Rafael Cediel

Sensorineural hearing loss in insulin-like growth factor I-null mice: a new model of human deafness

It has been reported that mutations in the gene encoding human insulin‐like growth factor‐I (IGF‐I) cause syndromic hearing loss. To study the precise role of IGF‐I in auditory function and to hypothesize the possible morphological and electrophysiological changes that may occur in the human inner ear, we have analysed the auditory brainstem response in a mouse model of IGF‐I deficiency. We show here that homozygous Igf‐1–/– mice present an all‐frequency involved bilateral sensorineural hearing loss. Igf‐1–/– mice also present a delayed response to acoustic stimuli; this increases along the auditory pathway, indicating a contribution of the central nervous system to the hearing loss in Igf‐1–/– mice. These results support the use of the Igf‐1–/– mouse as a new model for the study of human syndromic deafness.

Melanin precursors prevent premature age‐related and noise‐induced hearing loss in albino mice

Strial melanocytes are required for normal development and correct functioning of the cochlea. Hearing deficits have been reported in albino individuals from different species, although melanin appears to be not essential for normal auditory function. We have analyzed the auditory brainstem responses (ABR) of two transgenic mice: YRT2, carrying the entire mouse tyrosinase (Tyr) gene expression‐domain and undistinguishable from wild‐type pigmented animals; and TyrTH, non‐pigmented but ectopically expressing tyrosine hydroxylase (Th) in melanocytes, which generate the precursor metabolite, L‐DOPA, but not melanin. We show that young albino mice present a higher prevalence of profound sensorineural deafness and a poorer recovery of auditory thresholds after noise‐exposure than transgenic mice. Hearing loss was associated with absence of cochlear melanin or its precursor metabolites and latencies of the central auditory pathway were unaltered. In summary, albino mice show impaired hearing responses during ageing and after noise damage when compared to YRT2 and TyrTH transgenic mice, which do not show the albino‐associated ABR alterations. These results demonstrate that melanin precursors, such as L‐DOPA, have a protective role in the mammalian cochlea in age‐related and noise‐induced hearing loss.

Trophic effects of insulin-like growth factor-I (IGF-I) in the inner ear

Insulin-like growth factors (IGFs) have a pivotal role during nervous system development and in its functional maintenance. IGF-I and its high affinity receptor (IGF1R) are expressed in the developing inner ear and in the postnatal cochlear and vestibular ganglia. We recently showed that trophic support by IGF-I is essential for the early neurogenesis of the chick cochleovestibular ganglion (CVG). In the chicken embryo otic vesicle, IGF-I regulates developmental death dynamics by regulating the activity and/or levels of key intracellular molecules, including lipid and protein kinases such as ceramide kinase, Akt and Jun N-terminal kinase (JNK). Mice lacking IGF-I lose many auditory neurons and present increased auditory thresholds at early postnatal ages. Neuronal loss associated to IGF-I deficiency is caused by apoptosis of the auditory neurons, which presented abnormally increased levels of activated caspase-3. It is worth noting that in man, homozygous deletion of the IGF-1 gene causes sensory-neural deafness. IGF-I is thus necessary for normal development and maintenance of the inner ear. The trophic actions of IGF-I in the inner ear suggest that this factor may have therapeutic potential for the treatment of hearing loss.

A Comparative Study of Age-Related Hearing Loss in Wild Type and Insulin-Like Growth Factor I Deficient Mice

