ABSTRACT
Although it is held that proinflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we demonstrate that FRS2ß, an adaptor protein expressed in a small subset of epithelial cells, triggers the proinflammatory changes that induce stroma in premalignant mammary tissues and is responsible for the disease onset. FRS2ß deficiency in mouse mammary tumor virus (MMTV)-ErbB2 mice markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2ß-deficient premalignant tissues. We found that colocalization of FRS2ß and the NEMO subunit of the IκB kinase complex in early endosomes led to activation of nuclear factor-κB (NF-κB), a master regulator of inflammation. Moreover, inhibition of the activities of the NF-κB-induced cytokines, CXC chemokine ligand 12 and insulin-like growth factor 1, abrogated tumorigenesis. Human breast cancer tissues that express higher levels of FRS2ß contain more stroma. The elucidation of the FRS2ß-NF-κB axis uncovers a molecular link between the proinflammatory changes and the disease onset.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Breast Neoplasms/etiology , Breast Neoplasms/metabolism , Mammary Neoplasms, Experimental/etiology , Mammary Neoplasms, Experimental/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Animals , Breast Neoplasms/immunology , Carcinogenesis , Cytokines/metabolism , Female , Humans , Inflammation/etiology , Inflammation/metabolism , Mammary Neoplasms, Experimental/immunology , Mammary Tumor Virus, Mouse , Mice , Mice, Knockout , NF-kappa B/metabolism , Pregnancy , Receptor, ErbB-2/metabolism , Retroviridae Infections , Tumor Microenvironment/immunology , Tumor Virus InfectionsABSTRACT
X-ray absorption near-edge structure (XANES) spectroscopy is a new method for the characterization of active pharmaceutical ingredients. XANES spectra show unique features depending on the electronic states of the X-ray absorbing elements and provide information about the chemical environment that affects the electronic states. In this study, six bisphosphonate hydrate crystals were used to investigate, for the first time, how the phosphorus K-edge XANES spectra are affected by the interatomic interactions and charged states of phosphonate moieties. Phosphorus K-edge XANES spectra showed several differences among the bisphosphonates. In particular, the chlorine atoms covalently bonded near the phosphonate and the number of electric charges of the phosphonate moieties seemed to have large effects on peak shape in XANES spectra. Unique shapes of the XANES spectra demonstrated that differences in interactions at the oxygen atoms of the phosphonate moieties could change the shapes of the XANES spectrum peaks to the extent that each material was distinguished based on the spectra. Since slight differences in interatomic interactions and charged states lead to variations in the spectra, XANES spectroscopy could be widely applied as the fingerprint method to evaluate active pharmaceutical ingredients.
Subject(s)
Diphosphonates , X-Ray Absorption Spectroscopy , Diphosphonates/chemistry , Phosphorus/chemistry , Crystallization , Molecular StructureABSTRACT
Sulfur K-edge X-ray absorption near-edge spectroscopy (XANES) measurements were performed to characterize the crystal polymorphs of the active pharmaceutical ingredients (APIs) containing sulfur atoms. Cimetidine (CIM) was used as a model API. Each crystal form of CIM has its own XANES spectrum, so we can discriminate the crystal form by its spectrum. The analysis of the crystal structure of CIM revealed that the difference in the shape of XANES spectra was ascribable to the difference in the C-S-C bond angle of CIM molecules and the intermolecular hydrogen bonds, such as C-H···S and N-H···S, and S-S interaction. It was found that the peak shape of the XANES spectrum is gentle when the C-S-C bond angle is large, while the peak shape can be steep when the C-S-C bond angle is small. Furthermore, it was found that the peak energy values varied depending on the hydrogen bonds and S-S interaction. By linear combination fitting using XANES spectra, it was possible to quantify the ratio of CIM form A crystal in mixed powders of form A and monohydrate crystals. These results indicate that XANES measurements can be a useful technique to evaluate the crystal polymorphism of APIs containing S atom in pharmaceutical formulation.
