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Biomedical subjects

A F Ryan

Publications and source records attributed to A F Ryan.

At least 73 records · Page 4Linked to original sources
PubMed1994

Nicotinic acetylcholine receptor subunits expressed in rat cochlea detected by the polymerase chain reaction.

Poly(A)+ RNA was extracted from rat cochleae using guanidinium thiocyanate and oligo(dT)-cellulose, and converted into cDNA by reverse transcriptase using an oligo(dT) primer. Oligonucleotides complementary to conserved 5' and 3' regions of alpha and beta subunits of the neuronal nicotinic acetylcholine receptor subunit (nAChR) family were then used as primers to screen the cochlear cDNA via the polymerase chain reaction (PCR) procedure. PCR products of approximately 900 bp length, purified by agarose gel electrophoresis, were nick translated to produce [32P]-dCTP labelled probes for Southern Blot screening of nAChR cDNAs. Of the four alpha and three beta subunits screened, only alpha 5 and beta 4 nAChR cDNAs hybridized. The alpha 5 PCR product was cloned and sequenced and proved to be identical to published sequence for alpha 5. The detection of alpha 5 and beta 4 nAChR subunit expression in cochlear tissue supports previous electrophysiological and immunocytochemical evidence for nAChR-mediated centrifugal control of hearing function.

Animals↗
PubMed1994

Protection from noise-induced hearing loss by prior exposure to a nontraumatic stimulus: role of the middle ear muscles.

Recent evidence suggests that prior exposure to a moderate-level acoustic stimulus can reduce damage due to later exposure to the same stimulus at high intensity [Canlon et al., Hear. Res. 34, 197-200 (1988)]. To test the role of the middle ear muscles (MEMs) in this phenomenon, Mongolian gerbils were conditioned by exposure to a two-octave band of noise (1414-5656 Hz) at 81 dB SPL for 3 weeks. Either immediately afterward, or following a one week rest period, they were exposed to the same stimulus at 110 dB SPL for one hour. The ABR thresholds of these animals were compared to those seen in animals exposed at 110 dB SPL without conditioning. The MEMs of one ear in each subject were cut, to determine their role in any noise trauma protection effects. In the unoperated ears, conditioning without a recovery period did not alter the effects of the 110 dB stimulus. Conditioning followed by a one week recovery period reduced both temporary (TTS) and permanent (PTS) threshold shift. MEM section had no effect on either TTS or PTS in unconditioned subjects, and did not alter the reduction in TTS or PTS seen with conditioning. It is concluded that the noise trauma resistance provided by acoustic conditioning is not mediated by the MEMs.

Acoustic Stimulation↗
PubMed1994

Acidic and basic FGF mRNA expression in the middle ear mucosa during experimental acute and chronic otitis media.

Fibroblast growth factors (FGFs) induce the proliferation and differentiation of cells of mesodermal and neuroectodermal origin. Using in situ hybridization, messenger ribonucleic acid encoding acidic FGF, basic FGF and FGF receptor 1 (FGFR1) were localized in the middle ear mucosa of experimental animals with acute and chronic immune-mediated otitis media with effusion (OME). Basic FGF-labeled cells were seen in the subepithelial connective tissue layer (SE) preferentially near the epithelial basement membrane. Acidic FGF-labeled cells were seen in the SE, preferentially near blood vessels and occasionally in the cellular middle ear effusion (CE). FGFR1-labeled cells were seen in the SE and in the CE. The distribution of labeled cells in the middle ear suggests that basic FGF is produced by fibroblasts, acidic FGF is produced by leukocytes, and FGFR1 is produced by both fibroblasts and leukocytes. A role is proposed for these peptides in the proliferation and maintenance of the middle ear submucosa during otitis media.

Acute Disease↗
PubMed1993

Cloning and in situ hybridization of type 2A and 2B rat skeletal muscle myosin tail region: implications for filament assembly.

