This is the first genome-wide association study with the Hybrid Mouse Diversity Panel (HDMP) to define the genetic landscape of the variation in the suprathreshold wave 1 amplitude of the auditory ...brainstem response (ABR) both pre- and post-noise exposure. This measure is correlated with the density of the auditory neurons (AN) and/or the compliment of synaptic ribbons within the inner hair cells of the mouse cochlea. We analyzed suprathreshold ABR for 635 mice from 102 HMDP strains pre- and post-noise exposure (108 dB 10 kHz octave band noise exposure for 2 h) using auditory brainstem response (ABR) wave 1 suprathreshold amplitudes as part of a large survey (Myint et al., Hear Res 332:113–120, 2016). Genome-wide significance levels for pre- and post-exposure wave 1 amplitude across the HMDP were performed using FaST-LMM. Synaptic ribbon counts (Ctbp2 and mGluR2) were analyzed for the extreme strains within the HMDP. ABR wave 1 amplitude varied across all strains of the HMDP with differences ranging between 2.42 and 3.82-fold pre-exposure and between 2.43 and 7.5-fold post-exposure with several tone burst stimuli (4 kHz, 8 kHz, 12 kHz, 16 kHz, 24 kHz, and 32 kHz). Immunolabeling of paired synaptic ribbons and glutamate receptors of strains with the highest and lowest wave 1 values pre- and post-exposure revealed significant differences in functional synaptic ribbon counts. Genome-wide association analysis identified genome-wide significant threshold associations on chromosome 3 (24 kHz; JAX00105429;
p
< 1.12E-06) and chromosome 16 (16 kHz; JAX00424604;
p
< 9.02E-07) prior to noise exposure and significant associations on chromosomes 2 (32 kHz; JAX00497967;
p
< 3.68E-08) and 13 (8 kHz; JAX00049416; 1.07E-06) after noise exposure. In order to prioritize candidate genes, we generated
cis
-eQTLs from microarray profiling of RNA isolated from whole cochleae in 64 of the tested strains.
This is the first report of a genome-wide association analysis, controlled for population structure, to explore the genetic landscape of suprathreshold wave 1 amplitude measurements of the mouse ABR. We have defined two genomic regions associated with wave 1 amplitude variation prior to noise exposure and an additional two associated with variation after noise exposure.
ABR wave I amplitude represents the synapse of auditory nerve fibers with the inner hair cell and is highly correlated with synapse counts. Cochlear synaptopathy, the loss of synaptic connections ...between inner hair cells and auditory nerve fibers, has been well-demonstrated in animal models of noise-induced hearing loss. The peak-to-peak wave I amplitude was determined at baseline and 2 weeks after noise exposure. We determined the ABR wave I amplitude at 80 dB SPL at the frequencies of 8, 12, 16, 24, and 32 kHz. A total of 69 strains (1–8 mice/strain) were analyzed. A statistically significant post-noise reduction in wave I amplitude was observed in all the tested frequencies (
p
< 0.00001). We identify distinct patterns of noise susceptibility and make this complete phenotypic dataset available for general use. This data establishes a new resource for the study of NIHL in mice and we hope this database will be a useful tool to expand the research in this field.
The separate and collective role of soil nematodes and protists at community level in soil biogeochemical processes remains poorly quantified due to the lack of appropriate methodologies to study ...them independently from other soil biota under conditions emulating natural soil. Here we present a methodological exploration for the selective removal of nematodes and protists using gamma irradiation and the subsequent reinoculation with metazoan-free soil powder inoculum that allows reconstructing a complex microbial food web comprising the native microflora and soil protist communities, without other fauna. Nematodes, and culturable flagellates and ciliates were removed from 6 kGy onwards, while a small fraction of the amoebae survived (8% of the entire protist population) at 16 kGy but were totally removed at 27 kGy. Reinoculation with pulverized soil powder successfully established a protist community of similar size and composition as the control soil as assessed by both amplicon sequencing-based detection and microscopic observation after cultivation. A viable native microflora community remained from 6 to 16 kGy doses, but microbial activity was completely inhibited at 27 kGy. We suggest that, in ecological relevant experiments where selective removal of nematodes or other fauna is the purpose and protist community is meant to be kept intact, gamma irradiation needs to be followed routinely by soil powder inoculation in order to restore the suppressed protist community. This experiment also opens up new perspectives for studying the ecological roles of the entire protist community or specific groups thereof (flagellates and ciliates) in real soil and in the presence of a viable native microflora community.
•Gamma irradiation was applied to selectively eliminate soil nematodes and/or protists in soil microcosms.•Nematodes, and culturable flagellates and ciliates were removed from 6 kGy onwards,•Soil amoebae showed higher resistance to gamma irradiation than flagellates and ciliates.•A viable native microflora community remained after gamma irradiation up to 16 kGy.•Soil powder reinoculation successfully established a representative protist community in (partially) sterilized soil.