Subject(s)
Cimetidine , Sulfur , X-Rays , X-Ray Absorption Spectroscopy , Sulfur/chemistryABSTRACT
Polymorphic crystals of ambroxol, forms I and II, and form A ambroxol hydrochloride crystals were characterized with bromine K-edge X-ray absorption near-edge structure (XANES) spectroscopy and single-crystal X-ray structure analysis. The XANES spectra had unique shapes depending on the crystal forms. Refined single-crystal structures revealed different interatomic interactions around bromine atoms, such as C-H Br and N-H Br hydrogen bonds, Br O halogen bonds, and N-H π interactions. Differences in these weak interactions could affect the electronic states of the bromines, resulting in differences in the XANES spectra. The results demonstrated that weak non-conventional interatomic interactions could alter the shape of XANES spectra. Hence, the spectra could be used for evaluating polymorphs of active pharmaceutical ingredients.
Subject(s)
Ambroxol , Bromine , X-Rays , X-Ray Absorption Spectroscopy , Hydrochloric AcidABSTRACT
Nobiletin (NOB) is a flavonoid with attractive pharmaceutical characteristics, including anti-Alzheimer's, anti-inflammation, and anti-cancer properties, but it has low solubility in water, resulting in reduced bioavailability. Its solubility must be improved to develop NOB as a drug. Cocrystal engineering can change the physicochemical properties of an active pharmaceutical ingredient and generate remarkable drug candidates that are superior in drug formulation. In this report, extensive co-crystal screening of NOBs with 31 cocrystal formers (coformers) with various functional groups was carried out by the liquid-assisted grinding method. As a result, four cocrystals (NOB with urea (URE), oxalic acid, gallic acid and salicylic acid) and one solvate crystal (NOB with formic acid (FOR)) were found. Powder X-ray diffraction and thermal analysis revealed the unique crystal morphology of all the obtained samples. In addition, the crystal structures of two of them (NOB-URE and NOB-FOR) were determined by single crystal X-ray diffraction. The results revealed that NOB-URE and NOB-FOR are new cocrystals or solvate crystals consisting of molar ratios of 1 : 2 and 1 : 0.73, respectively. In NOB-URE, we could observe a transient increase in solubility due to supersaturation, suggesting that URE is one of the better coformers of NOB.
Subject(s)
Citrus , Flavonoids , Crystallization , X-Ray Diffraction , Solubility , Pharmaceutical PreparationsABSTRACT
Programmed cell death protein (PD)-1 is a coinhibitory molecule that suppresses immune response and maintains immune homeostasis. Moreover, the PD-1 pathway blocks cancers from being attacked by immune cells. Anti-PD-1 antibody therapy such as nivolumab improves survival in cancer patients. However, the occurrence of autoimmune inflammatory disorders in various organs has been increasingly reported as an adverse effect of nivolumab. Of the disorders associated with nivolumab, Sicca syndrome occurs in 3% to 11% of cases and has unknown pathologic mechanisms. Whether the absence of the PD-1 pathway causes functional and morphologic disorders in lacrimal glands was determined by analyzing PD-1 gene-knockout (Pdcd1-/-) mice. Histopathologic analysis showed that Pdcd1-/- mice developed dacryoadenitis beginning at 3 to 4 months of age, and deteriorated with age. Flow-cytometric analysis confirmed that cells infiltrating the affected lacrimal glands consisted mainly of CD3+ T cells and only a small proportion of CD19+ B cells. Among infiltrating T cells, the CD4+ Th-cell subset consisted of Th1 cells producing interferon-γ in an early stage of dacryoadenitis in Pdcd1-/- mice. Experiments of lymphocyte transfer from Pdcd1-/- into irradiated wild-type mice confirmed that CD4+ T cells from Pdcd1-/- mice induced dacryoadenitis. These results indicate that PD-1 plays an important role in the prevention of autoimmune inflammatory disorders in lacrimal glands caused by activated CD4+ Th1 cells.
