title: Improved cryosections and specific immunohistochemical methods for detecting hypoxia in mouse and rat cochleae.
abstract: The present study was undertaken to develop an improved cryoembedding method for analysis of mice and rat cochleae, which permits high-quality cryosections and preserves overall structure and cellular resolution as shown by hematoxylin/eosin staining. The preservation of morphology and antigenicity is mandatory to achieve optimal results. A total of 20 male cd/1 mice and 14 male Sprague-Dawley rats were used in experiments for optimization of preservation, fixative, decalcification, embedding and cryosectioning of cochleae from adult and aged rodents. In addition, a novel immunohistochemical procedure (using Hydroxyprobe-1 kit) was developed for detecting regions of hypoxia in mice and rat cochlea. This method employs a primary fluorescent-conjugated monoclonal antibody directed against pimonidazole protein adducts that are created in hypoxic tissues. Subsequent studies of hypoxia inducible factor-1alpha (HIF-1alpha) by immunofluorescence in the cochlea of these animals were performed in order to confirm that immunochemical detection of pimonidazole protein is representative of a hypoxic environment. We conclude that the present method results in high-quality cryosections of cochlear tissues presenting good anatomical and histological preservation. Furthermore, our optimized procedures provide novel tools for the investigation of neuro-sensory-epithelium in physio-pathological situations associated with hypoxia and/or ischemia, such as inner ear development, plasticity, regeneration and senescence.
authors.affiliation: Laboratoire d'Otologie Neuro-otologie et Micro-Endoscopie, IFR Jean Roche, Faculté de Médecine Nord, Université de la Méditerranée, Bd Pierre Dramard, 13916 Marseille cedex 20, France.
authors.forename: Emilie
authors.initials: E
authors.lastname: Donadieu
authors.forename: Wissal
authors.initials: W
authors.lastname: Hamdi
authors.forename: Arnaud
authors.initials: A
authors.lastname: Deveze
authors.forename: Michel
authors.initials: M
authors.lastname: Lucciano
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Lavieille
authors.forename: Jacques
authors.initials: J
authors.lastname: Magnan
authors.forename: Catherine
authors.initials: C
authors.lastname: Riva
mesh_terms: D000818:Animals; D021461:Animals, Outbred Strains; D015415:Biomarkers / Q000378:metabolism; D015834:Cochlear Diseases / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D019084:Fluorescent Antibody Technique, Indirect; D005629:Frozen Sections; D000860:Hypoxia / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D008297:Male; D051379:Mice; D009593:Nitroimidazoles; D051381:Rats; D017207:Rats, Sprague-Dawley; D016610:Tissue Embedding / Q000379:methods
chemical_list: D015415:Biomarkers; C497443:Hif1a protein, mouse; C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009593:Nitroimidazoles; C033815:pimonidazole