Celloidin-embedded Human Temporal Bone Sections Research Articles

Immunohistochemical location of Na+, K+-ATPase α1 subunit in the human inner ear

Na+, K+-ATPase (Na,K-ATPase) is an ubiquitous enzyme in the inner ear and a key factor in the maintenance of the osmotic gradient of the endolymph. This study uses Na,K-ATPase α1 subunit immunoreactivity (IR) to identify cellular structures in the normal and disease human cochlea. Formalin-fixed celloidin-embedded (FFCE) human temporal bone sections were immunoreacted with mouse monoclonal antibodies against Na,K-ATPase α1 subunit. Na,K-ATPase α1 IR was examined in the cochlea of 30 patients: four with normal hearing, 5 with Meniere's disease, and 21 with other inner ear diseases: 11 male, 19 female; ages 42 to 96 years-old (yo), average age of 77 yo. Na,K-ATPase α1 IR area was quantified using the ImageJ software program. Na,K-ATPase α1 IR was located in the stria vascularis, and in type I, II and IV fibrocytes of the spiral ligament in the cochlea from patients with normal hearing. Na,K-ATPase α1 IR was seen in Deiters's cells and inner phalangeal cells of the organ of Corti. Na,K-ATPase α1 IR was present in satellite cells that surround the neurons of the spiral ganglia. In the inner ear of pathological specimens, Na,K-ATPase IR area was decreased (compared to the normal) in the stria vascularis, supporting cells in the organ of Corti and satellite cells of the spiral ganglia. These results show that Na,K-ATPase α1 IR is a good marker to identify cellular structures of the human inner ear and may be used to study cellular changes in the cochlea associated with aging and disease. The ubiquitous localization of Na,K-ATPase α1 in the human cochlea is consistent with the Na,K-ATPase role in ionic homeostasis and osmolarity, similar to that seen in animal models.

Immunohistochemical localization of Nrf2 in the human cochlea

Oxidative stress plays an important role in several inner ear diseases and normal aging. Nuclear (erythroid-derived 2)-like 2, also known as Nrf2, is a transcription factor encoded by the NFE2L2 gene that controls the expression of genes necessary to reduce oxidative stress. There are no studies to the date on the localization of Nrf2 in the human inner ear in normal or pathological conditions. Therefore, we investigated the immunohistochemical localization of Nrf2 in the human cochlea and vestibule using formalin-fixed celloidin-embedded human temporal bone sections. Nrf2 immunoreactivity (IR) was found in the inner and outer hair cells and supporting cells of the organ of Corti throughout the cochlea. Nfr2-IR was also found in hair cells and supporting cells of the maculae and cristae vestibular sensory epithelia. Nrf2-IR was decreased in the organ of Corti of older age individuals. The immunolocalization of Nrf2 in both auditory and vestibular sensory epithelia suggest that this transcription factor may play a relevant role in protecting sensory hair cells from oxidative stress.

R450 – Manganese Superoxide Dismutase in Spiral Ganglion Cells

Problem Manganese superoxide dismutase (Mn SOD2) is a key metabolic anti-oxidant enzyme of the superoxide dismutase family for detoxifying the free radical cascade inside the mitochondria of the cochlea via activation of downstream uncoupling proteins. Copper/zinc superoxide dismutase (Cu/Zn SOD1) is localized in the cytoplasm. This study examined whether the pattern of expression of these SODs in the cochlea is correlated with the differential cellular vulnerability found in basal versus apical turn of the cochlea. Methods Immunohistochemical methods were used to identify the distribution of Mn SOD2 and Cu/Zn SOD1 in paraffin embedded sections of paraformaldehyde fixed formic acid decalcified temporal bones from mice, rats, and macaques; and special archival celloidin-embedded human temporal bone sections. Results In mice, rats and macaques, both the proportion of Mn SOD2 immunopositive type 1 spiral ganglion cells and the intensity of immunoreactivity were elevated near the cochlear apex. Strongly stained Mn SOD2 type 1 spiral ganglion cells were also observed in archival human temporal bone sections. In contrast, the Cu/Zn SOD1 immunopositive type 1 spiral ganglion cells were distributed identically across cochlear turns in rats and macaques. Conclusion These findings suggest that spiral ganglion cellular responses to ROS exposure may vary along the cochlear spiral, with a lower response capacity in the basal turn. Significance Hair cells and spiral ganglion cells appear to be more vulnerable to ototoxins at the base of the cochlea than at the apex. Our data raises the general hypothesis that a lower Mn SOD2 anti-oxidative capacity at the cochlear base could contribute to the high frequency hearing loss seen in presbycusis and ototoxin-induced hearing loss. The conservative pattern of Mn SOD2 immunostaining across species further suggests that it may be a fundamental mechanism in ROS metabolism and signaling. Support PA Lions Hearing Research Foundation, American Otologic Society Research Fellowship.

Inflammatory response to chronic otitis media in DiGeorge syndrome: a case study using immunohistochemistry on archival temporal bone sections.

