Mitochondria-activated cisternae generate the cell specific vesicles in auditory hair cells.

A dense population of vesicles largely fills the infranuclear compartment of gerbil inner hair cells (IHCs). Although the nature of the cargo in these vesicles has not been determined, the absence of a Golgi apparatus from the IHC's basal compartment suggests that the vesicles lack the glycosylated protein that Golgi cisternae would provide. Instead, they likely possess neurotransmitter and function as synaptic vesicles. The morphologic mechanism for generating the vesicles also remains unexplained. Ultrastructural examination revealed a few discrete clusters of mitochondria in the IHC's basal compartment. The clustered mitochondria made contact either with intermingling single cisternae or with one end of an unique set of polarized parallel cisternae. Both of these cisternal forms belong to a novel, mitochondria-activated category of cisternae which transforms into aligned segments where contacting mitochondria. Mitochondria-activated cisternae also envelope the vesicles in Hensen bodies of outer hair cells (OHCs). Coexistence of the mitochondria-activated cisternae with a specialized population of cytoplasmic vesicles in both IHCs and OHCs implicated this type of cisterna in synthesis of the cell specific vesicles. Assumedly, the mitochondria-activated cisternae possess an ATPase of the Class IV type. This class of enzymes, also designated flippases, translocates aminophospholipid from the outer to inner leaflet of the lipid bilayer and appears thereby to induce a lipid asymmetry which leads to cisternal segmentation and then vesiculation. In support of such an interpretation, RT-PCR analysis demonstrated the presence of Class IV ATPase in the Organ of Corti.

Cell-specific expression of neutral glycosphingolipids in vertebrate brain: immunochemical localization of 3-O-acetyl-sphingosine-series glycolipid(s) in myelin and oligodendrocytes.

The tissue- and cell-specific expression of three neutral glycosphingolipids, gangliotetraosylceramide (GA1), gangliopentaosylceramide (GalNAc-GA1), and the novel 3-O-acetyl-sphingosine-series glycolipid (FMC-5), were examined with monospecific polyclonal antibodies. Immunohistochemical studies of rodent brain cross-sections indicated that both GA1 and FMC-5 antibodies stained myelin. In contrast, GalNAc-GA1 antibody distinctly stained neurons in cerebral cortex, but only partially delineated Purkinje cells and other neurons in cerebellum. Preliminary studies of mixed glial cultures suggested the following: 1) both FMC-5 and GA1 antibodies stained oligodendrocytes and oligo progenitors, and 2) GalNAc-GA1 antibody did not stain any cells in the culture. Because the GalNAc-GA1 was associated with neurons, we examined the immunoreactivity of GalNAc-GA1 antibody in primary neuronal cultures. Further studies using primary cultures of rat brain oligodendrocytes, and dissociated cerebellar neuronal cultures indicated that both GA1 and FMC-5 are specifically expressed by oligodendrocytes, whereas GalNAc-GA1 is primarily localized in interneurons and to some extent in Purkinje neurons.

Nuclear factor kappaB deficiency is associated with auditory nerve degeneration and increased noise-induced hearing loss.

Degeneration of the spiral ganglion neurons (SGNs) of the auditory nerve occurs with age and in response to acoustic injury. Histopathological observations suggest that the neural degeneration often begins with an excitotoxic process affecting the afferent dendrites under the inner hair cells (IHCs), however, little is known about the sequence of cellular or molecular events mediating this excitotoxicity. Nuclear factor kappaB (NFkappaB) is a transcription factor involved in regulating inflammatory responses and apoptosis in many cell types. NFkappaB is also associated with intracellular calcium regulation, an important factor in neuronal excitotoxicity. Here, we provide evidence that NFkappaB can play a central role in the degeneration of SGNs. Mice lacking the p50 subunit of NFkappaB (p50(-/-) mice) showed an accelerated hearing loss with age that was highly associated with an exacerbated excitotoxic-like damage in afferent dendrites under IHCs and an accelerated loss of SGNs. Also, as evidenced by immunostaining intensity, calcium-buffering proteins were significantly elevated in SGNs of the p50(-/-) mice. Finally, the knock-out mice exhibited an increased sensitivity to low-level noise exposure. The accelerated hearing loss and neural degeneration with age in the p50(-/-) mice occurred in the absence of concomitant hair cell loss and decline of the endocochlear potential. These results indicate that NFkappaB activity plays an important role in protecting the primary auditory neurons from excitotoxic damage and age-related degeneration. A possible mechanism underlying this protection is that the NFkappaB activity may help to maintain calcium homeostasis in SGNs.

