Michael Anne Gratton

Age-related decreases in endocochlear potential are associated with vascular abnormalities in the stria vascularis.

The density and diameter of strial capillaries were assessed in whole-mount preparations of the cochlear lateral wall from 18 gerbils aged in quiet for at least 36 months. Following morphometric analysis, histopathologic changes in selected regions of the lateral wall were examined by light and transmission electron microscopy. Alterations in strial vasculature were compared with the endocochlear potential (EP) measurements from the same ear. Vascular degeneration occurred in a segmental fashion in that regions of atrophic capillaries were found throughout the cochlea but primarily in the apical and lower basal turns and in the hook. The amount of stria with normal capillaries varied greatly among the aged ears, ranging from 19 to 87%. The resting EP also varied markedly, ranging from 23 to 83 mV. Little correlation was found between vascular alterations and the corresponding EP value from individual cochlear turns. However, significant correlations were found between the total strial area with normal vasculature and both the mean EP value and that recorded at either the round window or first turn in that ear.

Alterations in microvasculature are associated with atrophy of the stria vascularis in quiet-aged gerbils

Age-related changes in the integrity of the stria vascularis and its microvasculature were assessed in whole mount preparations of the gerbil cochlear lateral wall. Small focal regions containing few or no capillaries were present at the extreme ends of the stria vascularis in most 5-9 month-old gerbils. A few ears in this age range also contained small regions devoid of capillaries at the extreme basal end of the stria. These degenerate foci expanded in a systematic fashion toward the middle turn of the cochlea with increasing age. Gerbils aged 33 months or older exhibited a normal strial vasculature pattern only in portions of the middle and upper basal turns. The remainder of the stria in these older gerbils contained degenerate regions which showed both loss of capillaries and atrophy of strial marginal cells. Quantification via computer-aided image analysis confirmed that the areas of strial atrophy correlated well with the loss of strial capillaries at all ages. However, regions containing capillaries with decreased diameter were not necessarily accompanied by atrophic changes in marginal cells. The results suggest that degeneration of the stria vascularis begins prior to onset of auditory threshold shift and is preceded and possibly initiated by changes in the strial microvasculature.

Age-related thickening of basement membrane in stria vascularis capillaries

Ultrastructural examination was undertaken to investigate the pathogenesis of age-related atrophy of the stria vascularis (StV). Basement membrane (BM) thickness was increased in 65-85% of strial capillaries in gerbils aged 33 months or older and often exceeded by several-fold that observed in young controls. In an early stage of thickening the BM expanded slightly around the full capillary profile, after which nodular expansions of BM encircling slender cell processes were often observed at or near one or both poles of the elliptical vessel profile. As widening progressed, the BM consisted of 2-3 layers separated by cell processes in the nodules but fewer strata elsewhere. Association of slender processes of both endothelial cells and pericytes with focal thickening outside the process suggested their participation in genesis of the capillary lesion. In later stages of atrophy, pericytes degenerated and disappeared, while endothelial cells remained intact. Eventually, thick multilayered BM devoid of endothelial cells surrounded a narrow lumen occluded by debris. The age-related change in BM in the inner ear was confined to StV capillaries. Degenerative changes in StV epithelial cells occurred apparently as a secondary consequence of the capillary lesion. The pathologic alterations in marginal cells included extrusion of blebs from the luminal surface, separation and loss of basolateral interfoldings, alteration and depletion of mitochondria and nuclear pyknosis. At the end-stage of degeneration, the StV consisted of a simple or multiple layer of squamous cells lining the scala media.

