phalloidine and Disease-Models--Animal

Merosin deficient congenital muscular dystrophy (MDC1A) is a severe neuromuscular disorder with onset in infancy that is associated with severe morbidities (particularly wheelchair dependence) and early mortality. It is caused by recessive mutations in the LAMA2 gene that encodes a subunit of the extracellular matrix protein laminin 211. At present, there are no treatments for this disabling disease. The zebrafish has emerged as a powerful model system for the identification of novel therapies. However, drug discovery in the zebrafish is largely dependent on the identification of phenotypes suitable for chemical screening. Our goal in this study was to elucidate novel, early onset abnormalities in the candyfloss (caf) zebrafish, a model of MDC1A. We uncovered and characterize abnormalities in spontaneous coiling, the earliest motor movement in the zebrafish, as a fully penetrant change specific to caf mutants that is ideal for future drug testing.

Topics: Animals; Animals, Genetically Modified; Disease Models, Animal; Heterozygote; Laminin; Muscle, Skeletal; Muscular Dystrophies; Mutation; Phalloidine; Phenotype; Zebrafish

This study utilized pharmacological activation of Nrf2 with oleanolic acid (OA, 22.5mg/kg, sc for 4 days) and the genetic alteration of Nrf2 (Nrf2-null, wild-type, and Keap1-HKO mice) to examine the role of Nrf2 in protection against phalloidin hepatotoxicity. Mice were given phalloidin (1.5mg/kg, ip for 8h) to examine liver injury and the expression of toxicity-related genes. Phalloidin increased serum enzyme activities and caused extensive hepatic hemorrhage and necrosis in Nrf2-null and wild-type mice, but less injury was seen in Keap1-HKO mice and OA-pretreated mice. Phalloidin increased the expression of neutrophil-specific chemokine mKC and MIP-2 in Nrf2-null and WT mice, but such increases were attenuated in Keap1-HKO and OA-pretreated mice. Phalloidin increased, while Nrf2 activation attenuated, the expression of genes involved in acute-phase response (Ho-1) and DNA-damage response genes (Gadd45 and Chop10). Phalloidin is taken up by hepatocytes through Oatp1b2, but there was no difference in basal and phalloidin-induced Oatp1b2 expression among Nrf2-null, wild-type, and Keap1-HKO mice. In contrast, OA decreased phalloidin-induced Oatp1b2. Phalloidin activated MAPK signaling (p-JNK), which was attenuated by activation of Nrf2. In conclusion, this study demonstrates that protection against phalloidin hepatotoxicity by OA involves activation of Nrf2 and suppression of Oatp1b2.

Topics: Animals; Chemical and Drug Induced Liver Injury; Cytoprotection; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Gene Expression Regulation; Inflammation Mediators; Kelch-Like ECH-Associated Protein 1; Lipid Peroxidation; Liver; Mice, Inbred C57BL; Mice, Knockout; NF-E2-Related Factor 2; Oleanolic Acid; Organic Anion Transporters, Sodium-Independent; Phalloidine; RNA, Messenger; Signal Transduction; Solute Carrier Organic Anion Transporter Family Member 1B3

The Mongolian gerbil, Meriones unguiculatus, has been widely employed as a model for studies of the inner ear. In spite of its established use for auditory research, no robust protocols to induce ototoxic hair cell damage have been developed for this species. In this paper, we demonstrate the development of an aminoglycoside-induced model of hair cell loss, using kanamycin potentiated by the loop diuretic furosemide. Interestingly, we show that the gerbil is relatively insensitive to gentamicin compared to kanamycin, and that bumetanide is ineffective in potentiating the ototoxicity of the drug. We also examine the pathology of the spiral ganglion after chronic, long-term hair cell damage. Remarkably, there is little or no neuronal loss following the ototoxic insult, even at 8 months post-damage. This is similar to the situation often seen in the human, where functioning neurons can persist even decades after hair cell loss, contrasting with the rapid, secondary degeneration found in rats, mice and other small mammals. We propose that the combination of these factors makes the gerbil a good model for ototoxic damage by induced hair cell loss.

