Morphological and Proliferative Responses of Rat Microglia Cells to Lipopolysaccharide and Β-Amyloid

Microglia are the residential macrophages of the central nervous system (CNS) and are sensitive to any changes in their environment. There is rapid microglial activation in most pathological conditions in CNS. The association of activated microglia with various inflammatory and neurodegenerative diseases are well documented. In this study we evaluated the in vitro responses of microglia to lipopolysaccharide (LPS) or beta amyloid (Aβ) in an attempt to further define the activation of microglia. LPS is a potent activator of monocytes and macrophages and Aβ is used to activate microglia in vitro into a neurotoxic phenotype distinguished by secretion of proinflammatory molecules. Microglia were cultured from Sprague-Dawley neonatal rats and purity of culture determined using a common marker for microglia, lectin. Morphological changes following LPS or Aβ treatment were evaluated using fluorescent and confocal microscope. Treated microglia assumed a deramified shape with a condensed cytoplasm, typical of activated microglia. Stimulation of microglia with LPS or Aβ also resulted in significant ultrastructural changes. Transmission electron microscopy revealed an increased number of enlarged, elongated and swollen mitochondria. There were also some changes in the appearance of the endoplasmic reticulum. Untreated microglia displayed mainly smooth endoplasmic reticulum (SER) whereas LPS-treated cells displayed polyribosomes and rough endoplasmic reticulum (RER). It is therefore assumed that LPS-treated microglia have an increased ability in synthesising protein, some of which may be secretory molecules necessary for inflammation. Aβ-treated microglia also displayed more RER compared to control, but lesser compared to LPS. The nucleus in treated microglia appeared enlarged in comparison to untreated cells. The number of cells following treatment revealed that microglia are more viable following LPS treatment compared to Aβ. Immunophenotyping assays demonstrated upregulation of MHC II and CD40 by microglia following treatment. Both MHC II and CD40 are implicated in antigen-presenting and this result indicates that in comparison to LPS, Aβ may have more ability to induce antigen-presenting capabilities in microglia. As shown by viability counts, carboxy fluorescein succinimidyl ester (CFSE) staining also revealed no microglia proliferation following treatment with LPS and Aβ. In conclusion, our study illustrates activation-induced alterations in the morphology, ultrastructure and cell surface phenotype of microglia, which were not accompanied by proliferation of microglia. These changes represent the range of effects that occur in microglia following activation. In an attempt to culture microglia from adult rats for the purpose of determining the effects of aging on microglia responses, modifications were made on the established protocol for neonatal microglia cultures. This includes growing adult cells on poly-L-lysine-coated tissue culture flasks and substituting insulin in cell culture media with insulin-transferrin-selenium (ITS). We were able to successfully support the in vitro growth of adult microglia, which was also enhanced with the addition of the growth factor M-CSF. Key words: microglia, lipopolysacharide, beta amyloid, morphology, proliferation, CD40/MHC I

Effect of cystathionine-gamma-lyase gene silencing with siRNA on inflammation in acute pancreatitis

