Analysis of the Hyalinization Reaction in Otosclerosis.

IMPORTANCE: Otosclerotic bone has been observed to penetrate the endosteal layer of the cochlea, resulting in direct contact with the soft-tissue structures of the inner ear. Sensorineural hearing loss has been observed in some, but not all, of these cases. The development of histologic changes occurring in the cochlear soft tissues at the site of otosclerotic endosteal penetration has been descriptively referred to as a hyalinization reaction. The role of the hyalinization reaction in the development of hearing loss is unknown. OBJECTIVE: To evaluate the composition of these hyalinized soft tissues using immunostaining techniques. DESIGN, SETTING, AND PARTICIPANTS: Retrospective review in a human temporal bone histopathology research laboratory of 3 specimens from patients with endosteal otosclerotic involvement. INTERVENTIONS: Evaluation of human temporal bone pathology findings. MAIN OUTCOMES AND MEASURES: Human temporal bone sections with endosteal otosclerotic involvement were studied using immunostaining techniques to identify collagen I, chondroitin sulfate, and keratan sulfate deposition in the hyalinization reaction tissue. RESULTS: Intense collagen I staining was demonstrated within the hyalinization reaction in an onionskin-like layered fashion. In addition, dual immunofluorescence-stained sections for proteoglycans revealed both chondroitin sulfate and keratan sulfate deposition in the hyalinized tissue. CONCLUSIONS AND RELEVANCE: The tissue of the hyalinization reaction appears to be composed of collagen I, chondroitin sulfate, and keratan sulfate, which are known to act as molecular barriers. This observation suggests that the hyalinization reaction may limit the diffusion of toxic substances produced by otosclerotic bone into the soft tissues and fluids of the cochlea.

Cochlear nucleus neuron analysis in individuals with presbycusis.

OBJECTIVES/HYPOTHESIS: The aim of this study was to analyze the cochlear nucleus neuron population in individuals with normal hearing and presbycusis. STUDY DESIGN: Retrospective study of archival human temporal bone and brain stem tissues. METHODS: Using strict inclusion criteria, the temporal bones and cochlear nuclei from six normal hearing individuals and four individuals with presbycusis were selected for analysis. The spiral ganglion cell population, the cochlear nucleus neuron population, and the cell body size of the neurons were quantified in these cases. RESULTS: A relationship was not observed between age and the spiral ganglion cell population in the normal hearing group. Presbycusis subjects exhibited a reduced spiral ganglion cell population. The mean cochlear nucleus neuron population was observed to be significantly higher in the presbycusis group (mean ± standard deviation: 114,170 ± 10,570) compared to the normal hearing group (91,470 ± 9,510) (P = .019). This difference was predominantly the result of greater multipolar and granule cell neuron populations. Only the fusiform neuron type exhibited a significantly different mean cell body cross-sectional area between the normal hearing group (242 ± 27) and the presbycusis group (300 ± 37) (P = .033). CONCLUSIONS: This investigation is the first time, to our knowledge, that the populations of the eight neuron types in the cochlear nucleus have been quantified in both normal hearing individuals and individuals with presbycusis. The data support the concept that presbycusis is not an effect of aging alone but instead may be a condition that predisposes one to hearing loss with advancing age and is characterized by a congenitally elevated cochlear nucleus neuron population.

Duplex real-time PCR assay for quantifying mitochondrial DNA deletions in laser microdissected single spiral ganglion cells.

Laser microdissection (LMD) has been used to isolate groups of cells and single cells from numerous tissues. In this chapter, we describe a technique for isolating individual spiral ganglion cells from archival formalin-fixed, celloidin-embedded (FFCE) human temporal bone sections. The DNA isolated from these single cells is suitable for analysis with a duplex real-time polymerase chain reaction (PCR) methodology to quantify the mitochondrial DNA (mtDNA) deletion level present.

Dual immunofluorescence staining of proteoglycans in human temporal bones.