Subject(s)
Autoimmune Diseases/immunology , Dacryocystitis/immunology , Dacryocystitis/metabolism , Programmed Cell Death 1 Receptor/deficiency , Th1 Cells/immunology , Animals , Autoimmune Diseases/metabolism , Autoimmunity/immunology , Disease Models, Animal , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Programmed Cell Death 1 Receptor/immunology , Sjogren's Syndrome/immunologyABSTRACT
Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) regulate the growth and morphogenesis of various exocrine glands with branched morphologies. Their roles in lacrimal gland (LG) development remain unknown, but fibroblast growth factor (FGF) 10 is crucial for early LG organogenesis. To clarify the roles of EGF, HGF, and FGF10 in LG development, LG epithelial cells were isolated from late-embryonic and neonatal mice; cultured; and treated with EGF, HGF, or FGF10 and their respective receptor tyrosine kinase (RTK) inhibitors AG1478, PHA665752, or SU5402. EGF and HGF increased the number of viable cells by enhancing DNA synthesis, FGF10 and SU5402 showed no such effect, and RTK inhibitors exhibited the opposite effect. EGF and HGF receptors were immunostained in cultured late-embryonic LG epithelial cells and terminal LG acini from late embryos and adult mice. HGF was detected in neonatal LG epithelial cell culture supernatants by western blotting. In the absence of EGF and HGF RTK inhibitors, growth factor addition increased the number of viable cells and suppressed cell death. However, when one RTK was inhibited and a growth factor targeting an intact RTK was added, the number of dead cells increased as the number of viable cells increased. No cells survived when both RTKs were inhibited. In explant cultures of LGs from embryos, AG1478 or PHA665752 decreased the number of Ki67-positive proliferating epithelial cells in terminal acini. Thus, EGF and HGF may function in a cooperative autocrine manner, supporting cell proliferation and survival during LG development in late-embryonic and neonatal mice.
Subject(s)
Epidermal Growth Factor , Lacrimal Apparatus , Animals , Cell Proliferation , Cells, Cultured , Epidermal Growth Factor/metabolism , Epidermal Growth Factor/pharmacology , Epithelial Cells , Fibroblast Growth Factors/metabolism , Lacrimal Apparatus/metabolism , MiceABSTRACT
Diabetic retinopathy is a major cause of blindness in developed countries, and is characterized by deterioration of barrier function causing vascular hyperpermeability and retinal edema. Vascular endothelial growth factor (VEGF) is a major mediator of diabetic macular edema. Although anti-VEGF drugs are the first-line treatment for diabetic macular edema, some cases are refractory to anti-VEGF therapy. Osteopontin (OPN) is a phosphoglycoprotein with diverse functions and expressed in various cells and tissues. Elevated OPN level has been implicated in diabetic retinopathy, but whether OPN is involved in hyperpermeability remains unclear. Using streptozotocin-induced diabetic mice (STZ mice) and human retinal endothelial cells (HRECs), we tested the hypothesis that up-regulated OPN causes tight junction disruption, leading to vascular hyperpermeability. The serum and retinal OPN concentrations were elevated in STZ mice compared to controls. Intravitreal injection of anti-OPN neutralizing antibody (anti-OPN Ab) suppressed vascular hyperpermeability and prevented decreases in claudin-5 and ZO-1 gene expression levels in the retina of STZ mice. Immunohistochemical staining of retinal vessels in STZ mice revealed claudin-5 immunoreactivity with punctate distribution and attenuated ZO-1 immunoreactivity, and these changes were prevented by anti-OPN Ab. Intravitreal injection of anti-OPN Ab did not change VEGF gene expression or protein concentration in retina of STZ mice. In an in vitro study, HRECs were exposed to normal glucose or high glucose with or without OPN for 48 h, and barrier function was evaluated by transendothelial electrical resistance and Evans blue permeation. Barrier function deteriorated under high glucose condition, and was further exacerbated by the addition of OPN. Immunofluorescence localization of claudin-5 and ZO-1 demonstrated punctate appearance with discontinuous junction in HRECs exposed to high glucose and OPN. There were no changes in VEGF and VEGF receptor-2 expression levels in HRECs by exposure to OPN. Our results suggest that OPN induces tight junction disruption and vascular hyperpermeability under diabetic conditions. Targeting OPN may be an effective approach to manage diabetic retinopathy.
