Biomedical subjects

Allen F Ryan

Publications and source records attributed to Allen F Ryan.

32 records · Page 2Linked to original sources

PubMed2003

Clostridium difficile toxin B, an inhibitor of the small GTPases Rho, Rac and Cdc42, influences spiral ganglion neurite outgrowth.

OBJECTIVE: Neurotrophins and extracellular matrix (ECM) molecules are involved in neurite guidance during the development of spiral ganglion (SG) neurons. Several intracellular signaling molecules can be activated by ECMs and neurotrophins via their cognate receptors. In other systems these include the Rho small GTPases, which influence reorganization of the actin cytoskeleton that is required for axon growth. The aim of this study was to determine whether neurotrophin-3 (NT-3)-mediated SG neurite outgrowth on laminin-1 (LN) is dependent on the activation of the small GTPases Rho/Rac/Cdc42. MATERIAL AND METHODS: SG explants from postnatal day 4 rats were cultured on LN with and without NT-3 and increasing concentrations of Clostridium difficile Toxin B, an inhibitor of Rho GTPases. After fixation and immunocytochemical labeling, neurite growth was evaluated. RESULTS: Treatment with C. difficile Toxin B without NT-3 led to a dose-dependent decrease in the length and number of processes on LN. In contrast, C. difficile Toxin B had no significant effect on NT-3-mediated stimulation of neurite growth on LN, in terms of either number or length. CONCLUSION: The results suggest that the Rho GTPases (Rho, Rac and Cdc42) are not involved in the pathways linking NT-3 signals to neurite outgrowth, but appear to be involved in LN signaling in these neurons. However, NT-3 can override or bypass LN signaling to promote neurite extension.

Animals↗

PubMed2003

Gentamicin-induced hair cell death is not dependent on the apoptosis receptor Fas.

OBJECTIVES/HYPOTHESIS: The hair cells are the most vulnerable elements in the cochlea, and damage to them is the most common cause of sensorineural hearing loss. Understanding the intracellular events that lead to the death of hair cells is a key to developing protective strategies. The Fas death receptor-mediated apoptotic pathway is well studied and plays an important role in the elimination of damaged cells in a number of different cellular systems. We have studied the role of the Fas receptor in aminoglycoside-mediated toxicity in vitro. We employed the MRL/MpJ-Fas mouse, which does not express a functional Fas receptor. STUDY DESIGN: Response of Fas-deficient hair cells to gentamicin was compared with the response of normal hair cells in vitro. METHODS: Basal turn organ of Corti explants from p3-5 mice were maintained in tissue culture and treated with gentamicin for 72 hours. The explants were fixed and were stained with phalloidin, and counting was performed. RESULTS: There was no difference in hair cell loss between Fas mutant mice and control MRL/MpJ mice with a functional Fas receptor. CONCLUSION: The gentamicin-mediated hair cell death is not dependent on a functional Fas receptor.

Animals↗

PubMed2003

Branching of spiral ganglion neurites is induced by focal application of fibroblast growth factor-1.

OBJECTIVES/HYPOTHESIS: During the terminal innervation of the developing organ of Corti, fibroblast growth factor-1 (FGF-1) messenger RNA has been shown to be transiently expressed in the sensory epithelium of the rat, suggesting that this growth factor may contribute to developmental processes such as innervation and synaptogenesis of the inner and outer hair cells. In a previous study it has been demonstrated that exogenous FGF-1 supports rat spiral ganglion neurite extension in vitro, whereas a secreted form of FGF-1 produced by transfected fibrocytes induces neurite branching and targeting. STUDY DESIGN: Response of spiral ganglion neurites to FGF-1-coupled beads was compared with the response to noncoupled control beads. METHODS: Effects of multiple focal sources of FGF-1 to outgrowing spiral ganglion neurites were investigated on explants from postnatal day 4 rat spiral ganglion samples that were cultured in the presence of FGF-1 covalently coupled to polybead microspheres. After fixation and immunocytochemical labeling of the explants the growth patterns of the extending neuronal processes were evaluated. RESULTS: When spiral ganglion neurites were observed near clusters of FGF-1 beads, they formed a plexus-like network characterized by significantly higher branching in the vicinity of the beads. However, fibers did not appear to terminate on the beads. Plexus-like formations were not seen at a distance from FGF-1 coupled beads or in the vicinity of control beads lacking FGF-1 on their surface. CONCLUSION: The results of the study indicate that spiral ganglion neurites branch in response to focal sources of FGF-1, suggesting an important role of this growth factor for hair cell innervation during the terminal development of the sensory epithelium.

