment and STZ groups compared with that of control group (P < 0.05), and the similar results were revealed between treatment group and STZ. The amount of expression of NT protein at different time points in different groups as described above was also significant difference (P < 0.001). Peroxynitrite plays an important role in the formation of diabetic rat cataract and is a participant in the pathogenesis of oxidative stress induced cataract. Puerarin is an effective antioxidative agent in the treatment of early diabetic rat cataract.

For Bacillus licheniformis SJ4628, an organism widely used in the enzyme industry, methods for determination of cell vitality at a single cell level using 5(6)-carboxyfluorescein diacetate succinimidyl ester in combination with fluorescence ratio imaging microscopy and flow cytometry were developed. Immediately after inoculation and during growth, changes in intracellular pH values determined by fluorescence ratio imaging microscopy and in green fluorescence intensities determined by flow cytometry were observed. Correlations between the capacity to multiply and intracellular pH or green fluorescence intensity were demonstrated. Populations of cells not having a pH gradient or exhibiting low fluorescence intensities had significantly longer lag phases than populations of cells with a pH gradient and high fluorescence intensities.

Flow cytometric analysis of cell suspensions exposed to shock waves in the presence of the radical sensitive dye hydroethidine

To explore the protective effect of sodium channels antagonists HOE642 on lung ischemia reperfusion and the role of the p38 mitogen activated protein kinase (p38MAPK) signaling pathway in this process. Methods: A total of 36 mice were randomly divided into a sham operation group (SHAM group), a lung ischemia reperfusion group (I/R group) and a lung ischemia reperfusion+HOE642 group (HOE group). The water content was detected by electronic scales, and the lung tissue pathological changes were observed under optical microscope. The inflammatory cytokines including IL-6 and TNF-α were examined by ELISA. The intracellular calcium fluorescence intensity was examined and observed under fluorescence microscope, and the protein expression of p38MAPK was detected by Western blot. Results: Lung water content in the HOE group was lower than that in the I/R group, but higher than that in the SHAM group (both P<0.05). Lung interstitial edema, hemorrhage, lung tissue inflammatory cells infiltration were significantly alleviated in the HOE group than those in the I/R group, while the injury in the HOE group was aggravated than those in the SHAM group (both P<0.05). The IL-6 and TNF-α in lung tissues in the HOE group were lower than those in the I/R group, but higher than those in the SHAM group (both P<0.05). Intracellular calcium fluorescence intensity in the HOE group was lower than that in the I/R group, but higher than that in the SHAM group (both P<0.05). The protein expression of p38MAPK in lung tissues in the HOE group was lower than that in the I/R group, but higher than that in the SHAM group (both P<0.05). Conclusion: HOE642 may exert protective effect on pulmonary I/R injury through regulation of the p38MAPK signaling pathway, resulting in reduction of intracellular calcium ion concentration and calcium overload, and decrease of inflammatory response.

Objective To investigate the triggering mechanisms of retinal ganglion cells (RGCs)alterations in retinally degenerative rats.Methods Retinal dystrophic Royal College of Surgeons (RCS-p+)and control RCS rats (RCS-rdy+-p+) were divided into three groups according to postnatal days,including postnatal 21 days (P21d),P60d and P90d,with six rats in each group. Fluorogold was injected into superior colliculus and lateral geniculate body for retrograde labeling RGCs.Then retinal flat mounts were observed under fluorescence microscope to investigate the morphological and RGC changes in density during retinal degeneration.Living retinal mounts or sections were prepared to investigate the calcium concentration of RGCs using a laser confocal microscope. Results At P90d RGCs of control rats were distributed throughout entire retina and appeared uniform.The soma and processes were apparent and clear.But at P90d RGCs of RCS-p+ rats were distributed sparsely in retina.The soma seemed messy and the number and dendritic fields decreased greatly,and some baculiform or broken cells appeared. RGC density was5421.0±72.1/mm2 at P21d,4195.0±136.4/mm2 at P60dand2906.0±133.2/mm2 at P90d.At P21d there were no obvious differences in RGCs density between RCS-p+ and control rats (t=-1.301,P＞0.05).At P60d and P90d there were significant differences in RGC density (t=16.172,30.131; P＜0.01).At P2ld there was no significant differences in RGC intracellular calcium fluorescence intensity between RCS-p+ and control rats (t=-1.545,P＞0.05).At P60d and P90d the intracellular calcium fluorescence intensity decreased obviously and the differences were significant (t=-18.058,-15.015; P＜0.01).Conclusions RGCs were secondarily impaired following the loss of photoreceptors at late stages of retinal degeneration,and the morphological and cell density of RGCs were affected.The overloading of intracellular calcium concentration may be the triggering factor of degeneration and death of RGCs in retinally dystrophic rats. Key words: Retinal degeneration/pathophysiology; Retinal ganglion cells/physiology; Calcium/adverse effects; Animal experimentation

Nanoscale polymeric micelles have attracted more and more attention as a promising nanocarrier for controlled delivery of antineoplastic drugs. Herein, the doxorubicin (DOX)-loaded poly(D-lactide)-based micelle (PDM/DOX), poly(L-lactide)-based micelle (PLM/DOX), and stereocomplex micelle (SCM/DOX) from the equimolar mixture of the enantiomeric four-armed poly(ethylene glycol)-polylactide (PEG-PLA) copolymers were successfully fabricated. In phosphate-buffered saline (PBS) at pH 7.4, SCM/DOX exhibited the smallest hydrodynamic diameter (Dh) of 90 ± 4.2 nm and the slowest DOX release compared with PDM/DOX and PLM/DOX. Moreover, PDM/DOX, PLM/DOX, and SCM/DOX exhibited almost stable Dhs of around 115, 105, and 90 nm at above normal physiological condition, respectively, which endowed them with great potential in controlled drug delivery. The intracellular DOX fluorescence intensity after the incubation with the laden micelles was different degrees weaker than that incubated with free DOX · HCl within 12 h, probably due to the slow DOX release from micelles. As the incubation time reached to 24 h, all the cells incubated with the laden micelles, especially SCM/DOX, demonstrated a stronger intracellular DOX fluorescence intensity than free DOX · HCl-cultured ones. More importantly, all the DOX-loaded micelles, especially SCM/DOX, exhibited potent antineoplastic efficacy in vitro, excellent serum albumin-tolerance stability, and satisfactory hemocompatibility. These encouraging data indicated that the loading micelles from nonlinear enantiomeric copolymers, especially SCM/DOX, might be promising in clinical systemic chemotherapy through intravenous injection.