Deterioration of the Medial Olivocochlear Efferent System Accelerates Age-Related Hearing Loss in Pax2-Isl1 Transgenic Mice.

The development, maturation, and maintenance of the inner ear are governed by temporal and spatial expression cascades of transcription factors that form a gene regulatory network. ISLET1 (ISL1) may be one of the major players in this cascade, and in order to study its role in the regulation of inner ear development, we produced a transgenic mouse overexpressing Isl1 under the Pax2 promoter. Pax2-regulated ISL1 overexpression increases the embryonic ISL1(+) domain and induces accelerated nerve fiber extension and branching in E12.5 embryos. Despite these gains in early development, the overexpression of ISL1 impairs the maintenance and function of hair cells of the organ of Corti. Mutant mice exhibit hyperactivity, circling behavior, and progressive age-related decline in hearing functions, which is reflected in reduced otoacoustic emissions (DPOAEs) followed by elevated hearing thresholds. The reduction of the amplitude of DPOAEs in transgenic mice was first detected at 1 month of age. By 6-9 months of age, DPOAEs completely disappeared, suggesting a functional inefficiency of outer hair cells (OHCs). The timing of DPOAE reduction coincides with the onset of the deterioration of cochlear efferent terminals. In contrast to these effects on efferents, we only found a moderate loss of OHCs and spiral ganglion neurons. For the first time, our results show that the genetic alteration of the medial olivocochlear (MOC) efferent system induces an early onset of age-related hearing loss. Thus, the neurodegeneration of the MOC system could be a contributing factor to the pathology of age-related hearing loss.

Minimally invasive drug delivery to the cochlea through application of nanoparticles to the round window membrane.

Direct drug delivery to the cochlea is associated with the risk of irreversible damage to the ear. In this study, liposome and polymersome nanoparticles (NPs), both formed from amphiphilic molecules (lipids in liposomes and block copolymers in polymersomes), were tested as potential tools for drug delivery to the cochlea via application onto the round window membrane in adult mice (strain C3H). One day after round window membrane application, both types of NPs labeled with fluorescent markers were identified in the spiral ganglion in all cochlear turns without producing any distinct morphological or functional damage to the inner ear. NPs were detected, although to a lesser extent, in the organ of Corti and the lateral wall. The potential of liposome and polymersome NPs as therapeutic delivery systems into the cochlea via the round window membrane was evaluated using disulfiram, a neurotoxic agent, as a model payload. Disulfiram-loaded NP delivery resulted in a significant decrease in the number of spiral ganglion cells starting 2 days postapplication, with associated pronounced hearing loss reaching 20-35 dB 2 weeks postapplication as assessed through auditory brainstem responses. No changes in hair cell morphology and function (as assessed by recording otoacoustic emissions) were detected after disulfiram-loaded NP application. No effects were observed in controls where solution of free disulfiram was similarly administered. The results demonstrate that liposome and polymersome NPs are capable of carrying a payload into the inner ear that elicits a biological effect, with consequences measurable by a functional readout.

Calbindin and S100 protein expression in the developing inner ear in mice.

Calbindin (CB) and S100 are calcium-binding proteins expressed in the inner ear in vertebrates. Information about their developmental roles is incomplete. This study investigated the expression patterns of CB and S100 in C3H mice using immunohistochemistry, from embryonic day 11 (E11) to postnatal day 10 (P10). CB was expressed in the otocyst and vestibulocochlear ganglion (VCG) from E11. In the cochlea at E17, CB immunoreactivity clearly labeled the VCG, the outer and inner hair cells, and the stria vascularis. CB staining was also present in the vestibular sensory cells, including their nerve fibers. Two days later, to this expression pattern was added the labeling of Kölliker's organ. Early postnatal CB expression encompassed VCG neurons, auditory hair cells, their afferent nerve fibers, and cells of the cochlear lateral wall. The first signs of S100 immunostaining of cochlear and vestibular epithelial cells appeared at E14. At E17 S100 immunoreactivity was found in a restricted expression pattern in the cochlea. Immunostaining was also present in the sacculus and utriculus and their afferent fibers. The Deiters', pillar and inner hair cells, and the VCG were S100-positive from E19. Postnatally, S100 staining also appeared in the inner hair cells and Deiters' cells, in some VCG neurons, and, in addition, in the spiral limbus, the spiral prominence, and the intermediate cells of the stria vascularis. This study demonstrates that the sites of CB and S100 expression in the mouse inner ear during embryonic and early postnatal development do not overlap and signal independent developmental patterns.

Effect of low dose of vinclozolin on reproductive tract development and sperm parameters in CD1 outbred mice.