Animals↗

PubMed2002

Participation of Ras and extracellular regulated kinase in the hyperplastic response of middle-ear mucosa during bacterial otitis media.

Hyperplasia of middle-ear mucosa (MEM) during otitis media (OM) is thought to be partially mediated by the actions of growth factors and their receptors. The intracellular pathway leading from the small G-protein Ras to the extracellular regulated kinases (Erks) often links growth factor stimulation to cellular proliferation. This study assessed whether this pathway is involved in MEM hyperplasia during bacterial OM via the activation of Erk1/Erk2 in MEM of an in vivo rat bacterial OM model. Activation was maximal at 1 and 6 h and at 1 week after introduction of bacteria into the middle ear. Additionally, an in vitro model of rat MEM in bacterial OM was treated with farnesyl transferase inhibitor 277 or the Mek inhibitor U0126. MEM explants treated with either inhibitor demonstrated significant suppression of bacterially induced growth. These data support a role for Ras and Erk signaling in MEM hyperplasia during bacterial OM.

Animals↗

PubMed2002

Inflammatory signals increase Fas ligand expression by inner ear cells.

There is considerable evidence that hearing and vestibular function can be influenced by immune processes. The inner ear has evolved mechanisms, such as the blood-labyrinthine barrier that limit immune responses and autoimmune processes to reduce the potential for damage to cochlear cells. Recently, expression of Fas ligand (FasL) in some non-lymphoid tissue, as in the anterior chamber of the eye, has been hypothesized to play a role in protection of sensitive organs from activated T-cells. We show that under resting conditions, cochlear cells express little or no FasL. However, after exposure to interferon-gamma in vitro, FasL is induced in many neonatal cochlear cells. In addition, we show that FasL is upregulated in adult cochlear cells after induction of a sterile labyrinthitis in vivo. The induction of FasL by inflammation may serve to limit cochlear immune responses, and to protect sensorineural tissue from immune and autoimmune damage.

Animals↗

PubMed2002

The effects of laminin-1 on spiral ganglion neurons are dependent on the MEK/ERK signaling pathway and are partially independent of Ras.

Laminin-1 (LN) is expressed along the route of neural growth from spiral ganglion (SG) neurons towards the developing organ of Corti, and has been shown to enhance neurite outgrowth from SG neurons in vitro. Signal transduction pathways linking LN signaling at the cell membrane to the cell nucleus can involve a variety of signaling molecules. Data from other systems suggest the potential involvement of the small G protein Ras, and the mitogen-activated protein kinases (MAPKs) Erk and/or p38. To assess these possibilities, the length and number of processes extending from SG explants cultured on LN-coated surfaces were evaluated after treatment with the Ras inhibitor FTI-277, the p38 inhibitor SB203580 and MAPK kinase (MEK) inhibitor U0126, which operates immediately upstream of the Erk MAPK. Treatment with the Ras inhibitor at levels known to inhibit the H- and N-Ras isoforms had no effect, while FTI-277 levels known to inhibit K-Ras reduced only neurite length. Suppression of MEK resulted in a decrease of both parameters, while incubation with the p38 inhibitor had no effect. The results of this study suggest that MEK plays a central role in LN signaling in SG neurites. While K-Ras signaling may participate in MEK-dependent increases in neurite length, the MEK-dependent increase in neurite number appears to be activated by a different intracellular pathway.

Animals↗

PubMed2002

Interaction of spiral ganglion neuron processes with alloplastic materials in vitro(1).

The cochlear implant (CI) involves the introduction of alloplastic materials into the cochlea. While current implants interact with cochlear neurons at a distance, direct interactions between spiral ganglion (SG) neurites and implants could be fostered by appropriate treatment with neurotrophic factors. The interactions of fibroblasts and osteoblasts with alloplastic materials have been well studied in vitro and in vivo. However, interactions of inner ear neurons with such alloplastic materials have yet to be described. To investigate survival and growth behavior of SG neurons on different materials, SG explants from post-natal day 5 rat SG were cultured for 72 h in the presence of neurotrophin-3 (10 ng/ml) on titanium, gold, stainless steel, platinum, silicone and plastic surfaces that had been coated with laminin and poly-L-lysine. Neurite outgrowth was investigated after immunohistological staining for neurofilament, by image analysis to determine neurite extension and directional changes. Neurite morphology and adhesion to the alloplastic material were also evaluated by scanning electron microscopy (SEM). On titanium, SG neurites reached the highest extent of outgrowth, with an average length of 662 microm and a mean of 31 neurites per explant, compared to 568 microm and 21 neurites on gold, 574 microm and 24 neurites on stainless steel, 509 microm and 16 neurites on platinum, 281 microm and 12 neurites on silicone and 483 microm and 31 neurites on plastic. SEM revealed details of adhesion of neurites and interaction with non-neuronal cells. The results of this study indicate that the growth of SG neurons in vitro is strongly influenced by alloplastic materials, with titanium exhibiting the highest degree of biocompatibility with respect to neurite extension. The knowledge of neurite interaction with different alloplastic materials is of clinical interest, as development in CI technology leads to closer contact of implanted electrodes with surviving inner ear neurons.