OBJECTIVES/HYPOTHESIS: Immunofluorescence staining methods have been developed to study the distribution of macromolecules in archival formalin-fixed celloidin-embedded human temporal bone tissues. The aim of this study was to investigate the feasibility of utilizing this approach to evaluate the codistribution of more than one molecule of interest in a single tissue section. STUDY DESIGN: Retrospective study of proteoglycan codistribution in archival human temporal bone tissues. METHODS: The chondroitin sulfate and keratan sulfate proteoglycans were selected for evaluating this methodology. Human tissues with known proteoglycan staining patterns were studied as controls. Thirty-one formalin-fixed celloidin-embedded archival human temporal bones were evaluated, and the observations in 11 specimens are described. A dual immunofluorescence staining method was developed using primary antibodies of differing isotypes and secondary antibodies labeled with fluorophores having nonoverlapping emission characteristics. RESULTS: The specificity of the dual immunofluorescence technique for chondroitin sulfate and keratan sulfate proteoglycans was demonstrated in control tissues and confirmed through inhibition studies. The normal human tectorial membrane exhibited intense chondroitin sulfate staining. Cochlear and vestibular hair cells exhibited predominantly keratan sulfate staining. Keratan sulfate staining predominated in spiral ganglion cell bodies and fibers. Alterations in the normal distribution pattern of proteoglycans were observed in cases of presbycusis and otosclerosis. CONCLUSIONS: The dual immunofluorescence staining methodology can be used to study archival formalin-fixed celloidin-embedded human temporal bone tissues. This technique may be applied to the evaluation of other molecules in archival human temporal bone tissues and lead to improvement in our understanding of the function of these molecules and their role in disease processes.

Proteomic analysis of formalin-fixed celloidin-embedded whole cochlear and laser microdissected spiral ganglion tissues.

CONCLUSION: The results of this study demonstrate that proteomic analysis can be successfully performed on formalin-fixed celloidin-embedded (FFCE) archival human cochlear tissues. OBJECTIVE: To investigate the feasibility of analyzing protein expression in archival cochlear tissues. MATERIAL AND METHODS: A new methodology, referred to as Liquid Tissue(TM), was used to extract proteins from human cochlear tissue sections and spiral ganglion tissue isolated by laser microdissection (LMD). Protein identification was performed by bioinformatic analysis of high resolution tandem mass spectrometric data from fractionated tryptic peptide samples. RESULTS: Twenty-six proteins were identified with a minimum of 2 unique peptides and 450 proteins were identified with 1 unique peptide at a confidence level of 95% in cochlear tissue. Ten proteins were identified with a minimum of 2 unique peptides and 485 proteins were identified with 1 unique peptide at a confidence level of 95% in spiral ganglion tissue.

Major arc mitochondrial DNA deletions in cytochrome c oxidase-deficient human cochlear spiral ganglion cells.

CONCLUSIONS: This study suggests that cytochrome c oxidase subunit 3 (COX 3) expression is diminished in spiral ganglion cells from individuals with presbycusis. In addition to the mitochondrial DNA (mtDNA) common deletion (CD), other deletions involving the mtDNA major arc contribute to the observed deficit in COX 3 expression. OBJECTIVES: To assess COX 3 deficiency in spiral ganglion cells from individuals with presbycusis and to determine whether deletions other than the CD contribute to this deficiency. METHODS: COX 3 immunofluorescence staining of archival human temporal bone tissue sections from individuals with presbycusis and from age-matched normal-hearing individuals was performed and the intensity of spiral ganglion cell immunostaining was measured. Single COX 3-deficient spiral ganglion cells were isolated by laser microdissection (LMD) and the DNA was analyzed with duplex real-time PCR assays to assess the CD level and the total mtDNA major arc deletion level. RESULTS: A statistically significant difference (p = 0.021) in the mean intensity of COX 3 immunofluorescence staining of spiral ganglion cells was observed between individuals with presbycusis and normal-hearing controls. The total mtDNA major arc deletion level was greater than the CD level in COX 3-deficient spiral ganglion cells.

