Biomedical subjects

B A Schulte

Publications and source records attributed to B A Schulte.

At least 19 recordsLinked to original sources

PubMed1996

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.

Acoustic Stimulation↗

PubMed1996

Identification and cloning of site C splice variants of plasma membrane Ca-ATPase in the gerbil cochlea.

Plasma membrane Ca-ATPase (PMCA) gene products were identified in the gerbil cochlea by reverse-transcription polymerase chain reaction (RT-PCR). Cochlear cDNA was amplified using PMCA isoform-specific primers from splice site C, the calmodulin binding domain. PCR products were cloned and sequenced. The putative housekeeping PMCA genes, 1b and 4b, as expected, were present in the gerbil cochlea and shared 98.6 and 100% amino acid homology with published rat sequences, at splice site C, respectively. PMCA2b, 3a and 3b splice variants also were detected in cochlear cDNAs and shared 95, 94.3 and 98% amino acid homology with their rat counterparts. PMCA isoforms 2 and 3 have been shown to occur in highly specialized tissues, such as muscle and brain, that require finely tuned regulation of intracellular free Ca2+ levels. The presence of several isoforms and splice variants of PMCA in the cochlea most probably reflects their differential expression among the several cell types that have been shown to contain immunoreactive PMCA. This suggests that the cochlea has developed complex mechanisms that finely tune intracellular Ca2+ levels in different highly specialized cell types.

Amino Acid Sequence↗

PubMed1996

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.

Animals↗

PubMed1996

Differentiation of glycoconjugates localized to sensory terminals and selected sites in brain.

Distribution of complex carbohydrates in the peripheral and central nervous systems was investigated cytochemically with a lectin that binds specifically to terminal alpha GalNAc and with monoclonal antibodies against carbohydrate epitopes, including glucuronic acid 3-SO4 and chondroitins 6-SO4 and 4-SO4. Comparative staining with these methods differentiated and partially characterized several glycoconjugates in various sites and allowed a comparison of chemical heterogeneity to neural specialization. Distal terminals of sensory neurons concerned with hearing, balance, taste, touch, and sight expressed glucuronyl 3-SO4, which apparently was present in an undefined glycoprotein. Some neurons in sensory nuclei of the brainstem exhibited a similar constituent on their surfaces. Retinal rod outer segments and the cerebellar granular layer possessed masked glucuronyl 3-SO4 that became immunopositive after digestion with chondroitinase ABC and that occurred in chondroitin 6-SO4 and chondroitin 4-SO4, respectively. The surface of neurons in the eighth nerve root and in neighboring nodes of Ranvier stained for unmasked glucuronic acid 3-SO4 and chondroitin 6-SO4. Some neurons of the cerebral cortex expressed unmasked glucuronyl 3-SO4, chondroitin 6-SO4, and terminal alpha GalNAc on their surfaces. Certain cortical neurons and nerve tracts with chondroitin 6-SO4 and terminal alpha GalNAc lacked glucuronyl 3-SO4, and other neurons possessing chondroitin 6-SO4 failed to express either glucuronyl 3-SO4 or terminal alpha GalNAc. Lability of lectin affinity to hyaluronidase suggested the presence of terminal alpha GalNAc in the chondroitin 6-SO4 on cortical neurons. The findings document further the heterogeneity of neural glycoconjugates, expand knowledge about the diversity of neurons with respect to their content of partially characterized glycoconjugates, and link glucuronyl 3-SO4 with or without chondroitin 6-SO4 spatially to sites of active Na+ transport in sensory nerves.

Animals↗

PubMed1996

Focal expression of A-CAM on pillar cells during formation of Corti's tunnel in gerbil cochlea.

BACKGROUND: The organ of Corti in the mammalian cochlea develops from a simple epithelium into a complex structure consisting of as many as ten highly specialized cell types. The purpose of the present study was to examine the possible role of a cell adhesion specific molecule (A-CAM) in mediating morphogenesis of the organ of Corti. METHOD: The expression of A-CAM was studied in a series of inner ears collected from neonatal gerbils at two days intervals from 0 to twenty days after birth. Immunostaining was performed with a mouse monoclonal antibody raised against A-CAM isolated from chicken cardiac muscle. RESULTS: The adhesion molecule appeared just prior to opening of the tunnel of Corti and disappeared after full expansion of tunnel space. A-CAM was detected in the basal turn of the cochlea as early as two days after birth and its expression moved progressively toward the apex of the cochlear spiral. It was observed in all three cochlear turns at eight days after birth but disappeared after postnatal day twelve. Immunostaining for A-CAM was localized only to the region where inner and outer pillar cells separate to form the tunnel of Corti. CONCLUSIONS: A-CAM apparently plays a role in the formation of the tunnel space by modulating apical adhesion above the separation between inner and outer pillar cells.