Changes in fast myosin expression play a critical role in skeletal muscle adaptation. Two fast myosin isoforms, type 2A and type 2B, are commonly expressed by fast muscle fibers but their sequences have not been determined to allow mRNA expression studies. A complete set of rat skeletal muscle myosins was amplified by PCR of cDNAs derived from skeletal muscle mRNA, cloned in a TA cloning vector, and sequenced. Specificity was demonstrated by in situ hybridization against skeletal muscle and myosin protein identification using monoclonal antibodies. Two novel sequences were cloned: A type 2A myosin which consisted of a 642 bp segment from the 3' end and a type 2B myosin which consisted of a 624 bp segment also from the 3' end. This region encodes that portion of the myosin molecule implicated in the control of filament assembly. The two fast myosins showed 88% homology in the open reading frame and 95% homology at the amino acid level. Based on this homology, it is unlikely that selective myosin filament assembly occurs during muscle fiber type transformation between type 2A and 2B.

Amino Acid Sequence↗
PubMed1993

Fibronectin-like immunoreactivity of the basilar membrane of young and aged rats.

Dysfunction of cochlear mechanics has been hypothesized to be a source of age-related hearing loss and the basilar membrane mass and stiffness contribute to normal cochlear mechanics. Fibronectin, a large, extracellular matrix protein and a major component of the basilar membrane, may contribute to both the mass and stiffness of the membrane. Mesothelial cells underlying the basilar membrane may produce the fibronectin and also contribute to the mass of the membrane. Changes in either the fibronectin or the mesothelial cells might, therefore, have an effect on cochlear mechanics. In order to assess basilar membrane changes in aged animals, young adult (2-4 months) and aged (24-26 months) Sprague-Dawley rats were evaluated for the presence of fibronectin-like protein and mesothelial cells. The basilar membrane in the young animals had strong fibronectin-like immunoreactivity throughout its length. The old animals, on the other hand, showed normal fibronectin immunoreactivity in the basilar membrane of the basal turn, but little or no reactivity in the apical cochlear turn. The number of mesothelial cells was reduced throughout the length of the membrane in aged animals, with the greatest loss in the basal turn (60% fewer cells). These two degenerative changes, which appear to be independent of each other, may contribute to the observed threshold shifts in aged cochleas.

Aging↗
PubMed1993

Cloning genes from an inner ear cDNA library.

A rat inner ear complementary DNA (cDNA) library containing 1.9 x 10(6) recombinants was constructed and evaluated. Inserts averaged 2.0 (+/- 2.1) kilobases in length. A subset of inserts was screened for site of expression. Two cDNA transcripts were isolated based on cochlear expression restricted to the spiral ganglion. One transcript showed a high degree of homology to several long interspersed DNA elements, neuron-specific nuclear transcripts thought to be involved in gene regulation. The second transcript showed no homology to known sequences and appears to encode a neuron-specific protein of about 248 amino acids. The library can be used to identify proteins important for inner ear function and disease.

Animals↗
PubMed1993

Acidic and basic FGF mRNA expression in the adult and developing rat cochlea.

In situ hybridization was used to document the distribution of mRNA encoding acidic and basic fibroblast growth factor (aFGF and bFGF) in the rat cochlea from embryonic day (E) 16 to postnatal day (P) > 60. bFGF mRNA was not detected in the cochlea at any age. In the adult, aFGF mRNA was strongly expressed in spiral ganglion (SG) neurons, and this expression increased from base to apex. The stria vascularis (SV) and spiral prominence (SP) showed lesser expression which was equal in all turns. Developmentally, low level expression of aFGF mRNA was first seen in the SG at E-20, and remained low until P-4. Expression increased from P-6 to P-14, when adult levels were reached. aFGF mRNA was also observed in the developing hair cells of all turns at E-20. This expression increased after birth but disappeared after P-6. Expression in the SV and SP was first noted at E-20 and reached adult levels by P-16 and P-10, respectively. High levels of aFGF mRNA in the adult SG suggest that aFGF is important for the maintenance of SG neuron function and structure. aFGF in hair cells during the first postnatal week may be involved in the establishment of cochlear innervation.

Animals↗
PubMed1993

Growth factors during proliferation of the middle ear mucosa.

The presence of growth factors during otitis media with effusion (OME), induced by antigenic stimulation of the middle ear, was explored in guinea pigs. There was a significant increase in heparin-binding proliferative activity detected in mucosal tissue. This heparin-binding activity was identified as basic fibroblast growth factor (FGF) using several criteria. First, the majority of this activity was eluted from heparin-Sepharose gels with 1.3-1.8 M NaCl, consistent with basic FGF. Second, these fractions are potent stimulators of endothelial cell proliferation, characteristic of basic FGF. Third, the fractions contain basic FGF-like immunoreactivity based on radioimmunoassay. Finally, Western blotting of tissue extracts reveals the presence of an 18 kDa protein that is indistinguishable from basic FGF. We conclude that basic FGF plays a major role in the proliferation of the middle ear mucosa during OME.