Animals↗

PubMed2002

Immune-mediated hearing loss: basic mechanisms and options for therapy.

Immune responses are an important component of the defense of the inner ear against infection. However, immunity appears to be restricted in the labyrinth, as the inflammation associated with immune responses can damage the delicate cellular structures that mediate hearing and balance. When immunoregulation is compromised, either experimentally or through disease, immune-mediated inner ear damage and hearing loss can result. Because there are few resident leukocytes within the labyrinth, immunity and inflammation are mediated primarily by cells that enter the inner ear following the activation of post-capillary venules, which in the cochlea are the spiral modiolar veins. Activated lymphocytes are an important component of the infiltrating leukocytes, and their interactions with antigen mediate inner ear immunity. While evidence of autoimmunity specific to the inner ear is incomplete, an autoimmune origin is often suspected in idiopathic, bilateral, rapidly progressive hearing loss. Systemic immunosuppressive drugs can be effective in reversing such hearing loss, although at the cost of occasionally severe side-effects. Experimental evidence suggests that local therapy may be effective in treating this condition, if it were to target the infiltration of leukocytes into the labyrinth.

Animals↗

PubMed2002

Growth factors and their receptors in the middle ear mucosa during otitis media.

OBJECTIVE: The hyperplastic response of the middle ear mucosa during bacterial otitis media is thought to be mediated by the actions of growth factors and their respective receptors. The purpose of the study was to explore the expression of growth factors known to stimulate epithelial cells in other systems, as well as their receptors, in the middle ear mucosa during otitis media. STUDY DESIGN: Expression of mRNA growth factors and receptors was measured over time after inoculation of the rat middle ear with bacteria. METHODS: The middle ears of 12 male Sprague-Dawley rats were injected with 10(5)/mL Haemophilus influenzae strain 3655 (nontypeable, biotype II). Three rats were killed at 6, 24, 48, and 72 hours. Three untreated rats were also killed to serve as negative controls. The middle ear mucosa samples were surgically removed and homogenized. Reverse transcription-polymerase chain reaction was performed on each sample with primers for rat epidermal growth factor, epidermal growth factor receptor (ErbB), heparin binding epidermal-like growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, keratinocyte growth factor, betacellulin, amphiregulin, and neuregulin-alpha. RESULTS: Hepatocyte growth factor and epidermal growth factor receptor primers demonstrated polymerase chain reaction products of the expected size that were not displayed in the normal middle ear mucosa. Keratinocyte growth factor and hepatocyte growth factor receptor demonstrated polymerase chain reaction products at all time points tested. Betacellulin and neuregulin-alpha products were present at all time points except 72 hours after infection. CONCLUSIONS: The results of the study support a role for growth factors in the middle ear mucosa during otitis media. These bioactive ingredients contribute to mucosal hyperplasia.

Animals↗

PubMed2002

Inhibition of the c-Jun N-terminal kinase signaling pathway influences neurite outgrowth of spiral ganglion neurons in vitro.

OBJECTIVES: Inhibitors of the c-Jun N-terminal kinase (JNK) signaling pathway have been demonstrated to protect hair cells of the auditory system and different types of neurons from various insults, and their use for future therapeutic applications has been proposed. In the study, we evaluated the effects of inhibition of the JNK pathway on process outgrowth from spiral ganglion neurons. METHODS: Spiral ganglion explants from rats (postnatal days 3-5) that were cultured on laminin were treated with neurotrophin-3 and/or the JNK signaling pathway inhibitor CEP-11004. Both neurite length and number of the explants were evaluated and statistically analyzed by analysis of variance. RESULTS: Inhibition of the JNK signaling pathway reduced process outgrowth from spiral ganglion explants. The reduction, both in length and number of neurites, was reversed by the application of neurotrophin-3. CONCLUSIONS: The results indicate that an intact JNK signaling pathway is important for process outgrowth of spiral ganglion neurons. However, neurotrophin-3 stimulates process extension by a JNK independent pathway. Our results demonstrate that inhibition of the JNK pathway can have adverse effects on the extension of spiral ganglion neurons, but that the negative effects can be ameliorated by appropriate treatment.

