2'-hydroxy C16-ceramide induces apoptosis-associated proteomic changes in C6 glioma cells.

Ceramide is a bioactive sphingolipid involved in regulation of numerous cell signaling pathways. Evidence is accumulating that differences in ceramide structure, such as N-acyl chain length and desaturation of sphingoid base, determine the biological activities of ceramide. Using synthetic (R)-2'-hydroxy-C16-ceramide, which is the naturally occurring stereoisomer, we demonstrate that this ceramide has more potent pro-apoptotic activity compared to its (S) isomer or non-hydroxylated C16-ceramide. Upon exposure to (R)-2'-hydroxy-ceramide, C6 glioma cells rapidly underwent apoptosis as indicated by caspase-3 activation, PARP cleavage, chromatin condensation, and annexin V stain. A 2D gel proteomics analysis identified 28 proteins whose levels were altered during the initial 3 h of exposure. Using the list of 28 proteins, we performed a software-assisted pathway analysis to identify possible signaling events that would result in the observed changes. The result indicated that Akt and MAP kinase pathways are among the possible pathways regulated by (R)-2'-hydroxy-ceramide. Experimental validation confirmed that 2'-hydroxy-ceramide significantly altered phosphorylation status of Akt and its downstream effector GSK3ß, as well as p38, ERK1/2, and JNK1/2 MAP kinases. Unexpectedly, robust phosphorylation of Akt was observed within 1 h of exposure to 2'-hydroxy-ceramide, followed by dephosphorylation. Phosphorylation status of MAPKs showed a complex pattern, in which rapid phosphorylation of ERK1/2 was followed by dephosphorylation of p38 and ERK1/2 and phosphorylation of the 46 kDa isoform of JNK1/2. These data indicate that (R)-2'-hydroxy-ceramide regulates multiple signaling pathways by affecting protein kinases and phosphatases with kinetics distinct from that of the extensively studied non-hydroxy-ceramide or its unnatural stereoisomer.

Renal Glomerular Mitochondria Function in Salt-Sensitive Hypertension.

Salt-sensitive (SS) hypertension is accompanied with an early onset of proteinuria, which results from the loss of glomerular podocytes. Here, we hypothesized that glomerular damage in the SS hypertension occurs in part due to mitochondria dysfunction, and we used a unique model of freshly isolated glomeruli to test this hypothesis. In order to mimic SS hypertension, we used Dahl SS rats, an established animal model. Animals were fed a 0.4% NaCl (normal salt, NS) diet or challenged with a high salt (HS) 4% NaCl diet for 21 days to induce an increase in blood pressure (BP). Similar to previous studies, we found that HS diet caused renal hypertrophy, increased BP, glomerulosclerosis, and renal lesions such as fibrosis and protein casts. We did not observe changes in mitochondrial biogenesis in the renal cortex or isolated glomeruli fractions. However, Seahorse assay performed on freshly isolated glomeruli revealed that basal mitochondrial respiration, maximal respiration, and spare respiratory capacity were lower in the HS compared to the NS group. Using confocal imaging and staining for mitochondrial H2O2 using mitoPY1, we detected an intensified response to an acute H2O2 application in the podocytes of the glomeruli isolated from the HS diet fed group. TEM analysis showed that glomerular mitochondria from the HS diet fed group have structural abnormalities (swelling, enlargement, less defined cristae). Therefore, we report that glomerular mitochondria in SS hypertension are functionally and structurally defective, and this impairment could eventually lead to loss of podocytes and proteinuria. Thus, the glomerular-mitochondria axis can be targeted in novel treatment strategies for hypertensive glomerulosclerosis.

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.

Identification of ClC-2 and CIC-K2 chloride channels in cultured rat type IV spiral ligament fibrocytes.

