phalloidine and Cicatrix

Topics: Actins; Blotting, Western; Burns; Cell Differentiation; Cells, Cultured; Cicatrix; Collagen; Collagen Type I; Collagen Type III; Fibroblasts; Humans; Myofibroblasts; Phalloidine; Rhodamines; Transforming Growth Factor beta1

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

Intracochlear infusion of the KHRI-3 monoclonal antibody results in in vivo binding to guinea pig inner ear supporting cells, loss of hair cells and hearing loss. To further characterize the basis for KHRI-3-induced hearing loss, antibody was produced in a bioreactor in serum-free medium, affinity purified, and compared to conventionally prepared antibody by infusion into the scala tympani using mini-osmotic pumps. In vivo antibody binding was observed in 10 of 11 guinea pigs. A previously unreported pattern of KHRI-3 antibody binding to cells involved in scar formation was noted in five guinea pigs. All but one of the KHRI-3-infused animals demonstrated a hearing loss of > 10 dB in the treated ear. In five of 11 animals the threshold shift was 30 dB or more, and all had hair cell losses. In one guinea pig infused with 2 mg/ml of antibody, the organ of Corti was absent in the basal turn of the infused ear. This ear had a 45-50 dB threshold shift but, curiously, no detectable antibody binding in the residual organ of Corti. Organ of Corti tissue was fragile in antibody-infused ears. Breaks within the outer hair cell region occurred in 5/11 infused ears. The contralateral ears were normal except for one noise-exposed animal that demonstrated hair cell loss in the uninfused ear. Three animals were exposed to 6 kHz noise (108 dB) for 30 min on day 7. Antibody access to the organ of Corti may be increased in animals exposed to noise, since the strongest in vivo binding was observed in noise-exposed animals. Loss of integrity of the organ of Corti seems to be the primary mechanism of inner ear damage by KHRI-3 antibody. The binding of KHRI-3 antibody in new scars suggests a role of the antigen in scar formation. Antibodies with binding properties similar to KHRI-3 have been detected in 51% of patients diagnosed with autoimmune sensorineural hearing loss; thus, it seems likely that such autoantibodies also may have pathologic effects resulting in hearing loss in humans.

Topics: Animals; Antibodies, Monoclonal; Autoimmune Diseases; Cicatrix; Ear, Inner; Guinea Pigs; Hair Cells, Auditory; Hearing Loss, Sensorineural; Hearing Loss, Sudden; Humans; Mice; Microscopy, Fluorescence; Organ of Corti; Phalloidine

Hair cell degeneration and the repair process due to differing types of trauma have been studied extensively in the organ of Corti. It has been determined that, during scar formation, after differing types of trauma to the auditory sensory system, the reticular lamina is maintained with adherens junctions and tight junctions. We investigated the repair process within the vestibular epithelium. Hair cell degeneration was induced by the unilateral application of streptomycin to the inner ears of guinea pigs. Whole mount preparations of all five vestibular organs were processed and examined by fluorescence, light and electron microscopy. Scar formation was seen as early as 4 days post-treatment with streptomycin and was noted to coincide with hair cell degeneration. Neighboring supporting cells swelled and filled the space beneath the degenerating hair cell. Between three and five supporting cells participate in the reparative process. The distribution of cytokeratin is also altered during scar formation. The area once occupied by the hair cell becomes filled with cytokeratin-rich processes of supporting cells. It appears that differing numbers of supporting cells are involved in the reparative process within the vestibular sensory epithelium as compared to the auditory system. The reticular lamina remains intact at all times. This may possibly prevent mixing of fluids between different compartments in the inner ear and dysfunction of the vestibular sensory organs.

Topics: Actins; Animals; Cicatrix; Cochlea; Epithelium; Guinea Pigs; Hair Cells, Auditory; Histocytochemistry; Keratins; Labyrinth Supporting Cells; Microscopy, Confocal; Microscopy, Electron; Microscopy, Fluorescence; Organ of Corti; Phalloidine; Saccule and Utricle; Streptomycin; Vestibular Nerve; Vestibule, Labyrinth