Hydrogen sulfide (H2S) is an endogenous inflammatory mediator produced by the activity of cystathionine γ–lyase (CSE) in mammals. Macrophages are a key element of the immune system and play a crucial role in inflammation. To determine the role of H2S and macrophages in inflammation, the expression of CSE in the murine macrophage cell line and human primary macrophages was investigated. The results showed that H2S is produced by the activity of CSE in macrophages. Acute pancreatitis is an inflammatory disorder in the pancreas which develops a cascade of immunological events and results in the activation of inflammatory cells including macrophages. After confirming that H2S is produced by the activity of CSE in macrophages the primary aim was to demonstrate the importance of hydrogen sulfide in the activation of macrophages by inhibiting CSE expression in macrophages during inflammation. Previous studies using pharmacological inhibition of CSE with (DL-propargylglycine) PAG produced conflicting results that may be due to the lack of specificity of this pharmacological agent. To overcome this problem, specific small interfering RNA (siRNA) molecules were used to silence CSE and inhibit the production of H2S by this enzyme in both in vitro and in vivo experiments. SiRNA treatment reduced the levels of CSE mRNA and protein in LPS-activated macrophages compared with controls and non-transfected cells. Furthermore, the levels of pro-inflammatory cytokines by LPS activated macrophages were significantly lower in siRNA transfected cells compared to untransfected controls. For example, increased levels of TNF-α (ng/ml) were observed by LPS-treated cells (11.22 ± 3.340SD) compared to control cells (0.03165 ± 0.00998SD) while there was a significant reduction (One-way ANOVA, p<0.01) in the levels of TNF-α in the siRNA transfected cells (7.782 ± 2.289SD). However, the production levels of NO by the transfected cells was higher, suggesting that CSE activity had an inhibitory effect on NO production. These findings suggest that the CSE enzyme has a crucial role in the activation of macrophages and its activity has an inhibitory effect on NO production by these cells. The mechanism by which H2S acts as an inflammatory mediator in LPS-induced macrophages (transfected with anti CSE siRNA) was investigated further. LPS-induced activation of transcription factor nuclear factor-κB (NF-κB) was reduced compared with untransfected cells and phosphorylation and activation of mitogen-activated protein kinase (ERK) 1/2 increased in LPS-induced macrophages. Phosphorylation of ERK in LPS-induced RAW 264.7 cells reached a peak at 30 minutes after activation. Silencing the CSE gene by siRNA reduced phosphorylation and activation of ERK1/2 in LPS-induced RAW 264.7 cells. These findings suggest that siRNA reduces the inflammatory effects of CSE through the ERK-NF-κB signalling pathway. To determine the role of common signalling pathway in biosynthesis of CSE in human primary macrophages, specific inhibitors were used to block NF-κB, ERK, p38 and JNK. Inhibition of NF-κB, ERK and JNK resulted in reduced levels of CSE expression in these cells but inhibition of p38 did not reduce the CSE expression in macrophages. To investigate the role of CSE expressed by macrophages, an in vivo mouse model of caerulein-induced acute pancreatitis was employed. SiRNA was injected into the tail vein to transfect blood monocytes. Higher levels of the pro-inflammatory cytokines TNF-α, IL-6, IL-1β and MIP-2 were found in the pancreas and the lung, as well as increased activity of pancreatic and lung MPO and plasma amylase levels were observed following caerulein-induced acute pancreatitis in mice. The siRNA treatment attenuated inflammation in the pancreas and lungs of mice following acute pancreatitis, as shown by MPO activity, plasma amylase activity and histology. Caerulein treatment increased pancreatic MPO activity (fold increase over saline group) (16.95 ± 3.22SD), but administration of siRNA before caerulein treatment reduced MPO activity significantly (3.97 ± 2.29SD) (One-way ANOVA, p<0.0001). There was also significantly reduced lung MPO activity following administration of siRNA. Caerulein treatment increased lung MPO activity (fold increased over saline group) (3.71 ± 0.84SD) while siRNA administration prior to caerulein treatment reduced lung MPO activity significantly (0.84 ± 0.38SD) (One-way ANOVA, p<0.0001). SiRNA treatment also reduced levels of pro-inflammatory mediators including IL-6, MCP-1, TNF-α, and IL-1β in the pancreas and the lung. These results were supported by histological experiments of the pancreas and the lung which demonstrated reduced infiltration of inflammatory leukocytes following silencing of CSE expression in macrophages compared to the mice without siRNA treatment. These findings indicate a crucial pro-inflammatory role for H2S synthesized by CSE in macrophages in acute pancreatitis and suggest a therapeutic role for siRNA to specifically silence CSE. The results from this thesis showed that CSE silencing with siRNA reduced macrophage activation and this has resulted in attenuation of inflammation in acute pancreatitis

5,6-Dioxo-1,10-phenanthrolin-1-ium nitrate

In the title salt, C12H7N2O2 +·NO3 −, the monoprotonated cation is connected to the nitrate anion by a hydrogen bond. Weak C—H⋯O hydrogen bonds hold the planar cations together in a layer structure

An Efficient Synthesis of Tetrahydrobenzo[ b

Tetrahydrobenzo[b]pyran derivatives were efficiently synthesized by the reaction of appropriated aromatic aldehydes, malononitrile and dimedone in the presence of SiO2-Pr-SO3H as a nanoporous and recoverable solid acid catalyst, in good to excellent yields. Single crystal x-ray analysis conclusively confirmed the structure of the 2-amino-3-cyano-7,7-dimethyl-4-(4-methylphenyl)-5-oxo-4H-5,6,7,8-tetrahydro-benzopyran

