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Biomedical subjects

Allen F Ryan

Publications and source records attributed to Allen F Ryan.

At least 19 recordsLinked to original sources
PubMed2006

A PI3K pathway mediates hair cell survival and opposes gentamicin toxicity in neonatal rat organ of Corti.

Gentamicin is well known to promote hair cell death in inner ear, but it also appears to activate opposing pathways that promote hair cell survival. In combination with others, our previous work has indicated that a K-Ras/Rac/JNK pathway is important for hair cell death and an H-Ras/Raf/MEK/Erk pathway is involved in promoting hair cell survival (Battaglia et al., Neuroscience 122(4):1025-1035, 2003). However, these data also suggested that a Ras-independent survival pathway for activation of MEK might be stimulated by gentamicin. To investigate alternatives to the Ras/Raf/MEK/Erk pathway in promoting hair cell survival, cochlear explants were exposed to gentamicin combined with several inhibitors of alternative pathways (LY294002, calphostin C, SH-6, U73122). When exposed to gentamicin with the PI3K inhibitor LY294002 (10, 50 microM), the protein kinase C (PKC) inhibitor calphostin C (50, 100 nM) or the PKB/Akt inhibitor SH-6 (5, 10 microM), hair cell damage was significantly increased compared to gentamicin alone. By Western blotting, strong PKB/Akt activation was observed in the organ of Corti following exposure to 50 microM gentamicin for 6 h. In addition, PKC activation by 12-O-tetradecanoylphorbol-13-acetate protected outer hair cells from gentamicin induced cell death. In contrast, the phospholipase C-gamma (PLCgamma) inhibitor U73122 (2, 5 microM) did not affect hair cell damage when combined with gentamicin. Also, phosphorylation of PLCgamma was not increased in the organ of Corti following gentamicin treatment, as evaluated by Western blot. The results indicate that PI3K promotes hair cell survival via its downstream targets, PKC and PKB/Akt. This suggests that both Ras-dependent and Ras-independent survival pathways are involved during gentamicin exposure. In contrast, PLCgamma activation of PKC does not appear to play a role.

Animals↗
PubMed2006

Mouse models of induced otitis media.

The mouse has seen limited use as a model for experimental otitis media, due primarily to the small size of its middle ear. However, the genetic resources of this species offer substantial potential benefits. These include detailed genomic information, a wealth of genetic models, and gene arrays that represent virtually all mouse genes. This has led to the development of methods for inducing otitis in mice. These include surgical approaches to the middle ear, documentation of the murine middle ear response to various pathogens and inflammatory factors, as well as characterization of induced otitis media in several mouse strains. The results indicate that induced otitis media in the normal mouse is in most respects comparable to that observed in other animal models and in humans. They further suggest that the considerable genetic resources of this species can be harnessed to increase our understanding of this disease.

Animals↗
PubMed2006

Environmental micro-patterning for the study of spiral ganglion neurite guidance.

The projection of neuronal processes is guided by a variety of soluble and insoluble factors, which are sensed by a fiber's growth cone. It is the differential distribution of such guidance cues that determine the direction in which neurites grow. The growth cone senses these cues on a fine scale, using extensible filopodia that range from a few to tens of mum in length. In order to study the effects of guidance cues on spiral ganglion (SG) neurites, we have used methods for distributing both soluble and insoluble cues on a scale appropriate for sensing by growth filopodia. The scale of these methods are at the micro, rather than nano, level to match the sensing range of the growth cone. Microfluidics and transfected cells were used to spatially localize tropic factors within the fluid environment of extending neurites. Micro-patterning was used to present neurites with stripes of insoluble factors. The results indicate that differentially distributed permissive, repulsive and stop signals can control the projection of SG neurites. Implications for future micro-patterning studies, for SG development and for the growth of deafferented SG dendrites toward a cochlear implant are discussed.

Animals↗
PubMed2005

Developmentally regulated expression of the P2X3 receptor in the mouse cochlea.

ATP-gated non-selective cation channels assembled from P2X(3) receptor subunits contribute to transduction and neurotransmitter signaling in peripheral sensory systems and also feature prominently in the development of the central nervous system. In this study, P2X(3) receptor expression was characterized in the mouse cochlea from embryonic day 18 (E18) using confocal immunofluorescence. From E18 to P6, spiral ganglion neuron cell bodies and peripheral neurites projecting to the inner and outer hair cells were labeled. The inner spiral plexus associated with the inner hair cell synapses had a stronger fluorescence signal than outer spiral bundle fibers which provide the afferent innervation to the outer hair cells. Labeling in the cell bodies and peripheral neurites diminished around P6, and was no longer detected after the onset of hearing (P11, P17, adult). In opposition to the axiom that P2X(3) expression is neuron-specific, inner and outer sensory hair cells were labeled in the base and mid turn region at E18, but at P3 only the outer hair cells in the most apical region of the cochlea continued to express the protein. These data suggest a role for P2X(3) receptor-mediated purinergic signaling in cochlear synaptic reorganization, and establishment of neurotransmission, which occurs just prior to the onset of hearing function.

