Growth hormone promotes hair cell regeneration in the zebrafish (Danio rerio) inner ear following acoustic trauma

Abstract

Background: Previous microarray analysis showed that growth hormone (GH) was significantly upregulated following acoustic trauma in the zebrafish (Danio rerio) ear suggesting that GH may play an important role in the process of auditory hair cell regeneration. Our objective was to examine the effects of exogenous and endogenous GH on zebrafish inner ear epithelia following acoustic trauma.

Methodology/principal findings: We induced auditory hair cell damage by exposing zebrafish to acoustic overstimulation. Fish were then injected intraperitoneally with either carp GH or buffer, and placed in a recovery tank for either one or two days. Phalloidin-, bromodeoxyuridine (BrdU)-, and TUNEL-labeling were used to examine hair cell densities, cell proliferation, and apoptosis, respectively. Two days post-trauma, saccular hair cell densities in GH-treated fish were similar to that of baseline controls, whereas buffer-injected fish showed significantly reduced densities of hair cell bundles. Cell proliferation was greater and apoptosis reduced in the saccules, lagenae, and utricles of GH-treated fish one day following trauma compared to controls. Fluorescent in situ hybridization (FISH) was used to examine the localization of GH mRNA in the zebrafish ear. At one day post-trauma, GH mRNA expression appeared to be localized perinuclearly around erythrocytes in the blood vessels of the inner ear epithelia. In order to examine the effects of endogenous GH on the process of cell proliferation in the ear, a GH antagonist was injected into zebrafish immediately following acoustic trauma, resulting in significantly decreased cell proliferation one day post-trauma in all three zebrafish inner ear end organs.

Conclusions/significance: Our results show that exogenous GH promotes post-trauma auditory hair cell regeneration in the zebrafish ear through stimulating proliferation and suppressing apoptosis, and that endogenous GH signals are present in the zebrafish ear during the process of auditory hair cell regeneration.

Figure 1. Effect of GH on hair cell bundle density.

(A) Phalloidin-labeled saccular epithelia of baseline, buffer-injected, and GH-injected zebrafish at post-sound exposure day 2 (psed2). The upper image shows the five locations of hair cell counts along the rostral-caudal axis of the saccule. The enlarged images to the right of the saccules are representative 100X images of saccules at 75% along the rostral-caudal axis. Scale bar  = 100 µm, D = dorsal, R = rostral, * = presumed newly formed hair cell bundles, ∧ = scar formation characteristic of hair cell loss. (B) Mean (±S.E.) number of hair cell bundles/900 µm² at specified locations along the rostral-caudal axis of the saccule of baseline and buffer- or GH-injected zebrafish. N = 6; * P<0.001.

Figure 2. Effect of GH on hair cell type.

Phalloidin-labeled saccular epithelia of (A) baseline, (B) buffer-injected, and (C) GH-injected zebrafish at post-sound exposure day 2 (psed2) at 25% along the rostral-caudal axis of the saccule. Arrow = hair cell with missing and splayed stereocilia; triangle = bundleless hair cell, * = presumed newly formed hair bundles. Mean (±S.E.) number of hair cell bundles/900 µm² at (D) 25% and (E) 75% along the rostral-caudal axis of the saccule of buffer- or GH-injected zebrafish. A similar pattern was evident at the 50% rostral-caudal location. N = 6; * P<0.001.

Figure 3. Effect of GH on cell proliferation.

(A) 100X images of BrdU and DAPI labeling in the saccules of buffer- and GH-injected zebrafish. (B) Mean (±S.E.) number of BrdU-labeled cells in the saccules, lagenae, and utricles of baseline and buffer- or GH-injected zebrafish. N = 6; * P<0.001. (C) BrdU-labeling in the saccules of baseline, buffer- or GH-injected zebrafish at post-sound exposure day 1 (psed1). Scale bar  = 100 µm. Rostral-caudal orientation is the same as Fig. 1A.

Figure 4. Effect of GH on apoptosis.

(A) 100X images of TUNEL and DAPI labeling in the saccule of a buffer-injected zebrafish. (B) Mean (±S.E.) number of TUNEL-labeled cells in the saccules, lagenae, and utricles of baseline and buffer- or GH-injected zebrafish. N = 6; * P<0.01, ** P<0.001. (C) TUNEL-labeling in the saccules of baseline, buffer- or GH-injected zebrafish at post-sound exposure day 1 (psed1). Scale bar  = 100 µm. Rostral-caudal orientation is the same as Fig. 1A.

Figure 5. Expression of GH mRNA in zebrafish saccules.

Photomicrographs of saccules of zebrafish labeled with probes against GH mRNA via fluorescent in situ hybridization (FISH); green = FITC conjugated probe, blue = DAPI. Saccules from (A) a baseline control (not exposed to sound) and (B–E) sound-exposed zebrafish. (D) 100X image of the boxed area in (C), showing that GH mRNA appears to be localized perinuclearly in blood cells. (A–C) show only the caudal portion, while (E) shows a whole saccule. Caudal is to the right as in previous figures. Scale bars (D = 10 µm; E = 100 µm).

Figure 6. Effect of GH antagonist on cell proliferation.

(A) Mean (±S.E.) number of BrdU-labeled cells in the saccules, lagenae, and utricles of buffer- or GH-antagonist-injected zebrafish. N = 6–8; * P<0.001. (B) BrdU-labeling in representative saccules of buffer- or GH-injected (two examples are provided) zebrafish at post-sound exposure day 1 (psed1). Rostral-caudal orientation is the same as Fig. 1A.