The Digital Cytocochleogram.

The Mouse Cochlea Database (MCD) is a collection of resources that include digital images and bibliographic information on the mouse cochlea and is available at: http://mousecochlea.ccgb.umn.edu. The purpose of this communication is to report on the development of one MCD resource: the Digital Cytocochleogram. A cytocochleogram is a graphic representation of the anatomical state of the hair cells along the complete width and length of the organ of Corti. The Digital Cytocochleogram provides Internet users with a complete collection of digital images of one or more surface preparations of the mouse organ of Corti from which morphometric information can be obtained. By moving a mouse driven, screen cursor over a digital image, the location and approximate frequency region of the anatomical structure is displayed. Users can also measure the straight-line distance between any two structures on the image. The Digital Cytocochleogram resource uses two software programs, the Coordinate Finder and Viewer, which are written as CGI scripts. The Coordinate Finder program maps each digital image to an X,Y coordinate system. The total length of the organ of Corti from all tissue segments is computed using an arc-distance approximation formula, with the lateral border of the inner pillar cell headplates serving as a trace line or reference location. After all of the digital images of the tissue segments are mapped, they are placed on the MCD Website where users can use the Viewer program to view and morphometrically assess structures using a web browser. A single, complete surface preparation from a normal mouse is presently available on the MCD website. As the MCD grows, additional images of surface preparations at different magnifications from normal, mutant, and experimentally altered mouse cochleas will become available.

Immunolocalization of tenascin in the chinchilla inner ear.

Tenascin was immunolocalized in the chinchilla cochlea and vestibular system to better understand the functional morphology of the inner ear. Inner ear tissues were fixed with acetone, decalcified and cryosectioned. Indirect immunofluorescence, using antibodies directed against human tenascin epitopes, were used to detect tenascin. As a positive control, tenascin immunoreactivity was found in kidney, cortical mesangial cells and the extracellular matrix of glomeruli and medullary tubule interstitial spaces, concurring with previously reported results. In the cochlea, tenascin immunoreactivity was present in osteocytes, the mesothelial cells underlying the basilar membrane (BM) and within the fibrous matrix of the BM. Greater reactivity was observed in the mesothelial cells than in the fibrous matrix of the BM. In the vestibular system, tenascin immunoreactivity formed a diffuse band directly beneath the basal lamina of the ampullary and otoconial organs. Tenascin immunoreactivity was also observed in cup-shaped regions between the type I vestibular hair cells and their surrounding VIII nerve calyces in the ampullary and otoconial organs. This is the first report of the anatomical distribution of tenascin in the adult, mammalian inner ear, other than our previously published abstract P.A. Santi and D. Swartz, Soc. Neurosci. Abstr. 23 (1997) 731.

Immunohistochemical localization of keratan sulfate in the chinchilla inner ear.

Keratan sulfate (KS) was immunolocalized in the chinchilla cochlea and vestibular system using indirect immunohistochemistry and a monoclonal antibody (clone 5-D-4) directed against a proteoglycan core antigen. As a positive control, anti-KS Mab reactivity was found in the pericellular matrix and lacuna walls of temporal bone osteocytes. In the cochlea, anti-KS Mab reactivity was abundant in the basal cell layer of the stria vascularis and in the marginal band and Hensen's stripe of the tectorial membrane. Less anti-KS Mab reactivity was present in the cover net, Hardesty's membrane and the upper fibrous zone of the limbal layer of the tectorial membrane. In the vestibular system, anti-KS Mab reactivity was immunolocalized to a portion of the epithelium overlying the cupula of the crista ampullaris, in the apical surface of crista ampullaris epithelium, crista ampullaris stereocilia and in the otoconia. Elucidating the distribution of KS in the cochlea will improve our understanding of cochlear anatomy and is a first step toward understanding the etiology of hearing loss observed in diseases involving KS metabolism, namely, mucopolysaccharidosis type IV (Morquio's syndrome).