Insulin-like growth factor-I (IGF-I) belongs to the family of insulin-related peptides that fulfils a key role during the late development of the nervous system. Human IGF1 mutations cause profound deafness, poor growth and mental retardation. Accordingly, Igf1−/− null mice are dwarfs that have low survival rates, cochlear alterations and severe sensorineural deafness. Presbycusis (age-related hearing loss) is a common disorder associated with aging that causes social and cognitive problems. Aging is also associated with a decrease in circulating IGF-I levels and this reduction has been related to cognitive and brain alterations, although there is no information as yet regarding the relationship between presbycusis and IGF-I biodisponibility. Here we present a longitudinal study of wild type Igf1+/+ and null Igf1−/− mice from 2 to 12 months of age comparing the temporal progression of several parameters: hearing, brain morphology, cochlear cytoarchitecture, insulin-related factors and IGF gene expression and IGF-I serum levels. Complementary invasive and non-invasive techniques were used, including auditory brainstem-evoked response (ABR) recordings and in vivo MRI brain imaging. Igf1−/− null mice presented profound deafness at all the ages studied, without any obvious worsening of hearing parameters with aging. Igf1+/+ wild type mice suffered significant age-related hearing loss, their auditory thresholds and peak I latencies augmenting as they aged, in parallel with a decrease in the circulating levels of IGF-I. Accordingly, there was an age-related spiral ganglion degeneration in wild type mice that was not evident in the Igf1 null mice. However, the Igf1−/− null mice in turn developed a prematurely aged stria vascularis reminiscent of the diabetic strial phenotype. Our data indicate that IGF-I is required for the correct development and maintenance of hearing, supporting the idea that IGF-I-based therapies could contribute to prevent or ameliorate age-related hearing loss.

The role of insulin-like growth factor-I in the physiopathology of hearing

Insulin-like growth factor-I (IGF-I) belongs to the family of polypeptides of insulin, which play a central role in embryonic development and adult nervous system homeostasis by endocrine, autocrine, and paracrine mechanisms. IGF-I is fundamental for the regulation of cochlear development, growth, and differentiation, and its mutations are associated with hearing loss in mice and men. Low levels of IGF-I have been shown to correlate with different human syndromes showing hearing loss and with presbyacusis. Animal models are fundamental to understand the genetic, epigenetic, and environmental factors that contribute to human hearing loss. In the mouse, IGF-I serum levels decrease with aging and there is a concomitant hearing loss and retinal degeneration. In the Igf1−/− null mouse, hearing loss is due to neuronal loss, poor innervation of the sensory hair cells, and age-related stria vascularis alterations. In the inner ear, IGF-I actions are mediated by intracellular signaling networks, RAF, AKT, and p38 MAPK protein kinases modulate the expression and activity of transcription factors, as AP1, MEF2, FoxM1, and FoxP3, leading to the regulation of cell cycle and metabolism. Therapy with rhIGF-I has been approved in humans for the treatment of poor linear growth and certain neurodegenerative diseases. This review will discuss these findings and their implications in new IGF-I-based treatments for the protection or repair of hearing loss.

Comparison of different aminoglycoside antibiotic treatments to refine ototoxicity studies in adult mice

Hearing and balance receptors in the inner ear are highly susceptible to damage caused by a wide variety of toxic substances, including aminoglycosides. This class of antibiotics is commonly used in medicine, even though they may produce irreversible bilateral neurosensorial deafness. To identify potential ototoxic agents and novel therapeutic targets, it is necessary to generate standardized animal models of aminoglycoside ototoxicity, which will also serve to explore otic cell repair and regeneration. Although the mouse is the species most often used in biomedical research, due to the genetic information and genetically-modified strains available, there are few standard models of aminoglycoside ototoxicity in adult mice. Most protocols to produce ototoxicity in adult mice employ high doses of aminoglycosides for long periods of time, which causes systemic toxicity, side-effects and high mortality rates. Here, we compare the effects of systemic treatment with four different, yet common, aminoglycoside antibiotics in two mouse strains, evaluating their effects on mortality, cochlear morphology and auditory brainstem responses. Our data indicate that gentamicin and neomycin caused high mortality in the adult mouse without significantly changing the auditory threshold. Amikacin produced a tolerable rate of mortality but at doses that did not exhibit ototoxicity. Finally, intramuscular injection of kanamycin in C57BL/6JOlaHsd mice induced significant dose-dependent bilateral hearing loss with a moderate rate of mortality and less discomfort than following subcutaneous administration.