Neuropilin-1 (Nrp1) encodes the transmembrane cellular receptor neuropilin-1, which is associated with cardiovascular and neuronal development and was within the peak SNP interval on chromosome 8 in ...our prior GWAS study on age-related hearing loss (ARHL) in mice. In this study, we generated and characterized an inner ear-specific Nrp1 conditional knockout (CKO) mouse line because Nrp1 constitutive knockouts are embryonic lethal. In situ hybridization demonstrated weak Nrp1 mRNA expression late in embryonic cochlear development, but increased expression in early postnatal stages when cochlear hair cell innervation patterns have been shown to mature. At postnatal day 5, Nrp1 CKO mice showed disorganized outer spiral bundles and enlarged microvessels of the stria vascularis (SV) but normal spiral ganglion cell (SGN) density and presynaptic ribbon body counts; however, we observed enlarged SV microvessels, reduced SGN density, and a reduction of presynaptic ribbons in the outer hair cell region of 4-month-old Nrp1 CKO mice. In addition, we demonstrated elevated hearing thresholds of the 2-month-old and 4-month-old Nrp1 CKO mice at frequencies ranging from 4 to 32kHz when compared to 2-month-old mice. These data suggest that conditional loss of Nrp1 in the inner ear leads to progressive hearing loss in mice. We also demonstrated that mice with a truncated variant of Nrp1 show cochlear axon guidance defects and that exogenous semaphorin-3A, a known neuropilin-1 receptor agonist, repels SGN axons in vitro. These data suggest that Neuropilin-1/Semaphorin-3A signaling may also serve a role in neuronal pathfinding in the developing cochlea. In summary, our results here support a model whereby Neuropilin-1/Semaphorin-3A signaling is critical for the functional and morphological integrity of the cochlea and that Nrp1 may play a role in ARHL.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ototoxicity is a common side effect of cisplatin chemotherapy. This study was undertaken to determine the potential protective effects of a systemic administration of dexamethasone against ...cisplatin-induced ototoxicity. A prospective controlled trial conducted in an animal model. The setting was Animal care research facilities of the Montreal Children’s Hospital Research Institute. An experimental guinea pig model was used. The animals were divided as follows: group 1 (
n
= 10): 12 mg/kg intraperitoneal (IP) cisplatin, group 2 (
n
= 14): 15 mg/kg/day dexamethasone IP for 2 days followed by cisplatin 12 mg/kg IP, group 3 (
n
= 14): 10 mg/kg/day dexamethasone IP for 2 days, on day 3, they received cisplatin 12 mg/kg IP followed by 20 mg/kg/day dexamethasone for 2 days and group 4 (
n
= 5): 10 ml of saline IP twice a day for 3 days. Auditory brainstem response (ABR) threshold shifts were measured at four frequencies (8, 16, 20 and 25 kHz) for groups 1, 2 and 3. Histological changes in the organ of Corti, the stria vascularis, the spiral ligament and the spiral ganglion neurons as well as scanning electron microscopy for outer hair cells were completed. Immunohistochemistry for tumour necrosis factor-alpha (TNF-α) was performed. ABR threshold shifts were similar in all groups. Histological and scanning electron findings demonstrate that dexamethasone has greater protective effect on the stria vascularis. Systemic dexamethasone administration in a guinea pig model did not provide significant protection against cisplatin-induced ototoxicity. Dexamethasone may be useful in future applications as a complementary treatment.
The discovery of environmentally specific genetic effects is crucial to the understanding of complex traits, such as susceptibility to noise-induced hearing loss (NIHL). We describe the first ...genome-wide association study (GWAS) for NIHL in a large and well-characterized population of inbred mouse strains, known as the Hybrid Mouse Diversity Panel (HMDP). We recorded auditory brainstem response (ABR) thresholds both pre and post 2-hr exposure to 10-kHz octave band noise at 108 dB sound pressure level in 5-6-wk-old female mice from the HMDP (4-5 mice/strain). From the observation that NIHL susceptibility varied among the strains, we performed a GWAS with correction for population structure and mapped a locus on chromosome 6 that was statistically significantly associated with two adjacent frequencies. We then used a "genetical genomics" approach that included the analysis of cochlear eQTLs to identify candidate genes within the GWAS QTL. In order to validate the gene-by-environment interaction, we compared the effects of the postnoise exposure locus with that from the same unexposed strains. The most significant SNP at chromosome 6 (rs37517079) was associated with noise susceptibility, but was not significant at the same frequencies in our unexposed study. These findings demonstrate that the genetic architecture of NIHL is distinct from that of unexposed hearing levels and provide strong evidence for gene-by-environment interactions in NIHL.
Cisplatin, one of the most effective and widely used chemotherapeutic agents in the treatment of head and neck malignancies, has severe dose-limiting side effects including ototoxicity. This study ...evaluates the effectiveness of nanoencapsulated curcumin and dexamethasone in preventing degenerative changes in inner ear cells caused by cisplatin.
Prospective study, animal experiment.