The effect of a low dose of vinclozolin within the development of the reproductive tract during gestation (VIN-GD 15-22) and puberty (VIN-PND 23-44) in CD1 mice was tested. We found a decrease in the anogenital distance, prostate weight and pathology of testes in both experimental groups. Sperm counts decreased to 46% (VIN-GD) and to 81% (VIN-PND), and also the acrosomal state (evaluated by antiacrosomal antibody) decreased in both groups to 89% in comparison to the control group (100%). Sperm head abnormalities increased by approximately 18% and 13%, respectively. In this connection, the expression of some genes was changed (arosome-related gene (Acr), apoptosis related genes (p53, p21)). In conclusion, a low dose of vinclozolin affected the reproductive tract, sperm parameters and expression of selected genes in both experimental groups.

Aging cochleas in the F344 rat: morphological and functional changes.

The Fischer 344 rat strain has been frequently used as an animal model of rapid aging. The present study was aimed at evaluating the incidence of apoptotic cells in the inner ear of 20-24-month-old F344 rats and to correlate it with cochlear function using otoacoustic emissions. Staining with cresyl violet and the enzymatic labeling (terminal deoxynucleotidyl transferase, TdT) of fragmented DNA revealed large numbers of apoptotic cells in the marginal and basal layers of the stria vascularis and in adjacent cells of the spiral ligament. The amplitudes of distortion products otoacoustic emissions (DPOAEs), which reflect functional state of the outer hair cells, were significantly reduced or totally absent in these animals. In contrast to old F344 rats, no marked DPOAE amplitude reduction and smaller numbers of apoptotic cells were found in young 4-month-old F344 rats or in aged 24-28-month-old Long Evans rats. The accumulation of apoptotic cells, mainly in the basal layer of the stria vascularis and in adjacent cells of the spiral ligament, leads to a detachment of the stria vascularis from the spiral ligament and results in the impairment of outer hair cell function. This specific type of strial deterioration suggests that aged F344 rats can serve as an animal model of strial presbycusis.

Collagen changes in the cochlea of aged Fischer 344 rats.

Hearing function in the Fischer 344 (F344) albino inbred strain of rats deteriorates with aging faster than in other strains, in spite of the small hair cell loss in old F344 animals [Popelar, J., Groh, D., Pelanova, J., Canlon, B., Syka, J., 2005. Age-related changes in cochlear and brainstem auditory function. Neurobiol. Aging, in press.]. This study was aimed at elucidating the structural changes in the inner ear of this rat strain during aging. Cochlear histopathology was examined in 20-24-month-old F344 rats and compared with that of young F344 rats (4 months) and of old rats of the Long-Evans (LE) strain. Hematoxylin/eosin staining in aged F344 rats showed degenerative changes in the organ of Corti, consisting of a damaged layer of marginal cells, reduced vascularization of the stria vascularis and a distorted tectorial membrane detached from the organ of Corti. Age-related changes in collagen distribution were observed with Masson's trichrome staining in the spiral ligament of old F344 rats. The results of immunohistochemical staining for type II collagen revealed a marked decrease in collagen fibers in the area connecting the spiral ligament and stria vascularis and a decrease in area IV fibrocytes in old F344 but not in LE rats. These findings may contribute to an explanation of the substantial hearing loss found in old F344 rats.

Development of the inner ear in Splotch mutant mice.

The Splotch mouse, a Pax 3 mutation, represents a model of Waardenburg syndrome I. We show that the homozygous Splotch mutation (Sp(2H)) is associated with severe defects that prevent the formation of the cochlea and vestibulo-cochlear ganglion. To clarify the role of Pax 3 in inner ear formation, we examined the expression of polysialic acid (PSA) associated with neural cell adhesion molecule (NCAM). In accordance with the occurrence of phenotypic abnormalities, PSA NCAM was expressed early in otocyst development in the otic epithelium and the vestibulo-cochlear anlage. During the period of vestibular and cochlear ganglia formation, PSA NCAM expression was decreased. In the late phase of embryonic development, the expression of calcium binding proteins (S100) in the vestibulo-cochlear ganglion was also decreased. Minor differences in S100 immunostaining were found postnatally between the cochleas of heterozygous and wild type animals.

Genetic analysis of X-linked hybrid sterility in the house mouse.