Glycolipidic component of the epithelial cell coat of the endolymphatic sac.

The endolymphatic sac (ES) is thought to synthesize and secrete glycoconjugates such as sulfated glycoproteins into the endolymphatic lumen. Ganglioside Gm1 is a specialized glycolipid containing one sialic acid molecule, which is generally found in the outer leaflet of the cell membrane. This glycolipid, which is known to be a specific receptor for cholera toxin (CT), acts as a membrane transducer and is involved in the modulation of cell metabolism, growth and regeneration. In the present study we identified Gm1 by studying the distribution of the FITC-labeled CT-subunit B in the ES epithelium of adult guinea pigs. Our findings indicate the presence of this ganglioside in the ES, with a predominant localization in the basolateral aspect of the epithelial cell layer. No detectable differences between ES cell types could be identified, whilst the ES distal and intermediate portions showed more reactivity than the proximal portion. This study, which represents the first description of a lipidic glycoconjugate component in the ES, provides evidence in support of the role of the ES in the turnover and regulation of inner ear fluids.

Identification and localization of the GM1 ganglioside in the cochlea using thin-layer chromatography and cholera toxin.

Using high-performance thin-layer chromatography, we identified GM1, GM3, GD3, GD1a, GT1b, GQ1b, and other gangliosides in chinchilla cochlea and cerebellum. GM1 was also identified on chromatograms with the B-subunit of cholera toxin (BCT). BCT was also used to determine the distribution of GM1 in fixed and unfixed tissues from cochlea, cerebellum, and sciatic nerve. Positive control tissues showed expected labeling of GM1 by BCT. Negative controls showed expected suppression of BCT binding to GM1 after GM1 extraction and GM1 absorption. In the cochlea, GM1 appeared abundant in plasma membranes of most epithelial cells lining the endolymphatic surface of the scala media, including the interdental, inner supporting, pillar, Deiters, Hensen, Claudius, Boettcher, spiral prominence, and external sulcus. GM1 appeared less abundant in cells of the stria vascularis, Reissner's membrane, and in nerve fibers. In hair cells, the stereocilia appeared to contain GM1; however, the endolymphatic surface of the cuticular plate and the body of the outer hair cells appeared to contain little GM1. In addition, the tectorial membrane, connective tissue of the spiral limbus, and amorphous layer of the basilar membrane also appeared to contain little GM1. Enzymatic degradation of glycoproteins and transformation of polysialogangliosides to GM1 increased the reactivity of BCT to cochlear GM1. This further supported the presence of GM1 and other gangliosides in the cochlea. Although the functional significance of GM1 and other gangliosides in the cochlea is not yet known, they are likely to play important roles in membrane function.

Localization of the GM1 ganglioside in the vestibular system using cholera toxin.

Cholera toxin is an ubiquitous activator of intracellular adenylate cyclase and is divided in two major components: A and B. The B-component consists of several subunits that specifically bind to the external cell membrane. The receptor for the toxin, the GM1 ganglioside, is concentrated in nervous tissues. The B subunit of the cholera toxin, conjugated to different molecules (i.e., choleragenoid) is therefore a sensitive anatomical tracer and has been used to detect the presence of GM1 in mammalian tissues. Using choleragenoid, unlabeled and labeled with FITC, we have determined the distribution of the GM1 ganglioside in the vestibular system of the chinchilla. Vestibular tissues were fixed in 4% paraformaldehyde in phosphate buffer, decalcified in 10% EDTA and prepared as either whole-mount, surface-preparations, or for radial cryosections. Positive control tissue consisted of binding to normal brain tissues. Negative controls consisted of several treatments: masking of the GM1 receptors with unlabeled choleragenoid, tissue extraction of GM1 using ethanol, and preabsorbing the choleragenoid with bovine GM1. In addition, to exclude staining of glycoproteins that may have a carbohydrate structure similar to GM1, tissues were digested with trypsin prior to choleragenoid exposure. In the vestibular system, a strongly positive reaction was observed in: the sensory stereocilia and supporting cells of the maculae and cristae, epithelial cells of the planum semilunatum, and polygonal cells of the semicircular canal. Positive but less strong reactivity was observed in the sensory cell body of maculae and cristae, nerve fibers, epithelial cells of utricle and ampulla walls and flattened epithelial cells of the semicircular canals. No reactivity was present in the supporting connective tissue cells and fibrils, blood vessels, gelatinous cupula of the cristae ampullaris and statoconial membranes. Brain tissue showed strong choleragenoid reactivity. The negative controls showed no or greatly reduced reactivity to choleragenoid. Trypsin digestion did not decrease reactivity to choleragenoid.