Topics: Aminoglycosides; Animals; Cochlea; Disease Models, Animal; Female; Gerbillinae; Hair Cells, Auditory, Outer; Hearing; Hearing Loss; Humans; Immunohistochemistry; Kanamycin; Male; Myelin Sheath; Phalloidine; Regeneration; Spiral Ganglion

Nemaline myopathy is an inherited muscle disease that is mainly diagnosed by the presence of nemaline rods in muscle biopsies. Of the nine genes associated with the disease, five encode components of striated muscle sarcomeres. In a genetic zebrafish screen, the mutant träge (trg) was isolated based on its reduction in muscle birefringence, indicating muscle damage. Myofibres in trg appeared disorganised and showed inhomogeneous cytoplasmic eosin staining alongside malformed nuclei. Linkage analysis of trg combined with sequencing identified a nonsense mutation in tropomodulin4 (tmod4), a regulator of thin filament length and stability. Accordingly, although actin monomers polymerize to form thin filaments in the skeletal muscle of tmod4(trg) mutants, thin filaments often appeared to be dispersed throughout myofibres. Organised myofibrils with the typical striation rarely assemble, leading to severe muscle weakness, impaired locomotion and early death. Myofibrils of tmod4(trg) mutants often featured thin filaments of various lengths, widened Z-disks, undefined H-zones and electron-dense aggregations of various shapes and sizes. Importantly, Gomori trichrome staining and the lattice pattern of the detected cytoplasmic rods, together with the reactivity of rods with phalloidin and an antibody against actinin, is reminiscent of nemaline rods found in nemaline myopathy, suggesting that misregulation of thin filament length causes cytoplasmic rod formation in tmod4(trg) mutants. Although Tropomodulin4 has not been associated with myopathy, the results presented here implicateTMOD4 as a novel candidate for unresolved nemaline myopathies and suggest that the tmod4(trg) mutant will be a valuable tool to study human muscle disorders.

Topics: Actinin; Actins; Alleles; Animals; Animals, Genetically Modified; Cytoplasm; Disease Models, Animal; Genetic Linkage; Male; Muscles; Muscular Diseases; Mutation; Myofibrils; Neuromuscular Diseases; Phalloidine; Phenotype; Sarcomeres; Tropomodulin; Zebrafish; Zebrafish Proteins

Recent studies have shown that mutations in PTPRQ, a gene encoding a receptor-like inositol lipid phosphatase, cause recessive, nonsyndromic, hereditary hearing loss with associated vestibular dysfunction. Although null mutations in Ptprq cause the loss of high-frequency auditory hair cells and deafness in mice, a loss of vestibular hair cells and overt behavioral defects characteristic of vestibular dysfunction have not been described. Hair bundle structure and vestibular function were therefore examined in Ptprq mutant mice. Between postnatal days 5 and 16, hair bundles in the extrastriolar regions of the utricle in Ptprq(-/-) mice become significantly longer than those in heterozygous controls. This increase in length (up to 50%) is accompanied by the loss and fusion of stereocilia. Loss and fusion of stereocilia also occurs in the striolar region of the utricle in Ptprq(-/-) mice, but is not accompanied by hair bundle elongation. These abnormalities persist until 12 months of age but are not accompanied by significant hair cell loss. Hair bundle defects are also observed in the saccule and ampullae of Ptprq(-/-) mice. At ∼3 months of age, vestibular evoked potentials were absent from the majority (12 of 15) of Ptprq(-/-) mice examined, and could only be detected at high stimulus levels in the other 3 mutants. Subtle but distinct defects in swimming behavior were detected in most (seven of eight) mutants tested. The results reveal a distinct phenotype in the vestibular system of Ptprq(-/-) mice and suggest similar hair bundle defects may underlie the vestibular dysfunction reported in humans with mutations in PTPRQ.

Topics: Acoustic Stimulation; Actins; Age Factors; Animals; Animals, Newborn; Disease Models, Animal; Evoked Potentials, Auditory; Hair Cells, Auditory; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Microscopy, Electron; Mutation; Phalloidine; Psychoacoustics; Receptor-Like Protein Tyrosine Phosphatases, Class 3; Receptors, G-Protein-Coupled; Stereocilia; Vestibular Diseases

The purpose of this study was to assess the ability of quantitative in vivo confocal microscopy to characterize the natural history and detect changes in crystal volume in corneas from a novel animal model of cystinosis, the cystinosin (Ctns(-/-)) mouse.. Two Ctns(-/-) mice and one C57Bl/6 mouse were examined at each of the following time points: 2, 3, 5, 7, 10, 12, and 14 months of age. In vivo confocal microscopy scans were performed in 4 different regions of the cornea per eye. After, animals were sacrificed and cornea blocks evaluated for cell morphology using phalloidin and lymphocytic infiltration using CD45 antibodies by ex vivo confocal microscopy. Cystine crystal content in the cornea was measured by calculating the pixel intensity of the crystals divided by the stromal volume using Metamorph Image Processing Software.. Corneal crystals were identified in Ctns(-/-) eyes beginning at 3 months of age and increased in density until 7-12 months, at which time animals begin to succumb to the disease and corneas become scarred and neovascularized. Older Ctns(-/-) mice (7 months and older) showed the presence of cell infiltrates that stained positively for CD45 associated with progressive keratocyte disruption. Finally, at 12 months of age, decreased cell density and endothelial distortion were detected.. Confocal microscopy identified corneal crystals starting at 3 month old Ctns(-/-) eyes. Cystine crystals induce inflammatory and immune response with aging associated with loss of keratocyte and endothelial cells. These findings suggest that the Ctns(-/-) mouse can be used as a model for developing and evaluating potential alternative therapies for corneal cystinosis.

