Hearing Improvement in A/J Mice via the Mouse Nerve Growth Factor

Abstract

Objectives: To investigate the otoprotective effects of mouse nerve growth factor (mNGF) in A/J mice.

Methods: The mice at postnatal day 7 (P7) were randomly separated into a mNGF treated group (mNGF group) and a distilled water (for injection) treated group (control group). The mNGF dissolved in distilled water or distilled water alone was given to the mice once every other day from P7 by intramuscular injection in the hips. The otoprotective effects of mNGF in A/J mice were observed in a time course manner. The thresholds of auditory-evoked brainstem response (ABR) were tested from the age of the 3rd to the 8th week. Sections of the inner ears were stained by hematoxylin and eosin, and spiral ganglion neurons (SGNs) were observed at the age of the 3rd, the 6th,and the 8th week. Counts of whole mount outer hair cells (OHCs) in the cochleae were made at the age of 8 weeks. Expression of apoptosis related genes was determined by quantitative real-time polymerase chain reaction and Western blotting.

Results: ABR thresholds of the mNGF group were significantly lower than those of the control group at the age of the 6th and the 8th week. Moreover, the mNGF preserved OHC and SGN in the mouse cochleae in this period. Further experiments showed that the expression of caspase genes (including caspase-3) was inhibited in the mouse inner ears in the mNGF group.

Conclusion: The mNGF improves hearing in A/J mice by preserving SGN and OHC in the cochleae.

Keywords: Hair Cells; Nerve Growth Factor; Neurons; Protection.

Fig. 1.

Time course observation of auditory-evoked brainstem response (ABR) thresholds in the mouse nerve growth factor (mNGF) treated mice. A/J mice in the control and mNGF groups showed increasing ABR thresholds to auditory click (A) and sound frequencies of 8 kHz (B), 16 kHz (C), and 32 kHz (D) from the age of 3 to 8 weeks. However, ABR thresholds in the mNGF group were lower than those of the control group from the age of 4 to 8 weeks (n=14 for control group, and n=12 for mNGF group at each time point). SPL, sound pressure levels; dB, decibel. Error bars represent the standard error of the mean. *P<0.05. **P<0.01.

Fig. 2.

Preservation of outer hair cell (OHC) in the cochleae of mice in mouse nerve growth factor (mNGF) group. Hair cells were stained for Factin with Alexa Fluor 488-labeled phalloidin. (A) Representative OHC images in the three turns of mNGF and control mice at the age of 8 weeks. OHC loss at middle turns or basal turns of the control group was more severe than that of the mNGF group (scale bar=50 µm). (B) Percentages of OHC loss in the three cochlear turns of both mouse groups at the age of 8 weeks. Percentage of OHC loss at middle turns or basal turns in mNGF group was significantly lower than that in the control group. There was only a small number of OHC loss at the apical turns in both mouse groups (n=4 for each group). Error bars represent the standard error of the mean. *P<0.05. **P<0.01.

Fig. 3.

Preservation of spiral ganglion neurons (SGNs) in the mice of mouse nerve growth factor (mNGF) group. (A–C) Densities of SGN at the three cochlear turns in the mNGF and control groups. At the age of 3 weeks, there were no significant differences of SGN densities between the two mouse groups at any turn (P>0.05). Whereas the densities in mNGF group were significantly higher than those in the control group at the age of 6 weeks (*P= 0.021, P=0.032, and P=0.035, respectively, between apical, middle, and basal turns) and 8 weeks (**P =0.010, P =0.021, and P =0.023, respectively, between apical, middle, and basal turns) (n=5 for each group at each time point). Error bars represent the standard error of the mean. (D) Representative images of SGN at basal turns of the mNGF and control groups at the age of 8 weeks. The control mice showed relative sparse SGN, which proved that mNGF preserved SGN in A/J mice. Scale bar=50 µm.

Fig. 4.

Gene expression profiles in the inner ears of mice in the mouse nerve growth factor (mNGF) group. mRNA levels of Bak, Bax, Caspase-3, Caspase-9, and Caspase-12 in the inner ears of the mNGF group were lower than those of the control group, with significant differences of the 5 genes at the age of 2 weeks (n=6 for each group) (A), 4 weeks (n=6 for each group) (B), and with significant differences of Caspase-3, Caspase-9, and Caspase-12 at the age of 8 weeks (n=5 for each group) (C). Error bars represent the standard error of the mean. *P<0.05. **P<0.01. (D) Western blotting images of the cleaved caspase-3 (17 kD) in the inner ears of the two mouse groups at the age of 8 weeks. The results showed that the levels of cleaved caspase-3 in the mouse inner ears of mNGF group were downregulated compared with the levels of the control group.