Cochlear delivery of fibroblast growth factor 1 and its effects on apoptosis and cell cycling in noise-exposed guinea pig ears.
ABSTRACT Acidic fibroblast growth factor 1 (FGF-1) is a mitogen and antiapoptotic factor synthesized by cochlear neurons and transported to the organ of Corti. The objectives of this investigation were threefold: (1) to develop an animal model to study the cochlear effects of intratympanic delivery of FGF-1; (2) to determine the distribution, in the mature mammalian cochlea, of FGF-1 and the receptor, FGFR3, to which it binds with high affinity; and (3) to examine the effect of exogenous FGF-1 on cochlear apoptotic and cell-cycling markers in noise and non-noise-exposed guinea pigs ears.
Fifteen adult Hartley guinea pigs were divided into three groups. Group 1 animals (n = 5) underwent direct placement of FGF-1 in phosphate buffered saline (PBS) (20 pg/mL) soaked Gelfoam pledgets to the right round window membrane. Phosphate buffered saline-soaked Gelfoam pledgets were placed on the left round window membrane as a control. In group 2 animals (n = 5), surgical placement of either FGF-1 or PBS was followed by exposure to 120 dB of white noise for 2 hours. Group 3 animals (n = 5) were subjected to identical noise conditions prior to undergoing round window application of either FGF-1 or PBS. All groups were allowed to recover in a noise-controlled environment for 12 hours following surgery. Anti-FGF-1-stained Western blots and optical densitometry analyses were used to quantitate passage of FGF-1 into cochlear perilymph. Standard in situ immunohistochemical techniques were used to stain each cochlea for FGF-1 and FGFR3, apoptotic markers p53 and p21, Bcl-2, and the cell-cycling marker proliferating cell nuclear antigen (PCNA). Tissue sections were subjected to the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling technique (TUNEL) for apoptosis.
Western blot and optical densitometry analyses of cochlear perilymph showed increased concentrations of FGF-1 in 10 of 14 experimental cochleas. Cochlear perilymph FGF-1 was consistently bound to heparan sulphate proteoglycan (HSPG). Immunoreactivity of both FGF-1 and FGFR3 was observed in spiral ganglion neurons, inner and outer hair cells, pillar cells, and Dieter and Hensen's cells. Specific FGF-1 immunostaining to the distal portion of the pars pectinata of the basilar membrane was noted in noise-exposed animals only. Bcl-2 and PCNA immunostaining was not detected in any group. There was no significant nuclear immunoreactivity to proapoptotic markers, p53 and p21, in any group. Semiquantitative analysis of TUNEL staining in block sections of all cochleas demonstrated a 340% increase in nuclear immunoreactivity of noise-exposed outer hair cells and organ of Corti cells. There was no difference between FGF-1 treated and control ears subjected to TUNEL staining.
Exogenous FGF-1 crosses the round window membrane and is bound to HSPG in cochlear perilymph. The specific immunoreactivity of the pars pectinata to FGF-1 may represent a unique reservoir for cochlear FGF-1 in noise-exposed ears of the guinea pig. Noise induces apoptosis of organ of Corti cells as demonstrated with the TUNEL technique. PCNA, Bcl-2, p53, and p21 in noise-exposed and non-noise-exposed guinea pig cochleas are not affected by exogenous FGF-1. Noise-induced hair cell apoptosis appears to be independent of the p53 pathway. Lack of immunoreactivity to Bcl-2 supports the concept that the apoptotic mechanism is likely to involve C-Jun-N-terminal kinase- or caspase-dependent pathways. Exogenous FGF-1 does not alter apoptosis or cell cycling in the mature guinea pig cochlea within 12 hours of acute acoustic trauma.
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ABSTRACT: Recent research has shown the essential role of reduced blood flow and free radical formation in the cochlea in noise-induced hearing loss (NIHL). The amount, distribution, and time course of free radical formation have been defined, including a clinically significant late formation 7-10 days following noise exposure, and one mechanism underlying noise-induced reduction in cochlear blood flow has finally been identified. These new insights have led to the formulation of new hypotheses regarding the molecular mechanisms of NIHL; and, from these, we have identified interventions that prevent NIHL, even with treatment onset delayed up to 3 days post-noise. It is essential to now assess the additive effects of agents intervening at different points in the cell death pathway to optimize treatment efficacy. Finding safe and effective interventions that attenuate NIHL will provide a compelling scientific rationale to justify human trials to eliminate this single major cause of acquired hearing loss.Hearing Research 05/2007; 226(1-2):22-43. · 2.54 Impact Factor