Topics: Age Factors; Amino Acid Transport Systems, Neutral; Animals; Cicatrix; Cornea; Crystallization; Cystine; Cystinosis; Disease Models, Animal; Endothelial Cells; Female; Keratinocytes; Leukocyte Common Antigens; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Neovascularization, Pathologic; Phalloidine

Structural and functional neuroimaging studies suggest abnormal activity in the striatum of patients with the fragile X syndrome (FXS), the most common form of inherited mental retardation.. Neurophysiological and immunofluorescence experiments in striatal brain slices. We studied the synaptic transmission in a mouse model for FXS, as well as the subcellular localization of fragile X mental retardation protein (FMRP) and brain cytoplasmic (BC1) RNA in striatal axons.. Our results show that absence of FMRP is associated with apparently normal striatal glutamate-mediated transmission, but abnormal gamma-aminobutyric acid (GABA) transmission. This effect is likely secondary to increased transmitter release from GABAergic nerve terminals. We detected the presence of FMRP in axons of striatal neurons and observed a selective increase in the frequency of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs, mIPSCs) in fmr1-knockout mice. We also observed reduced paired-pulse ratio of evoked IPSCs, a finding that is consistent with the idea that transmitter release probability from striatal GABAergic nerve terminals is higher than normal in these mutants. Finally, we have identified the small noncoding BC1 RNA as a critical coplayer of FMRP in the regulation of striatal synaptic transmission.. Understanding the physiologic action of FMRP and the synaptic defects associated with GABA transmission might be useful to design appropriate pharmacologic interventions for FXS.

Topics: Animals; Cerebral Cortex; Corpus Striatum; Disease Models, Animal; Electric Stimulation; Fragile X Mental Retardation Protein; Fragile X Syndrome; gamma-Aminobutyric Acid; Gene Expression Regulation; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Neural Inhibition; Patch-Clamp Techniques; Phalloidine; Ribonucleoproteins, Small Cytoplasmic; RNA, Long Noncoding; RNA, Untranslated; Synaptic Transmission; Vesicular Inhibitory Amino Acid Transport Proteins

Choroidal neovascularization (CNV) is the end point of several ocular diseases that lead to blindness. The authors developed an imaging technique for visualizing and quantifying morphologic changes associated with experimental laser-induced CNV.. CNV was induced using laser energy to disrupt Bruch's membrane. Rats were euthanatized immediately after laser injury and at 1, 2, 3, 4, 7, 14, and 60 days. Nonlasered eyes were used as the control. Eyes were enucleated and fixed, and the posterior eye cups were fluorescently labeled with markers for nuclei (DAPI; 4',6'-diamino-2-phenylindole), endothelial cells (isolectin IB4), microglia (CD11b), and filamentous actin (phalloidin). FITC-dextran perfusion was compared with our technique. A confocal microscope was used to evaluate flatmounted specimens. Computer software generated three-dimensional reconstructions for qualitative and quantitative analysis of confocal image stacks.. In nonlasered areas, RPE cells were visualized as a uniform hexagonal array. Immediately after laser exposure, a circular area devoid of fluorescent labeling was observed, indicating disruption of the choroid-Bruch's membrane-RPE complex. One day after laser exposure, cellular debris and fragmented nuclei were present, and an autofluorescent ring was visible at the site of Bruch's membrane disruption. The ring correlated with bubble formation and CNV induction. Three days after laser injury, phalloidin-labeled RPE cells and isolectin-labeled endothelial cells increased significantly, reflecting cell proliferation and migration. By day 4, isolectin-positive cells forming vascular tubes were visualized. The volume of CNV vessels increased exponentially during the next 3 days. By 7 days, a well-defined isolectin-labeled CNV network was present, and its volume was preserved for several weeks. CNV volumes calculated on the basis of FITC-dextran perfusion were significantly lower than volumes obtained using lectin-labeled samples.. A novel imaging technique was developed that allows a three-dimensional reconstruction and measurement of laser-induced CNV lesions in rat choroid/RPE flatmounts. This technique provides excellent morphologic detail and facilitates the study of critical early events in CNV, including the rupture of Bruch's membrane and the formation of endothelial clusters before vessel formation. CNV complexes are labeled at an earlier stage and more reproducibly than with FITC-dextran perfusion, providing a more accurate preclinical evaluation of antiangiogenic molecules.

