Improved cryosections and specific immunohistochemical methods for detecting hypoxia in mouse and rat cochleae

Donadieu, Emilie; Hamdi, Wissal; Devèze, Arnaud; Lucciano, Michel; Lavieille, Jean‐Pierre; Magnan, Jacques; Riva, Catherine

doi:10.1016/j.acthis.2007.01.004

Cited by 9 publications

(9 citation statements)

References 32 publications

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“…We estimated the ROS formation in the cochlea in response to the chronic hypoxia condition. We found nuclear immunoreactivity for 8-oxoG corresponding to genomic oxidative DNA damage according to Donadieu et al,2 specifically in the SG and the SV of cochlea of hypoxic C57BL/6 mice, while there was no labeling for 8-oxoG in cochleae of 3-, 4-and 6-month mice. The regional and temporal patterns of alteration due to oxidative damage and hypoxia were quite consistent.…”

Section: Discussionmentioning

confidence: 80%

“…An important transcription factor involved in the signaling and adaptation of hypoxia/ischemia is the hypoxia inducible factor (HIF). 35 Using a technique developed previously, 2 we did not reveal any significant HIF-1a expression in cochlea from mice aged three, four and six months. On the contrary, HIF-1a was detected in cochleae of the 4-month hypoxic C57BL/6 mice with a differential region specific pattern of HIF activation that may indicate that HIF expression plays a role in inner ear homeostasis.…”

Section: Discussionmentioning

confidence: 99%

“…In our in vivo study, we demonstrated clearly that HIF-1a protein, effectively expressed in specific areas, is the cogent demonstration of the presence of a hypoxic environment in the cochlea, as previously reported. 2 It has been proposed that increased ROS generation would allow the hypoxic response 39 by inducing the post-translational stabilization of HIF-1a during hypoxic incubation of cells and may further cause perturbation of the downstream transactivation of HIF-1a target genes. We estimated the ROS formation in the cochlea in response to the chronic hypoxia condition.…”

Section: Discussionmentioning

confidence: 99%

“…1,2 In fact, it has been shown that hypoxia can cause reversible hearing loss, 3 as well as damages in brain development 4 and muscle degeneration. 5 There may be unique physiological signal associated with transitions between normoxia and hypoxia statuses, such as the formation of reactive oxygen species (ROS).…”

Section: Introductionmentioning

confidence: 99%

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Hypoxia-inducible factor 1-alpha target protein up-regulation in Hypoxic cochlear neurons is associate with aged-related hearing loss in C57BL/6 mice

Donadieu

Riva

2012

Ageing Res

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Molecular mechanisms underlining hypoxia-induced aged-hearing loss were studied. 3-months C57BL/6 mice were subjected to four weeks of hypoxia (10% 0 2 ), whereas, controls were kept under normoxic condition for up to six months. Auditory function was explored by CAP and Preyer's reflex measurements and correlated with histological analysis of the cochlea. The presence of oxidative damage, HIF-1 responsive target genes regulation involved in cell death, inflammation and neovascularization were assessed by immunofluorescence analysis. Hypoxia was associated to severe hearing loss at 4-8 and 16 KHz and degeneration of the cochlea, with significant cell loss (30%) in the spiral ganglion, the lateral wall, and the hair cells with a basal-apical alteration gradient. This was correlated with ROS formation and HIF-1a overexpression. Cochlear degeneration was due to apoptosis via activated caspase-3, P53, Bax and Bcl-2 protein differential expression in spiral ganglion, modiolus and spiral ligament. On the other hand, Hsp70, NF-kB transcription factor pathway and inflammatory mediators (caspase-1 and TNF-a) were induced in the stria vascularis. Furthermore, a phenomenon of neovascularization was observed with significant thickening of stria vascularis and increased expression of VEGF. In total, we demonstrated that the tandem-HIF-ROS is responsible for the caspase-3 and Bax-mediated apoptosis via P53 protein accumulation in the cochlear neurons, while inflammatory response mediated by Hsp70 stress protein and NF-kB transcription factor generating a neovascularization phenomenon occurred in stria vascularis.