Voltage-gated chloride channels (ClCs) are important mediators of cellular ion homeostasis and volume regulation. In an earlier study, we used immunohistochemical, Western blot, and reverse transcriptase PCR (RT-PCR) approaches to identify ClC-K variants in types II, IV, and V fibrocytes of the rodent spiral ligament. We have now confirmed the expression of ClC-K2 in these cells by in situ hybridization. All three of these fibrocyte subtypes are thought to be involved in cochlear K(+) recycling; thus, it is important to understand the precise mechanisms regulating their membrane conductance and the role played by ClCs in this process. In this study, we report the characterization of a secondary cell line derived from explants from the region of the rat spiral ligament underlying and inferior to the spiral prominence. The cultured cells were immunopositive for vimentin, Na,K/ATPase, Na,K,Cl-cotransporter, carbonic anhydrase isozyme II, and creatine kinase isozyme BB, but not for cytokeratins or Ca/ATPase, an immunostaining profile indicative of the type IV subtype. Evaluation of the cultures by RT-PCR and Western blot analysis confirmed the presence of both ClC-2 and -K2. Whole-cell patch clamp recordings identified two biophysically distinct Cl(-) currents in the cultured cells. One, an inwardly rectifying Cl(-) current activated by hyperpolarization or decreasing extracellular pH corresponded with the properties of ClC-2. The other, a weak outwardly rectifying Cl(-) current regulated by extracellular pH, Cl(-), and Ca(2+) resembled the channel characteristics of ClC-K2 when expressed in Xenopus oocytes. These findings suggest that at least two functionally different chloride channels are involved in regulating membrane anion conductance in cultured type IV spiral ligament fibrocytes.

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.

Macrophage-Mediated Glial Cell Elimination in the Postnatal Mouse Cochlea.

Hearing relies on the transmission of auditory information from sensory hair cells (HCs) to the brain through the auditory nerve. This relay of information requires HCs to be innervated by spiral ganglion neurons (SGNs) in an exclusive manner and SGNs to be ensheathed by myelinating and non-myelinating glial cells. In the developing auditory nerve, mistargeted SGN axons are retracted or pruned and excessive cells are cleared in a process referred to as nerve refinement. Whether auditory glial cells are eliminated during auditory nerve refinement is unknown. Using early postnatal mice of either sex, we show that glial cell numbers decrease after the first postnatal week, corresponding temporally with nerve refinement in the developing auditory nerve. Additionally, expression of immune-related genes was upregulated and macrophage numbers increase in a manner coinciding with the reduction of glial cell numbers. Transient depletion of macrophages during early auditory nerve development, using transgenic CD11bDTR/EGFP mice, resulted in the appearance of excessive glial cells. Macrophage depletion caused abnormalities in myelin formation and transient edema of the stria vascularis. Macrophage-depleted mice also showed auditory function impairment that partially recovered in adulthood. These findings demonstrate that macrophages contribute to the regulation of glial cell number during postnatal development of the cochlea and that glial cells play a critical role in hearing onset and auditory nerve maturation.

Contribution of bone marrow hematopoietic stem cells to adult mouse inner ear: mesenchymal cells and fibrocytes.

Bone marrow (BM)-derived stem cells have shown plasticity with a capacity to differentiate into a variety of specialized cells. To test the hypothesis that some cells in the inner ear are derived from BM, we transplanted either isolated whole BM cells or clonally expanded hematopoietic stem cells (HSCs) prepared from transgenic mice expressing enhanced green fluorescent protein (EGFP) into irradiated adult mice. Isolated GFP(+) BM cells were also transplanted into conditioned newborn mice derived from pregnant mice injected with busulfan (which ablates HSCs in the newborns). Quantification of GFP(+) cells was performed 3-20 months after transplant. GFP(+) cells were found in the inner ear with all transplant conditions. They were most abundant within the spiral ligament but were also found in other locations normally occupied by fibrocytes and mesenchymal cells. No GFP(+) neurons or hair cells were observed in inner ears of transplanted mice. Dual immunofluorescence assays demonstrated that most of the GFP(+) cells were negative for CD45, a macrophage and hematopoietic cell marker. A portion of the GFP(+) cells in the spiral ligament expressed immunoreactive Na, K-ATPase, or the Na-K-Cl transporter (NKCC), proteins used as markers for specialized ion transport fibrocytes. Phenotypic studies indicated that the GFP(+) cells did not arise from fusion of donor cells with endogenous cells. This study provides the first evidence for the origin of inner ear cells from BM and more specifically from HSCs. The results suggest that mesenchymal cells, including fibrocytes in the adult inner ear, may be derived continuously from HSCs.

Topical application of mitomycin C to the middle ear is ototoxic in the gerbil.