Synthesis and Characterization of Nanoporous Carbon Materials; The Effect of Surfactant Concentrations and Salts

Abstract: Nanoporous carbon framework was synthesized using phenol and formaldehyde as carbon precursors and triblock copolymer (pluronic F127) as soft template via evaporation induced self-assembly. Hexagonal mesoporous carbon with specific surface area of 350 m 2 /g through optimizing the situation was obtained. The effects of different surfactant/phenol molar ratio and presence of salts on specific surface area, pore size and pore volume for all the prepared samples were studied by means of the Brunauer-Emmett-Teller (BET) formalism, powder X-ray diffraction technique and FT-IR spectroscopy

Designer hydrogenated wrinkled Yolk@Shell TiO2 architectures: towards advanced room temperature visible light selective photocatalysts

Smart architectures of TiO2 are attracting increasing attention due to their outstanding properties in a broad range of fields. Herein, we report the preparation of an unprecedented yolk/shell wrinkled TiO2 architecture with excelling photocatalytic activities under visible light irradiation. This method includes solvothermal, partial etching and hydrogen treatment sequential preparation steps. The solvothermal step lead to yolk@shell TiO2 (Y@S-TiO2) structures which can generate a multi reflection of incident light so as to promote an efficient light harvesting due to an enhanced surface area and light scattering ability based on the hydrothermal alkaline partial etching. The hydrogen treatment process generated Ti3+ species on the surface of TiO2 which facilitate electron-hole separation, decreasing the band gap of titania to visible region. The resultant yolk@hydrogenated wrinkled shell TiO2 architecture exhibited a high efficiency in visible light oxidation of alcohols to the corresponding aldehydes (up to 90 in conversion, 97% in selectivity)

Knoevenagel condensation of isatins with malononitrile/ethyl cyanoacetate in the presence of sulfonic acid functionalized silica (SBA-Pr-SO3H) as a new nano-reactor

The Knoevenagel condensation between isatins and active methylene compounds like malononitrile and ethyl cyanoacetate to prepare 2-oxoindolin-3-ylidene malononitrile/ cyanoacetates is described. The reactions occur in the presence of sulfonic acid functionalized silica SBA-15 (SBA-Pr-SO3H) in an aqueous medium giving excellent yields of the products in short reaction times. SBA-Pr-SO3H with a pore size of 6 nm is found to be an efficient and environmentally benign catalyst for this reaction

Application of SBA-Pr-SO3H in the synthesis of benzoxazole derivatives

Propylsulfonic acid functionalized SBA-15 (SBA-Pr-SO3H) catalyzed the synthesis of 2-aryl benzoxazoles from 2-aminophenol and benzoyl chloride derivatives in good yields under reflux condition in acetic acid. In solvent free condition, hydroxybenzanilide derivatives were obtained

Establishing a culture system that supports in vitro expansion of adult microglia

Introduction: The vast majority of in vitro research on microglia are based on cells isolated from neonatal animals (3-5 days of age). Studying microglia of adults has been limited by the lack of a suitable culture system that supports their growth. In this study, we describe a protocol for growing microglia of adults based on modifications of the technique for culturing microglia isolated from neonatal rats. Methods: Mixed glia isolated from adult rats (age range of 1 month to 3 years old) were seeded in culture flasks coated with poly-L-lysine. Cells were maintained in DMEM media supplemented with insulin-transferrin-selenium (ITS) and recombinant human macrophage colony-stimulating factor (M-CSF). Mild trypsinisation was carried out to isolate microglia from mixed glia culture. Results: Microglia cells of adult rats were successfully grown in vitro. For the expansion of adult microglia, it was observed that coating the cell culture flasks with poly-L-lysine was crucial to encourage cell adherence. The substitution of insulin in culture media with ITS was found to improve cell yield and reduced the number of days required for culture from 28 days to 14 days. Addition of M-CSF to cell culture medium, along with the improvisations described above provided the best adult microglia cell yield (2.91 ± 0.56 × 10 6 cells) compared to the technique of replating cells (0.91 ± 0.65 × 10 6 cells; p<O.O5). Conclusion: Optimisation of primary cell culture technique by coating culture flasks with poly-L-lysine, supplementation of culture medium with ITS and M-CSF allowed microglia of adult rats to be successfully cultured in vitro