Topics: Actins; Animals; Biomarkers; CD11b Antigen; Cell Nucleus; Choroidal Neovascularization; Dextrans; Disease Models, Animal; Endothelium, Vascular; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Glycoproteins; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Indoles; Lectins; Male; Microglia; Microscopy, Confocal; Phalloidine; Pigment Epithelium of Eye; Rats; Rats, Inbred BN; Versicans

Biliary glutathione is an important generator of the bile-salt independent flow, and is known to be regulated by the hepatic glutathione availability. We investigated, in an acute model of phalloidin-induced cholestasis, biliary glutathione secretion and the role of hepatic glutathione, oxidative stress, and protein kinase c activation, which have been implicated in many hepatotoxin-induced hepatic dysfunctions.. Rats were given a single dose of 80 microg/100 g body weight of phalloidin and the hepatic thiols and glutathione content, redox state and vesicular activity were evaluated during both development of and recovery from cholestasis. The prophylactic effect of N-acetylcysteine (a precursor of glutathione synthesis and an antioxidant) was also examined. In addition, in the isolated perfused rat liver, we studied the prophylactic effect of the PKc inhibitor H7 on phalloidin-induced cholestasis.. In the early stages of cholestasis, phalloidin induced a decline in bile flow, mainly related to a disruption of biliary glutathione secretion. The decline in biliary glutathione content was not associated with increased glutathione degradation, indicated by a parallel decline in biliary non-protein thiols and by the lack of an increase in biliary gamma-glutamyltranspeptidase. There was also no evidence of hepatic depletion of glutathione or of oxidative stress, as measured by the oxidized-to-reduced glutathione ratio. Moreover, phalloidin resulted in inhibition of vascular transcytosis as assessed by horseradish peroxidase labeling. Pre-treatment with N-acetylcysteine did not counteract the decline in biliary glutathione secretion and bile flow produced by phalloidin, supporting the view that the hepatic availability of glutathione and oxidative stress injury are not implicated in the early stages of cholestatic injury. Moreover, treatment with H-7 did not alter the biliary glutathione output, or the decline in bile flow induced by the toxin.. This study suggests that the phalloidin-induced inhibition of bile formation may be attributed to rapid disruption of the hepatocanalicular transport of glutathione.

Topics: Animals; Cholestasis; Disease Models, Animal; Glutathione; Male; Oxidative Stress; Phalloidine; Rats; Rats, Sprague-Dawley

Dystrophin is the product of the gene that is mutated in Duchenne muscular dystrophy (DMD), a progressive neuromuscular disease for which no treatment is available. Mice carrying a mutation in the gene for dystrophin (mdx mice) display only a mild phenotype, but it is aggravated when combined with a mutation in the MyoD gene. The nematode worm Caenorhabditis elegans has a dystrophin homologue (dys-1), but null mutations in dys-1 do not result in muscle degeneration.. We generated worms carrying both the dys-1 null mutation cx18, and a weak mutation, cc561ts, of the C. elegans MyoD homologue hlh-1. The double mutants displayed a time-dependent impairment of locomotion and egg laying, a phenotype not seen in the single mutants, and extensive muscle degeneration. This result allowed us to look for genes that, when misexpressed, could suppress the dys-1; hlh-1 phenotype. When overexpressed, the dyc-1 gene - whose loss-of-function phenotype resembles that of dys-1 - partially suppressed the dys-1; hlh-1 phenotype. The dyc-1 gene encodes a novel protein sharing similarities with the mammalian neural nitric oxide synthase (nNOS)-binding protein CAPON, and is expressed in the muscles of the worm.. As a C. elegans model for dystrophin-dependent myopathy, the dys-1; hlh-1 worms should permit the identification of genes, and ultimately drugs, that would reverse the muscle degeneration in this model.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Carrier Proteins; Disease Models, Animal; Dystrophin; Genes, Reporter; Helminth Proteins; Mice; Molecular Sequence Data; Muscle Proteins; Muscles; Muscular Dystrophy, Animal; Mutagenesis; Myogenic Regulatory Factors; Nuclear Proteins; Phalloidine; Phenotype; Rats; Sequence Alignment; Suppression, Genetic; Time Factors; Transcription Factors

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions "cytocauds." The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.

