Intranasal delivery of NGF rescues hearing impairment in aged SAMP8 mice

Abstract

Hearing loss impacts the quality of life and affects communication resulting in social isolation and reduced well-being. Despite its impact on society and economy, no therapies for age-related hearing loss are available so far. Loss of mechanosensory hair cells of the cochlea is a common event of hearing loss in humans. Studies performed in birds demonstrating that they can be replaced following the proliferation and transdifferentiation of supporting cells, strongly pointed out on HCs regeneration as the main focus of research aimed at hearing regeneration. Neurotrophins are growth factors involved in neuronal survival, development, differentiation, and plasticity. NGF has been involved in the interplay between auditory receptors and efferent innervation in the cochlea during development. During embryo development, both NGF and its receptors are highly expressed in the inner ears. It has been reported that NGF is implicated in the differentiation of auditory gangliar and hair cells. Thus, it has been proposed that NGF administration can decrease neuronal damage and prevent hearing loss. The main obstacle to the development of hearing impairment therapy is that efficient means of delivery for selected drugs to the cochlea are missing. Herein, in this study NGF was administered by the intranasal route. The first part of the study was focused on a biodistribution study, which showed the effective delivery in the cochlea; while the second part was focused on analyzing the potential therapeutic effect of NGF in senescence-accelerated prone strain 8 mice. Interestingly, intranasal administration of NGF resulted protective in counteracting hearing impairment in SAMP8 mice, ameliorating hearing performances (analyzed by auditory brainstem responses and distortion product otoacoustic emission) and hair cells morphology (analyzed by microscopy analysis). The results obtained were encouraging indicating that the neurotrophin NGF was efficiently delivered to the inner ear and that it was effective in counteracting hearing loss.

Fig. 1. Biodistribution study scheme.

Schematic representation of the biodistribution of NGF in mice upon intranasal administration at different time points.

Fig. 2. Aged animal study and NGF treatment scheme.

NGF was administered intranasally in SAMP8 mice and then different tests were performed at different time points.

Fig. 3. Graphical representation of the human NGF concentration in cochlear perilymph at 12, 24, and 48 h post-treatment in rhNGF- treated (red) and PBS-treated (black) animals.

Data are mean ± SEM. *<0.05; ***<0.0001 vs PBS-sampling 12 h. N = 4.

Fig. 4. Graphical representation of ABR threshold at 1 and 3 months old.

Data are mean ± SEM. N = 8.Graphical representation of ABR threshold at 1 and 3 months old for the SAMR1 (left), SAMP8+vehicle (mid), and SAMP8+rhNGF (right) groups. Data are mean ± SEM. *<0.05; ***<0.0001 vs SAMR1. ^##<0.005; ^###<0.0001 vs SAMP8 + vehicle. N = 8.

Fig. 5. Graphical representation of DPOAE amplitudes at 1 and 3 months old.

Data are mean ± SEM. N = 8. Graphical representation of DPOAE amplitudes at 1 and 3 months old for the SAMR1 (left), SAMP8+vehicle (mid), and SAMP8+rhNGF (right) groups. Data are mean ± SEM. *<0.05; ***<0.0001 vs SAMR1. ^#<0.05; ^###<0.0001 vs SAMP8 + vehicle. ***<0.0001 vs SAMR1. ^###<0.0001 vs SAMP8 + vehicle. N = 8.

Fig. 6. Graphical representation of plasma prestin concentration.

Data are mean ± SEM. ***<0.0001 vs SAMR1. ^###<0.0001 vs SAMP8 + vehicle. N = 8.

Fig. 7. Cytocochleogram analysis and relative graphical representations.

A Representative images for cochlear segments (scale bar: 100 µm) stained with Myosin VIIa of SAMP8 mice control, vehicle and hrNGF-treated are shown. B Graphical representation of the number of IHC (left) and OHC (right) at 3 months. N = 8. Bar = 100 μm.

Fig. 8. SEM images of the cochlea mid-turn of SAMR1 SAMP8+vehicle and SAMP8+rhNGF groups.

Arrows indicate hair cell loss. Asterisks indicate a fusion of stereocilia.

Fig. 9. Immunofluorescence analysis in mice cochlea.

A Immunofluorescence images of cochlea mid-turn of SAMR1, SAMP8+vehicle, and SAMP8+rhNGF groups at 3 months old. Myosin-VIIa marks hair cells (magenta), GluR2 marks ribbon synapses (green), and Tuj1 marks efferent and afferent neurons of the cochlea (blue). Merge compositions on the right. Bar = 35 μm. B The number of ribbon synapses of the IHCs per 100 µm of sensory epithelium. Data are mean ± SEM. ***<0.0001 vs SAMR1. ^###<0.0001 vs SAMP8 + vehicle. N  = 8.