FIGURES AND TABLES
FROM:
Adenovirus-mediated overexpression of a gene prevents hearing loss and progressive inner hair cell loss after transient cochlear ischemia in gerbils
N Hakuba, K Watabe, J Hyodo, T Ohashi, Y Eto, M Taniguchi, L Yang, J Tanaka, R Hata and K Gyo
BACK TO ARTICLE
Figure 1.
Stereoscopic photomicrograph of X-gal staining. A stereoscopic photomicrograph of a cochlea obtained 4 days after administering Ad- Lac Z (a). After X-gal staining, transfected cells with blue cytosol are seen throughout the cochlea from the basal to the apical turn. The most conspicuous blue regions correspond to the mesothelial cells of the scala tympani and lateral wall tissue. A whole-mount specimen from a cochlea processed 4 days after administering Ad-Lac Z (b). All the staining-positive cells with blue cytosol are located in the supporting cell layer of the organ of Corti beneath the basilar membrane.
Full figure and legend (42K)
Figure 2.
Cryosection of X-gal staining. A light photomicrograph of a 10-
m-thick cryosection obtained 4 days after administering Ad- Lac Z shows positive-stained cells located in the mesothelial cell layer of the organ of Corti (arrow), Reissner membrane (double arrow) (a), spinal ganglion cells (arrowhead), and around hair cells (double-headed arrow) (b).
Figure 3.
Western blot band of GDNF protein production. Representative Western blot showing a GDNF band to evaluate the GDNF protein production when we induced ischemia insult 4 days after injection of Ad-GDNF, Ad-LacZ, or artificial perilymph. A single band of GDNF protein is seen between the 30- and 46-kDa bands in all groups. No band was detected when the blots were incubated without primary antibody.
Full figure and legend (12K)
Figure 4.
Densitometric analysis of GDNF protein production. The integrated optical density was obtained from digitized Western blot data using the program NIH Image. The data were normalized to internal standards (no-treatment animals) on each gel and given as percentage values. No significant change in the GDNF protein level was seen in the Ad-LacZ or artificial perilymph groups. In contrast, there was a significant increase in GDNF immunoreactivity in the Ad-GDNF group 4 days following inoculation (*P<0.01).
Full figure and legend (12K)
Figure 5.
Western blot band of GDNF protein production by Ad-GDNF. Representative Western blot showing a GDNF band to confirm the duration of GDNF protein production by Ad-GDNF vector; 24 animals were examined 0, 4, 8, and 11 days after inoculation. A single band of GDNF protein is seen between the 30- and 46-kDa bands. No band was detected when the blots were incubated without primary antibody.
Full figure and legend (14K)
Figure 6.
Densitometric analysis of the duration of GDNF protein production by Ad-GDNF. The integrated optical density was obtained from digitized Western blot data using the program NIH Image. The data were normalized to internal standards (no-treatment animals) on each gel and given as percentage values. The result shows a significant increase in the GDNF protein level following the inoculation of the Ad-GDNF vector until 11 days following inoculation.
Full figure and legend (15K)
Figure 7.
Changes in the CAP threshold after virus inoculation. CAP threshold after injection of Ad-GDNF vector, Ad-LacZ vector, or artificial perilymph at a rate of 0.5 l/min for 4 min into the cochlea. The CAP threshold before vertebral artery occlusion was defined as 0 dB. There were no significant differences among the three groups, suggesting that the virus has no toxic effects. All values are presented as the mean
s.d. Statistical analysis consisted of two-way ANOVA followed by Dunnett's multiple comparison test.
Figure 8.
The rates of inner and outer hair cell loss 11 days after injection of Ad-GDNF vector, Ad-LacZ vector, or artificial perilymph. No cell damage was detected in any group. These results show that at the dose used in this experiment, the virus vector had no toxic effects, even 11 days after virus inoculation.
Full figure and legend (25K)
Figure 9.
Changes in the CAP threshold following transient cochlear ischemia. The CAP threshold before vertebral artery occlusion was defined as 0-dB. In the Ad-GDNF group, the CAP threshold recovered to the pre-ischemic level within 1 day after reperfusion. The average increase in the CAP threshold on the seventh day was significantly less in the Ad-GDNF group than in the Ad-LacZ or artificial perilymph group (*P<0.05, **P<0.01, respectively). All values are presented as the means.d. Statistical analysis consisted of two-way ANOVA followed by Dunnett's multiple comparison test.
Figure 10.
Hair cells 7 days after transient ischemia in the Ad-LacZ group. Representative inner and outer hair cells stained with rhodamine-phalloidin (Figure 6a) and Hoechst 33342 (Figure 6b) seen seven days following transient cochlear ischemia/reperfusion in the Ad-LacZ group. Damaged hair cells lacking both stereocilia and nuclei (arrowhead) were observed sporadically. In contrast, all cells were intact in the Ad-LacZ group. Scale bars: 20 m.
Figure 11.
Hair cells 7 days following transient ischemia in the Ad-GDNF group. Representative inner and outer hair cells stained with rhodamine-phalloidin (Figure 7a) and Hoechst 33342 (Figure 7b) seen 7 days after transient cochlear ischemia/reperfusion in the Ad-GDNF group. All cells were intact in the Ad-LacZ group. Scale bars: 20 m.
Figure 12.
Rates of IHC loss following transient cochlear ischemia/reperfusion. The rates of inner hair cell (IHC) loss 1, 4, and 7 days after transient cochlear ischemia–reperfusion. In the Ad-LacZ and artificial perilymph groups, the mean IHC loss rates increased gradually until 4 days after reperfusion and remained constant thereafter. On the seventh day, the rates of inner and outer hair cell loss in the Ad-GDNF group were significantly smaller than those in both the Ad-LacZ and artificial perilymph groups (*P<0.05, *P<0.01, respectively). All values are presented as the means.d. Statistical analysis consisted of two-way ANOVA followed by Dunnett's multiple comparison test.
Figure 13.
Rates of OHC loss following transient cochlear ischemia/reperfusion. The rates of outer hair cell (OHC) loss 1, 4, and 7 days following transient cochlear ischemia/reperfusion. There were no statistical differences among the three groups. All values are presented as the means.d. (n=6 for each group). Statistical analysis consisted of two-way ANOVA followed by Dunnett's multiple comparison test.
Figure 14.
Rates of inner and outer hair cell loss on the nontreated side. The rates of inner and outer hair cell loss 7 days after transient cochlear ischemia/reperfusion on the nontreated side. There were no statistical differences in the rates of IHC and OHC loss among the three groups. These data indicated that the severity of ischemic insult was roughly the same in all three groups. All values are presented as the means.d. (n=6 for each group). Statistical analysis consisted of two-way ANOVA followed by Dunnett's multiple comparison test.
