Polyester wax: a new embedding medium for the histopathologic study of human temporal bones

BACKGROUND: Celloidin and paraffin are the two common embedding mediums used for histopathologic study of the human temporal bone by light microscopy. Although celloidin embedding permits excellent morphologic assessment, celloidin is difficult to remove, and there are significant restrictions on success with immunostaining. Embedding in paraffin allows immunostaining to be performed, but preservation of cellular detail within the membranous labyrinth is relatively poor. OBJECTIVES/HYPOTHESIS: Polyester wax is an embedding medium that has a low melting point (37 degrees C), is soluble in most organic solvents, is water tolerant, and sections easily. We hypothesized that embedding in polyester wax would permit good preservation of the morphology of the membranous labyrinth and, at the same time, allow the study of proteins by immunostaining. METHODS: Nine temporal bones from individuals aged 1 to 94 years removed 2 to 31 hours postmortem, from subjects who had no history of otologic disease, were used. The bones were fixed using 10% formalin, decalcified using EDTA, embedded in polyester wax, and serially sectioned at a thickness of 8 to 12 mum on a rotary microtome. The block and knife were cooled with frozen CO2 (dry ice) held in a funnel above the block. Sections were placed on glass slides coated with a solution of 1% fish gelatin and 1% bovine albumin, followed by staining of selected sections with hematoxylin and eosin (H&E). Immunostaining was also performed on selected sections using antibodies to 200 kD neurofilament and Na-K-ATPase. RESULTS: Polyester wax-embedded sections demonstrated good preservation of cellular detail of the organ of Corti and other structures of the membranous labyrinth, as well as the surrounding otic capsule. The protocol described in this paper was reliable and consistently yielded sections of good quality. Immunostaining was successful with both antibodies. CONCLUSION: The use of polyester wax as an embedding medium for human temporal bones offers the advantage of good preservation of morphology and ease of immunostaining. We anticipate that in the future, polyester wax embedding will also permit other molecular biologic assays on temporal bone sections such as the retrieval of nucleic acids and the study of proteins using mass spectrometry-based proteomic analysis.     

Cochlear wall erosion after cochlear implantation

OBJECTIVES: Cochlear implantation is a clinically satisfactory procedure, but it is associated with a variable degree of histologic intracochlear trauma. We report a new histologic finding in a cochlear implant specimen from the House Ear Institute collection. METHODS: An analysis of 34 temporal bones with single-channel (n = 23) or multichannel (n = 11) cochlear implants was performed. All temporal bones had been fixed for a month in 10% buffered formalin, progressively decalcified in ethylenediaminetetraacetic acid, and embedded in celloidin. After electrode removal, the bones were cut into 20-microm sections and stained. RESULTS: In 1 specimen, the implanted electrode had caused erosion of the bone through the endosteum into the marrow spaces, at the superior-anterior portion of the basal turn. This area showed an intense lymphocytic infiltration surrounded by some new bone formation. CONCLUSIONS: Trauma may provoke an inflammatory reaction due to the presence of the foreign body after violation of the endosteum.     

Ante mortem ruptures of the endolymphatic membranes in children: real finding or artifact?

We undertook this study to determine whether or not ante mortem ruptures of the endolymphatic membranes occurred naturally in the infantile labyrinth, what were the most frequent locations and to assess the statistical correlation of an earlier finding of bulging of Reissner's membrane in pediatric temporal bones. We examined 128 temporal bones from 80 children between the ages of newborn to 12 years with an average age of 15.4 months. All temporal bones were prepared by the celloidin technique and studied by light microscopy. The endolymphatic membranes of the cochlear duct, saccule, utricle and semicircular ducts were examined for ruptures. A rupture of the saccule and utricle was considered to be an ante mortem event if its edges were rolled and bound with fine adhesions and could be identified in adjacent sections. Ante mortem ruptures were seen in 11.1% of the specimens and occurred in 2.4% in Reissner's membrane, in 6.3% in the saccular membrane and in 2.4% in the utricular membrane. In these specimens, the histopathological changes included atrophy of the stria vascularis and collapse of the organ of Corti, which provided further evidence of an ante mortem event. Infants with extralabyrinthine congenital anomalies or children who were treated with chemotherapy were more likely to develop ruptures of the endolymphatic membranes if they also received aminoglycosides.     

