Recent Advances in Laser Microprobe Mass Analysis (LAMMA) of Inner Ear Tissue

Maintenance of ionic gradients within the various fluids compartments of the inner ear requires transport active cellular systems at different locations. LAMMA analysis is ideally suited for detection of ions in microquantity on cellular levels overcoming many technical difficulties. The present paper summarizes the results of microprobe analysis obtained with laser induced mass spectrometry (LAMMA) supplemented by X-ray microprobe analysis of epithelial cell layers adjacent to the endolymphatic space (a) in the cochlear duct, (b) in the vestibular organ and (c) in the endolymphatic sac. The possible role of inner ear as well as ocular melanin in the mechanisms of active ion transport is discussed

Early-onset liver mtDNA depletion and late-onset proteinuric nephropathy in Mpv17 knockout mice

In humans, MPV17 mutations are responsible for severe mitochondrial depletion syndrome, mainly affecting the liver and the nervous system. To gain insight into physiopathology of MPV17-related disease, we investigated an available Mpv17 knockout animal model. We found severe mtDNA depletion in liver and, albeit to a lesser extent, in skeletal muscle, whereas hardly any depletion was detected in brain and kidney, up to 1 year after birth. Mouse embryonic fibroblasts did show mtDNA depletion, but only after several culturing passages, or in a serumless culturing medium. In spite of severe mtDNA depletion, only moderate decrease in respiratory chain enzymatic activities, and mild cytoarchitectural alterations, were observed in the Mpv17−/− livers, but neither cirrhosis nor failure ever occurred in this organ at any age. The mtDNA transcription rate was markedly increased in liver, which could contribute to compensate the severe mtDNA depletion. This phenomenon was associated with specific downregulation of Mterf1, a negative modulator of mtDNA transcription. The most relevant clinical features involved skin, inner ear and kidney. The coat of the Mpv17−/− mice turned gray early in adulthood, and 18-month or older mice developed focal segmental glomerulosclerosis (FSGS) with massive proteinuria. Concomitant degeneration of cochlear sensory epithelia was reported as well. These symptoms were associated with significantly shorter lifespan. Coincidental with the onset of FSGS, there was hardly any mtDNA left in the glomerular tufts. These results demonstrate that Mpv17 controls mtDNA copy number by a highly tissue- and possibly cytotype-specific mechanism

Techniques for the observation and measurement of red blood cell velocity in vessels of the guinea pig cochlea

Fluorescence techniques combined with intravital microscopy provide a powerful approach to the study of cochlear blood microcirculation. In the current study, fluorescein isothiocyanate conjugated to high molecular weight dextrans was added to plasma to enhance the visual contrast of flowing blood in microscopic images from the guinea pig cochlea. Photometric signals, obtained from video pictures of the blood vessels, provided a means to continuously measure red cell velocity by using crosscorrelation algorithms to extract the time delay for moving features of the image. Alternatively, a small amount of fluorescently-labeled red blood cells (RBCs) were added to the vascular volume to serve as natural indicators of whole blood flow. The speed of these cells was measured by video photometric detection of the time required for the cells to pass between two predetermined positions in the television image. RBCs can be made fluorescent by chemical bonding of a fluorochrome to the cell membrane or by internal loading of the cell with an inert fluorochrome. Labeled RBCs provide a means to determine blood velocity in capillaries having extremely poor optical contrast, a situation which is generally the case for relatively thick tissues such as the lateral wall of the membranous labyrinth.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26885/1/0000451.pd

Phenotypes of the ovarian follicular basal lamina predict developmental competence of oocytes

BACKGROUND: The ovarian follicular basal lamina underlies the epithelial membrana granulosa and maintains the avascular intra-follicular compartment. Additional layers of basal lamina occur in a number of pathologies, including pili annulati and diabetes. We previously found additional layers of follicular basal lamina in a significant percentage of healthy bovine follicles. We wished to determine if this phenomenon existed in humans, and if it was related to oocyte function in the bovine. METHODS: AND RESULTS: We examined follicles from human ovaries (n = 18) by electron microscopy and found that many follicles had additional layers of basal lamina. Oocytes (n = 222) from bovine follicles with normal or unusual basal laminas were isolated and their ability to undergo in vitro maturation, fertilization and culture to blastocyst was compared. Healthy bovine follicles with a single layer of basal lamina had oocytes with significantly (P < 0.01) greater developmental competence than healthy follicles with additional layers of follicular basal lamina (65 versus 28). CONCLUSIONS: These findings provide direct evidence that the phenotype of the follicular basal lamina is related to oocyte competence

A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion

Degenerative Veränderungen im alternden Innenohr, mit besonderer Berücksichtigung der vasculären Veränderungen, in Flächenpräparaten der menschlichen Cochlea dargestellt

Temporal bones from 150 patients, ranging in age from fetuses and newborn to 97 years, were studied by the technique of microdissection and the use of surface specimens stained with OSO 4 . Hair cell and nerve degeneration were seen in the extreme basal turn of the cochlea even in children. In the fetal cochlea vascularization is very dense, in the newborn and infant somewhat less so. A gradual involution of blood vessels occurs postnatally and continues with maturity and aging. Involution is seen especially in the membranous wall of the cochlea and in the system of spiral vessels of the basilar membrane and vestibular lip. During the first decade the radiating arterioles and the outer spiral vessel in the basal turn are reduced to their adult size. In presbycusis material we observed a marked loss of capillaries and of some of the radiating arterioles in the spiral ligament. Other arterioles had thickened walls. This devascularization was accompanied by atrophy and acellularity of the spiral ligament and atrophy of the stria. Atrophy of the spiral vessels was seen, especially in the lower half of the basal turn. Most of the cochlear blood vessels have clearly distinguishable perivascular spaces. Vessels which had become occluded and disappeared left behind them intervascular strands and/or avascular channels . Such channels were seen to connect the perivascular space of one capillary with that of another, representing the perivascular space of the vessel which had atrophied. The hair cell and nerve degeneration seen in presbycusis may be caused, at least in part, by microangiopathy of this type. Similar vascular changes have been observed in retinal vessels. It is possible that the gradual reduction of blood supply through the disappearance of capillaries occurs in many tissues of the body and plays an important role in the aging process. Mit Hilfe der Oberflächenpräparation wurden von uns 150 menschliche Labyrinthe aller Altersgruppen untersucht. Es wurde eine Haarzellen-und Nervendegeneration schon im Kindesalter beobachtet. Blutgefäße in der Schnecke haben perivasculdre Spalten; Gefäße atrophieren and werden zu „avascular channels”, leeren Spaltrdumen oder Striingen. Bei Presbyakusis fällt neben der Haarzell- and Nervendegeneration in der Basalwindung der Schnecke die Gefäßatrophie im Ligamentum spirale und der Membrana basilaris auf. Dazu kommt noch eine beträchtliche Atrophie des Ligamentum spirale und der Stria vascularis. Bemerkenswert ist, daß die Devascularisation schon im Kindesalter beginnt.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47255/1/405_2004_Article_BF00373313.pd