HYPOTHESIS: Mitomycin C is ototoxic when applied topically to the structures of the middle ear. BACKGROUND: Mitomycin C is a topically applied medication widely used in a variety of surgical procedures to prevent excessive scar tissue formation. Its safety for use during otologic procedures has not been fully evaluated. METHODS: A laboratory study was undertaken using the Mongolian gerbil as an animal model. Both acute and chronic effects on cochlear function of mitomycin C were assessed with measurements of compound action potential (CAP) thresholds of the auditory nerve, CAP input/output functions, distortion product otoacoustic emissions, and endocochlear potentials. Morphologic changes were assessed with light microscopy using hematoxylin-eosin staining as well as transmission electron microscopy. RESULTS: Five-minute applications of mitomycin C (0.5 mg/ml) to the entire surface of the middle ear adversely affected CAP thresholds, input/output functions, distortion product otoacoustic emissions, and the endocochlear potential. Ninety-minute exposures of mitomycin C solely to the round window produced similar changes. Histologic evaluation of animals 1 week after treatment showed damage to cochlear hair cells, the stria vascularis, and spiral ganglion neurons when compared with controls. CONCLUSION: Mitomycin C can produce substantial sensorineural hearing loss when applied topically to the gerbil middle ear for even brief periods. Consequently, its safety for topical use in the human middle ear is highly questionable.

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).

Chronic reduction of endocochlear potential reduces auditory nerve activity: further confirmation of an animal model of metabolic presbyacusis.

Gerbils aged in quiet show a decline of the endocochlear potential (EP) and elevated auditory nerve compound action potential (CAP) thresholds. However, establishing a direct relationship between an age-related reduction in the EP and changes in the activities of primary auditory neurons is difficult owing to the complexity of age-related histological changes in the cochlea. To address this issue, we developed a young gerbil model of "metabolic" presbyacusis that uses an osmotic pump to deliver furosemide into the round window niche for 7 days, resulting in a chronically reduced EP. In this model, the only major histopathologic changes were restricted to the hook region of the cochlea and consisted of loss of strial intermediate cells and massive edema in the lateral wall. The morphological and physiological evidence suggests that the cochlea can adapt to furosemide application over time. The morphology of spiral ganglion cells and hair cells appeared normal throughout the cochlea. CAP responses and EP values in this model are similar to those of quiet-aged ears. The spontaneous activity of single auditory fibers (n = 188) was assessed in 15 young gerbils treated with furosemide for 7 days. The percentage of recorded low-spontaneous rate (SR) fibers at characteristic frequencies (CFs) > or = 6 kHz was significantly lower in furosemide-treated than in control ears. Recovery function tests of CAP responses after prior stimulation also showed a decline in activity of the low-SR population with CFs > or = 6 kHz in the treated cochleas. A similar loss in the activity of low-SR fiber has been previously shown in quiet-aged gerbils. These results suggest that dysfunction of the cochlear lateral wall and subsequent chronic reduction in the EP can directly affect the activity patterns of primary auditory neurons in a manner similar to that seen in aged gerbils.

Unmyelinated auditory type I spiral ganglion neurons in congenic Ly5.1 mice.

With the exception of humans, the somata of type I spiral ganglion neurons (SGNs) of most mammalian species are heavily myelinated. In an earlier study, we used Ly5.1 congenic mice as transplant recipients to investigate the role of hematopoietic stem cells in the adult mouse inner ear. An unanticipated finding was that a large percentage of the SGNs in this strain were unmyelinated. Further characterization of the auditory phenotype of young adult Ly5.1 mice in the present study revealed several unusual characteristics, including 1) large aggregates of unmyelinated SGNs in the apical and middle turns, 2) symmetrical junction-like contacts between the unmyelinated neurons, 3) abnormal expression patterns for CNPase and connexin 29 in the SGN clusters, 4) reduced SGN density in the basal cochlea without a corresponding loss of sensory hair cells, 5) significantly delayed auditory brainstem response (ABR) wave I latencies at low and middle frequencies compared with control mice with similar ABR threshold, and 6) elevated ABR thresholds and deceased wave I amplitudes at high frequencies. Taken together, these data suggest a defect in Schwann cells that leads to incomplete myelinization of SGNs during cochlear development. The Ly5.1 mouse strain appears to be the only rodent model so far identified with a high degree of the "human-like" feature of unmyelinated SGNs that aggregate into neural clusters. Thus, this strain may provide a suitable animal platform for modeling human auditory information processing such as synchronous neural activity and other auditory response properties.

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.