Age Factors↗

PubMed1996

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.

Actins↗

PubMed1996

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

Aging↗

PubMed1996

The fine structure of spiral ligament cells relates to ion return to the stria and varies with place-frequency.

Ultrastructural analysis of cells in the cochlea's lateral wall was undertaken to investigate morophologic features relevant to the route of K+ cycling from organ of Corti (OC) to stria vascularis (StV) and to the question of a transcellular versus an extracellular path. The fine structure of outer sulcus cells (OSCs) evidenced their capacity for uptake of K+ from Claudius cells and from perilymph in inferior spiral ligament. Plasmalemmal amplification and mitochondrial density together with known content of Na,K-ATPase testified to activity of type II, IV and V fibrocytes in resorbing K+. Location and fine structure afforded a basis for distinguishing subtypes among the type I, II and IV cells. The type II, IV and V fibrocytes can be viewed as drawing K+ from surrounding perilymph and from OSCs and generating an intracellular downhill diffusion gradient for K+ flow through gap junctions to subtype Ib and Ia fibrocytes and strial basal cells. Pumping action enabled by extreme structural specialization of type II fibrocytes is considered to mediate K+ translocation across the interruption between the gap junction connected epithelial and gap junction connected fibrocyte systems and to explain ion flow directed toward StV through OSCs and fibrocytes despite their lack of polarity. The OSC bodies shrank, their root bundles expanded and the gap junction contact between OSCs and Claudius cells increased toward the base of the cochlea. Expanding root bundles and type I and IIb fibrocyte populations contrasted with shrinking OHCs and Deiters and tectal cells from the apex to the base of the cochlea. These differences indicated an increased magnitude and alternate route of K+ transport toward the StV in high as compared to low-frequency regions. The augmented K+ transport through spiral ligament in basal cochlea correlates with and provides a possible basis for the larger endocochlear potential in the base. The findings appear consistent with current flow extracellularly through scalae tympani and vestibuli and transcellularly through OC, OSCs and class I, II, IV and V fibrocytes.

Acoustic Stimulation↗

PubMed1996

Glycosylation of secretory proteins in salivary glands and saliva studied by lectin-probes.

Lectin-probes were used histochemically to study glycosylation patterns in prepackaged secretory material in cat and rat submandibular glands and to assess the secretory changes induced by nerve stimulations. The same probes were also used for correlative biochemical assessments of the constituents in glandular extracts and in saliva from the same experiments, after electrophoretic separations and immobilization on nitrocellulose membranes. These studies provided a broader understanding than the use of either procedure alone. Lectin binding patterns in saliva were more complex than anticipated from the histochemistry and this probably relates to the presence of different constituents in secretory granules and a greater availability of binding sites biochemically. In cats, parasympathetic stimulation caused depletion of lectin staining in acini and to some extent in demilunes, whereas sympathetic stimulation caused depletion of lectin binding in striated ducts and reduction in demilunar size. Biochemically after SDS-PAGE of saliva similar secretory effects were observed but each nerve also evoked some secretion from the cells not showing histochemical change. In rats, sympathetic stimulation caused depletion of lectin positive granules from both acini and granular tubules. After SDS-PAGE of the saliva one zone of lectin binding 94-150 kD (Mr) was identified as acinar mucin. Granular tubule proteins consisting mainly of kallikreins occurred in a zone 25-35 kD (Mr) and lectin bindings suggested that different kallikreins may be differently glycosylated. This unexpected possibility was confirmed on slot-blot preparations of separated kallikreins, which also revealed the novel finding that some kallikreins are O- as well as N-glycosylated.

Animals↗

PubMed1996

Immunolocalization of keratan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate in periprosthetic breast capsules exhibiting synovial metaplasia.

The distribution of various proteoglycans and basement membrane components within 10 breast capsules with synovial metaplasia was assessed immunohistochemically. Immunoreactive keratan sulfate and chondroitin-4-sulfate were present in many of the synovial metaplasia lining cells, suggesting active secretion of these proteoglycans into the intraprosthetic space. In contrast, chondroitin-6-sulfate was confined to the extracellular matrix of the underlying supporting fibrous capsule. Type IV collagen and laminin were not associated with the synovial metaplasia lining, thus confirming the absence of a basement membrane, as previously indicated by morphologic analysis. As with tendon reconstruction, the development and maintenance of a synovial metaplasia lining that secretes lubricating factors such as proteoglycans may be beneficial for decreased capsular contracture.