Folic acid deficiency induces premature hearing loss through mechanisms involving cochlear oxidative stress and impairment of homocysteine metabolism

Nutritional imbalance is emerging as a causative factor of hearing loss. Epidemiologic studies have linked hearing loss to elevated plasma total homocysteine (tHcy) and folate deficiency, and have shown that folate supplementation lowers tHcy levels potentially ameliorating age‐related hearing loss. The purpose of this study was to address the impact of folate deficiency on hearing loss and to examine the underlying mechanisms. For this purpose, 2‐mo‐old C57BL/6J mice (Animalia Chordata Mus musculus) were randomly divided into 2 groups (n = 65 each) that were fed folate‐deficient (FD) or standard diets for 8 wk. HPLC analysis demonstrated a 7‐fold decline in serum folate and a 3‐fold increase in tHcy levels. FD mice exhibited severe hearing loss measured by auditory brainstem recordings and TUNEL‐positive‐apoptotic cochlear cells. RT‐quantitative PCR and Western blotting showed reduced levels of enzymes catalyzing homocysteine (Hcy) production and recycling, together with a 30% increase in protein homocysteinylation. Redox stress was demonstrated by decreased expression of catalase, glutathione peroxidase 4, and glutathione synthetase genes, increased levels of manganese superoxide dismutase, and NADPH oxidase‐complex adaptor cytochrome b‐245, α‐polypeptide (p22phox) proteins, and elevated concentrations of glutathione species. Altogether, our findings demonstrate, for the first time, that the relationship between hyperhomocysteinemia induced by folate deficiency and premature hearing loss involves impairment of cochlear Hcy metabolism and associated oxidative stress.—Martínez‐Vega, R., Garrido, F., Partearroyo, T., Cediel, R., Zeisel, S. H., Martínez‐Álvarez, C., Varela‐Moreiras, G., Varela‐Nieto, I., and Pajares, M. A. Folic acid deficiency induces premature hearing loss through mechanisms involving cochlear oxidative stress and impairment of homocysteine metabolism. FASEB J. 29, 418‐432 (2015). www.fasebj.org

Treatment with N- and C-Terminal Peptides of Parathyroid Hormone-Related Protein Partly Compensate the Skeletal Abnormalities in IGF-I Deficient Mice

Insulin-like growth factor-I (IGF-I) deficiency causes growth delay, and IGF-I has been shown to partially mediate bone anabolism by parathyroid hormone (PTH). PTH-related protein (PTHrP) is abundant in bone, and has osteogenic features by poorly defined mechanisms. We here examined the capacity of PTHrP (1–36) and PTHrP (107–111) (osteostatin) to reverse the skeletal alterations associated with IGF-I deficiency. Igf1-null mice and their wild type littermates were treated with each PTHrP peptide (80 µg/Kg/every other day/2 weeks; 2 males and 4 females for each genotype) or saline vehicle (3 males and 3 females for each genotype). We found that treatment with either PTHrP peptide ameliorated trabecular structure in the femur in both genotypes. However, these peptides were ineffective in normalizing the altered cortical structure at this bone site in Igf1-null mice. An aberrant gene expression of factors associated with osteoblast differentiation and function, namely runx2, osteoprotegerin/receptor activator of NF-κB ligand ratio, Wnt3a , cyclin D1, connexin 43, catalase and Gadd45, as well as in osteocyte sclerostin, was found in the long bones of Igf1-null mice. These mice also displayed a lower amount of trabecular osteoblasts and osteoclasts in the tibial metaphysis than those in wild type mice. These alterations in Igf1-null mice were only partially corrected by each PTHrP peptide treatment. The skeletal expression of Igf2, Igf1 receptor and Irs2 was increased in Igf1-null mice, and this compensatory profile was further improved by treatment with each PTHrP peptide related to ERK1/2 and FoxM1 activation. In vitro, PTHrP (1–36) and osteostatin were effective in promoting bone marrow stromal cell mineralization in normal mice but not in IGF-I-deficient mice. Collectively, these findings indicate that PTHrP (1–36) and osteostatin can exert several osteogenic actions even in the absence of IGF-I in the mouse bone.

Corrigendum to: Swept-sine noise-induced damage as a hearing loss model for preclinical assays.

[This corrects the article on p. 7 in vol. 7, PMID: 25762930.].

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures. Chitosan-glycerophosphate (CGP) and Ringer´s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear. The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.