Subject(s)
Diabetes Mellitus, Experimental , Diabetic Retinopathy , Macular Edema , Osteopontin , Tight Junctions , Animals , Blood-Retinal Barrier , Claudin-5/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetic Retinopathy/metabolism , Endothelial Cells/metabolism , Glucose/pharmacology , Macular Edema/metabolism , Mice , Osteopontin/genetics , Osteopontin/metabolism , Retina/metabolism , Retinal Vessels/metabolism , Streptozocin , Tight Junctions/metabolism , Tight Junctions/pathology , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Bromine K-edge X-ray absorption near-edge structure (XANES) spectroscopy analyses were used to evaluate the crystals of the active pharmaceutical ingredients, eletriptan hydrobromide, dextromethorphan hydrobromide and scopolamine hydrobromide salts and the solid dispersion of eletriptan hydrobromide. The crystals and the solid dispersion of the active pharmaceutical ingredient (API) salts could be discriminated based on the shape of the XANES spectra. The differences in the shape of XANES spectra was ascribable to the differences in the interatomic interactions of the bromine ions based on the crystal structures. Ratio of the eletriptan hydrobromide α-form crystal in mixed powders of α-form and monohydrate crystals could be quantified by the linear-combination fitting using their XANES spectra. These results indicated that the XANES spectroscopy are a potent method for evaluating the APIs of pharmaceutical formulations even at the higher energy region around the bromine K-edge of 13470 eV.
Subject(s)
Bromine/chemistry , Hydrocarbons, Brominated/analysis , Pharmaceutical Preparations/analysis , Catalysis , Molecular Structure , Salts/analysis , X-Ray Absorption SpectroscopyABSTRACT
Sulfur K-edge X-ray absorption near-edge structure (XANES) spectroscopy was evaluated for its ability to detect non-conventional C-HâªâªâªS hydrogen bonds in crystals of the sulfur-containing penam antibiotics ampicillin and amoxicillin. The XANES spectra of the nearly isomorphous crystals of ampicillin trihydrate and amoxicillin trihydrate were very similar, whereas that of ampicillin anhydrate displayed unique features. Single-crystal X-ray structure analyses revealed that the C-HâªâªâªS hydrogen bond geometries and the chemical types of the hydrogen donors differed between the isomorphous trihydrate crystals and ampicillin anhydrate crystal. These observations demonstrate that the shapes of the sulfur K-edge XANES spectra are dependent on the nature of the C-HâªâªâªS hydrogen bonds. Sulfur K-edge XANES spectroscopy shows promise for use in the detection and analysis of non-covalent interactions, including hydrogen bonds to sulfur atoms, within active pharmaceutical ingredients.
Subject(s)
Amoxicillin , Sulfur , Ampicillin , Anti-Bacterial Agents , Hydrogen , Hydrogen Bonding , Pharmaceutical Preparations , Sulfur/chemistry , X-Ray Absorption Spectroscopy/methods , X-RaysABSTRACT
Tear fluid secreted from the exocrine lacrimal gland (LG) has an essential role in maintaining a homeostatic environment for a healthy ocular surface. Tear secretion is regulated by the sympathetic and parasympathetic components of the autonomic nervous system, although the contribution of each component is not fully understood. To investigate LG innervation, we identified sympathetic and parasympathetic postganglionic nerves, specifically innervating the mouse LG, by injecting a retrograde neuronal tracer into the LG. Interruption of neural stimuli to the LG by the denervation of these postganglionic nerves immediately and chronically decreased tear secretion, leading to LG atrophy along with destruction of the lobular structure. This investigation also found that parasympathetic, but not sympathetic, innervation was involved in these alterations.
