title: Imaging and cell count in cleared intact cochlea in the Mongolian gerbil using laser scanning confocal microscopy.
abstract: OBJECTIVE
  To draw up a clearing protocol for Mongolian gerbil cochlea, and to assess the feasibility of quantifying and analyzing 3D cell architecture in the transparent cochleae.
  
  
  METHODS
  Freshly dissected inner ears were prepared on a 13-day protocol: fixation, microdissection, post-fixation, decalcification, pretreatment (signal enhancement, permeabilization and blocking), fluorescent labeling (indirect immunolabeling and direct labeling), dehydration, clearing in Spalteholz solution (MSBB: methyl salicylate and benzyl benzoate) and mounting. Image acquisition used laser scanning confocal microscopy. ImageJ software was used to measure the length of the organ of Corti thus available for analysis and to count inner and outer hair cells.
  
  
  RESULTS
  Four cochleas underwent imaging. 3D reconstruction enabled organ of Corti length to be measured, at a mean 1269±346μm. Mean inner and outer hair-cell count per organ of Corti length was 142±44 and 400±122, respectively.
  
  
  CONCLUSIONS
  Cochlear clearing by MSBB was feasible in Mongolian gerbils and provided high-resolution immunofluorescence-labeled inner-ear images. To our knowledge, this was the first application of the technique in this species. Cell count could thus be performed along the organ of Corti length without traumatic dissection.
authors.affiliation: Department of Otology and Neurotology, CHU Lille, 59000 Lille, France; Inserm U1008, Controlled Drug Delivery Systems and Biomaterials, University Lille 2, CHU Lille, 59000 Lille, France. Electronic address: michael.risoud@gmail.com.
authors.forename: M
authors.initials: M
authors.lastname: Risoud
authors.affiliation: Department of Otology and Neurotology, CHU Lille, 59000 Lille, France.
authors.forename: J
authors.initials: J
authors.lastname: Sircoglou
authors.affiliation: Department of Otology and Neurotology, CHU Lille, 59000 Lille, France.
authors.forename: G
authors.initials: G
authors.lastname: Dedieu
authors.affiliation: BioImaging Center Lille-Nord de France (BICeL), University Lille 2, 59000 Lille, France.
authors.forename: M
authors.initials: M
authors.lastname: Tardivel
authors.affiliation: Department of Otology and Neurotology, CHU Lille, 59000 Lille, France; Inserm U1008, Controlled Drug Delivery Systems and Biomaterials, University Lille 2, CHU Lille, 59000 Lille, France.
authors.forename: C
authors.initials: C
authors.lastname: Vincent
authors.affiliation: Department of Otology and Neurotology, CHU Lille, 59000 Lille, France; Inserm U1008, Controlled Drug Delivery Systems and Biomaterials, University Lille 2, CHU Lille, 59000 Lille, France.
authors.forename: N-X
authors.initials: NX
authors.lastname: Bonne
mesh_terms: D000818:Animals; D002452:Cell Count / Q000379:methods; D003051:Cochlea / Q000166:cytology*; D005240:Feasibility Studies; D005849:Gerbillinae; D006199:Hair Cells, Auditory, Inner / Q000166:cytology; D018072:Hair Cells, Auditory, Outer / Q000166:cytology; D018613:Microscopy, Confocal / Q000379:methods; D023421:Models, Animal
keywords: Clearing; Cochlea; Hair cells; Laser scanning confocal microscopy; Mongolian gerbil; Transparent