Inner ear damage and passage through the round window membrane of Pseudomonas aeruginosa exotoxin A in a chinchilla model.

By the use of computer-assisted morphometric analysis of the organ of Corti and/or measurements of action potential threshold changes, inner ear changes in chinchillas were evaluated 4 weeks after application on the round window membrane of a Pseudomonas aeruginosa exotoxin A solution. Severe inner ear damage was detected after application of 50 ng (5 microL at a concentration of 10 micrograms/mL) exotoxin A, whereas application of 5 ng exotoxin did not cause measurable inner ear damage. Perilymph concentrations of exotoxin A were measured with an enzyme-linked immunosorbent assay 1.5 to 19 hours after 50 ng, 0.5 micrograms, or 5 micrograms of exotoxin A was applied on the round window membrane. Only the highest concentration produced measurable levels of exotoxin in the inner ear fluids. It is concluded that exotoxin A present on the round window membrane of the chinchilla has the ability to penetrate into the inner ear and cause irreversible inner ear changes.

Ultrastructure of proteoglycans in the tectorial membrane.

The ultrastructure of proteoglycans (PGs) in the tectorial membrane (TM) of the mature chinchilla cochlea was investigated using the cationic dye Cuprolinic blue. When used at a high critical electrolyte concentration, Cuprolinic blue has been shown specifically to bind to the glycosaminoglycan residues of sulfated PGs. After Cuprolinic blue treatment, PGs were observed in the TM which were represented as rod-shaped, electron-dense structures. A perifibrillar, primarily orthogonal, array of PGs was associated with the type A protofibrils. These PGs were distributed in 50 nm intervals along the length of the type A protofibrils. A less common orientation was parallel to the axis of the type A protofibrils. PGs did not appear to be associated with the type B protofibrils. Based upon previous results by other investigators, the TM contains types II and IX collagen, and it appears likely that the type A protofibrils are composed of collagen type II. PGs visualized in the TM in this study thus may represent the glycosaminoglycan residue of type IX collagen which is associated with the type II collagen fibrils. Alternatively, the TM PGs may be small dermatan or chondroitin sulfate PGs.

Distribution of familial nephritis antigen in normal tissue and renal basement membranes of patients with homozygous and heterozygous Alport familial nephritis. Relationship of familial nephritis and Goodpasture antigens to novel collagen chains and type IV collagen.