Animals↗
PubMed1992

Selective retrograde transport of nipecotic acid, a GABA analog, labels a subpopulation of gerbil olivocochlear neurons.

Perfusion of the gerbil cochlea with micromolar quantities of 3H-gamma-aminobutyric acid (GABA) results in rapid, selective labeling of 50-60% of the olivocochlear (OC) efferent terminals on afferent dendrites beneath the inner hair cells, and all of the efferent terminals beneath the outer hair cells. In order to identify the neurons from which these GABA-accumulating terminals originate, the cell bodies were localized by using retrograde transport of 3H-nipecotic acid, a metabolically inert GABA analog. With survival times of 6-30 hours after cochlear injection, myelinated OC efferent fibers and cell bodies were well labeled, with the greatest number being labeled at 12-18 hours. All of the labeled neurons belonged to the medial OC system, and no lateral OC neurons were labeled. It is concluded that the GABA-accumulating endings in the gerbil cochlea arise from medial OC neurons, and therefore that medial OC efferent neurons in this species project to both inner and outer hair cell regions.

Animals↗
PubMed1992

Immunohistochemical localization of fibronectin-like protein in the inner ear of the developing gerbil and rat.

Immunohistochemistry was used to demonstrate the distribution of fibronectin-like protein within the developing inner ear of two species of altricial rodents: gerbils and rats. While there were temporal differences between the two species, the developmental sequence of immunostaining was virtually identical. Most notably, in rats from embryonic day 18 through day 1 postpartum, and in gerbils from birth through day 4 postpartum, intense, discrete fibronectin-like immunoreactivity was observed in the cochlea immediately beneath the inner and outer hair cells, sites of active auditory nerve fiber growth and nerve-hair cell synaptogenesis at these ages. The results suggest that fibronectin is appropriately positioned spatially and temporally to play a significant role in promoting, guiding and/or maintaining neural innervation within the developing organ of Corti. The temporo-spatial pattern of immunostaining in Schwann cells and auditory (VIIIth cranial) nerve neurons implies that fibronectin also plays a significant role in the early formation of myelin. In non-neural elements of the cochlea, fibronectin is a major structural component within the basilar membrane at all of the developmental stages investigated.

Animals↗
PubMed1992

Lymphocyte subsets in immune-mediated otitis media with effusion.

To examine the role of T-cell subsets in immune-mediated otitis media with effusion induced by keyhole limpet hemocyanin (KLH), we used immunohistochemical methods to investigate the kinetics of immunocytes of the middle ear (ME) and eustachian tube (ET) in healthy BALB/c mice. Antibodies against murine macrophages and granulocytes (anti-Mac-1), helper T cells (anti-Lyt-1), suppressor T cells (anti-Lyt-2), immunoglobulins (anti-IgG, -IgM, -IgA), secretory component (SC) and KLH were used. The ME exhibited a substantial immune response, whereas the response of the ET was minor and was associated with a secondary ME immune response. After KLH challenge, an effusion with an extensive infiltration of inflammatory cells (Mac-1, IgG+ and IgM+ cells) was observed at days 1 and 3 in the ME cavity and rapidly disappeared by day 7. Within the ME mucosa, a large number of cells was observed at days 1 and 3, peaking on day 7 when a submucosal lymphoid infiltration was detected. In the immune response of the ME mucosa, Mac-1 cells were the predominant cell type followed by helper T cells, IgG+ cells, IgA+ cells and then IgM+ cells. Suppressor T cells were rarely detected after KLH challenge. SC was present within ME epithelial cells from days 1 to 14.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Central auditory metabolic activity induced by intense noise exposure.