Expression of CLC-K chloride channels in the rat cochlea.

Current models of the lateral K+ recycling pathway in the mammalian cochlea include two multicellular transport networks separated from one another by three interstitial gaps. The first gap is between outer hair cells and Deiters cells, the second is between outer sulcus cells and type II spiral ligament fibrocytes and the third is between intermediate and marginal cells in the stria vascularis. K+ taken up by cells bordering these interstitial spaces is accompanied by Cl-. Maintaining appropriate electrolyte balance and membrane potentials in these cells requires a mechanism for exit of the resorbed Cl-. One possible candidate for regulating this Cl- efflux is ClC-K, a chloride channel previously thought to be kidney specific. Here, we demonstrate the expression of both known isoforms of ClC-K in the organ of Corti, spiral ligament and stria vascularis of the rat cochlea by immunohistochemical, Western blot and RT-PCR analysis. These results indicate a role for ClC-K in mediating Cl- recycling in the cochlea. The widespread expression of both ClC-K isoforms in the cochlea may help to explain the symptoms of Bartter's syndrome Type III, a mutation in the hClC-Kb gene (human homologue of ClC-K2), which results in renal salt wasting without deafness. These data support the hypothesis that both isoforms of ClC-K are co-expressed in some cell membranes and account for the preservation of hearing in the presence of a mutation in only one channel isoform.

Expression of oncostatin M and its receptors in normal and cirrhotic human liver.

BACKGROUND/AIMS: In the cirrhotic liver, gene expression of the multifunctional cytokine oncostatin M (OSM) is up-regulated, but its cellular origin is unknown. Therefore, we investigated the expression of OSM protein and its specific receptor subunits, OSMRbeta and LIFRbeta in normal and cirrhotic human liver using immunohistochemical and Western blot analysis. RESULTS: OSM protein was expressed in Kupffer cells, variably in normal liver but consistently in cirrhosis. OSMRbeta was expressed at low level in hepatocytes of all normal livers examined, but in no cirrhotic sample. In contrast, LIFRbeta receptor was expressed weakly in normal livers, but much more intensely in cirrhosis, in reactive ductules, bile duct epithelial cells and perisinusoidal areas. Double immunostaining showed co-localization of LIFRbeta with cytokeratin 7, proliferating cell nuclear antigen (PCNA) and leukemia inhibitory factor (LIF), in bile duct epithelial cells, but not with alpha-smooth muscle actin, a myofibroblast marker. CONCLUSIONS: In human liver, OSM protein is expressed in Kupffer cells, variably in normals but universally in cirrhosis. The differential expression pattern of OSM and its receptors could allow for differential OSM signaling by alternative utilization of receptors to promote hepatocyte proliferation in acute injury and, with its homologue LIF, for the bile ductular reaction in cirrhosis.

Presence of surfactant lamellar bodies in normal and diseased sinus mucosa.

BACKGROUND: Pulmonary surfactant originates from phospholipid lamellar bodies secreted from the type II epithelial cell of the alveolus. In the lower airway, surfactant optimizes surface tension and oxygen exchange, decreases mucus viscosity and aids in mechanical elimination of inhaled pathogens. In addition to the lung, lamellar bodies have been identified in many other cell types throughout the human body. However, no prior studies have identified lamellar bodies in human sinus mucosa. OBJECTIVES: We performed ultrastructural studies to assess whether lamellar bodies are present in the human sinus in a variety of diseased and normal epithelium. METHODS: We biopsied sinus mucosa from 5 subjects, 1 each with allergic fungal sinusitis, eosinophilic mucin rhinosinusitis, cystic fibrosis, frontal sinus mucocele, and cerebrospinal fluid leak (healthy control). Mouse lung served as a positive control. Specimens were prepared using ferrocyanide-reduced osmium tetroxide and thiocarbohydrazide for fixation (R-OTO method) to avoid extraction of phospholipids during dehydration and were viewed with transmission electron microscopy. RESULTS: We identified lamellar bodies in the sinus mucosa of all patients. Additionally, preservation of mouse lung lamellar bodies confirms that the R-OTO method is a valid technique to preserve these structures. CONCLUSIONS: We describe a simpler, faster technique for identification of cellular phospholipid components than those used previously. Definitive identification of these lamellar bodies within ciliated pseudostratified epithelium of the upper airway indicates that surfactant may have a role in sinus function and pathophysiology.