Topics: Actin Cytoskeleton; Animals; Animals, Newborn; Cilia; Deafness; Disease Models, Animal; Evoked Potentials, Auditory, Brain Stem; Hair Cells, Auditory; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Microscopy, Electron; Microscopy, Electron, Scanning; Organ of Corti; Phalloidine; Vestibular Diseases; Vestibule, Labyrinth

Environmental heat stress may result in loss of fluid from the vascular space, which can lead to circulatory shock. Since the endothelium serves as the blood vessel barrier between the vascular and interstitial spaces, direct heat damage to this tissue may contribute to such fluid loss. This study modeled heat influences on the actin cytoskeletal proteins that provide the tensile forces that sustain endothelial junctional integrity or barrier function. Heat effects on bovine aortic endothelial cell (BAEC) F-actin and F-actin stress fibers (FASFs) were correlated with intercellular permeability (IP). F-actin concentration and FASF distribution were analyzed by quantitation of the specific binding of rhodamine phalloidin (RP) to F-actin and by observing the fluorescence of RP-FASF complexes, respectively. Dextran fluorescein IP was determined. The IP was elevated (p < 0.05) at 43 degrees C, but not at 41 degrees C. At 43 degrees C, BAECs were rounded and had disrupted FASFs and diminished cell-to-cell apposition. Similar cells were seen at 41 degrees C, but these were interspersed among FASF-containing cells to sustain apposition. Thus, disruption of FASFs correlated with increases in IP. F-actin was increased (p < 0.05) after hyperthermia. Since G-actin is more susceptible to irreversible heat denaturation, F-actin sustainment may function to preserve the actin pool and prevent irrevocable loss of the blood vessel barrier after heat stress.

Topics: Actins; Animals; Aorta; Cattle; Disease Models, Animal; Endothelium, Vascular; Heat Stress Disorders; Intercellular Junctions; Microscopy, Fluorescence; Phalloidine

To compare and contrast the in vivo mechanism of wound healing after mechanical scrape and transcorneal freeze (TCF) injury in a rabbit eye model by examining changes in the cytoskeletal organization of contractile, filamentous actin (f-actin) microfilaments as relates to differences in cell migration or translocation during endothelial repair.. Endothelial wound healing after mechanical scrape and transcorneal freeze injury was studied in rabbit eyes using laser scanning confocal microscopy (LSCM). Central corneal mechanical scrape injury was made using an olive tip cannula, and TCF injury was made using a 3-mm diameter stainless steel probe cooled with liquid nitrogen. Cytoskeletal changes in f-actin stained with phalloidin-FITC were observed during wound healing using LSCM.. At 6 hours after mechanical scrape, the leading edge of the migrating sheet showed a decrease in the intensity of phalloidin-FITC staining, suggesting a decrease in cortical f-actin. Migrating endothelial cells in vivo did not appear to develop stress fibers after mechanical scrape, which is consistent with an in vitro cell spreading mechanism of endothelial wound healing. By 24 hours, the denuded area was almost fully resurfaced by migrating endothelial cells. On the other hand, TCF injury produced fibroblastic changes in the endothelial cells with extension and elongation of spindle-shaped endothelial cells at the leading edge by 24 hours after injury. Fibroblastic endothelial cells developed prominent actin stress-fibers, which is consistent with an in vitro cell migration mechanism of endothelial wound healing. Three days after TCF, the wounded area was resurfaced with two cell types: rough, fibroblast-like cells forming a retrocorneal fibrous membrane having prominent f-actin bundles or stress fibers with few cell-cell junctions, and smooth, polygonal-shaped endothelial cells having tight cell junctions with a cortical distribution of f-actin. After 28 days the retrocorneal fibrous membrane was posteriorly covered with normal endothelium.. These data support the hypothesis that endothelial wound healing involves two separate, injury-dependent, mechanisms--cell spreading and cell migration.

Topics: Actins; Animals; Cell Division; Cell Movement; Cryosurgery; Cytoskeleton; Disease Models, Animal; Endothelium, Corneal; Fluorescein-5-isothiocyanate; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Phalloidine; Rabbits; Wound Healing

Topics: Alkaline Phosphatase; Animals; Cholestasis, Intrahepatic; Disease Models, Animal; Female; Hyperbilirubinemia; Liver; Male; Mitosis; Oligopeptides; Phalloidine; Rats; Rats, Inbred Strains; Sex Factors