The glomerular basement membranes (GBM) of Alport familial nephritis (FN) are laminated and split and fail to bind Goodpasture autoantibodies by indirect immunofluorescence. The Goodpasture antigen has been localized to multiple peptides of the noncollagenous C terminal (NC1) domain of type IV collagen. The principal target antigen is a 28-kDa peptide (M28) that coisolates with type IV collagen NC1 and which is derived from a larger collagenous molecule. We have shown that two novel 28-kDa peptides found in normal GBM (M28M28+) are absent from collagenase digests of X-linked dominant Alport FN GBM and that monoclonal antibodies specific for these collagen chains fail to bind to Alport GBM. In normal tissue these chains have a distribution restricted to specific basement membranes of kidney, eye, inner ear, lung, and brain, the former three of which are affected in Alport FN. Epitopes on a 26-kDa NC1 peptide identified by an antibody from a transplanted Alport patient (FN antibody) colocalized with the 28-kDa components in these tissues. The FN antibody did not bind to the GBM of homozygous Alport males. Antibodies to the 28-kDa peptides and the FN antibody colocalized in a segmental pattern in heterozygous Alport GBM by indirect immunofluorescence and were unrelated to the normal distribution of type IV collagen. Three of eight homozygous Alport FN tissues showed the presence of the 28-kDa components in Bowman's capsule in a focal distribution, and in four of eight tissues reactive antigen was present in the cytoplasm of some parietal and visceral epithelial cells. These observations support the hypothesis that the genetic abnormality in Alport FN is a defective parent chain of the 26-kDa peptide, which results in failure of normal 28-kDa collagen chain integration.

Immunohistochemical localization of fibronectin in the chinchilla cochlea.

The purpose of this study was to better understand the molecular composition of the cochlea. Fibronectin (FN), a well characterized adhesive glycoprotein, was localized by immunofluorescence microscopy in fresh and fixed cochlear tissues, and in fixed kidney tissue, using a polyclonal, affinity-purified, rabbit, anti-fibronectin antibody and a secondary antibody coupled to FITC. The FN antibody was free from cross-reactivity with other known basement membrane and cell matrix molecules. FN reactivity in the cochlea was most intense in the basilar membrane, latero-basal borders of Boettcher's cells, otic capsule, endothelial basement membranes (particularly those of the stria vascularis), and as a diffuse, fan-shaped network radiating into the spiral ligament. Little FN labelling was present in the epithelial basement membranes. Negative control tissue showed no immunoreactivity; whereas, positive kidney control tissue showed appropriate FN immunoreactivity in the mesangium of the glomerulus. The most significant finding of this study was that FN is a major component of the basilar membrane and its distribution appears to correspond to the amorphous ground substance. FN was not localized in the organ of Corti or at the tips of the hair-cell stereocilia.

Human tissue distribution of novel basement membrane collagen.

The authors have defined the specificity of monoclonal antibodies to collagen fragments of basement membrane (BM) and have used these highly specific antibodies to study the human tissue distribution of two novel 28 kd noncollagenous (NC1) peptides (M28 , M28+) compared with those derived from type IV collagen (alpha 1[26 kd] and alpha 2[24 kd] NC1). A limited distribution of the 28 kd peptides was observed in specialized BM of the kidney, eye, cochlea, lung, and brain, whereas type IV collagen is found in all human BM. These novel peptides, which colocalize with each other, are found in BM that also contain type IV collagen but do not, in all cases, colocalize with type IV collagen. The presence of the 28 kd peptides in the BM of the kidney, cochlea, and eye is in keeping with abnormalities involving these components in BM of patients with Alport familial nephritis (FN), who frequently have hearing loss, anterior lenticonus and retinal flecks in addition to renal disease. These 28 kd peptides are distinct, biochemically and immunochemically, from the alpha 1 and alpha 2 chain NC1 peptides of type IV collagen, and represent either peptide fragments of genetically distinct BM collagen molecules or additional molecules originating from the same gene family as type IV collagen.

Secretion of endolymph by semicircular canals of the shark.