Pathologic changes of presbycusis begin in secondary processes and spread to primary processes of strial marginal cells.

Strial atrophy underlying age-related hearing loss was investigated by ultrastructural comparisons in young and senescent gerbils. In young animals strial marginal cells (MCs) projected primary processes which gave rise to and were connected by numerous ultrathin secondary processes. In 30-36-month-old gerbils, the MC secondary processes degenerated into lamellar or amorphous profiles as the first manifestation of strial atrophy. Some short primary processes shorn of projecting and connecting secondaries coalesced to form mitochondria-filled lobules. Strial involution appeared to progress with transformation of the degenerating processes and lobules into permanent residues of laminated amorphous substance. A second apparently unique form of degeneration was observed in which areas filled with homogeneous granular material replaced the processes that comprise the basal half of the normal MC. An abrupt line of transition separated this structureless degradation product below from the viable upper half of the MC. The terminally involuted stria consisted of MC bodies lining scala media, along with vestigial remnants of MC processes, nearby normal appearing intermediate cells (ICs) and unaltered basal cells. The only age-related change in ICs involved incorporation of melanosomes into very large, matrix-filled lysosomes. A profile of one MC in apparent necrosis provided evidence for an infrequent occurrence of MC death. These data support a progression of pathologic changes beginning with the demise of MC secondary processes and ending with ablation of secondary and primary processes. The initial injury apparently occurs as a result of oxidative self-damage to mitochondria in the MCs primary processes, leading to insufficient ATP for the Na,K-ATPase of the secondary processes. The reduced ATP level may cause cytotoxic alteration of the cytosolic Na(+)/K(+) ratio first in MC secondary processes and later in the primaries, with consequent degeneration of these structures.

Novel structures in marginal and intermediate cells presumably relate to functions of apical versus basal strial strata.

Prior ultrastructural studies showed that K+ supplied to the stria vascularis came from recycling ions from the organ of Corti or perilymph to strial basal cells. A newly distinguished basal subtype of intermediate cell (BIC) completely covered the basal cells with a leaf-like horizontal process and appeared situated to absorb from them all of the recycled K+. The basal region of marginal cells (MCs) projected foot-like and enlarged processes to border BICs opposite an unique ca. 150 angstroms space. These basal MC processes appeared positioned to resorb part of the K+ recycled to BICs. A second, upper subtype of IC (UIC), occupying middle to upper strial strata, contacted BIC's extensively. UICs were thus located to resorb from BICs the portion of the recycled K+ not forwarded to basal MC processes. The apical segment of MCs projected mitochondria-filled primary processes and numerous associated secondary processes. The Na,K-ATPase-rich secondary processes populated mid to upper stria where they could siphon K+ from UICs and resorb and secrete the ions thus generating the 150 mM [KCl] of endolymph. The morphologic relationship of basal marginal cell processes to BICs differed so strikingly from the relation of upper MC processes to UICs as to suggest a different function for basal stria, one possibly concerned with generating the endocochlear potential.

Comparative studies of oncostatin M expression in the tissues of adult rodents.

Oncostatin M (OSM), a member of the interleukin-6 family of cytokines, is thought to be expressed mostly by activated T-lymphocytes and monocytes in adult animals. However, here we report specific constitutive tissue expression of OSM in the pancreas, kidney, testes, spleen, stomach, and brain, but not liver or lung, of three adult rodent species.

Ultrastructure indicative of ion transport in tectal, Deiters, and tunnel cells: differences between gerbil and chinchilla basal and apical cochlea.

