Julio Contreras

Delayed inner ear maturation and neuronal loss in postnatal Igf-1-deficient mice.

Insulin-like growth factor-1 (IGF-1) has been shown to play a key role during embryonic and postnatal development of the CNS, but its effect on a sensory organ has not been studied in vivo. Therefore, we examined cochlear growth, differentiation, and maturation in Igf-1 gene knock-out mice at postnatal days 5 (P5), P8, and P20 by using stereological methods and immunohistochemistry. Mutant mice showed reduction in size of the cochlea and cochlear ganglion. An immature tectorial membrane and a significant decrease in the number and size of auditory neurons were also evident at P20. IGF-1-deficient cochlear neurons showed increased caspase-3-mediated apoptosis, along with aberrant expression of the early neural markers nestin and Islet 1/2. Cochlear ganglion and fibers innervating the sensory cells of the organ of Corti presented decreased levels of neurofilament and myelin P0 in P20 mouse mutants. In addition, an abnormal synaptophysin expression in the somata of cochlear ganglion neurons and sensory hair cells suggested the persistence of an immature pattern of synapses distribution in the organ of Corti of these animals. These results demonstrate that lack of IGF-1 in mice severely affects postnatal survival, differentiation, and maturation of the cochlear ganglion cells and causes abnormal innervation of the sensory cells in the organ of Corti.

Double bouquet cell in the human cerebral cortex and a comparison with other mammals.

Double bouquet cells (DBCs) are neocortical γ‐aminobutyric acid (GABA)ergic interneurons characterized by the vertical bundling of its axon, which are generally termed “bundles” or “horse‐tails.” Using immunocytochemistry for the calcium binding protein calbindin, we have analyzed the morphology, density, and distribution of DBC horse‐tails in different cortical areas of the human cortex (Brodmanns areas 10, 4, 3b, 22, 18, and 17). Although DBC horse‐tails were very numerous and regularly distributed in all cortical areas, variations were observed both in terms of morphology and density. We distinguished two major classes of DBC horse‐tails: the thicker complex type (type I) that had more axon collaterals; and the simple type (type II). The density of DBC horse‐tails was significantly higher in areas 17, 18, 22, and 4 than in areas 3b and 10. Moreover, the proportion of type I and type II DBC horse‐tails varied in the cortical areas studied. We also examined the distribution of DBC horse‐tails in frontal, parietal, and occipital areas of different mammalian species. We found DBCs to be present in carnivores but not in rodents, lagomorphs, or artiodactyls. In carnivores, relatively few DBC horse‐tails can be identified and they were generally found in the occipital cortex. Therefore, there is significant variability in the morphology and distribution of DBC horse‐tails in different species and cortical areas. We conclude that, although these interneurons may be an important element in the organization of cortical microcolumns in primates, this is not the case in other mammalian species. J. Comp. Neurol. 486:344–360, 2005.

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.

Effect of sildenafil on non-adrenergic non-cholinergic neurotransmission in bovine penile small arteries.

The purpose of the present study was to investigate the effect of the phosphodiesterase isoenzyme V inhibitor, sildenafil, on non-adrenergic non-cholinergic neurogenic relaxations of intracavernous isolated penile small arteries. Dense plexes of nerve fibres immunoreactive for neural nitric oxide (NO) synthase were observed in the adventitia-media junction of the penile small arteries. In 5-hydroxytryptamine-contracted preparations, the inhibitor of NO synthase, N(G)-nitro-L-arginine (L-NOARG), and of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), reduced the electrical field stimulation-induced relaxations. Sildenafil and exogenous NO induced relaxations of penile small arteries. Sildenafil enhanced NO and vasoactive intestinal peptide-induced relaxations. Moreover, sildenafil increased the duration of the relaxations elicited by electrical field stimulation in penile small arteries and corpus cavernosum tissue. In the presence of L-NOARG, sildenafil only at supratherapeutic concentrations reduced the prazosin-sensitive contractions elicited by EFS in penile small arteries. Neurogenic NO-mediated and guanylyl cyclase-dependent relaxations of penile small arteries and corpus cavernosum tissue, considered to be associated with the vasodilatation leading to erection, are selectively enhanced by an inhibitor of phosphodiesterase V.

Nitrergic relaxation of the horse corpus cavernosum. Role of cGMP.

The involvement of nitric oxide (NO) and the mechanisms mediating neurogenic relaxation were investigated in the horse corpus cavernosum. NADPH-diaphorase activity was expressed in nerve fibres around arteries and muscular bundles in the horse trabecular tissue. Relaxations in response to electrical field stimulation were tetrodotoxin (10(-6) M)-sensitive, indicating their neurogenic origin. The NO synthase inhibitor, L-NO-arginine (L-NO-Arg, 3 x 10(-5) M), abolished the electrically induced relaxations, which were significantly reversed by L-arginine (3 x 10(-3) M). Exogenous NO (10(-6)-10(-3) M) evoked relaxations which were unaffected by L-NO-Arg. 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 5 x 10(-6) M), an inhibitor of guanylate cyclase activation by NO, reduced the relaxations in response to electrical stimulation and exogenous NO. Iberiotoxin (3 x 10(-8) M) or apamin (5 x 10(-7) M), inhibitors of large and small conductance Ca2+-activated K+ channels, respectively, and glibenclamide (3 x 10(-6) M), a blocker of ATP-sensitive K+ channels, failed to modify the relaxations with NO. It is suggested that NO is present in nerve fibres of the horse corpus cavernosum and relaxes smooth muscle through a guanylate cyclase-dependent mechanism. Neither Ca2+-activated nor ATP-sensitive K+ channels seem to be involved in these relaxations.

Early Development of the Vertebrate Inner Ear

This is a review of the biological processes and the main signaling pathways required to generate the different otic cell types, with particular emphasis on the actions of insulin‐like growth factor I. The sensory organs responsible of hearing and balance have a common embryonic origin in the otic placode. Lineages of neural, sensory, and support cells are generated from common otic neuroepithelial progenitors. The sequential generation of the cell types that will form the adult inner ear requires the coordination of cell proliferation with cell differentiation programs, the strict regulation of cell survival, and the metabolic homeostasis of otic precursors. A network of intracellular signals operates to coordinate the transcriptional response to the extracellular input. Understanding the molecular clues that direct otic development is fundamental for the design of novel treatments for the protection and repair of hearing loss and balance disorders. Anat Rec, 2012.

Transforming growth factor β1 inhibition protects from noise-induced hearing loss

Excessive exposure to noise damages the principal cochlear structures leading to hearing impairment. Inflammatory and immune responses are central mechanisms in cochlear defensive response to noise but, if unregulated, they contribute to inner ear damage and hearing loss. Transforming growth factor β (TGF-β) is a key regulator of both responses and high levels of this factor have been associated with cochlear injury in hearing loss animal models. To evaluate the potential of targeting TGF-β as a therapeutic strategy for preventing or ameliorating noise-induced hearing loss (NIHL), we studied the auditory function, cochlear morphology, gene expression and oxidative stress markers in mice exposed to noise and treated with TGF-β1 peptidic inhibitors P17 and P144, just before or immediately after noise insult. Our results indicate that systemic administration of both peptides significantly improved both the evolution of hearing thresholds and the degenerative changes induced by noise-exposure in lateral wall structures. Moreover, treatments ameliorated the inflammatory state and redox balance. These therapeutic effects were dose-dependent and more effective if the TGF-β1 inhibitors were administered prior to inducing the injury. In conclusion, inhibition of TGF-β1 actions with antagonistic peptides represents a new, promising therapeutic strategy for the prevention and repair of noise-induced cochlear damage.