The semicircular canals of the vestibular labyrinth of the dogfish shark, Squalus acanthias, may serve as a simple in vitro system for the study of the ionic transport mechanisms involved in endolymph formation. Electron microscopy showed that the epithelium was made up of at least three distinct cell types divided into separate regions running the length of the canals. Secretion of endolymph was studied in isolated canals by the split droplet method; when the lumens of the canals were filled with shark Ringer a potassium-rich fluid was secreted into the lumen at a rate of 0.34 microliter.cm-2.min-1. The K concentration of the secreted fluid averaged 112 mM; the calculated rate of K secretion was 2.3 mumol.cm-2.h-1, comparable to recent measurements in mammalian utricle. Fluid secretion was dependent on active transport and was inhibited by ouabain, bumetanide, or methazolamide in the external bathing solution. Fluid secretion was unaffected by the K channel blocker, Ba, in the luminal droplet or by the adenylate cyclase stimulator, forskolin, in the external bathing solution. For electrophysiological analysis, isolated canals were perfused in a chamber designed for voltage/current clamping; an axial wire was inserted into the canal lumen and constant-current pulses were passed to determine tissue resistance. When the luminal fluid was high-K Ringer, transepithelial potential difference was -1.1 mV (lumen negative) and resistance 37 omega.cm2. Dilution and bi-ionic potential measurements showed that the epithelium of the canals exhibited only slight cation selectivity. These results are consistent with a model for endolymph secretion involving cotransport secondary to the ion gradients created by the Na+-K+-ATPase.

A newly identified surface coat on cochlear hair cells.

Routine electron microscope methods do not well preserve or stain the surface coat or glycocalyx on cochlear hair cells. In other tissues, enhanced preservation and staining of these glycoconjugates was obtained following fixation with glutaraldehyde containing a cationic dye (e.g., Alcian blue and ruthenium red). When cochleas were fixed with glutaraldehyde containing Alcian blue, the endolymphatic surface of hair cells, but not the supporting cells, displayed an extensive (approximately 90 nm thick) surface coat. Alcian blue positive material was also observed in the tectorial and basilar membranes and in a portion of the spiral ligament. In addition, acellular bands of Alcian blue positive material were observed between the tectorial membrane and the reticular lamina or inner sulcus cells. Although the function of these cochlear glycoconjugates is not yet known, it is proposed that they serve to attach the tectorial membrane to the organ of Corti, and they are involved in stereocilia fusion following sound exposure and ototoxic drug administration.

Organ of Corti surface preparations for computer-assisted morphometry.

This paper describes a relatively rapid method (67 h) for producing surface preparations of the organ of Corti that are suitable for a computer-assisted morphometric analysis of cochlear hair cells. On day 1, a cochlea was fixed in OsO4, dehydrated in ethanol and infiltrated with an Epon-like plastic (Medcast). On day 2, the Medcast within the cochlea was polymerized at 60 degrees C in an oven. On day 3, the complete organ of Corti was dissected into approximately 20 segments which were trimmed and mounted on a slide in Medcast. Two 400 mesh transmission electron microscope grids are mounted at the apical and basal ends of the organ of Corti. These grids served as reference points for the establishment of an independent X, Y coordinate system on the slide by a computer-assisted light microscope. The segments were permanently attached to the slide by polymerization of the Medcast at 60 degrees C for approximately 15 h. At the beginning of day 4, the organ of Corti surface preparation was ready for examination by light microscopy or could be further sectioned and examined by transmission electron microscopy.

Alcian blue staining of cochlear hair cell stereocilia and other cochlear tissues.

Using whole-mount surface preparations of the chinchilla organ of Corti, the anionic characteristics of cochlear tissues was investigated with the polycationic dye Alcian Blue. Our primary goal was to specifically stain the hair cell stereocilia for the purpose of producing cytocochleograms. To specifically stain hair cell stereocilia we investigated the effects of different fixatives and the 'critical electrolyte concentration' [Scott and Dorling (1965) Histochemie 5, 221-233] of MgCl2 on staining specificity. The stereocilia and the tectorial membrane were the most intensely stained cochlear structures and presumably have a strong negative charge. The most important factor in producing specific staining was fixation in osmium tetroxide rather than the addition of an electrolyte to the Alcian Blue staining solution. The use of Alcian Blue produced intense staining of hair cell stereocilia against an unstained background using standard, brightfield light microscopy.

A computer-assisted morphometric analysis of the organ of Corti.

