[Apoptosis and apoptosis-related genes in experimental autoimmune inner ear disease].

OBJECTIVE: To investigate the protein and mRNA expression patterns of apoptosis-related genes, together with evidence of apoptosis, in relation to experimental autoimmune inner ear disease (AIED). METHODS: Male C57BL/6 mice at 4 weeks age (n = 80) were randomly assigned to one of the five group (n = 16). The inbred mice were given a single subcutaneous injection of diluted solution of pertussis and an emulsion containing equal parts of complete Freund adjuvant (CFA) and inner ear antigens (IEAg) extracted form guinea pig. The animals were sacrificed for inner ear examination at a defined time after the immunization (7, 14, 21 or 28 days). An autoimmune inner ear diseases model was established. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP) nick end-laying (TUNEL) method. Using immunohistochemical techniques and reverse transcriptase polymerase chain reaction to clarify the profile of Fas, FasL, and bcl-2. RESULTS: Under normal conditions, no TUNEL-positive cell was observed in the cochlea except for a few positive cells in the supporting cells of Corti's organ and macula sacculi. Inner ear antigens administration induced TUNEL-positive reactions in a wide variety of cells such as inner hair cells, supporting cells, stria vascularis and spiral ligament fibrocytes. No positive staining was evident in outer hair cells, spiral ganglion cells and Scarpa's ganglion cells during the whole period. Fas proteins were expressed in a wide range of cells in inner ear. The levels of Fas mRNA were no significant differences between normal and AIED mice. FasL and bcl-2 proteins could be detected in spiral ganglion cells and Scarpa's ganglion cells both in normal and AIED mice. FasL positive cells increased in number in inner ear of AIED mice. bcl-2 positive cells were not detectable in inner hair cells, stria vascularis and spiral ligament both in normal and AIED mice. The mRNA of three kinds of apoptosis-related genes was detectable in the normal and AIED mice. FasL mRNA was expressed at low levels in normal, being maximal at 14 d post inoculation and decreased gradually to steady levels by 2 weeks. The levels of bcl-2 mRNA increased significantly during the period of AIED. CONCLUSION: Apoptosis mediated by Fas/FasL signal system may play a role in the initiation and maintenance of AIED. bcl-2 has a crucial role in the regulation of the process of apoptosis in the inner ear of AIED mice.

[An improved animal model of autoimmune inner ear disease].

OBJECTIVE: To establish an experimental autoimmune inner ear disease model, which could exhibit high reproducibility and be adopted for detailed immunological analysis. METHODS: Extraction of guinea pig inner ear antigens (IEAg). The inbred mice were given a single subcutaneous injection of diluted solution of pertussis and an emulsion containing equal parts of CFA and IEAg. The ABR threshold shifts were evaluated. The antibody level to IEAg in serum was detected by ELISA. Inner ear specimen were examined by light microscopy with hematoxylin and eosin staining. The infiltrated cells within cochlea were clarified with immunohistochemical techniques. RESULTS: The ABR thresholds of IEAg-sensitized animals were elevated significantly. Histological changes in cochlea were significant. Inflammatory cell infiltration was clearly observed in the cochlea of the animals following sensitization with IEAg. Degeneration of the spiral ganglion cells, which characterizes a decrease in cell numbers, and formation of endolymphatic hydrops were often seen too. Serum anti-IEAg levels after inoculation were significantly increased in the IEAg sensitised groups. Most of the infiltrated lymphocytes in scala tympani were CD4+ T cells. CONCLUSIONS: The experimental autoimmune inner ear disease can be induced by a single inoculation of IEAg-CFA emulsion and pertussis in inbred C57BL/6 mice.