Analysis of Variance↗

PubMed2002

Immunological damage to the inner ear: current and future therapeutic strategies.

There is considerable evidence to suggest that hearing and vestibular function can be influenced by immunity in the inner ear. While immunity can protect against infections of the labyrinth, immune response also has the capacity to damage the delicate tissues of the inner ear. Antigenic challenge of the inner ear of sensitized animals leads to rapid accumulation of leukocytes, antibody production, hearing loss and tissue damage. Moreover, a number of systemic autoimmune disorders include hearing loss and vertigo as part of their constellation of symptoms. It also appears that autoimmune damage can exist as an entity confined to the labyrinth. Immune disorders of the inner ear are of special interest since they are among the few forms of hearing loss that are currently amenable to medical treatment. In addition, recent developments in understanding the intracellular pathways that participate in damage to the inner ear provide new opportunities for pharmacotherapy of immune-mediated disorders of hearing and balance.

Animals↗

PubMedDate not supplied

Molecular studies of hair cell development and survival.

The development of hair cells in both the auditory and vestibular sensory epithelia is a complex process that involves the coordinated expression of many regulatory proteins. Among these is the POU-domain transcription factor Brn-3.1. This factor is expressed in hair cell precursors immediately after commitment to the hair cell fate, and continues throughout life. Deletion of this factor in mice leads to failure of hair cell differentiation during development, and to the death of a majority of the undifferentiated cells. Normal expression of Brn-3.1 is required for adult hair cell survival as well, since a mutation in this gene causes dominant, late-onset, inherited hearing loss in humans. The timing of the onset of Brn-3.1 expression suggests that factors regulating its expression may be involved in fate determination of hair cells. Moreover, genes that are themselves directly regulated by Brn-3.1 appear to play critical roles in hair cell development and survival.

Animals↗

PubMedDate not supplied

Induction of MHC class II antigens on cells of the inner ear.

Growing evidence supports the concept that immune reactions occur in the cochlea, where they can function either in protection or as a source of inflammation. Since immunity is generally initiated by antigen presentation of foreign substances to T cells, antigen-presenting cells expressing major histocompatibility complex (MHC) class II molecules are required. Under resting conditions, cochlear cells usually express no MHC class II. However, we show that exposure to -interferon in vitro induces an increase in MHC class II expression in neonatal cochlear cells of mice. In addition, MHC class II immunoreactivity was observed in the inner ear of adult mice after induction of sterile labyrinthitis in vivo. It is concluded that the induction of MHC class II molecules by inflammation may render cochlear cells competent to initiate and participate in immune reactions and may therefore contribute to both immunoprotective and immunopathological responses of the inner ear.

Animals↗

PubMedDate not supplied

Fas ligand expression in the organ of Corti.

We have previously demonstrated by FACS analysis and histochemistry that Fas ligand (FasL) increases on cochlear cell surfaces after immune response or stimulation with gamma-interferon (IFN-gamma). To determine whether the appearance of FasL on cochlear cell membranes is related to gene expression or to posttranslational events, cochlear cells were treated with IFN-gamma. They were evaluated for FasL gene expression by real-time PCR and for FasL protein localization by confocal microscopy of permeabilized and immunolabeled cells. Real-time PCR analysis of cDNAs generated from unstimulated or IFN-gamma-stimulated organ of Corti demonstrated no change in the transcription of the gene encoding FasL. In contrast, confocal microscopy revealed dramatic changes in the cellular distribution of FasL, consistent with movement from the endoplasmic reticulum to the cytoplasm and cell membrane. The results suggest that recruitment of preformed FasL from intracellular compartments, rather than its biosynthesis, is responsible for the increase in FasL on the cell surface following IFN-gamma stimulation. This is similar to the response of cytotoxic T lymphocytes in which gene expression is not involved in FasL surface appearance. Presumably, the use of preformed FasL increases the rapidity of this response. FasL localization to the membrane may be involved in protecting the inner ear from autoimmunity or inflammation. Alternatively it may be related to cochlear cell death in response to inflammatory stress.

Actins↗