Cochlear delivery of fibroblast growth factor 1 and its effects on apoptosis and cell cycling in noise-exposed guinea pig ears.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
David
Eytan A
EA
Department of Otolaryngology, University of Western Ontario, London, Ontario.
Jackson-Boeters
Linda
L
Daley
Tom
T
MacRae
Duncan L
DL
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology; D003051:Cochlea / Q000378:metabolism* / Q000473:pathology*; D004249:DNA Damage; D005346:Fibroblast Growth Factors / Q000378:metabolism*; D006168:Guinea Pigs; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D020287:In Situ Nick-End Labeling / Q000379:methods; D008297:Male; D009622:Noise / Q000009:adverse effects*; D009925:Organ of Corti / Q000378:metabolism / Q000473:pathology; D010446:Peptide Fragments / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011505:Protein-Tyrosine Kinases; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051498:Receptor, Fibroblast Growth Factor, Type 3; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D019812:Heparan Sulfate Proteoglycans; D010446:Peptide Fragments; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D017468:Receptors, Fibroblast Growth Factor; D016159:Tumor Suppressor Protein p53; D005346:Fibroblast Growth Factors; C052888:fibroblast growth factor (1-10); D011505:Protein-Tyrosine Kinases; D051498:Receptor, Fibroblast Growth Factor, Type 3; D020741:rho GTP-Binding Proteins