Animals↗
PubMed2005

Role of mast cells in otitis media.

BACKGROUND: New pathophysiologic concepts are needed to explain the clinically observed connection between the allergic diathesis and otitis media. Although mast cells, unlike lymphocytes, are common in the normal middle ear mucosa, their potential role in innate immunity of the middle ear and in the expression of inflammatory responses in that space to bacterial challenge, as opposed to allergy, has received relatively little attention. OBJECTIVE: In the current study, we examine the contributions of mast cells to the pathogenesis of bacterially induced inflammation in the middle ear and thus to otitis media. METHODS: Wild-type mice, mast cell-deficient mice, and mast cell-deficient mice whose mast cell populations were restored by transplantation of bone marrow-derived mast cells were challenged by using models of bacterial and allergic middle ear inflammation. RESULTS: Our results indicate that mast cells account for a substantial proportion of the innate immune response to bacteria in the middle ear. CONCLUSION: This mechanism may link responses to allergy and infection in the middle ear mucosa, and thus the mast cell may be a critical control element in the pathogenesis of otitis media.

Animals↗
PubMed2005

The role of Fas-mediated apoptosis in otitis media: observations in the lpr/lpr mouse.

Apoptosis, or programmed cell death, is a critical regulatory mechanism involved in the function, homeostasis and stimulus response of many organ systems. In the middle ear, apoptosis could participate in mucosal remodeling or leukocyte clearance during otitis media (OM). Fas is a death receptor that can contribute to apoptosis in a variety of cell types. To assess the role of Fas signaling in OM, we probed for expression of Fas and Fas ligand (FasL) by polymerase chain reaction (PCR) during bacterial OM in the rat. In addition, we assessed the response of the middle ear to endotoxin, an inflammatory bacterial product that has been used as a model for otitis media in the mouse, in normal and Fas deficient mice. We saw evidence of increased expression of Fas and Fas ligand during bacterial OM. Moreover, the intensity of the mucosal response to endotoxin was significantly greater and the resolution of the response was prolonged in Fas deficient mice. Prolonged resolution of mucosal hyperplasia may reflect reduced apoptosis of the hyperplastic mucosal cells. Elucidation of the pathways that regulate the mucosal hyperplastic response during otitis media brings us closer to manipulating them in the interest of reducing the chronic complications of this disease.

Animals↗
PubMed2004

A reusable microfluidic plate with alternate-choice architecture for assessing growth preference in tissue culture.

We present the design of a chamber to evaluate in vitro how species and concentrations of soluble molecules control features of cell growth-potentially including cell proliferation, cell motility, process extension, and process termination. We have created a reusable cell culture plate that integrates a microfluidic media delivery network with standard cell culture environment. The microfluidic network delivers a stream of cell culture media with a step-like concentration gradient down a 50-100 microm wide microchannel called the presentation region. Migrating cells or growing cell processes freely choose between the two distinct chemical environments in the presentation region, but they are forced to exclusively choose either one environment or the other when they grow past a physical barrier acting as a decision point. Our fabrication technique requires little specialized equipment, and can be carried out in approximately 4 days per plate. We demonstrate the effectiveness of our plates as neurites from spiral ganglion explants preferentially grow in media containing neurotrophin-3 (NT-3) as opposed to media without NT-3. Our design could be used without modification to study dissociated cell responses to soluble growth cues, and for behavioral screening of small motile organisms.

Animals↗
PubMed2004

Effects of amoxicillin on cytokine and osteocalcin expression in bone tissue during experimental acute otitis media.

There are indications that bone regulatory and immune systems are closely related. Of special interest in this context is the acute otitis media (AOM), which mainly affects immunologically immature patients and, when complicated, involves bone tissue. To explore host responses in bone tissue during Haemophilus influenzae-induced AOM modified by amoxicillin, a rat model and PCR techniques were used. The treatment eradicated the bacteria and induced changes in the expression profile of osteocalcin, a bone formation marker. The maximum levels of osteocalcin transcripts in the treatment group were delayed by about a week. The mRNA levels never reached the same high levels as in the untreated animal group, but the downregulation was slow and entailed higher osteocalcin mRNA levels for a longer time period in the treated animals. The expression of IL-6 and TNF-alpha, two cytokines associated with bone resorption, remained unaffected by the amoxicillin treatment, whereas the downregulation of IL-10, with suppressive effects on bone resorption, was slower than that during the natural course. By comparing the host responses on the molecular level in different tissues during treated and untreated AOM, new approaches to how to minimize the risks of severe AOM complications may evolve.