Cultured auditory cells House Ear Institute Organ of Corti-1 (HEI-OC1) and a guinea pig model were used for in vitro and in vivo experiments, respectively. Cell viability assays were conducted to compare the direct toxicity of cisplatin against auditory cells in the presence or absence of pretreatment with nanoencapsulated curcumin and dexamethasone. To recapitulate these effects in vivo, 68 guinea pigs received cisplatin either alone, or along with dexamethasone, nanoencapsulated curcumin, or the combination of both products. Outcome measures included auditory brainstem response, cochlear morphology under both light and scanning electron microscopy, and antioxidant enzyme assays.
Pretreatment of auditory cells with naonoencapsulated curcumin and dexamethasone resulted in significant attenuation of cisplatin toxicity. Similarly, in the corresponding animal model (guinea pig), cisplatin caused an average hearing loss of 50 dB, which was attenuated by nanoencapsulated curcumin and dexamethasone across all of the hearing frequencies. There was also greater preservation of histologic structures in this group. Superoxide dismutase and catalase activities were increased in cisplatin-treated animals, whereas the nanoencapsulated curcumin with dexamethasone led to a diminution of this effect.
Nanoencapsulated curcumin administered in combination with dexamethasone provides a partial but marked protection against cisplatin-induced hearing loss, likely because of reduced toxic damage to auditory cells.
Gene expression analysis is essential for understanding the rich repertoire of cellular functions. With the development of sensitive molecular tools such as single-cell RNA sequencing, extensive gene ...expression data can be obtained and analyzed from various tissues. Single-molecule fluorescence in situ hybridization (smFISH) has emerged as a powerful complementary tool for single-cell genomics studies because of its ability to map and quantify the spatial distributions of single mRNAs at the subcellular level in their native tissue. Here, we present a detailed method to study the copy numbers and spatial localizations of single mRNAs in the cochlea and inferior colliculus. First, we demonstrate that smFISH can be performed successfully in adult cochlear tissue after decalcification. Second, we show that the smFISH signals can be detected with high specificity. Third, we adapt an automated transcript analysis pipeline to quantify and identify single mRNAs in a cell-specific manner. Lastly, we show that our method can be used to study possible correlations between transcriptional and translational activities of single genes. Thus, we have developed a detailed smFISH protocol that can be used to study the expression of single mRNAs in specific cell types of the peripheral and central auditory systems.
A cornerstone technique in the study of hearing is the Auditory Brainstem Response (ABR), an electrophysiologic technique that can be used as a quantitative measure of hearing function. Previous ...studies have published databases of baseline ABR thresholds for mouse strains, providing a valuable resource for the study of baseline hearing function and genetic mapping of hearing traits in mice. In this study, we further expand upon the existing literature by characterizing the baseline ABR characteristics of 100 inbred mouse strains, 47 of which are newly characterized for hearing function. We identify several distinct patterns of baseline hearing deficits and provide potential avenues for further investigation. Additionally, we characterize the sensitivity of the same 100 strains to noise exposure using permanent thresholds shifts, identifying several distinct patterns of noise-sensitivity. The resulting data provides a new resource for studying hearing loss and noise-sensitivity in mice.
•We conducted a superficial screening study for hearing function in 100 inbred strains of mice.•Several distinct patterns of baseline hearing impairment are observed, and possible avenues of research are discussed.•We also characterize the sensitivity of the same 100 strains to damaging levels of noise.•Several distinct patterns of noise-sensitivity are observed, and possible avenues of research are discussed.•The combined dataset is made available for general use.
Objectives/Hypothesis
Auralgan (benzocaine and antipyrine) is an over‐the‐counter otic drug commonly used for otalgia. Nevertheless, there is limited evidence about the effects of the drug on hearing ...function and cochlear morphology in the presence of a tympanic membrane perforation. The aim of the present study was to assess the cytotoxicity of Auralgan using cultured auditory cells (HEI‐OC1) and to examine its effects on hearing function and cochlear morphology after intratympanic administration in a chinchilla model.
Study Design
Animal experiment.
Methods
Cell viability and DNA labeling assays were conducted to investigate the cytotoxic effect of the drug on cultured auditory cells (HEI‐OC1). To examine the possible drug ototoxic effect in vivo, chinchillas received intratympanic injection of Auralgan in one ear, whereas the contralateral control ear received saline. Outcome measures included auditory brainstem response and postmortem cochlear morphology.
Results
A dose‐dependent toxic effect of Auralgan was noted on cultured cells. Animal experiments showed an inflammatory reaction in the experimental ears and facial paralysis in 80% of the animals on the side receiving transtympanic injection of Auralgan (
P
< .05). Auditory brainstem response testing demonstrated a 30‐ to 50‐dB hearing threshold shift across all frequencies tested (
P
< .05). Control ears showed no inflammation or significant threshold shift. Microscopy showed damage to the hair cells and stria vascularis with bleeding in the perilymphatic space in the experimental ears and damage to the hair cells.
Conclusions
Auralgan was cytotoxic to cultured auditory cells. It promoted an inflammatory reaction and seemed to be ototoxic when given via transtympanic injection in an animal model.
Level of Evidence
NA
Laryngoscope
, 125:1444–1448, 2015
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