Subject(s)
Lacrimal Apparatus/innervation , Lacrimal Apparatus/metabolism , Tears/metabolism , Animals , Female , Mice , Mice, Inbred C57BL , Parasympathetic Nervous System/anatomy & histology , Parasympathetic Nervous System/physiologyABSTRACT
Microglia and Müller cells (MCs) are believed to be critically involved in hypoxia-induced blood-retinal barrier (BRB) disruption, which is a major pathogenic factor of various retinopathies. However, the underlying mechanism remains poorly defined. The inner BRB (iBRB) is primarily formed of microvascular endothelial cells (ECs) with tight junction (TJ), which are surrounded and supported by retinal glial cells. We developed a novel in vitro iBRB model sheet by sandwiching Transwell membrane with layered mouse brain microvascular ECs (bEnd.3) and mouse retinal MCs (QMMuC-1) on each side of the membrane. Using this model, we tested the hypothesis that under hypoxic condition, activated microglia produce inflammatory cytokines such as interleukin (IL)-1ß, which may promote vascular endothelial growth factor (VEGF) production from MCs, leading to TJ disruption. The iBRB model cell sheets were exposed to 1% oxygen for 6 h with or without mouse brain microglia (BV2) or IL-1ß. TJ structure and function were examined by zonula occludens (ZO)-1 immunostaining and fluorescein isothiocyanate permeability assay, respectively. Relative gene expression of IL-1ß in BV2 under normoxic and hypoxic conditions was examined by real-time reverse transcription-polymerase chain reaction. VEGF protein concentration in QMMuC-1 supernatants was measured by enzyme-linked immunosorbent assay. The bEnd.3 cell sheet incubated with BV2 in hypoxic condition or with IL-1ß in normoxic condition showed abnormal localization of ZO-1 and aberrated barrier function. Under normoxic condition, EC-MC iBRB model cell sheet showed lower permeability than bEnd.3 cell sheet. Under hypoxic conditions, the barrier function of EC-MC iBRB model cell sheet was more deteriorated compared to bEnd.3 cell sheet. Under hypoxic condition, incubation of EC-MC iBRB model cell sheet with BV2 cells or IL-1ß significantly increased barrier permeability, and hypoxia-treated BV2 cells expressed significantly higher levels of IL-1ß mRNA. Incubation of QMMuC-1 with IL-1ß increased VEGF production. These results suggest that under hypoxic condition, microglia are activated to release proinflammatory cytokines such as IL-1ß that promote VEGF production from MCs, leading to disruption of iBRB function. Modulating microglia and MCs function may be a novel approach to treat hypoxia-induced retinal BRB dysfunction.
Subject(s)
Blood-Retinal Barrier/physiology , Capillary Permeability/physiology , Endothelium, Vascular/metabolism , Ependymoglial Cells/physiology , Hypoxia/metabolism , Microglia/physiology , Tight Junctions/metabolism , Animals , Cell Line , Cells, Cultured , Coculture Techniques , Enzyme-Linked Immunosorbent Assay , Gene Expression , Immunohistochemistry , Interleukin-1beta/genetics , Mice , Models, Biological , RNA, Messenger/genetics , Retinal Vessels/cytology , Vascular Endothelial Growth Factor A/metabolism , Zonula Occludens-1 ProteinABSTRACT
Chlorine K-edge X-ray absorption near-edge spectroscopy (XANES) measurements were performed to characterize the crystal polymorphs of identical active pharmaceutical ingredients (APIs) containing chloride atoms and their amorphous solid dispersions (ASDs). Indomethacin (IMC), of which three crystal forms (α, ß, and γ) have been reported, was used as a model API. The shape of XANES spectra was unique to each IMC crystal. The analysis of the crystal structures of IMC revealed that chlorine atoms of the IMCα form had unique intermolecular interactions and halogen bonds with oxygen atoms, while those of the IMCγ form do not have any notable interactions. This result showed that XANES measurements can detect weak interatomic interactions. The shapes of the ASD spectra were clearly different from those of the crystals, suggesting that the environment around the Cl atom of IMC was different from that of the crystals. A thermal stress test was then performed to study the transformation from the amorphous form to the crystalline form of IMC in the ASD. The powder X-ray diffraction (PXRD) patterns indicated that amorphous IMC transformed into crystals during the thermal stress test. In accordance with the PXRD results, the XANES spectra also transformed from ASD to crystalline form. These results indicate that the IMC transformation could be monitored by XANES measurement. Our findings led us to conclude that XANES measurement is a novel approach for the evaluation of crystal polymorphs of APIs and the crystalline state of APIs in ASDs.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/analysis , Chemistry, Pharmaceutical/methods , Indomethacin/analysis , X-Ray Absorption Spectroscopy , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Crystallization , Feasibility Studies , Indomethacin/chemistryABSTRACT