Breast↗

PubMed1996

Use of lectin-probes for correlative histochemical and biochemical assessments of the glycosylation patterns of secretory proteins, including kallikreins, in salivary glands and saliva.

Labelled lectins were used as probes to study the glycosylation and secretion of submandibular glycoproteins not only in sections of fixed glands but also in glandular extracts and in nerve-induced saliva, after electrophoretic separations and immobilization in nitrocellulose membranes. In cats the glycoproteins in sympathetic saliva differed considerably from those in parasympathetic saliva. In sympathetic saliva they were found to originate mainly from striated ducts, to some extent from demilunar cells and to a small extent from acinar cells, whereas in parasympathetic saliva they arose mainly from acinar cells and demilunes and only to a small extent from striated ducts. In rat submandibular glands sympathetic stimulation caused extensive depletion of lectin stainable granules from granular tubules. Corresponding strong binding occurred with the same lectins to constituents in saliva that ran between 25 and 35 kD on SDS gel electrophoresis and were shown to contain tissue kallikreins. Their binding patterns suggested that individual kallikreins from the same gland may be glycosylated in different ways. This possibility was tested on five different kallikreins after separation from submandibular extracts by isoelectric focussing. Lectin bindings on slot blot preparations of these kallikreins were tested before and after N-glycosidase F, sialidase or endo-alpha-N-acetylgalactosaminidase digestions. Results showed that, despite their close genetic and structural similarities, the kallikreins are in fact differently sialylated and fucosylated and the novel finding that some contain O-glycosidically linked side chains as well as the anticipated N-glycosidically linked side chains was revealed. Thus, correlative histochemical and biochemical assessments of bindings with lectin probes has provided important new information about differences in the glycosylation patterns of individual glycoproteins stored within the same secretory granules.

Animals↗

PubMed1995

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.

Acoustic Stimulation↗

PubMed1995

Postnatal expression of the facilitated glucose transporter, GLUT 5, in gerbil outer hair cells.

The fifth isoform from the family of facilitative glucose transporters (GLUT 5) was identified in the gerbil cochlea by light and electron microscopic immunohistochemistry. The immunostaining procedure employed a polyclonal antibody raised against a synthetic peptide representing the carboxy terminus of human GLUT 5. Immunoreactive GLUT 5 was abundant in the basolateral plasma membrane of outer hair cells (OHCs) in the mature gerbil cochlea. No immunostaining was observed in any other site in the cochlea or vestibular system. During postnatal development of the gerbil inner ear, OHCs were first observed to express GLUT 5 in a punctate pattern at 10 days after birth. Immunostaining intensity increased gradually and developed a linear pattern along the entire OHC basolateral plasmalemma between 10 and 16 days after birth when it approached adult levels. The developmental expression of GLUT 5 corresponded with the appearance of glycogen in OHCs and structural maturation of the organ of Corti. This time period also coincided with the onset and rapid maturation of auditory function in the gerbil. GLUT 5 apparently supports OHC function by facilitating uptake of glucose which enables these cells to sustain a high level of metabolic activity in a relatively anaerobic environment.

Animals↗

PubMed1995

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.

Aging↗

PubMed1995

Development of cochlear potentials in the neonatal gerbil.

The onset and maturation of hearing was examined in separate groups of sibling and nonsibling neonatal Mongolian gerbils (Meriones unguiculatus). Auditory nerve compound action potentials (CAP) and cochlear microphonics (CM) were measured at the round window, and the endocochlear potential (EP) was recorded at three different locations in pups aged 13 to 30 days after birth (DAB) and in 90 day-old animals. Maturational trends for the three potentials were similar to those previously reported for gerbil neonates. However, CAP thresholds continued to decrease, and CM and CAP input/output functions and EP continued to increase beyond 30 days of age, a time at which many investigators have considered hearing in the gerbil to be mature. The EP developed simultaneously throughout the cochlea and approached 80 mV by 20 DAB. CAP thresholds showed a highly correlated log-linear relationship with EP in groups of nonlittermates and in siblings studied at different ages. In contrast, maximum CAP and CM amplitudes increased with increasing EP, but did not show significant growth until the EP exceeded 70 mV.

Acoustic Stimulation↗

PubMed1995

Developmental expression of proteoglycans in the tectorial and basilar membrane of the gerbil cochlea.