This paper describes a method for a computer-assisted morphometric analysis of the organ of Corti and presents normal morphometric data on the chinchilla cochlea. The computer-assisted light microscope system that we developed consists of commercially available equipment: an Olympus light microscope, Boeckler X, Y motorized stage micrometers, an IBM PC computer and a computer program written by the second author. The system was developed for the analysis of whole-mount, surface preparations of the organ of Corti. The procedure for producing suitable surface preparations for a computer-assisted analysis of hair cells was described in a previous paper (Santi, 1986). The analysis procedure requires 2 passes along the length of the basilar membrane. During the first pass, the system establishes an X, Y slide coordinate system and maps basilar membrane distance onto the coordinate system. This procedure was performed in approximately 10 min using an efficient, 3-point arc approximation method. During the second pass, the user scans along the length of the basilar membrane and measures organ of Corti structures (e.g., hair cell dimensions) or records abnormal or missing hair cells. In addition to providing distance along the length of the basilar membrane, the system could also determine which hair cell row the user was examining. Thus, recording damaged and missing hair cells consisted of merely pressing the appropriate footpedal to indicate the type and degree of hair cell damage. After assessing hair cell damage, a cytocochleogram with an accompanying table of descriptive statistics was produced. Since the X,Y coordinate system for each slide was stored on a disk along with its corresponding basilar membrane distance map, slides could be removed and later reevaluated without the loss of previously obtained data. Normal morphometric data on the chinchilla organ of Corti consisted of cell dimensions within the reticular lamina and a two-dimensional graphical reconstruction of the curvature of the basilar membrane.

Cell volume density alterations within the stria vascularis after administration of a hyperosmotic agent.

The purpose of this study was to determine the effects of an intravenous injection of a hyperosmotic agent (mannitol) on the volume density (Vv) of the primary components of the stria vascularis (SV). Chinchillas received either a 2.0 g/kg injection of mannitol or an equal volume of saline as a control. At 1, 10 and 60 min after the injection, the right cochleas were fixed with osmium tetroxide and prepared for transmission electron microscopy. At a distance of 70% from the cochlear apex, the complete radial area of the SV was photographed and stereologically analyzed. Additional animals received mannitol or bumetanide for the purpose of measuring serum osmolality and the endocochlear potential (EP). The present results showed elevation of serum osmolality after mannitol but not after bumetanide and depression of the +EP after bumetanide but not after mannitol. Vv alterations of SV components after mannitol were similar to those Vv changes observed in a previous study, after bumetanide. After treatment with either diuretic, the Vv of the marginal cells decreased and the Vv of the intermediate cells and intercellular spaces increased. We conclude that since the Vv alterations of the SV components are so similar after both diuretics, none of these alterations is a morphological correlate of a depressed +EP which was observed after bumetanide. A model of the action of mannitol on the SV is proposed.

The effect of bumetanide on the stria vascularis: a stereological analysis of cell volume density.

The purpose of this research was to determine the effect of bumetanide on the volume density (Vv) of the cells, capillaries and intercellular spaces of the stria vascularis (SV). 29 chinchillas were divided into seven groups. There were 3 experimental groups, three control groups and one normal, untreated, group of animals. After either a 20 mg/kg intravenous injection of bumetanide or an injection of a control solution, the animals were killed at 10 min, 1 h and 24 h. One complete radial section of the SV was analyzed in each animal. This section was located at 70% of the length of the basilar membrane as measured from the cochlear apex. Marginal cell volume decreased by 24% and 15% at 10 min and 1 h, respectively, after bumetanide administration. Intermediate cell volume increased by 31% and 27% at 10 min and 1 h, respectively, after bumetanide administration. Intercellular space volume increased by 14% and 21% at 10 min and 1 h, respectively, after bumetanide administration. No significant alteration in the Vv was observed in the strial capillaries or basal cells. A hypothetical model of the ion transporting properties of the SV is presented.