PURPOSE: Disodium etidronate is a bisphosphonate, compounds that are widely used in the treatment of bone disorders such as osteoporosis and Paget's disease. We investigated the physical properties of disodium etidronate tetrahydrate crystal, form I. METHODS: We used X-ray powder diffraction (XRPD), thermal analysis, dynamic vapor sorption (DVS), X-ray single crystal structure analysis, and phosphorus K-edge X-ray absorption near-edge structure (XANES) spectroscopy for the first time. RESULTS: XRPD and thermal analyses demonstrated that form I was dehydrated and transformed to an amorphous form, to a crystalline form II, and finally to a form III by heating. DVS measurements revealed that the amorphous form, form II, and form III were rehydrated to form I by humidification, and form I was stable even at 0% relative humidity. These results indicate that form I is the most stable solid-state under ambient conditions and is suitable as an API for manufacture in solid formulations. The phosphorus K-edge XANES spectra differed among form I, the amorphous form, and form II, which may be ascribed to the difference in the coordinate bond schemes between the phosphate moieties and sodium ions. The results demonstrated that the phosphorus K-edge XANES spectroscopy could be applied to the identification or the discrimination of crystal forms of the APIs containing phosphate moieties. CONCLUSIONS: Acquired information about physical properties are crucial for manufacturing of solid formulations of disodium etidronate. XANES spectroscopy is a promising alternative method for evaluating the solid-state forms of APIs.
Subject(s)
Drug Compounding , Etidronic Acid/chemistry , Chemistry, Pharmaceutical/methods , Phosphorus/chemistry , X-Ray Absorption Spectroscopy , X-Ray DiffractionABSTRACT
Tearing maturates rapidly after birth, and external environmental challenges play a key role in promoting lacrimal functional maturation. However, little is known about the facilitative factors underlying this developmental process or the potential of application of these factors to treat hypofunction of the lacrimal gland. In this study, eye opening and the subsequent ocular surface sensory experience, which is thought to be involved in postnatal maturation of lacrimal function, were investigated. Our results demonstrated that eye opening after birth is essential for the maturation of neonatal tearing. The maturation process of lacrimal function is dependent on the ocular surface sensory experience via transient receptor potential cation channel subfamily member 1 after birth. This study provides, for the first time, important evidence of the sensory experience of the ocular surface in relation to the maturation of functional tear secretion during the postnatal period.
Subject(s)
Cornea/physiopathology , Lacrimal Apparatus Diseases/etiology , Rupture/etiology , TRPV Cation Channels/physiology , Animals , Animals, Newborn , Lacrimal Apparatus Diseases/metabolism , Lacrimal Apparatus Diseases/pathology , Mice, Inbred C57BL , Mice, Knockout , Rupture/metabolism , Rupture/pathologyABSTRACT
Elevated level of interleukin (IL)-17, predominantly produced by T helper (Th) 17 cells, has been implicated in diabetic retinopathy (DR), but it remains unclear whether IL-17 is involved in the pathogenesis of DR. Ins2Akita (Akita) mice spontaneously develop diabetes, and show early pathophysiological changes in diabetic complications. On the other hand, interferon-γ knock out (GKO) mice exhibit high differentiation and activation of Th2 and Th17 cells as a result of Th1 cell inhibition. In this study, Ins2Akita IFN-γ-deficient (Akita-GKO) mice were established by crossbreeding Akita mice with GKO mice, and Th17-mediated immune responses on DR were investigated. Blood glucose levels (BGL) of Akita mice and Akita-GKO mice were significantly higher than those of age-matched wild type (WT) or GKO mice, and there was no significant difference in BGL between Akita and Akita-GKO mice. Relative mRNA expression of ROR-γt that is a transcriptional factor of Th17 cells but not GATA-3 that is for Th2 cells was significantly upregulated only in Akita-GKO mice compared with WT mice, and the proportions of IL-17 and IL-22-producing splenic CD4+ cells were significantly higher in Akita-GKO mice than in wild type (WT), Akita, or GKO mice. In the retina, mRNA expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) were increased in Akita-GKO mice more than in Akita or GKO mice, and statistically significant differences were observed between Akita-GKO mice and WT mice. Leukostasis in retinal vessels and ocular level of VEGF protein increased significantly in Akita-GKO mice compared with the other groups. Edematous change in the retinal surface layer, retinal exudative lesions depicted as areas of hyperfluorescence in fluorescein angiography (FA), and vascular basement membrane thickening in all layers of the retina were also observed in Akita-GKO mice at 9-week-old but not in age-matched Akita or GKO mice. These results suggested that Th17 cell-mediated immune responses might be involved in promotion of functional and morphological changes in the retina of mice spontaneously developing diabetes.