Decline in the endocochlear potential corresponds to decreased Na,K-ATPase activity in the lateral wall of quiet-aged gerbils

The ion transport-mediating enzyme, Na,K-ATPase, is abundantly present in the cochlear lateral wall. This enzyme is essential for the generation and maintenance of the endocochlear potential. Diminished enzyme activity has been observed previously in the lateral wall of quiet-aged gerbils. The present study was designed to investigate the impact of the age-related decline in Na,K-ATPase specific activity upon auditory function. Measures of the resting endocochlear potential value and the level of Na,K-ATPase specific activity were made in cochleae obtained from gerbils aged in quiet conditions. Analysis revealed a high degree of correspondence between the level of lateral wall Na,K-ATPase specific activity and the value of the endocochlear potential measured in the round window/turn 1 region of the cochlea. Nonlinear regression models showed a strong relationship between the age-related reductions in enzyme activity and the magnitude of the endocochlear potential. The data suggest that during metabolic presbyacusis a decrease in Na,K-ATPase specific activity can explain most, but not all, of the decline in the endocochlear potential.

Expression patterns of ion transport enzymes in spiral ligament fibrocytes change in relation to strial atrophy in the aged gerbil cochlea.

Fibrocytes of the lateral wall function in conjunction with the stria vascularis (StV) to mediate cochlear ion homeostasis. Age-related changes in the expression patterns of ion transport enzymes in spiral ligament fibrocytes were investigated to ascertain their relation to metabolic presbyacusis in the gerbil. Immunoreactivity of fibrocytes for Na,K-ATPase (Na,K), carbonic anhydrase isozyme II (CA) and creatine kinase isozyme BB (CK) varied among and within cochleas from aged but not from young gerbils. The variable immunostaining was related to the extent and location of StV atrophy. Age-dependent degeneration and loss of Na,K in the StV occurred predominantly in the apex and lower base and hook of the cochlea, largely sparing more central regions. Immunostaining intensity for Na,K, CK, and CA in fibrocytes changed in relation to declines in strial marginal cell Na,K initially showing upregulation followed by downregulation. Spiral ligament fibrocytes in cochleas with more than two remaining normal turns often disclosed overexpression of CK in regions of strial atrophy. Conversely, CA in such cochleas was often increased in regions of normal StV adjacent to foci of atrophic StV. Senescent cochleas with two or fewer functional turns generally contained fibrocytes with diminished CK or CA immunoreactivity in regions of atrophic StV but in isolated instances exhibited fibrocytes with enhanced staining. Heightened staining for CK in type Ia fibrocytes underlying regions of complete or partial strial atrophy indicated an increased metabolic demand in fibrocytes in response to strial insufficiency. The findings provide further support for the role of spiral ligament fibrocytes in cochlear fluid and ion homeostasis.

Quantification of the stria vascularis and strial capillary areas in quiet-reared young and aged gerbils

The area of the stria vascularis (StV) and of StV capillaries was measured in radial sections from regions corresponding to 0.5, 2, 4, 10, 20 and 40 kHz. In young gerbils, StV area ranged from 3700 to 8500 microm2 and that of individual StV capillaries from 70 to 110 microm2. The maximal StV area as well as the largest number of capillaries occurred at the 20 kHz region. In quiet-aged gerbils, the StV area also varied with frequency and was 28-67% smaller than corresponding measures in young gerbils. The decrease in StV area was statistically significant at all but the 2 and 4 kHz regions. The area of individual StV capillaries declined also (8-29%) with age even when the StV area remained near normal. Reductions in capillary area were statistically significant at the 2, 20 and 40 kHz regions. The large variance in StV radial area among aged gerbils reflects the patchy nature of strial degeneration previously observed in this species. The data agree with those of our previous studies and indicate alterations in StV capillaries are a primary cause of presbyacusis in the gerbil.

Na,K-ATPase activity decreases in the cochlear lateral wall of quiet-aged gerbils.

Alterations in the distribution and activity of Na,K-ATPase have been implicated in declining cell function with age. However, the location, size and anatomical complexity of the cochlea have limited study of this essential enzyme. Here we describe a micro-colorimetric assay which measures Na,K-ATPase activity in subregions of individual cochleae. Na,K-ATPase activity was determined in lateral wall and organ of Corti tissues by measuring liberation of inorganic phosphate (Pi) from ATP against a standard phosphate curve. Na,K-ATPase specific activity, expressed as mu mol Pi liberated/mg protein/h, was calculated as the difference between total Pi liberated versus Pi liberated in the presence of ouabain. Na,K-ATPase specific activity and total protein content in the lateral wall significantly exceeded those of the organ of Corti. Although lateral wall protein content remained constant with age, Na,K-ATPase specific activity declined in some older gerbils, suggesting a basis for age-related reductions in magnitude of the endocochlear potential and confirming previous histochemical results. This microassay offers a sensitive, reliable means to assay enzyme activity in subregions or single turns of the cochlea that unlike other methods does not rely on use of radioisotopes, enzymatic cycling or sample pooling.