耳蜗火棉胶包埋冰冻切片技术的应用

目的通过改进耳蜗火棉胶切片技术,比较传统火棉胶切片和耳蜗火棉胶包埋冰冻切片技术,为耳科实验室制作高质量火棉胶切片,获得高质量耳蜗组织形态学图片提供方法。方法将豚鼠耳蜗组织进行常规的梯度酒精脱水、梯度火棉胶浸润、12%的火棉胶固定制作成火棉胶组织块,然后平行于蜗轴沿耳蜗侧面切取一个平面,将耳蜗其它组织修掉使得火棉胶包埋块成为一个长方体。用OCT将火棉胶组织块固定在冰冻切片机上,进行连续切片。结果用火棉胶将耳蜗包埋成块,OCT固定冰冻切片机的模具上进行切片。不用火棉胶切片机切片,同样可以获得传统火棉胶切片方法所获得的制片质量,并且具有切片效果良好、组织切片完整、厚薄均匀、不易形成皱折等优点。还可以减少火棉胶切片的费用开销,便于耳蜗火棉胶包埋制片技术的推广和应用。结论耳蜗火棉胶包埋冰冻切片技术是耳科实验室制作高质量耳蜗火棉胶切片,获得高质量耳蜗组织形态学图片简便有效的方法。     

A new technique for the study of temporal bone pathology

A new technique for the study of the temporal bone at autopsy is presented in which thin slices of the fixed bone are prepared on a special cutting machine which preserves the configuration of the delicate anatomical structures. Selected areas are then processed for light microscopy. There is thus opportunity for gross examination, only a short period for immersion in acid for decalcification if required, and special histological procedures may also be carried out. The method is quick and technically easier than serial sectioning and storage of the smaller numbers of sections from each case is less of a problem. Temporal bones which have been perfused through the perilymph within 3 h of death may also be cut in the same way in order to allow removal of samples of membranous labyrinth for surface preparation or electron microscopy, avoiding the need for drilling to reach the membranous labyrinth, and being quicker and less damaging.     

Immunohistochemistry and microwave decalcification of human temporal bones

Processing of human temporal bones is a long, expensive process and the resulting celloidin sections are difficult to use for immunohistochemistry. We tested the ability of immunohistochemical assays to work in human temporal bones that were decalcified using a microwave oven. Tissue was trimmed to an approximate cube (1.5-2 cm/side) containing only the cochlea and immersed in fresh EDTA with paraformaldehyde every 6 h. This sized block required 190-400 h to decalcify. The decalcified tissue was embedded in paraffin and sectioned. Sections were immunoassayed with anti-cytochrome c oxidase, anti-neurofilament or anti-peripherin. All three antibodies labeled the appropriate structures. This procedure may stimulate advancement in the understanding of human inner ear pathology.     

人膜迷路三维精确建模与虚拟可视化

目的建立精确的人内耳膜迷路的三维模型，以满足内耳可视化研究和生物力学建模的需要。方法制作全系列、不染色、层厚20μm的新鲜成人颞骨火棉胶连续切片，使用幻灯机和高像素数码相机结合图像拼合技术获取全部切片的高清晰度数字图像。在3D-Doctor软件中，将全系列图像进行对位、设定标度参数、分割后，采用表面重建法生成内耳膜迷路三维模型。结果建立了能清晰显示膜迷路精细结构的内耳三维模型，在3D-Doctor软件中实现了不同的显示效果，并利用Cortona虚拟现实建模语言软件在个人电脑上实现了模型的桌面虚拟可视化。结论全系列不染色的火棉胶连续切片、数码相机拍摄结合图像拼合和高性能图像处理软件的使用可以满足膜迷路精确建模的需要，并能提高建模的精确度。利用虚拟现实软件和设备可以实现内耳膜迷路的虚拟可视化，有利于形态学研究和教学。     