Quantification of the mitochondrial DNA common deletion in presbycusis.

OBJECTIVES/HYPOTHESIS: This study was conducted to evaluate the association between the mitochondrial DNA (mtDNA) common deletion (CD) level in cochlear tissue and the severity of hearing loss in individuals with presbycusis. STUDY DESIGN: Nineteen individuals with presbycusis, ranging from 60 to 87 years of age, who met strict audiometric criteria were compared with four age frequency-matched normal hearing controls ranging from 51 to 76 years of age. Five additional normal hearing individuals, ranging from 9 to 50 years of age, were also studied. METHODS: A duplex real time polymerase chain reaction assay was used to quantify the mtDNA in archival cochlear tissue samples. Linear regression models were used for comparison of the CD level between groups. RESULTS: The presbycusis group had a mean CD level of 32% with a standard deviation of 14%, and the normal hearing age matched control group had a mean CD level of 12% with a standard deviation of 2%. This difference in CD levels reached statistical significance (P = .011) and remained significant after adjusting for any differences in age between the two groups (age-adjusted P = .007). Furthermore, there was evidence for a significant association between the CD level and the severity of hearing loss based on audiometric thresholds at 8 kHz (r = 0.44, P = .034; age-adjusted partial correlation = 0.55, P = .007). CONCLUSIONS: For the first time, to our knowledge, these results demonstrate a relationship between quantitatively measured levels of the CD in human cochlear tissue and the severity of hearing loss in individuals with presbycusis. Laryngoscope, 2009.

Technical report: laser microdissection of cochlear structures from celloidin embedded human temporal bone tissues and detection of the mitochondrial DNA common deletion using real time PCR.

Laser microdissection (LMD) has been used to isolate groups of cells and single cells from numerous tissues. In this study, we describe a technique for isolating cochlear structures and individual spiral ganglion cells from archival celloidin embedded human temporal bone sections. The specimens isolated are suitable for quantifying the mitochondrial DNA (mtDNA) common deletion (CD) within these tissues using a real time polymerase chain reaction (PCR) assay. The results presented in this manuscript demonstrate the feasibility of using this LMD technique to study the accumulation of mtDNA deletions in diseases of the ear. To our knowledge, this approach to analyzing archival human temporal bone tissues has not been previously reported.

Technical report: immunofluorescence and TUNEL staining of celloidin embedded human temporal bone tissues.

The large archival human temporal bone collections of the world have been fixed in formalin and embedded in celloidin. These treatments have created challenges to the use of contemporary probes, which are routinely used in the evaluation of fresh and frozen tissues, for the analysis of archival temporal bone tissues. Formalin alters the configuration of proteins and can obscure antigens by modifying the epitopes recognized by antibodies. Celloidin embedding provides superior support of the delicate membranous structures of the inner ear to maintain tissue integrity during sectioning, however, inadequate removal of celloidin may limit tissue permeability resulting in poor penetration of large molecules. Methods are described in this manuscript that have allowed reproducible immunofluorescence and TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) staining results in these archival tissues. To our knowledge, successful immunofluorescence staining of type I collagen, immunofluorescence staining of cytochrome c oxidase subunit III (COX III), and TUNEL staining in archival human temporal bone tissues with confocal microscopy has not been previously reported. These results demonstrate the utility of developing techniques to evaluate the existing collections of archival temporal bones which remain our greatest source of tissue for investigating the causes of ear diseases.

Detection of mitochondrial DNA deletions in the cochlea and its structural elements from archival human temporal bone tissue.