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ABSTRACT: ZielLrmbelastungen in der Freizeit und dadurch bedingte Hrschden haben bei Kindern, Jugendlichen und jungen Erwachsenen in den letzten Jahren deutlich zugenommen. Meist handelt es sich um Innenohrschden nach Knalltraumata mit Spielzeugpistolen und Impulsschallbelastungen mit Rockmusik. Das Ziel der vorliegenden Arbeit war, die initialen und permanenten funktionellen und morphologischen Schden nach solchen Schalltraumata erstmals systematisch an Meerschweinchen zu erarbeiten.MethodenDie Knalltraumata haben wir mit Spielzeugpistolen (163 bis 188dB[lin], 8 Schsse, 1/min, 10cm Abstand zum linken Ohr, n=25) erzeugt, fr die Impulsschallbelastungen verwendeten wir Rockmusik (Mittelungspegel 106dB[A], 22,5h, n=17). Zum Vergleich wurden auch Schallbelastungen mit einem Breitbandrauschen (115dB[A], 22,5h, n=18) durchgefhrt. Die Hrschwellen haben wir mit der Hirnstammaudiometrie (f-BERA) bei 1,5; 2; 3; 4; 6; 8; 12 und 16kHz vor und unmittelbar nach den Schallexpositionen sowie am 1., 2., 3., 5., 7. und 21.Tag bestimmt. Zudem wurden Zytokochleogramme mit Hilfe der Fluoreszenzmikroskopie erstellt, um die Haarzellschden in 8 Frequenzregionen (<0,4kHz; 0,4–0,8kHz; 0,8–1,5kHz; 1,5–3 kHz; 3–5 kHz; 5–11,5kHz, 11,5–26kHz und >26kHz) unmittelbar nach den Schallbelastungen und am 1., 7. und 21.Tag zu quantifizieren.Ergebnisse Nach den Knalltraumata waren jeweils 55% der ueren Haarzellen (HZ) in allen drei Reihen und 15–18% der inneren Haarzellen (IHZ) in der 3–5 kHz-Region irreversibel geschdigt. Bis zum 3. Tag kam es zu einer partiellen Erholung des Gehrs, danach persisierte ein permanenter maximaler Hrverlust um 32–34 dB bei 3–4 kHz. Nach den Beschallungen mit Breitbandrauschen kam es nur innerhalb von 24 Stunden zu einer partiellen Hrerholung, danach verblieb ein permanenter Hrverlust um 24–32 dB bei 1,5–8 kHz, ohne dass in diesem Frequenzbereich morphologische Schden sichtbar waren. Auch nach den Impuls-Schallbelastungen mit Rockmusik kam es nur innerhalb von 24 Stunden zu einer partiellen Hrerholung, danach verblieb ein permanenter Hrverlust um 28–42 dB bei 6–16 kHz, wiederum ohne morphologische Schden. Alle Versuchsergebnisse waren hochsignifikant (p<0,001).SchlussfolgerungenAb dem 3.Tag nach einem Knalltrauma und ab dem 1.Tag nach den Beschallungen mit Breitbandrauschen oder Rockmusik kam es zu keiner weiteren spontanen Erholung des Gehrs. Den permanenten Hrverlusten nach Schallbelastungen mit Breitbandrauschen und Rockmusik liegen keine morphologisch sichtbaren Zilien- und Haarzellschden zugrunde, was auf neue Aspekte permanenter zellulrer Funktionsstrungen im Hrsystem deutet.The initial and permanent effects of leisure noise (toy pistols, rock music) compared to broadband noise were examined in 68 guinea pigs. Auditory threshold shifts at 1.5, 2, 3, 4, 6, 8, 12 und 16kHz were registered before and immediately after exposure as well as on days 1, 2, 3, 5,7 and 21 post-exposure using the auditory brain stem response (ABR) technique. In order to examine cilia and hair cell damage in eight cochlear frequency regions (<0,4kHz, 0,4–0,8kHz, 0,8–1.5kHz, 1.5–3 kHz, 3–5 kHz, 5–11.5kHz, 11.5–26kHz und >26kHz), cytocochleograms were performed immediately after exposure and on days1, 7 and 21.Frequency dependent functional or morphological damage was found which depended on the type of trauma tested. All results were highly significant (P<0.001).The results show that partial recovery of hearing occurred within 3days of acute acoustic trauma induced by toy pistols and within 1day after exposure to rock music or broadband noise. There was no further recovery of hearing within the following 18 and 20days, respectively. Furthermore, permanent threshold shifts after exposure to rock music or broadband noise were not associated with cilia and/or hair cell damage.HNO 03/2004; 52(4):301-310. · 0.42 Impact Factor
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ABSTRACT: Acoustic trauma, one of the leading causes of sensorineural hearing loss, induces sensory hair cell damage in the cochlea. Identifying the molecular mechanisms involved in regulating sensory hair cell death is critical towards developing effective treatments for preventing hair cell damage. Recently, microRNAs (miRNAs) have been shown to participate in the regulatory mechanisms of inner ear development and homeostasis. However, their involvement in cochlear sensory cell degeneration following acoustic trauma is unknown. Here, we profiled the expression pattern of miRNAs in the cochlear sensory epithelium, defined miRNA responses to acoustic overstimulation, and explored potential mRNA targets of miRNAs that may be responsible for the stress responses of the cochlea. Expression analysis of miRNAs in the cochlear sensory epithelium revealed constitutive expression of 176 miRNAs, many of which have not been previously reported in cochlear tissue. Exposure to intense noise caused significant threshold shift and apoptotic activity in the cochleae. Gene expression analysis of noise-traumatized cochleae revealed time-dependent transcriptional changes in the expression of miRNAs. Target prediction analysis revealed potential target genes of the significantly downregulated miRNAs, many of which had cell death- and apoptosis-related functions. Verification of the predicted targets revealed a significant upregulation of a target of miRNA-183. Moreover, inhibition of miR-183 with morpholino antisense oligos in cochlear organotypic cultures revealed a negative correlation between the expression levels of miR-183 and suggesting the presence of a miR-183/ target pair. Together, miRNA profiling as well as the target analysis and validation suggest the involvement of miRNAs in the regulation of the degenerative process of the cochlea following acoustic overstimulation. The miR-183/ target pair is likely to play a role in this regulatory process.PLoS ONE 01/2013; 8(3):e58471. · 3.73 Impact Factor