颞骨病理学研究的新技术应用

目的介绍用于颞骨病理学研究的分子生物学新技术.方法所有颞骨火棉胶切片均来自解放军总医院颞骨库,基于颞骨火棉胶切片的DNA提取、PCR扩增、原位杂交、原位PCR和免疫蛋白染色技术被用来研究老年性耳聋的分子病理机制.结果除了原位PCR技术,基于颞骨火棉胶切片的DNA提取、PCR扩增、原位杂交和免疫蛋白染色技术均取得了可接受的、有用的结果来帮助解释老年性耳聋的机制.结论存档的颞骨标本是非常珍贵的资源,加以利用可以揭示各型耳聋的分子病理机制.     

3-D reconstruction of the vestibular endorgans in pediatric temporal bones

We investigated the vestibular endorgans in three children using 3-D reconstructions from histological sections. The right temporal bone of a newborn child without peripheral vestibular pathology was used as reference model and the temporal bones from a child with Goldenhar syndrome and a child with Pierre Robin sequence with known peripheral vestibular pathology were studied. All five temporal bones were prepared by the celloidin technique and sectioned at 20 microm. Each available section was digitized with a slide scanner. The imaging data were layered anatomically correctly and rendered in a 3-D software. With this technique all vestibular endorgans were reconstructed and measured. The standard deviations in distances ranged between 0.5 and 1.2% and in angles between 0.1 and 2.9 degrees. Both maculae were curved in the longitudinal and transverse axes which described a curve of approximately 35 degrees. The angles between the semicircular ducts varied between 97 and 110 degrees. The pathological models demonstrated a distorted configuration of the semicircular canals and differed substantially from the reference model in most of the measured distances and angles. The method presented is capable of generating 3-D models of the vestibular system from histological sections with an acceptable precision without previously inserted reference marks. Archival celloidin sections are widely available and will be an important resource in understanding the detailed 3-D geometry of the vestibular system which has not yet been accomplished.     

Paget disease and sensorineural hearing loss associated with spiral ligament degeneration.

HYPOTHESIS: Previously unreported cystic degeneration of the spiral ligament in cases of Paget disease. BACKGROUND: About 70% of cases of Paget disease involve the skull, with hearing affected in approximately 50% of these. The hearing impairment may be sensorineural, mixed, or, rarely, only conductive. The etiology and pathogenesis of the hearing loss are not yet understood, and reports in the literature are inconsistent regarding the pathologic changes responsible for sensorineural hearing loss. Of six pairs of temporal bones from patients with Paget disease in the temporal bone collection of a research institution, two pairs have abnormalities not previously associated with sensorineural hearing loss or Paget disease. We report the histopathologic findings in these temporal bones. METHODS: The temporal bones were fixed in formalin, decalcified in ethylenediaminetetraacetic acid, embedded in celloidin, and sectioned in the horizontal plane at a thickness of 20 microm. Every 10th section was stained with hematoxylin-eosin and mounted on glass slides. The sections were examined by light microscopy. RESULTS: Cystic degeneration of the spiral ligament, primarily in the basal segment, was found in both cases. Endolymphatic hydrops and a small endolymphatic sac with calcification of the perisaccular tissue were found in one case. CONCLUSIONS: Cystic degeneration of the spiral ligament has not been previously reported and may be unique to Paget disease. This is consistent with recent literature showing a previously unsuspected role of the spiral ligament in sensorineural hearing loss.     

Morphometry of the human cochlear wall and implications for cochlear surgery

Objective—To study the lateral wall of the cochlea in human temporal bones (TBs), in order to evaluate the feasibility of performing micro-dissection of the spiral ligament and an endosteal electrode implantation.

Material and Methods—We reviewed the database of the TB bank of The House Ear Institute in search of bones that did not distort the otic capsule or spiral ligament, and selected 36 horizontally sectioned TBs. All bones had been removed using the en bloc technique, fixed in formalin, decalcified in EDTA, embedded in celloidin and cut into 20-μm serial sections. We evaluated sections stained with hematoxylin–eosin under a microscope, and made several measurements of the lateral wall of the cochlea using optical appliances. A total of 20 measurements were made at 3 levels: midmodiolar sections; sections at the round window niche; and sections containing the anterior border of the stapes footplate.