Hybrid sterility is a common postzygotic reproductive isolation mechanism that appears in the early stages of speciation of various organisms. Mus musculus musculus and Mus musculus domesticus represent two recently separated mouse subspecies particularly suitable for genetic studies of hybrid sterility. Here we show that the introgression of Chr X of M. m. musculus origin (PWD/Ph inbred strain, henceforth PWD) into the genetic background of the C57BL/6J (henceforth B6) inbred strain (predominantly of M. m. domesticus origin) causes male sterility. The X-linked hybrid sterility is associated with reduced testes weight, lower sperm count, and morphological abnormalities of sperm heads. The analysis of recombinant Chr Xs in sterile and fertile males as well as quantitative trait locus (QTL) analysis of several fertility parameters revealed an oligogenic nature of the X-linked hybrid sterility. The Hstx1 locus responsible for male sterility was mapped near DXMit119 in the central part of Chr X. To ensure full sterility, the PWD allele of Hstx1 has to be supported with the PWD allelic form of loci in at least one proximal and/or one distal region of Chr X. Mapping and cloning of Hstx1 and other genes responsible for sterility of B6-X PWD Y B6 males could help to elucidate the special role of Chr X in hybrid sterility and consequently in speciation.

Changes in sialylation in homozygous Sp2H mouse mutant embryos.

BACKGROUND: The splotch (Sp(2H)), Pax-3 mutant mice are characterized by neurulation defects, neural crest deficiencies, and altered somitogenesis. A link connecting all morphological abnormalities in the Pax-3 homozygous embryos is the composition of extracellular matrix and misexpression of cell adhesion molecules. The neural cell adhesion molecule (NCAM) is one of the Pax-3 target genes. Its adhesive properties depend on the attached polysialic acid (PSA). We have studied whether NCAM sialylation has been affected in the Pax-3 mutant embryos. METHODS: Genotyping of embryos was determined using polymerase chain reaction. The periodate-resorcinol method was used for quantitative determination of sialic acid. Immunoblotting was used to detect sialylated NCAM isoforms and sialic acids on the blot. This antibody was also used to detect PSA-NCAM spatial expression. RESULTS: Quantitative determination of sialic acid at days 10.5-13.5 showed decreased sialic acid content in Sp(2H) homozygotes. The results of both techniques used in evaluating the expression of PSA-NCAM isoforms in our study indicate that the 180-kDa isoform is decreased in homozygous Splotch (Sp(2H)) embryos. Immunohistochemistry showed decreased staining in the neural tube, ganglion VIII, (day 13.5), frontal lobe, olfactory bulb, and neuroblastic retinal cells (day 18.5). CONCLUSIONS: PSA-NCAM is present in Sp(2H) embryos of all genotypes, starting on developmental day 9.5. Sialylation of the 180-kDa NCAM isoform begins to decrease on day 12.5 in Sp(2H) homozygotes. Reduced NCAM sialylation could contribute to the decreased migration of cells in the sensory organs.

Body and organ weight, sperm acrosomal status and reproduction after genistein and diethylstilbestrol treatment of CD1 mice in a multigenerational study.

The effect of genistein (GEN) and diethylstilbestrol (DES) on body weight, weight of different organs, sperm acrosomal status and in vivo fertility of CD1 mice was tested in a multigenerational study. The adult parental generation of mice and F1 and F2 generations were exposed to selected drugs for all their life. GEN had effect on different body parameters of 30-day-old mice, but not of adult mice in the first generation. Contrary to that, treatment by DES had a strong effect on body weight, other body parameters and on the levels of serum hormones. In the first generation only sterile pairs of mice were observed. Monoclonal antibody against mouse intra-acrosomal sperm protein was used for analysis of the acrosome state and as biomarkers of sperm damage. In the control groups, about 93% of acrosome-reacted sperm was found, acrosome staining decreased to 78-84% (P<0.01). However, the GEN had no effect on fertility of CD1 mice. On the other hand, the fertility of mice exposed to DES was disrupted, especially in the first generation.

Effects of p-nonylphenol and resveratrol on body and organ weight and in vivo fertility of outbred CD-1 mice.

The aim of this study was to analyse the multigenerational effects of para-nonylphenol (NP) and resveratrol (RES) on the body weight, organ weight and reproductive fitness of outbred CD-1 mice. The data indicate that in male mice, NP had an effect on the weight of selected reproductive organs and the kidneys in the parental (P) generation males. Effects on selected reproductive organs, the liver and kidneys in the F1-generation males were also seen. In females, effects of NP on body weight and kidney weight were seen in the P generation, but no effects on any measured parameter were seen in the F1 generation. RES had no effect on body weight but did have some effect on selected male and female reproductive organs in the P generation. RES altered the spleen and liver weights of P-generation males and the kidney weight of F1-generation males. Acrosomal integrity (using a monoclonal antibody against intra-acrosomal sperm proteins) was assessed for both generations of NP- and RES-treated mice. A significant reduction in acrosomal integrity was seen in both generations of NP-treated, but not in RES-treated, mice. Fewer offspring were observed in the second litter of the F2 generation of mice treated with NP; no similar effect was seen in RES-treated mice. The litter sex ratio was not different from controls. Unlike RES, NP had a negative effect on spermatogenesis and sperm quality with a resultant impact on in vivo fertility.