Apoptosis in the developing rat cochlea and its related structures.

Mammalian development involves proliferation and programmed cell death (apoptosis). This study was undertaken to analyse the spatial and temporal organisation of apoptosis in developing rat cochlear and associated tissues using in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling of DNA fragments (TUNEL), and light and electron microscopy. Embryonic (E12-E19 days) and postnatal rats (P0-P21 days) were studied. Fixed tissues were stained for apoptosis using TUNEL technique and the cytomorphology of apoptosis was confirmed by light and electron microscopy. Apoptotic cells were detected predominantly during the embryonic and early postnatal development of the cochlea. Apoptosis occurred in embryonic precursors of the cochlear duct epithelium, mainly in the region of its outgrowth between E12 and E16. In the periotic mesenchyme, apoptosis occurred in areas committed to develop into the middle ear cavity (peaking at E16) and perilymphatic compartments (peaking around E18-E19). Apoptosis in the VIIIth nerve (statoacoustic) ganglion was detected throughout the embryonic and early postnatal periods, peaking at E18-E19, around the time when the cochlear neural connections are being established. At later postnatal days, apoptosis was seen only occasionally in cochlear tissues, predominantly in tissues lining the middle ear cavity and sporadically in cells of the otic capsule. Therefore, apoptosis appears to occur in areas of remodeling, in areas of cavitation and in areas of differentiation. These findings provide a template for studying the molecular mechanisms involved in the development of the rat inner ear.

Extracellular adenosine 5'-triphosphate (ATP) in the endolymphatic compartment influences cochlear function.

There is strong evidence for the presence of P2 purinoceptors on cochlear tissues, but the role of extracellular ATP in cochlear function is still unclear. Our previous studies have determined the presence of ATP in the cochlear fluids and indicated that the purinoceptors are substantially localized to the tissues lining the endolymphatic compartment. This implies that extracellular ATP may have an humoral role confined to the endolymphatic space. In order to study the influence of extracellular ATP in the endolymphatic space, a series of studies were undertaken in which ATP (10 microM to 10 mM) in artificial endolymph (EL) (test solution: 2-12.5 nl) was injected into the scala media and the effect on the cochlear microphonic (CM) and endocochlear potential (EP) evaluated. A double-barrelled pipette, with one barrel containing the test solution and the other artificial EL (control solution) was inserted into scala media of the third turn of the guinea-pig cochlea. A known volume (2-12.5 nl) of test or control solution was then pressure-injected into the space. ATP had a significant dose-dependent suppressive effect on both EP and CM with a threshold of approximately 2 x 10(-14) mol; the response was readily reversible, also in a dose-dependent fashion. Artificial EL of the same volume had no effect on EP and CM. The ATP effect on EP was blocked by the P2 purinoceptor antagonists suramin and reactive blue 2 (RB2). Neither adenosine (2 x 10(-13) to 2 x 10(-11) mol) nor suramin or RB2 on their own had any effect on EP and CM. This study provides the first evidence for an effect of extracellular ATP in the endolymphatic compartment on cochlear function which is mediated via P2 purinoceptors. This provides supporting evidence for an humoral role for extracellular ATP in the modulation of cochlear function.

Adenosine 5'-triphosphate (ATP) concentrations in the endolymph and perilymph of the guinea-pig cochlea.

The concentration of adenosine 5'-triphosphate (ATP) in endolymph (EL), perilymph (PL) and cerebrospinal fluid (CSF), collected from anesthetized guinea pigs was determined using the luciferase-luciferin reaction. The cochlea was exposed by a ventrolateral approach and the bone overlying scala media of the third turn (EL) or scala vestibuli of the first turn (PL) was shaved to a thin layer and a small fenestrum made. For EL sampling, a double-barrelled pipette was inserted through the spiral ligament-stria vascularis complex. One barrel was filled with 150 mM KCl to record the endocochlear potential (EP) and upon the appearance of the positive EP, 0.12-1.22 microliter of fluid was aspirated into the other barrel by gentle negative pressure. For PL sampling, a single-barrelled pipette was advanced into scala vestibuli and 0.3-1.6 microliter of fluid was collected by capillarity. CSF (0.36-1.75 microliter) was obtained from the cisterna magna. The cochleae were removed and processed for light microscopy to determine the extent of tissue damage from the sampling procedure. ATP concentrations (mean +/- SEM, nM) for EL, PL and CSF were 12.95 +/- 2.4 (n = 10), 10.5 +/- 3.9 (n = 11) and 16.1 +/- 5.4 (n = 11) respectively. Differences in ATP concentrations among fluids were not statistically significant. To test the effect of hypoxia on ATP levels, a group of guinea pigs was subjected to a 90 s period of respiratory anoxia prior to sampling of EL, PL or CSF. ATP concentrations were 14.4 +/- 3.5 (n = 11), 20.7 +/- 4.1 (n = 10) and 13.5 +/- 4.6 (n = 4) for EL, PL and CSF, respectively; only PL ATP concentrations were statistically different (P = 0.018, Wilcoxon rank sum test) to basal conditions. This is the first study which demonstrates the presence of free ATP in cochlear fluids. The results indicate that ATP is present in cochlear fluids at concentrations close to those known to cause hair cell depolarization in vitro.

Quinacrine staining of marginal cells in the stria vascularis of the guinea-pig cochlea: a possible source of extracellular ATP?

There is accumulating evidence for a purinergic humoral system involved in the control of cochlear function. Evidence of specific P2 purinoceptors on cochlear tissues implies a role for extracellular adenosine triphosphate (ATP) in the cochlea. To further this hypothesis a study was undertaken to determine if there was any specific source of purine compounds in cochlear tissues. Cochlear tissues (the sensory epithelium and lateral wall) from the guinea pig were incubated with the acridine derivative quinacrine dihydrochloride (5 x 10(-6) M in phosphate-buffered saline for 30 min at room temperature) which fluoresces on binding to high concentrations of ATP. Most cochlear tissues showed a diffuse green fluorescence slightly above the background level. However, a region of the marginal cells of the stria vascularis showed a specific punctate fluorescence. Optical sectioning of these cells by confocal microscopy revealed that the fluorescent structures in these marginal cells was confined to a region up to 10 microns from their endolymphatic surface. Similar cells studied by transmission electron microscopy showed membrane-bound vesicles located in the same region of the cell. These data imply that purine compounds are localized in discrete structures, perhaps vesicles, within the marginal cells which could serve as a source of extracellular ATP in the cochlea.

The nature and progression of injury in the organ of Corti during ischemia.

This study has defined the nature and sequence of ultrastructural changes in the organ of Corti following severe, total cochlear ischemia. Afferent nerve endings of IHC became swollen within 15 min and eventually ruptured. Outer hair cells were swollen within 30 min and showed alterations to mitochondria, endoplasmic reticulum and the nucleus whereas IHC remained unchanged for up to 60 min. Both efferent and afferent nerve endings of OHC were unaltered until after 60 min ischemia. Regardless of the type, cells in the base of the cochlea developed abnormalities more rapidly than those in the apical turns. These results imply a differential susceptibility to ischemic damage both among the different cell types and along the organ of Corti.