Immunohistochemical analyses were conducted on archival celloidin-embedded human temporal bone sections from an 8-month-old boy with chronic otitis media and DiGeorge syndrome. We employed antigen retrieval methods with saturated sodium hydroxide-methanol solution, microwave incubation, and proteolytic treatment to demonstrate the distribution of T-lymphocytes, B-lymphocytes, macrophages, and intercellular adhesion molecule 1 (ICAM-1) expression in the middle ear. B-lymphocytes and macrophages were observed predominantly within the middle ear mucosa. T-lymphocytes were rare. Further, ICAM-1 was expressed in the vascular endothelium of the lamina propria, as well as infiltrating mononuclear cells. This suggests that the expression of ICAM-1 can be induced in the middle ear with otitis media, even if T-lymphocytes are depressed in a cell-mediated immunodeficiency disorder such as DiGeorge syndrome.

Amplification of RNA from archival human temporal bone sections.

The amplification of DNA from celloidin-embedded human temporal bone sections by polymerase chain reaction (PCR) has been applied to some auditory diseases, such as herpes zoster oticus and hearing loss caused by the mutations of mitochondrial DNA. However, few studies have reported detection of RNA from temporal bone sections. Because RNA analysis from temporal bone sections may elucidate the development of the diseases in the auditory, vestibular, and facial nerves, the authors investigated whether RNA in these sections can be amplified by reverse transcription (RT)-PCR. Sections that were formalin-fixed, decalcified, and embedded between 1972 and 1986 were used. Nucleic acid was extracted from the celloidin-embedded temporal bone sections and subjected to RT-PCR. Human alpha-tubulin RNA was reverse transcribed to cDNA and amplified by nested PCR using two sets of primers that were designed to distinguish cDNA from genomic DNA based on the presence of an intron between the primers. Human alpha-tubulin RNA was detected in 11 of 14 temporal bone sections (79%) by RT-PCR. RNA was detected in even the oldest sections, which were processed in 1972. These results indicate that RNA can be analyzed from archival celloidin-embedded human temporal bone sections.

Application of labeling techniques to archival temporal bone sections.

Pathology of the human inner ear has traditionally been studied in celloidin-embedded, hematoxylin and eosin-stained sections of the temporal bone. Although the traditional histologic approach has yielded valuable information, it is now possible to extend these studies to include analysis of molecules using immunohistochemical and histochemical staining techniques. Fourteen antibodies and 6 lectins have been applied to 420 archival, celloidin-embedded human temporal bone sections. Tissues had been fixed in 10% formalin, embedded in celloidin, sectioned, and stored for as many as 40 years. The staining intensities varied among sections, so they were ranked from 'no label" to "dense label." To investigate the relationships between the extent of postmortem changes (PMCs), storage time, and staining intensity for each antibody, the sections were graded according to their PMCs, which ranged from good preservation of the temporal bone histologic structure to severe postmortem autolysis. Although statistical analysis indicated that both extent of PMCs and storage time in general decrease the staining intensity, both poorly fixed tissue and sections stored for a long time can yield good immunostaining results with some antibodies.

Molecular temporal bone pathology: IV. Analysis of DNA template length using mitochondrial PCR primers.

The focus of this study was to identify, via molecular biology techniques, the length of the DNA templates present in individual archival celloidin-embedded human temporal bone sections. Earlier studies have suggested that the maximum template length present in these tissues is on the order of 471 base pair (bp). Polymerase chain reaction (PCR) amplification of 92 bp, 121 bp, 471 bp, and 609 bp regions of mitochondrial DNA (mtDNA), extracted from single archival celloidin-embedded human temporal bone sections, was used to assess the length of the template DNA extracted. These data are crucial to determine the limits of applying PCR technology to amplify specific genomic DNA targets located within the human inner ear. The results described should be of value to those investigators extracting DNA from archival individual human temporal bone sections for polymerase chain reaction assays of specific genetic alterations or infectious agents associated with temporal bone pathologies.

Ribonucleases may limit recovery of ribonucleic acids from archival human temporal bones.

Messenger ribonucleic acid (mRNA) for actin was detected in celloidin-embedded archival human temporal bone sections with reverse transcription polymerase chain reaction (RT-PCR). Actin mRNA was detected in 10% of sections analyzed. One possible reason for this modest detection incidence is enzymatic degradation of RNA by exogenously introduced ribonucleases (RNases). We have identified steps of the temporal bone processing protocol for archival storage in which exogenous RNases could be introduced to the tissue, and have verified that the bone sections are exposed to these enzymes. We have demonstrated that implementing precautions to minimize exogenous RNase contamination during processing improves recovery of intact RNA. This study indicates that although gene expression analysis of archival human temporal bones may be limited by enzymatic degradation of RNA, simple modification of processing protocol can improve yield of informative data.

