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Therapeutic effect of electroacupuncture in a p75 knockout mouse model of progressive hearing loss
  1. Tameyasu Maeda1,
  2. Manabu Taniguchi1,
  3. Kenta Shingaki1,
  4. Shigeyuki Kanazawa2,
  5. Shingo Miyata3
  1. 1Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan
  2. 2Department of Plastic Surgery, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan
  3. 3Division of Molecular Brain Science, Research Institute of Traditional Asian Medicine, Kinki University, Osaka-sayama, Osaka, Japan
  1. Correspondence to Dr Shingo Miyata, Division of Molecular Brain Science, Research Institute of Traditional Asian Medicine, Kinki University, 337-2, Ohno-higashi, Osaka-sayama, Osaka 589-8511, Japan; smiyata{at}med.kindai.ac.jp

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Neurotrophin receptor p75 (p75NTR) knockout mice (p75(−/−) mice) provide a good animal model of progressive-onset hearing loss.1 Cell loss of the spiral ganglion neurons (SGNs) and hair cell degeneration at the basal turn of the cochlea are seen in p75(−/−) mice from 3 months of age. Furthermore, from 3 to 6 months of age, the hearing thresholds of p75(−/−) mice are gradually raised; and after 6 months of age, the mice mostly exhibit hearing loss.1

Several previous reports have indicated that electroacupuncture (EA) stimulation may improve subjective symptoms of tinnitus or hearing loss.2 ,3 However, the molecular mechanisms underlying the therapeutic effect of EA stimulation for hearing loss remain unclear. Thus, to determine whether EA stimulation is useful for the prevention of hearing loss, we used a p75(−/−) mouse model of progressive-onset hearing loss.

The Ting Gong (SI19; Small Intestine 19) and the Yifeng (TE17; Triple Energiser meridian 17) are acupuncture points commonly used to improve any kind of ear problem in Asian medical clinics —for example, hearing acuity and treating progressive hearing loss.2 ,3 We applied EA stimulation at SI19 and TE17 over 4 months (from 2 to 6 months of age). Acupuncture needles 0.16×4 mm were inserted perpendicularly as deep as 3–4 mm to the right and left of the SI19 and TE17 points, and electric stimulation of 1 Hz, 0.7 mA was performed twice a week for 30 min using a stimulator (Electrostimulator N-401; Ito Chotanpa Inc). Next, the mice were perfused transcardially with 50 mL of 4% paraformaldehyde solution under deep pentobarbital anaesthesia and the stimulated cochleae were dissected and post-fixed in 4% paraformaldehyde for 1 day. Thereafter, they were placed in a decalcifying solution (10% EDTA, pH 7.4) for 14 days. Paraffin-embedded 7 μm sections were then stained with haematoxylin and eosin. An Olympus BX50 microscope and a DP71 CCD digital colour camera unit (Olympus Co) was used for visual inspection and recording of images.

It was found that outer hair cells were lost and the organ of Corti was not present in the basal turns in the cochleae of the p75(−/−) mice without EA (−EA) at 6 months of age (figure 1A). However, the organ of Corti and outer hair cells remained normal in the p75(−/−) mice who had received EA (+EA), as is the case with wild-type mice (p75(+/+) mice) (figure 1A). Furthermore, the numbers of SGNs in the middle and basal turns of the cochlea were significantly reduced in the p75(−/−) mice (−EA) (figure 1B,C). By contrast, EA-stimulated p75(−/−) mice retained the same number of SGNs in these regions (figure 1B,C). Previous reports indicated that neurotrophin receptor signalling is important to prevent the degeneration of SGNs.4–7 We showed that EA stimulation significantly increased the expression of receptor tyrosine kinases in the SGNs of p75(−/−) mice cochleae (figure 2). However, elucidation of the functional roles of EA stimulation on progressive hearing loss remains a primary goal of future research.

Figure 1

Haematoxylin and eosin (H&E) staining of cochleae from mice with and without electroacupuncture (EA) stimulation. (A) H&E staining of the organ of Corti in the basal turns from p75(+/+) mice, p75(−/−) mice (−EA) and p75(−/−) mice (+EA). Arrowhead: inner hair cells; arrow: outer hair cells. Scale bar: 50 µm. (B) H&E staining of the spiral ganglion neurons (SGNs) in the basal turns from p75(+/+) mice, p75(−/−) mice (−EA), and p75(−/−) mice (+EA). Scale bar: 50 µm. (C) Quantification of the numbers of SGNs in the apical, middle and basal turns of the cochleae from p75(+/+) mice, p75(−/−) mice (−EA), and p75(−/−) mice (+EA). Asterisks (*) indicate a significant increase in the numbers of SGNs. Student t test, *p=0.02, **p=0.001.

Figure 2

Expression of neurotrophin receptor tyrosine kinases (Trks) in cochlear spiral ganglion neurons (SGNs) using pan-Trk antibody. Immunoreactivity for Trks increased significantly in the SGNs of p75(−/−) mice (+EA). Scale bar: 20 µm. EA, electroacupuncture.

Acknowledgments

We thank Ms Y Ohashi and Ms E Moriya for technical assistance; and Dr M Tohyama for valuable comments.

References

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Footnotes

  • TM and MT are contributed equally.

  • Contributors TM and MT contributed to the conclusions drawn from figures 1 and 2, and contributed to the writing of this manuscript. KS and SK carried out the immunohistochemistry. SM provided intellectual input, funding, coordination of the project and contributed to the writing of this manuscript.

  • Funding This work was supported in part by the Global COE Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

  • Competing interests None.

  • Ethics approval This work was approved by the Institutional Animal Care and Use Committee of Osaka University (No. 19-038-0).

  • Provenance and peer review Not commissioned; externally peer reviewed.

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