Neural activity in the central auditory system was mapped by measuring 2-deoxyglucose (2-DG) uptake during a one hour exposure to a two-octave (1414-5656 Hz) band of noise. Gerbils were exposed to 100, 110 or 120 dB SPL, intensities which can produce only temporary (100 dB) or both temporary and permanent (120 dB) hearing loss. Exposure to 100 dB SPL evoked high levels of neural activity throughout responsive regions of auditory nuclei. At 110 dB SPL, a central region of low neural activity was surrounded by areas exhibiting increased activity. At 120 dB SPL, neural activity was low in almost all areas of auditory nuclei. To study the effects of permanent hearing loss on auditory neuronal activity, other animals were given 2-DG during exposure to 65 dB SPL broad band noise as a test stimulus, two months after exposure to the noise band at 110 dB SPL. Central auditory nuclei showed a tonotopic region of low neural activity corresponding to an approximately 3 kHz pure tone, surrounded by regions of evoked activity. The deficits in evoked metabolic activity observed both during and long after noise exposure appear to exceed those predicted from the degree of temporary and permanent threshold shift produced by the same noise exposures.

Acoustic Stimulation↗
PubMed1992

Cochlear degeneration in aged rats of four strains.

Animals with various degrees of inbreeding, some of which are albino, are frequently used in biological research. Albinos do not produce melanin and it is therefore absent from the cochlea. While the function of melanin is unknown, it has been hypothesized that it is involved in cochlear homeostasis. It is possible then, that age-related degeneration may be affected by the presence or absence of melanin. We therefore evaluated young (2-6 months old) and aged (24-36 months old) cochleas in 4 different rat strains: albino Fischer 344 and Lewis rats and pigmented Lewis-Brown Norway F1 rats and Brown Norway rats. Cochlear morphology was the same across all strains of young adult animals with the exception that the pigmented animals had small, darkly stained granules in the stria vascularis. The aged pigmented animals all had large granules as well as small ones. Degeneration of spiral ganglion cells in the apical region of the ganglion had occurred in the old animals of all strains. Strial degeneration at the apex was also present in aged animals. There was no correlation between the presence or absence of melanin and the magnitude of cochlear degenerative changes in the aged animals. The presence or absence of melanin therefore, appears to have no effect on cochlear degeneration in the aged rat cochlea.

Aging↗
PubMed1992

Transgenic mice. Current applications to the study of the auditory and vestibular systems.

Understanding the variety of genetic disorders affecting the inner ear demands that we first comprehend the molecular mechanisms involved in its development. Mechanisms of development can best be understood by the study of mutant organisms. Progress in the study of mammalian development has been hampered by the difficulty inherent in the identification and isolation of mutated genes. Transgenic technology provides a direct experimental approach to the study of the control and tissue specificity of gene expression. Additionally, this technology allows new and exciting methods of human disease modeling. A by-product of this technology is the creation of insertional mutants, which provide a unique approach toward the identification of genes involved in a variety of developmental processes including those of hearing and balance.

Animals↗
PubMed1991

Enkephalin mRNA production by cochlear and vestibular efferent neurons in the gerbil brainstem.

Preproenkephalin mRNA production by efferent neurons projecting to the gerbil inner ear was assessed using combined in situ hybridization and retrograde labeling with fluorescent tracers. Virtually all vestibular efferent neurons were positive for preproenkephalin mRNA. Of the cochlear efferents, one-half of the medial olivocochlear neurons were positive for enkephalin. All lateral olivocochlear neurons were negative for enkephalin. The results suggest that there are two, biochemically distinct subpopulations of medial olivocochlear efferents in the gerbil.

Animals↗
PubMed1991

Preservation of mRNA during in situ hybridization in the cochlea.

Specific nucleic acid sequences can be identified within cells using in situ hybridization. Hybridization for mRNA can document the distribution and amount of specific gene transcripts. Decalcification protocols used for immunohistochemistry in the cochlea were evaluated for use with in situ mRNA hybridization. No loss of mRNA was detected following the use of decalcification solutions at 4 degrees C when paraformaldehyde was added to the EDTA solution. The primary determinant of mRNA preservation was paraformaldehyde.

Animals↗
PubMed1991

Occurrence and distribution of non-NMDA glutamate receptor mRNAs in the cochlea.

The expression of mRNAs encoding five putative non-NMDA glutamate receptors was investigated using in situ hybridization with radiolabeled riboprobes. Hybridization was observed in spiral ganglion neurons with probes complementary to mRNA products of the glutamate receptor genes GluR2 and GluR3. No specific hybridization was observed with probes for GluR1, GluR4 or GluR5. The results support the hypothesis that glutamate is the transmitter between cochlear inner hair cells and spiral ganglion neurons, and that it acts via non-NMDA glutamate receptors.

Cochlea↗