Results—The selected sections provide useful data when applied during surgery. All the numerical data were analyzed statistically and, although individual variability occurred, most of the measurements seemed to be quite homogeneous.

Conclusions—A greater understanding of the microscopic anatomy and dimensions of the human TB will help to understand the surgical relationships at the lateral wall of the cochlea. Our study provides some numerical references for the dimensions of the cochlea that may help in cochlear surgery. These dimensions would be of particular interest in cochlear implantation, especially in the design of endosteal electrodes.     

An approach for precise three-dimensional modeling of the human inner ear

For further morphological and physiological research, it is vital to establish precise three-dimensional models of the whole inner ear including the details of the membranous components. With the system of a projector and a high-resolution digital camera, 2 complete serial unstained celloidin sections of fresh human temporal bones were digitized as high-resolution images which were then sorted, calibrated, aligned and segmented using the 3D-Doctor software. Finally, 2 precise three-dimensional models of the inner ear were generated by simple surface rendering. The contours of tiny structures such as the crista ampullaris, the macula utriculi and the macula sacculi could be observed clearly. Our study suggests that it is technically feasible to employ complete serial unstained celloidin sections for precise three-dimensional reconstruction and that this helps reduce errors and laboratory workload. Moreover, the use of a high-resolution digital camera and the autoalignment function of 3D-Doctor further increase the accuracy of the models.     

The incidence and distribution of cupular deposits in the pediatric vestibular labyrinth.

OBJECTIVES: Deposits of basophilic material on the cupulae of the semicircular ducts have been described in adult human temporal bones, and such deposits have been implicated in the clinical phenomenon of benign paroxysmal positional vertigo (BPPV). Although relatively rare, BPPV has been reported to occur in children. The goal of this study was to evaluate the occurrence of cupular deposits in pediatric material. STUDY DESIGN: Basic science, two-center histopathologic temporal bone study. METHODS: We examined 186 temporal bones from 121 individuals between the ages of newborn and 10 years. The average age was 14.7 months. All temporal bones were acquired at autopsy, embedded in celloidin, and sectioned for light microscopic study. The cupulae of the semicircular ducts were identified and any deposits found were graded according to their size and position on the cupula. RESULTS: A total of 276 cupulae were identified, and in 35 (12.7%) of these a basophilic deposit was found. In many cases the deposits contained recognizable otoconial crystals. Most deposits were small and they were almost equally distributed between the three semicircular ducts. However, the lateral and posterior cupulae were somewhat more involved than the superior cupula. Our data showed that cupular deposits are less frequently found in the pediatric labyrinth, compared with adults as reported in previous studies. CONCLUSIONS: The lower occurrence in children versus adults suggests that the deposits may be a phenomenon of the aging vestibular labyrinth.     

Improved RNA analysis for immediate autopsy of temporal bone soft tissues

RNA analysis is essential for understanding biological activities of a cell or tissue. Unfortunately, retrieval of RNA from existing archives of human temporal bones has proven extremely difficult due to degradation of RNA molecules. The major factors that contribute to degradation of RNA in specimens from autopsied temporal bones are tissue autolysis due to time elapsed before autopsy, and technical problems in processing the bones after harvest. We therefore focused on improving the survival of RNA in human temporal bones by shortening the time to autopsy and through modification of the processing technique by removing targeted tissues directly from the temporal bones and by avoiding time-consuming decalcification and celloidin-embedding. Eight temporal bones collected at immediate autopsies were used in this study. Representative mRNAs, ranging from high (MUC5B, physically unstable) to low (beta-actin, physically stable) molecular weights, and from abundant (MUC5B) to non-abundant (MUC1) RNA, were studied by in situ hybridization, Northern blot technique, or both. Using this modified protocol in autopsies performed up to 6 h after death, the existence of mRNAs was demonstrated in all bones studied. This improved method demonstrates the feasibility of the use of autopsied temporal bone tissues for RNA analysis.     