The appearance and distribution of specific proteoglycans (PGs) was assessed during development and maturation of the tectorial (TM) and basilar membranes (BM) in the gerbil cochlea. At birth, monoclonal antibodies against keratan sulfate (KSPG) and chondroitin 4- or 6-sulfate (4S CSPG, 6S CSPG) reacted with the upper fibrous layer of the TM with staining for anti-KSPG predominating. Reactivity for 4S CSPG remained constant whereas that for 6S CSPG increased through day 20 when it exceeded that of 4S CSPG. The region of Köllikers organ near the developing tunnel of Corti stained positively with all three PG antibodies from birth through day 8. In contrast, cells in the developing inner spiral sulcus lacked immunoreactive KSPG but expressed CSPG. PGs were first detectable in the BM of the basal turn at day 8 and increased to near adult levels by 16 days after birth. Anti-KSPG again showed the strongest staining with labeling density for 4S and 6S CSPG being about equal at maturity. Staining with all three antibodies was localized along the margins of the BM. Reactivity of the TM and BM in the upper turns lagged behind that of the basal turns by 24-48 h. Our results show that the TM is relatively mature at birth, needing only minor changes in its PG content to reach adult levels. In contrast, the BM showed a marked increase in its content of PGs during a period corresponding to the onset and rapid development of auditory function.

Animals↗

PubMed1995

Cytological changes related to maturation of the organ of Corti and opening of Corti's tunnel.

Maturation of the organ of Corti in the gerbil was analyzed between 2 and 16 days after birth (DAB) by electron microscopy and immunostaining for beta-tubulin. At 2 DAB, the organ of Corti consisted of stratified epithelium bearing immature sensory hair cells (HCs) and supporting cells. Maturation of OHCs and Deiters cells progressed in a medial-to-lateral direction and cytoskeletal development in inner pillar cells preceded that in outer pillar cells at the single location studied along the frequency-place map. Pillar cell differentiation progressed through a unique stage characterized by the appearance and stratification of structural features apparently concerned with opening of Corti's tunnel and subsequently showed other structural changes related to maturity toward the adult form. Development of the microtubule cytoskeleton occurred first in the cell's apex and proceeded basally. Ruffling of a middle region of the cell surface by microvilli appeared to promote separation between inner and outer pillar cells and initiate tunnel opening at 4 DAB. Proliferation of distended cisternae of granular reticulum evidenced proteinaceous secretion by these cells between 4 and 8 DAB. Subsequent tunnel expansion at about 14 DAB coincided with appearance in outer pillar cells of tubulocisternal endoplasmic reticulum and associated Golgi complexes that are thought to mediate fluid and ion secretion. Sixteen days postnatally after disappearance of granular and tubulocisternal reticula and Golgi complexes and at the time of clearing of tunnel fluid, lysosomes interpreted as mediating catabolism of endocytosed protein congregated beneath the apical and apicolateral plasmalemmae of inner pillar cells. As with pillar cells, development of the microtubule system in Deiters cells proceeded from the cell's apex to base. Following differentiation of their microtubule system by 8 DAB, Deiters cells showed expansion of Golgi cisternae between 10 and 15 DAB and development of tubulocisternal endoplasmic reticulum at 15 DAB. Hair cells possessed abundant, distinctively large mitochondria from 4 to 10 DAB. The subsurface cisternae matured earlier in medial as opposed to lateral outer hair cells. Vesicles budding from underlying cisternae appeared associated with development of subsurface cisternae and at 16 DAB were still observed in third row but not in more mature first row HCs.

Animals↗

PubMed1995

Effect of postmortem autolysis on Na,K-ATPase activity and antigenicity in the gerbil cochlea.

Alterations in the enzymatic activity and antigenicity of Na,K-ATPase as well as changes in cochlear morphology were assessed in gerbil inner ears harvested at selected time intervals up to 18 h postmortem. Na,K-ATPase activity was assayed biochemically in one cochlea from each animal and the other cochlea was fixed and embedded in paraffin for evaluation by light microscopy. Na,K-ATPase antigenicity was assessed by immunostaining with a broad-spectrum antiserum reactive with all known isoforms of the enzyme, and structural preservation was evaluated on adjacent sections stained with hematoxylin and eosin. The results showed a downward trend in enzymatic activity of Na,K-ATPase in lateral wall tissues within 1 h of death. In contrast, Na,K-ATPase immunoreactivity was fairly well preserved with postmortem fixation delays up to 12 h, despite the considerable structural degradation of cochlear tissues which began 2-3 h postmortem. It is concluded that under controlled environmental conditions, cochleas collected up to 4 h postmortem are suitable for morphological and immunohistochemical study of Na,K-ATPase by light microscopy. Cochleas collected more than 5 h postmortem were useful only for relatively gross immunohistochemical studies. It is suggested that cochleas intended for biochemical assays of Na,K-ATPase and probably most other enzymes should be collected within 1 h of death.

Animals↗