Large-scale deletions of mitochondrial DNA (mtDNA) have been associated with aging and disease in post-mitotic tissues. These post-mitotic tissues, including skeletal muscle, heart and brain, are heavily dependent on intact functional mitochondria. The cochlear tissues are known to contain an abundance of mitochondria. This observation stimulated a search for mtDNA deletions in the cochlea and its elements using a sensitive nested PCR methodology and long range PCR to explain the functional deficits observed in age-related hearing loss. The presence of the so-called "common" deletion (CD) was detected in cochlear tissue from two individuals with age-related hearing loss, 73 and 78 years of age. Three additional deletions, that to our knowledge have not been previously reported, were also identified in these two individuals, including a 5354 bp deletion flanked with a 3 bp repeat, a 9682 bp deletion flanked by a 10 bp repeat and a 5142 bp deletion without a flanking repeat. The 9682 and 5142 bp deletions were also detected in an individual 39 years of age with normal hearing, however, these two deletions were not detected in a normal hearing individual 9 years of age. In contrast, the 5354 bp deletion was detected in all four of the individuals studied. To localize the deletions within the cochlea, the cochlear elements were removed by laser capture microdissection (LCM) and the mtDNA from these tissues was studied. The 5142 and 5354 bp deletions were detected in the organ of corti, spiral ligament, and ganglion cells, but not in the stria vascularis. These findings correlate with the reduction in the number of spiral ganglion cells and outer hair cells, and the normal stria vascularis volume observed in this individual. All four of these deletions involve the cytochrome c oxidase (COX) subunit III gene, encoded by mtDNA. These observations suggest that multiple mtDNA deletions may contribute to a deficit in mitochondrial function in the cochlea and result in hearing loss if a level of physiological significance is reached.

Presbycusis: a human temporal bone study of individuals with downward sloping audiometric patterns of hearing loss and review of the literature.

OBJECTIVE: The purpose of this retrospective case review was to identify patterns of cochlear element degeneration in individuals with presbycusis exhibiting downward sloping audiometric patterns of hearing loss and to correlate these findings with those reported in the literature to clarify conflicting concepts regarding the association between hearing loss and morphologic abnormalities. METHODS: Archival human temporal bones from individuals with presbycusis were selected on the basis of strict audiometric criteria for downward-sloping audiometric thresholds. Twenty-one temporal bones that met these criteria were identified and compared with 10 temporal bones from individuals with normal hearing. The stria vascularis volumes, spiral ganglion cell populations, inner hair cells, and outer hair cells were quantitatively evaluated. The relationship between the severity of hearing loss and the degeneration of cochlear elements was analyzed using univariate linear regression models. RESULTS: Outer hair cell loss and ganglion cell loss was observed in all individuals with presbycusis. Inner hair cell loss was observed in 18 of the 21 individuals with presbycusis and stria vascularis loss was observed in 10 of the 21 individuals with presbycusis. The extent of degeneration of all four of the cochlear elements evaluated was highly associated with the severity of hearing loss based on audiometric thresholds at 8,000 Hz and the pure-tone average at 500, 1,000, and 2,000 Hz. The extent of ganglion cell degeneration was associated with the slope of the audiogram. CONCLUSIONS: Individuals with downward-sloping audiometric patterns of presbycusis exhibit degeneration of the stria vascularis, spiral ganglion cells, inner hair cells, and outer hair cells that is associated with the severity of hearing loss. This association has not been previously reported in studies that did not use quantitative methodologies for evaluating the cochlear elements and strict audiometric criteria for selecting cases.

Questioning the relationship between cochlear otosclerosis and sensorineural hearing loss: a quantitative evaluation of cochlear structures in cases of otosclerosis and review of the literature.

BACKGROUND: The literature provides conflicting information regarding the prevalence and cause of sensorineural hearing loss (HL) in individuals with otosclerosis. OBJECTIVE: The purpose of this study was to further evaluate the association between involvement of the cochlear endosteal layer with otosclerosis and sensorineural HL. STUDY DESIGN: Retrospective case review. METHODS: Temporal bones and audiograms from five individuals with otosclerosis were evaluated. The cochlear elements were quantified. The location and extent of the cochlear element loss was correlated with the location and extent of endosteal involvement with otosclerosis. RESULTS: A reduction in the population of cochlear elements was observed in most individuals; however, the reduction was not proportional to the extent of endosteal involvement with otosclerosis. The cochlear elements remained normal adjacent to some areas of endosteal involvement with otosclerosis. One individual with extensive cochlear otosclerosis had normal hearing and predominantly normal cochlear elements. CONCLUSION: This study demonstrates a variable amount of degeneration of the cochlear elements in individuals with otosclerotic involvement of the endosteum. The reduction in the population of cochlear elements was not related to the extent and location of endosteal involvement with otosclerosis. These findings suggest that factors that limit the effect of otosclerotic endosteal involvement on the cochlear elements or processes that effect the cochlear elements directly and are independent of bone involvement may be present.