Amoxicillin↗
PubMed2004

Role of p38 mitogen-activated protein kinase in middle ear mucosa hyperplasia during bacterial otitis media.

Hyperplasia of the middle ear mucosa contributes to the sequelae of acute otitis media. Understanding the signal transduction pathways that mediate hyperplasia could lead to the development of new therapeutic interventions for this disease and its sequelae. Endotoxin derived from bacteria involved in middle ear infection can contribute to the hyperplastic response. The p38 mitogen-activated protein kinase (MAPK) is known to be activated by endotoxin as well as cytokines and other inflammatory mediators that have been documented in otitis media. We assessed the activation of p38 in the middle ear mucosa of an in vivo rat bacterial otitis media model. Strong activity of p38 was observed 1 to 6 h after bacterial inoculation. Activity continued at a lower level for at least 7 days. The effects of p38 activation were assessed using an in vitro model of rat middle ear mucosal hyperplasia in which mucosal growth is stimulated by nontypeable Haemophilus influenzae during acute otitis media. Hyperplastic mucosal explants treated with the p38 alpha and p38 beta inhibitor SB203580 demonstrated significant inhibition of otitis media-stimulated mucosal growth. The results of this study suggest that intracellular signaling via p38 MAPK influences the hyperplastic response of the middle ear mucosa during bacterial otitis media.

Acute Disease↗
PubMed2003

Rapid muscle-specific gene expression changes after a single bout of eccentric contractions in the mouse.

Eccentric contractions (ECs), in which a muscle is forced to lengthen while activated, result in muscle injury and, eventually, muscle strengthening and prevention of further injury. Although the mechanical basis of EC-induced injury has been studied in detail, the biological response of muscle is less well characterized. This study presents the development of a minimally invasive model of EC injury in the mouse, follows the time course of torque recovery after an injurious bout of ECs, and uses Affymetrix microarrays to compare the gene expression profile 48 h after ECs to both isometrically stimulated muscles and contralateral muscles. Torque dropped by approximately 55% immediately after the exercise bout and recovered to initial levels 7 days later. Thirty-six known genes were upregulated after ECs compared with contralateral and isometrically stimulated muscles, including five muscle-specific genes: muscle LIM protein (MLP), muscle ankyrin repeat proteins (MARP1 and -2; also known as cardiac ankyrin repeat protein and Arpp/Ankrd2, respectively), Xin, and myosin binding protein H. The time courses of MLP and MARP expression after the injury bout (determined by quantitative real-time polymerase chain reaction) indicate that these genes are rapidly induced, reaching a peak expression level of 6-11 times contralateral values 12-24 h after the EC bout and returning to baseline within 72 h. Very little gene induction was seen after either isometric activation or passive stretch, indicating that the MLP and MARP genes may play an important and specific role in the biological response of muscle to EC-induced injury.

Animals↗
PubMed2003

EphA4 provides repulsive signals to developing cochlear ganglion neurites mediated through ephrin-B2 and -B3.

The ephrins and Eph receptors make up two large families of bi-directional signaling molecules that are known to play a role in the development of the nervous system. Recently, expression of EphA4 in the developing cochlea was shown, with strong expression in cells lining the osseous spiral lamina (OSL) through which afferent dendrites must pass to reach the organ of Corti (OC). It was also demonstrated that ephrin-B2 and -B3, both of which are known to interact with EphA4, are expressed by spiral ganglion (SG) neurons. To investigate the functional role of EphA4 in the development of inner ear neurons, neonatal rat SG explants were cultured for 72 hours on uniformly coated surfaces or near stripes of EphA4/IgG-Fc-chimera. Control explants were cultured on or near IgG-Fc and EphA1/IgG-Fc-chimera. To assess the roles of ephrin-B2 and -B3 in EphA4 signaling, SG explants were cultured with or without anti-ephrin-B2 and/or -B3 blocking antibodies. Growth patterns of SG neurites at the border of EphA4 receptor stripes showed repulsion, characterized by turning, stopping and/or reversal. In the case of IgG-Fc and EphA1, the neurites grew straight onto the stripes. Treatment with either anti-ephrin-B2 or -B3 blocking antibodies significantly reduced the repulsive effect of an EphA4 stripe. Moreover, when both antibodies were used together, neurites crossed onto EphA4 stripes with no evidence of repulsion. The results suggest that EphA4 provides repulsive signals to SG neurites in the developing cochlea, and that ephrin-B2 and -B3 together mediate this response.