Characterization and development of an inner ear type I fibrocyte cell culture

A method has been developed that allows successful maintenance of secondary cell cultures derived from explants of the cochlear lateral wall of young adult gerbils. The secondary cultures were characterized morphologically with light and transmission electron microscopy and immunocytochemically with protein markers specific to various lateral wall cell types. Structural studies revealed fusiform-shaped cells with a paucity of cytoplasm surrounding the nucleus and slender processes. The cells showed little evidence of intercellular contact even when confluent. The cultures were immunopositive for vimentin, carbonic anhydrase isozyme II, creatine kinase isozyme BB and smooth endoplasmic reticulum Ca-ATPase, but lacked reactivity for cytokeratins and Na,K-ATPase. The results indicate that the cultures are comprised of type I fibrocytes from the spiral ligament. These findings are the first to demonstrate that inner ear spiral ligament cells can be isolated and maintained in secondary culture while retaining many of their in vivo characteristics. Based upon their location and content of ion transport enzymes, type I fibrocytes are thought to be involved in the recycling of potassium from perilymph into the stria vascularis. The establishment of this cell line provides a means to analyze the role of spiral ligament fibrocytes in maintenance of inner ear homeostasis.

Effects of nimodipine on noise-induced hearing loss.

The effects of nimodipine, a calcium channel blocker, on noise-induced hearing loss were examined in gerbils. Animals were implanted subcutaneously with a timed-release pellet containing either nimodipine (approximately 10 mg/kg/day) or placebo and exposed to either 102 or 107 dBA noise. Serum levels were tested in two subjects and were in the range known to protect humans from cerebral artery vasospasm and ischemia-related neurologic deficits. Nimodipine and control groups had similar amounts of noise-induced (a) permanent threshold shift; (b) reductions in distortion product otoacoustic emissions; (c) reductions in tuning and suppression of the compound action potential; and (d) loss of outer hair cells. The results suggest that nimodipine, at a dose which results in clinically relevant serum levels, does not provide protection from the effects of moderately intense noise exposures.

Localization of actin in basal cells of stria vascularis

The distribution of actin in the lateral wall of the cochlear duct was investigated in the gerbil, rat, mouse and hamster. A monoclonal antibody specific for muscle alpha and gamma actins, and a polyclonal antiserum reactive with smooth muscle gamma and non-muscle beta actins yielded strong immunostaining of basal cells in the stria vascularis and of smooth muscle cells in lateral wall blood vessels. Both cell types stained in all four genera. Diffuse cytosolic staining was observed along the full-length of the basal cell layer including the blunt cell processes which they extend toward strial marginal cells. Immunoreactive basal cells appeared continuous with morphologically similar cells investing vessels penetrating the stria from the spiral ligament. The basal cells failed to bind antibodies to smooth muscle alpha actin and sarcomeric actin. By electron microscopic immunocytochemistry, gold labeling for actin was observed on dense, fine fibrils in the cytoplasm of the basal cells. In paraffin sections adjacent to those stained for actin, antibody to vimentin stained intermediate and basal cells in the stria vascularis whereas antibody to isoform 1 of the facilitated glucose transporter protein family (GLUT1) labeled only the non-overlapping apical and basal plasmalemma of basal cells. Content of vimentin, GLUT1 and muscle gamma actin supports the derivation of basal cells from mesoderm. The presence of stress fibers containing muscle gamma actin points to a possible contractile activity of basal cells which conceivably could be related to transport of K+ to and within the intrastrial compartment or regulation of blood flow in the stria vascularis.