Subject(s)
Diabetes Mellitus, Experimental , Diabetic Retinopathy/diagnosis , Immunity, Cellular , Lymphocyte Activation/immunology , Th17 Cells/pathology , Animals , Cell Differentiation , Diabetic Retinopathy/immunology , Lymphocyte Count , Mice , Mice, Inbred C57BL , Th17 Cells/immunologyABSTRACT
Hypoxia-induced retinal edema primarily induced by vascular lesion is seen in various conditions such as diabetic retinopathy (DR) and retinal vein occlusion (RVO). The edematous changes in these conditions occur mainly in intermediate and deep layers of retina as a result of disruption of the inner blood-retinal barrier (iBRB). However, the effect of direct and acute hypoxia on iBRB remains to be elucidated. To investigate direct and acute hypoxia-induced changes in retina, especially in astrocytes/Müller cells that are involved in the maintenance of retinal structure and function, we developed an adult mouse model of hypoxia-induced retinal edema by 24-h exposure in a 6% oxygen environment. Immunohistochemical staining of glial fibrillary acidic protein (GFAP) was enhanced mainly in the superficial layer of the hypoxic retina, corresponding to edematous change. Electron microscopic observation of the hypoxic retina showed vacuole formation in astrocyte/Müller cell foot processes around capillaries in the superficial layer, while no abnormal findings in the perivascular areas were found in intermediate and deep layers. Increase in vascular leakage quantified by Evans blue dye and tight junction breakdown detected by electron-dense tracer were observed in the hypoxia group. In the hypoxic retina, microglia was activated and relative gene expressions of pro-inflammatory cytokines were significantly upregulated. Dexamethasone suppressed these hypoxia-induced pathological reactions. Thus, unlike DR and RVO that induce iBRB breakdown in deeper retinal layers, atmospheric hypoxia induced iBRB disruption with subsequent edematous change mainly in the superficial layer of the retina, and that dexamethasone prevented these pathological changes. In this mouse model, direct and acute hypoxia induces retinal edema in the superficial layer of the retina with morphological changes of astrocytes/Müller cells, and is potentially useful for ophthalmic research in the field related to retinal hypoxia and its treatment.