Animals↗
PubMed2003

Noise induces up-regulation of P2X2 receptor subunit of ATP-gated ion channels in the rat cochlea.

Regulation of P2X(2) receptor (P2X(2)R) expression in the rat cochlea in response to noise was analysed. Sustained loud sound (90-120 dB white noise, > 6 h), increased P2X(2)R mRNA and protein levels in rat organ of Corti and spiral ganglion (primary auditory neurones). P2X(2)R expression by the type I spiral ganglion neurones, which innervate the inner hair cells via the inner spiral plexus, was confirmed by confocal immunofluorescence. This also revealed increased P2X(2)R labelling of outer hair cell (OHC) stereocilia and cuticular plates, reflecting trafficking of greater numbers of ATP-gated ion channels assembled with P2X(2)R subunits to the transducer site. Whole-cell voltage clamp of OHC confirmed the noise-induced up-regulation of ATP-gated inward currents. These data indicate that regulation of P2X(2) receptor gene expression in the cochlea is adaptive, with sustained loud sound promoting increased transcription and translation specifically at sites regulating hearing sensitivity and auditory neurotrans-mission.

Adenosine Triphosphate↗
PubMed2003

Contribution of BK Ca2+-activated K+ channels to auditory neurotransmission in the Guinea pig cochlea.

Large-conductance calcium-activated potassium (BK) channels are known to play a prominent role in the hair cell function of lower vertebrates where these channels determine electrical tuning and regulation of neurotransmitter release. Very little is known, by contrast, about the role of BK channels in the mammalian cochlea. In the current study, we perfused specific toxins in the guinea pig cochlea to characterize the role of BK channels in cochlear neurotransmission. Intracochlear perfusion of charybdotoxin (ChTX) or iberiotoxin (IbTX) reversibly reduced the compound action potential (CAP) of the auditory nerve within minutes. The cochlear microphonics (CM at f1 = 8 kHz and f2 = 9.68 kHz) and their distortion product (DPCM at 2f1-f2) were essentially not affected, suggesting that the BK specific toxins do not alter the active cochlear amplification at the outer hair cells (OHCs). We also tested the effects of these toxins on the whole cell voltage-dependent membrane current of isolated guinea pig inner hair cells (IHCs). ChTX and IbTX reversibly reduced a fast outward current (activating above -40 mV, peaking at 0 mV with a mean activation time constant tau ranging between 0.5 and 1 ms). A similar block of a fast outward current was also observed with the extracellular application of barium ions, which we believe permeate through Ca2+ channels and block BK channels. In situ hybridization of Slo antisense riboprobes and immunocytochemistry demonstrated a strong expression of BK channels in IHCs and spiral ganglion and to a lesser extent in OHCs. Overall, our results clearly revealed the importance of BK channels in mammalian cochlear neurotransmission and demonstrated that at the presynaptic level, fast BK channels are a significant component of the repolarizing current of IHCs.

Acoustic Stimulation↗
PubMed2003

A mouse model for acute otitis media.

To induce acute otitis media in the mouse and to describe the clinical and bacteriological course of the infection, middle ears of BALB/c, Swiss-Webster and C57BL/6 mice were inoculated with Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. Systemic and local changes were monitored by clinical observations, otomicroscopy, and analysis of bacterial samples from blood and middle ears. Agglutination of mouse erythrocytes by M. catarrhalis was also tested. Depending on bacterial strain, bacterial dose, and mouse strain three responses were identified: acute otitis media, otitis media with serous effusion, or no reaction. BALB/c mice were the most susceptible animals. On day 3, 76% of the BALB/c mice had developed middle ear infection, 50% had a positive middle ear culture, 56% were bacteremic, and 10% had succumbed to a disseminated infection. The local infections lasted approximately a week. Animals which survived recovered without permanent deterioration or otomicroscopically discernible changes. In no case did M. catarrhalis induce a culture-positive middle ear infection, possibly due to an inability to agglutinate the mouse erythrocytes. The mouse model can become a useful tool in studies of pneumococcal and H. influenzae-induced otitis media, but the bacterial dose has to be carefully titrated and adjusted to the chosen mouse strain.

Acute Disease↗