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Section: Discussionmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Hypoxia-inducible factor 1-alpha target protein up-regulation in Hypoxic cochlear neurons is associate with aged-related hearing loss in C57BL/6 mice

Donadieu

Riva

2012

Ageing Res

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“…Although gelatin embedding has been used to achieve good morphology and immunochemical staining of the adult rat cochlea, the thinnest sections produced using this method were reportedly 20 μm (Hurley et al, 2003). More recently, 5 μm frozen sections were obtained from both the mouse and the rat cochlea embedded in OCT compound (Donadieu et al, 2007). To date, there are no detailed reports describing methods to preserve good morphology for immuno-analysis in the adult guinea pig cochlea.…”

Section: Introductionmentioning

confidence: 99%

A protocol for cryoembedding the adult guinea pig cochlea for fluorescence immunohistology

Coleman

Rickard

Silva

et al. 2009

Journal of Neuroscience Methods

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Green fluorescent protein (GFP) has been used extensively to label cells in vitro and to track them following their transplantation in vivo. During our studies using the mouse embryonic stem cell line R1 B5-EGFP, we observed variable levels of fluorescence intensity of the GFP within these transfected cells. The variable fluorescence of this protein coupled with the innately autofluorescent nature of several structures within the cochlea collectively made the in vivo identification of these transplanted stem cells difficult. We have modified previously published protocols to enable the discrimination of an authentic GFP signal from autofluorescence in the adult guinea pig cochlea using fluorescence-based immunohistochemistry. The protocol described can also be used to label tissues of the cochlea using a chromogen, such as 3,3′-diaminobenzidine tetrahydrochloride (DAB). Moreover, the described method gives excellent preservation of structural morphology making the tissues useful for both morphological and quantitative studies in combination with robust immunohistochemistry in the adult guinea pig cochlea. KeywordsCochlea; stem cells; GFP; cryosectioning; cell transplantation; deafness IntroductionThe structural heterogeneity of tissues in the mammalian cochlea, its small size, the large volumes of fluid spaces within it and the fact that is embedded in the temporal bone make the collection of well preserved morphological sections challenging. This is particularly true for the adult guinea pig (GP) cochlea, which contains four fluid-filled turns (the most of any mammalian species), is spherical in shape, and is approximately 4 mm high × 2 mm wide. Previously, the processing and collection of cochlear tissues for immunohistochemistry has been described as a compromise between the maintenance of morphology and preservation of antigenicity (Hurley et al., 2003). Although paraffin sections are generally considered superior to frozen sections in terms of their preservation of cochlear morphology, this technique involves high temperatures and treatment with strong chemicals. This often results in decreased immunogenicity of tissues and increased autofluorescence, making authentic signals difficult to detect. Conversely, frozen sections generally result in excellent antigenic preservation but

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Relative Time Course of Degeneration of Different Cochlear Structures in the CD/1 Mouse Model of Accelerated Aging

Mahendrasingam

MacDonald

Furness

2011

JARO

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Presbycusis (age-related hearing loss) can result from various cochlear pathologies. We have studied the time course of degeneration in a mouse that shows accelerated presbycusis, the CD/1 mouse, as a possible model to investigate stem-cell strategies to prevent or ameliorate presbycusic changes. CD/1 mice from 0 to 72 weeks old were examined by light and electron microscopy. Early pathological changes were detected in basal turn spiral ligament fibrocytes and spiral ganglion, but the latter was variable as both satellite cells and neurons were normal in some cochleae. Light microscopic counts in the spiral ligament of 20-week-old mice revealed that of the five main types (types I-V), only type V fibrocytes showed no reduction in numbers compared with 3-week-old animals, and type IV showed the greatest losses. However, all types of fibrocyte showed subtle damage when examined using electron microscopy, in the form of swollen mitochondria, as early as 2 weeks. The extent of mitochondrial damage showed a degree of correspondence with the light microscopic pattern of fibrocyte loss in that types III and IV fibrocytes had the most abnormal mitochondria and type V the least, especially at early stages. By 10-15 weeks, ultrastructural features of fibrocyte damage were similar to longer term changes reported in gerbils. Stria vascularis, spiral ganglion and hair cells showed few consistent early signs of damage but became increasingly affected, lagging behind the fibrocyte damage. Our data suggest that fibrocyte pathology may precede other presbycusic changes; breakdown of homeostatic mechanisms to which they contribute may cause the subsequent degeneration of the hair cells. Overall, there were many similarities to presbycusic changes in other rodents and humans. Therefore, the features of accelerated aging in this mouse make it a suitable model for rapidly assessing possible strategies to prevent or ameliorate presbycusic changes.

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Improved cryosections and specific immunohistochemical methods for detecting hypoxia in mouse and rat cochleae

Cited by 9 publications

References 32 publications

Hypoxia-inducible factor 1-alpha target protein up-regulation in Hypoxic cochlear neurons is associate with aged-related hearing loss in C57BL/6 mice

Hypoxia-inducible factor 1-alpha target protein up-regulation in Hypoxic cochlear neurons is associate with aged-related hearing loss in C57BL/6 mice

A protocol for cryoembedding the adult guinea pig cochlea for fluorescence immunohistology

Relative Time Course of Degeneration of Different Cochlear Structures in the CD/1 Mouse Model of Accelerated Aging

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