Presbycusis: a human temporal bone study of individuals with flat audiometric patterns of hearing loss using a new method to quantify stria vascularis volume.

OBJECTIVE: The purpose of this study was to determine the prevalence of stria vascularis atrophy in individuals with presbycusis and flat audiometric patterns of hearing loss. Individuals with presbycusis have historically been categorized by the shape of their audiograms, and flat audiometric thresholds have been reported to be associated with atrophy of the stria vascularis. Stria vascularis volume was not measured in these studies. STUDY DESIGN: Retrospective case review. METHODS: Archival human temporal bones from individuals with presbycusis were selected on the basis of strict audiometric criteria for flat audiometric thresholds. Six temporal bones that met these criteria were identified and compared with 10 temporal bones in individuals with normal hearing. A unique quantitative method was developed to measure the stria vascularis volume in these temporal bones. The hair cell and spiral ganglion cell populations also were quantitatively evaluated. RESULTS: Only one of the six individuals with presbycusis and flat audiometric thresholds had significant atrophy of the stria vascularis. This individual with stria vascularis atrophy also had reduced inner hair cell, outer hair cell, and ganglion cell populations. Three of the individuals with presbycusis had spiral ganglion cell loss, three individuals had inner hair cell loss, and all six individuals had outer hair cell loss. CONCLUSIONS: The results of this investigation suggest that individuals with presbycusis and flat audiometric patterns of hearing loss infrequently have stria vascularis atrophy. Outer hair cell loss alone or in combination with inner hair cell or ganglion cell loss may be the cause of flat audiometric thresholds in individuals with presbycusis.

Lactancia en la media hora postparto y su efecto en la lactancia materna exclusiva / Effect of one-half-hour post-partum breastfeeding on exclusive breastfeeding

Objetivo: Estudiar porque disminuye la frecuencia de lactancia materna exclusiva (LME) y determinar el efecto de la lactancia precoz (LP) y contacto piel a piel (CPP) en LME. Procedimiento: Grupo de estudio (n=30): contacto piel a piel y lactancia materna en la primera media hora postparto y control de LME a los 5 y 15 dias. Grupo control (n=30): igual seguimiento pero sin CPP ni LP. Estudio longitudinal prospectivo. Resultados: En el grupo de estudio la LME estaba presente en el 93,33 por ciento a los 5 días, y en el 73,33 por ciento a los 15 dias; en el grupo control la LME estaba presente en el 76,67 por ciento y 30 por ciento (p=0,05) respectivamente. Grupos de riesgo que disminuyen LME: menores de 21 años, educación secundaria, procedencia urbana marginal y primíparas. Conclusión: La LP y el CPP es determinante para garantizar LME a corto plazo.

Objective: To study the diminishing frequency of exclusive breast feeding and the effect of precocious breast feeding (PBF) and skin- skin contact (SSC) in exclusive breast feeding (EBF). Procedures: Study group (n=30): Skin to skin contact and breast feeding in the first half hour of the child birth and EBF control within 5-15 days. Control group (n=30): The same procedure but without SSC and PBE Prospective longitudinal study. Results: In the study group EBF was present in 93,33% in the first 5 days and in 73,3% in the first 15 days; in the control group EBF was present in 76,67% and 30% (p<0,05), respectively. Risk groups in which EBF decreases: Less than 21 years old, high school urban-marginal procedence and primigravid. Conclusion: The PBF and SSC is determinant to guarantee EBF in a short period.