Application of labeling techniques to archival temporal bone sections

Otolaryngology–Head and Neck SurgeryVolume 117, Issue 2 p. P83-P83 Original Article Application of Labeling Techniques to Archival Temporal Bone Sections Qing Tian MD, Qing Tian MD (presenter) Los Angeles and La Jolla, Calif.Search for more papers by this authorFred H. Linthicum MD, Fred H. Linthicum MD Los Angeles and La Jolla, Calif.Search for more papers by this authorElizabeth Keithley PhD, Elizabeth Keithley PhD Los Angeles and La Jolla, Calif.Search for more papers by this author Qing Tian MD, Qing Tian MD (presenter) Los Angeles and La Jolla, Calif.Search for more papers by this authorFred H. Linthicum MD, Fred H. Linthicum MD Los Angeles and La Jolla, Calif.Search for more papers by this authorElizabeth Keithley PhD, Elizabeth Keithley PhD Los Angeles and La Jolla, Calif.Search for more papers by this author First published: 01 August 1997 https://doi.org/10.1016/S0194-59989780104-0Read the full textAboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume117, Issue2August 1997Pages P83-P83 RelatedInformation

Polymerase chain reaction amplification of DNA from archival celloidin-embedded human temporal bone sections.

A method was developed for fast and efficient isolation of DNA from formalin-fixed, decalcified, celloidin-embedded human temporal bone sections for subsequent use in polymerase chain reaction (PCR) DNA amplification. The method relies on the use of an enzymatic digestion with proteinase K to release and solubilize the patient's DNA from an individual 20- to 25-microns temporal bone section. The method described should be of great value to those investigators extracting DNA from archival individual human temporal bone sections for polymerase chain reaction assays of specific genetic alterations associated with temporal bone pathologies. The molecular characterization of viral infections, oncogenes, or other etiological agents of disease using PCR could provide important information regarding the etiopathogenesis of many auditory, vestibular, and facial nerve disorders, such as autoimmune hearing loss, congenital hearing losses, Meniere's disease, otosclerosis, or Bell's palsy.

Immunohistochemical study of intermediate filament proteins on routinely processed, celloidin-embedded human temporal bone sections by using a new technique for antigen retrieval.

Although immunohistochemical studies of intermediate filament proteins have been carried out on temporal bone sections by using modified fixation/embedding techniques to preserve antigenicity, there have been no light microscopic studies concerning immunohistochemical staining on routinely formalin-fixed celloidin-embedded human temporal bone sections. A method for immunostaining routinely processed celloidin-embedded tissues would be extremely valuable in that it would permit study of the extensive collections of formalin-celloidin temporal bone specimens that exist in major centers of otopathologic research. Recently, we have developed a new technique which can be used to retrieve the antigenicity masked by formalin fixation and decalcification. This method requires immersing slides for 30 min at room temperature in a solution of saturated sodium hydroxide in methanol before immunostaining. Using this method, 45 celloidin-embedded human temporal bone sections were stained with monoclonal antibodies to keratin, vimentin, neurofilament, glial fibrillary acidic protein and desmin as primary antibodies using a sensitive streptavidin-biotin procedure. The results obtained by using this technique are at least equivalent to those obtained with modified fixatives, cryosections or immuno-electron microscopy. This new method may provide a useful approach for studying routinely processed, celloidin-embedded human temporal bone sections and open a new field in immuno-otopathology.

S-100 protein in human inner ear: use of a novel immunohistochemical technique on routinely processed, celloidin-embedded human temporal bone sections.

The authors recently developed a new antigen retrieval technique which allows immunohistochemical detection of proteins in routinely processed, celloidin-embedded human temporal bone sections. This technique was used in the present study, which reports the occurrence of S-100 protein in the human inner ear. Fifteen celloidin-embedded human temporal bone sections, obtained from the Eastern Temporal Bone Bank at the Massachusetts Eye and Ear Infirmary, were tested with the monoclonal antibody to S-100. This protein was found in the spiral ligament, in Reissner's membrane, in the spiral limbus, and in the basement membrane of the cochlea. S-100-positive, thin fibers could be seen as supporting elements in the acoustic nerve and the facial nerve. This protein was localized along the surface of vestibular wall. The new technique provides an immunohistochemical approach to studying routinely processed human temporal bone sections and may prove useful in the field of immuno-otopathology.

A technique for retrieving antigens in formalin-fixed, routinely acid-decalcified, celloidin-embedded human temporal bone sections for immunohistochemistry.

The application of immunohistochemistry to routinely decalcified, celloidin-embedded human temporal bone sections has been hampered because of antigen loss during processing of the specimens. To our knowledge, there has been no published report to date describing immunohistochemical staining of such tissues suitable for examination by light microscopy. Here we report a novel antigen retrieval technique which can be successfully used to stain a variety of antigens in routinely formalin-fixed, trichloroacetic acid-decalcified, celloidin-embedded human temporal bone sections. The new procedure reported here for decalcified human temporal bone tissues simply requires immersing slides for 30 min at room temperature in an antigen retrieval solution. A total of 60 decalcified, celloidin-embedded human temporal bone tissues were tested with monoclonal antibodies (MAb) to 15 different antigens. Of these, 12 MAb showed definite positive staining, while three were negative. This technique may prove very useful in studying the expression of various antigens by immunohistochemistry in formalin-fixed, acid-decalcified, celloidin-embedded tissues.