Ultrastructural examination revealed an epithelium of about five tectal cells (TCs) roofing the outer tunnel (OT) in the mid to upper, but not the basal, region of gerbil and chinchilla cochlea. Structures in TCs that are apparently specialized for retrieval of K(+) released into tunnel fluid from outer hair cells (OHCs) include surface fimbriae in the gerbil and canalicular reticulum in the chinchilla. A tunnel roof of organelle-rich TCs appeared to be better equipped for ion resorption than a roof composed of organelle-poor Hensen cells (HCs). Fimbriae, filopodia, and the cell body of TCs descended to contact the third Deiters cell (DC3) in the gerbil, and the hypertrophied DC3 phalanx rose to contact TCs in the chinchilla, which suggests a solute exchange between TCs and DCs. Previously unrecognized structures that are speculated to provide ATP ligand for cochlear purinoreceptors occurred in the chinchilla DC and gerbil TC. The observation of a microtubule stalk in DCs indicated that they also function in cochlear mechanics. A newly delineated lateral tunnel cell (LTC) intervened between the DC3 and HC in both species. The apicomedial plasmalemma of all DCs fitted closely to the base of OHCs and enveloped afferent nerves. The morphologic specializations reported here provide further support for the proposed transcellular lateral flow route for K(+) currents generated by sound exposure and neural activity. The previously demonstrated expansion of Boettcher cells, outer sulcus cell roots, type Il and IV fibrocytes, and apical microvilli on HCs and Claudius cells (CCs) in the base of the cochlea is postulated here to mediate a basal parallel current that could supply the increased K(+) transport required for the basally elevated electric potential (EP).

Inspissation of pancreatic zymogen material in cystic fibrosis.

Cystic fibrosis is characterized by the elaboration of abnormal, thick, tenacious mucus resulting in obstructive disease in sites such as the lung and pancreas. In the pancreas, acinar plugs of mucus have been reported as the earliest recognizable morphologic lesion in cystic fibrosis. Since mucus is not normally elaborated within the pancreatic lobular tissue, the mechanism of accumulation of mucus in acini is enigmatic. To investigate this phenomenon, well-preserved autopsy pancreatic tissue was studied ultrastructurally. This study demonstrated very prominent mucous metaplasia in these diseased organs. Acinar plugs, though, developed before mucous metaplasia. Subsequent histochemical study was performed, which demonstrated that the early acinar plugs exhibited the same staining properties as zymogen granules and were distinct from the staining pattern of mucus in pancreatic tissue of cystic fibrosis patients. These findings, then, indicate that zymogen material, not mucus, becomes inspissated in the acini of the pancreas in early cystic fibrosis, and that subsequent mucous metaplasia occurs as the obstruction and exocrine atrophy progress.

Spiral ligament pathology in quiet-aged gerbils.

The ultrastructure of the spiral ligament was compared in aged and young gerbils to assess the involvement of connective tissues in the lateral wall and particularly the fibrocytes in development of presbyacusis. Pathologic features in fibrocytes of senescent gerbils spanned a wide range reflecting different stages of lateral wall involution. All of the type II, IV and V fibrocytes selectively developed cytosolic vacuoles in an early degenerative phase showing minimal strial involvement. Clear spaces indicative of interstitial edema separated the vacuolated cell bodies and their plasmalemmal processes. As a presumed intermediate phase, profiles of amorphous substance apparently derived from apoptosis/necrosis of type II fibrocytes infiltrated the type II fibrocyte area among nearly normal appearing cells. In cochlear turns with advanced strial degeneration, type II fibrocytes disappeared from the spiral prominence area leaving only type I-like fibrocytes occasionally accompanied by a collagen infiltrate. Type V fibrocytes disappeared similarly from the suprastrial area. The extent of atrophy in type II fibrocytes corresponded in general with that in the neighboring stria vascularis. Age-dependent atrophy in the lateral wall largely spared type I fibrocytes except that they often enclosed discrete amorphous foci lacking organelles. The involution thus affected principally the Na,K-ATPase-positive fibrocytes functioning in active uptake rather than passive conductance of K(+). The vacuolization and degeneration exclusive to ATPase-rich fibrocytes and the associated intercellular edema are interpreted as secondary responses, possibly as a result of impaired diffusion of K(+) through downstream marginal cells.