Volume 4, Issue 6, Pages 575-585 (December 2001) The Functional and Structural Outcome of Inner Ear Gene Transfer via the Vestibular and Cochlear Fluids in Mice  Kohei Kawamoto, Seung-Ha Oh, Sho Kanzaki, Nadine Brown, Yehoash Raphael  Molecular Therapy  Volume 4, Issue 6, Pages 575-585 (December 2001) DOI: 10.1006/mthe.2001.0490 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 A diagram of the two tube placements (cochleostomy and canalostomy) in the left inner ear of the mouse. In the cochleostomy the hole was drilled in the otic capsule below the stapedial artery, and in the canalostomy the tube was introduced into a hole drilled in the posterior semicircular canal and was directed toward the crus commune. Molecular Therapy 2001 4, 575-585DOI: (10.1006/mthe.2001.0490) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Fluorescence immunohistochemistry on frozen sections of mouse inner ears inoculated via the canalostomy with adenovirus expressing bacterial lacZ. (A) The posterior crista ampullaris (arrowheads delineate the luminal border of the sensory epithelium) shows β-galactosidase expression in the sensory cells. (B-F) Transgene expression in different regions of the inner ear follows a similar pattern with prominent expression in the mesothelial cells lining the perilymphatic spaces (arrows), whereas the sensory epithelium is negative (arrowheads). This is shown in the posterior semicircular canal and ampulla (B, and C at higher magnification), the utricle (D), saccule (E), and cochlea (F). e, Endolymphatic space; p, perilymphatic space. (G–J) The contralateral ears show no gene expression in the sensory epithelium (arrowheads) or the mesothelial cells, as demonstrated in the posterior ampulla (G), utricle (H), saccule (I), and cochlea (J). Bars, 1 mm in (B) and 0.1 mm in (F) (for A, and C-F), and 0.1 mm in (J) (for G–J). Molecular Therapy 2001 4, 575-585DOI: (10.1006/mthe.2001.0490) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Cryosections of mouse inner ears in the cochleostomy group stained with antibodies to β-galactosidase and a secondary fluorescent antibody. (A) Transgene expression is detected in the saccular membranous labyrinth, including supporting cells (arrowheads), and in the surrounding mesothelial cells (arrows). (B) Transgene expression is detected in the sensory epithelium of the organ of Corti (arrowheads), and mesothelial cells lining the perilymphatic space (arrows). e, Endolymphatic fluid space; p, perilymphatic fluid space. Bar, 0.01 mm in (B) (for A and B). Molecular Therapy 2001 4, 575-585DOI: (10.1006/mthe.2001.0490) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 ABR threshold shift (mean ± SD) at each frequency tested for 0.5 μl versus 1.0 μl Ad.CMV-lacZ inoculation in the canalostomy group (A) and the cochleostomy group (B). (A) In the canalostomy group, threshold shifts at all three measured frequencies were 2 or 3 dB in the 0.5 μl inoculated group and less than 10 dB in the 1.0 μl inoculated group. (B) In the cochleostomy group, the 0.5 μl inoculated group had mean threshold shifts of 50, 36, and 45 dB at 6, 12, and 24 kHz, respectively. The 1.0 μl inoculated group had 73, 43, and 63 dB mean threshold shifts at these frequencies. Molecular Therapy 2001 4, 575-585DOI: (10.1006/mthe.2001.0490) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 ABR threshold shift (mean ± SD) at each frequency tested for the Ad.CMV-lacZ inoculation via the canalostomy versus the cochleostomy. A significant difference (*P < 0.05) was found between threshld shift in the canalostomy and cochleostomy groups at all the frequencies tested, in both volume groups (0.5 μl and 1.0 μl). Molecular Therapy 2001 4, 575-585DOI: (10.1006/mthe.2001.0490) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 6 ABR threshold shift (mean ± SD) at each frequency for the 0.5 μl inoculation of the Ad.CMV-lacZ versus Ringer's solution via cochleostomy. There were no significant differences between the Ad.CMV-lacZ inoculated group and the Ringer's solution inoculated group. Molecular Therapy 2001 4, 575-585DOI: (10.1006/mthe.2001.0490) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 7 Average score of the vestibular function tests (swimming, circling behavior, and head tilt) performed in the nine mice in the 13-day survival group, after the inoculation of Ad.CMV-lacZ via the canalostomy (0.5 μl), showing recovery of vestibular function by day 13. A score of 1 indicates no obvious deviation from normal. Molecular Therapy 2001 4, 575-585DOI: (10.1006/mthe.2001.0490) Copyright © 2001 American Society for Gene Therapy Terms and Conditions