Subject(s)
Dexamethasone/pharmacology , Disease Models, Animal , Glucocorticoids/pharmacology , Hypoxia/complications , Papilledema/prevention & control , Animals , Blood-Retinal Barrier/physiology , Cytokines/metabolism , Fluorescein Angiography , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry , Mice , Mice, Inbred C57BL , Microscopy, Electron, Transmission , Oxygen/toxicity , Papilledema/etiology , Papilledema/metabolism , Papilledema/pathology , Real-Time Polymerase Chain ReactionABSTRACT
BACKGROUND: A report examining whether clinically available antidepressants increase urethral smooth muscle contraction via antagonistic effects on the α2-adrenoceptor (α2-AR) is lacking. OBJECTIVES: The present study was performed to evaluate the potential of clinically available antidepressants to reverse α2-AR-mediated contractile inhibition in rat vas deferens, in order to predict whether they can induce voiding impairment. METHOD: The effects of 18 antidepressants of different classes on electrical field stimulation (EFS)-induced contractions suppressed by 10-8 mol/L clonidine (a selective α2-AR agonist) in isolated rat vas deferens were investigated and related to their respective clinical blood concentrations. RESULTS: The EFS-induced contractions suppressed by clonidine were recovered by amitriptyline (a tricyclic antidepressant), mirtazapine (a noradrenergic and specific serotonergic antidepressant), and trazodone (a serotonin 5-HT2A receptor antagonist) at concentrations close to the clinical blood levels. EFS-induced contractions were also recovered by trimipramine, clomipramine (tricyclic antidepressants), mianserin (a tetracyclic antidepressant), sertraline (a selective serotonin reuptake inhibitor [SSRI]), and sulpiride (a dopamine D2-receptor antagonist), albeit at concentrations that substantially exceeded their clinically-achievable blood levels. EFS-induced contractions were not significantly affected by imipramine, nortriptyline, amoxapine (tricyclic antidepressants), maprotiline (a tetracyclic antidepressant), fluvoxamine, paroxetine, escitalopram (SSRIs), milnacipran, duloxetine (serotonin and noradrenaline reuptake inhibitors), and aripiprazole (a dopamine partial agonist). CONCLUSIONS: These findings suggest that amitriptyline, mirtazapine, and trazodone induce voiding impairment caused by increased urethral resistance by enhancing sympathetic nerve activities attributed to α2-AR antagonism.
Subject(s)
Adrenergic alpha-2 Receptor Agonists/pharmacology , Antidepressive Agents/toxicity , Clonidine/pharmacology , Dysuria/chemically induced , Muscle Contraction , Muscle, Smooth/drug effects , Vas Deferens/drug effects , Animals , Antidepressive Agents/classification , Dose-Response Relationship, Drug , Dysuria/physiopathology , Electric Stimulation , In Vitro Techniques , Male , Muscle, Smooth/physiopathology , Rats, Wistar , Risk Assessment , Vas Deferens/physiopathologyABSTRACT
The morphology and stability of amorphous nanoparticles of glibenclamide (GLB) prepared by the antisolvent method using different methods of adding hypromellose (HPMC) were evaluated. Nano-A was prepared by the injection of a dimethyl sulfoxide (DMSO) solution of GLB into the HPMC solution, whereas nano-B was obtained by the injection of a DMSO solution of GLB and HPMC into water. Cryogenic transmission electron microscopy, field-emission scanning electron microscopy, and field-emission transmission electron microscopy, including energy dispersive X-ray spectrometry, revealed that the particles of the nano-A and nano-B samples are hollow spheres and nonspherical nanoparticles, respectively. Powder X-ray diffraction and solid-state NMR measurements showed that GLB is present in an amorphous state in both nano-A and nano-B. The weight ratios of HPMC in the GLB/HPMC nanoparticles were 11 and 16% for nano-A and nano-B, respectively, as determined by solution-state NMR. The glass transition temperatures ( Tg) of nano-A and nano-B evaluated using differential scanning calorimetry were lower by about 10 °C compared to that of amorphous GLB, presumably because of a Tg confinement effect and the surface coverage and mixing of HPMC, as suggested by the inverse gas chromatography experiment. GLB crystallization during storage was suppressed more strongly in nano-B than nano-A, owing to the higher amount of HPMC and the higher miscibility between GLB and HPMC. It is suggested that the diffusion rate of the solvent during nanoprecipitation determined the nanoparticle properties. In nano-A, the precipitation of GLB first occurred at the outer interface because of the rapid diffusion of the solvent. Thus, hollow spherical particles with HPMC preferentially located near the surface were formed. On the other hand, the diffusion of the solvent in nano-B was suppressed because of the presence of HPMC, yielding small nonspherical nanoparticles with a high miscibility of GLB and HPMC.