Argon Laser Irradiation to the Semicircular Canal

In order to elucidate the effects of argon laser irradiation on the lateral semicircular canal of the guinea pig, the vestibular labyrinth was histologically studied after irradiation, using the conventional celloidin method. Irrigation of the external meatus with ice water was used to evaluate the function of the semicircular canal by recording caloricnystagmus. When irradiation was performed, a laser probe was approximated to the lateral canal, 0.5 to 1 mm away from the surface of the canal. Each time, power applied was 1.0 W on the dial of the laser machine. The duration of irradiation was 0.5 s. The lateral canal was irradiated one to 15 times. Twenty-five to 87 days after irradiation, the temporal bones were fixed in Heidenhein-Susa solution, removed, and subjected to celloidin processing. The irradiated bony wall of the lateral canal demonstrated charring. Lucent areas were observed around and under the charred area. The semicircular duct showed shrinkage with disappearance of the trabecular mesh. New bone formation was observed along the endosteum of the irradiated area. The lateral canal was completely occluded by ossification with or without fibrosis when sufficient energy was applied. The anterior and posterior canals were normal. Caloric tests using 5 mL of ice water for 5 s failed to elicit nystagmus on the irradiated side.     

Ultrastructural characterization of otospongiotic lesions in re-embedded celloidin sections

A technique of using re-embedded celloidin sections for ultrastructural analysis was used for the study of otospongiosis in human temporal bones. Celloidin sections stored in 80% alcohol with active lesions of cochlear otospongiosis were processed and re-embedded in epoxy resin. Semithin and thin sections were cut and analysed for a characterization of the ultrastructural cellular histopathology. The predominant cell types were found to be osteoblasts/osteocytes and macrophages. Lymphocytes were also noted but were rare. Several osteoblasts showed signs of active collagen and bone matrix production, indicative of ongoing new bone formation and repair. Macrophages often interacted physically to form cell clusters. The macrophages were frequently observed to endocytose the non-mineralized bone matrix as well as to degrade mononuclear cells presumed to represent osteoblasts. The observations may support the notion that increased osteolysis in active ostospongiosis is partly caused by a recruited osteoclast activity and partly by an impaired bone repair mechanism due to a macrophage digestion of osteoblast-deposited non-mineralized bone matrix. These two conditions may act in concert with cellular degradation of bone-producing cells.     

The pathology of the temporal bones of a child with acquired cytomegalovirus infection: studies by light microscopy, immunohistochemistry and polymerase-chain reaction

STUDY DESIGN: The first case of an acquired cytomegalovirus (CMV) infection of the inner ear is reported in a 3-year-old girl in remission from acute lymphocytic leukemia. METHODS: Horizontal sections of the temporal bones were studied by light microscopy and immunohistological staining by avidin-biotin-complex-technique was performed on selected archival sections. Three sections were processed for detection of the virus genome by the polymerase chain reaction (PCR). RESULTS: By light microscopy the epithelium of the endolymphatic sac, the utricle and the semicircular canals showed deeply stained acidophilic inclusions and the stria vascularis had a loose structure especially in the intermediate layer. The changes were limited to the non-sensory parts of the labyrinth and no CMV type cells were observed in the organ of Corti. There was a loss of inner and outer hair cells and loss of cochlear ganglion cells caused by either the virus or treatment with gentamicin. Standard immunohistochemistry failed to demonstrate staining with CMV antibodies, but PCR, demonstrated CMV-DNA in one section. CONCLUSION: Molecular techniques may be able to detect acquired CMV infections in archival pediatric bones temporal bones. The histologic findings in the labyrinth were milder, however showed some similarity to children with congenital CMV labyrinthitis.     

Antigen retrieval immunohistochemistry used for routinely processed celloidin-embedded human temporal bone sections: standardization and development

The use of immunohistochemistry (IHC) in routinely processed celloidin-embedded human temporal bone section has created a fruitful field in understanding the pathogenesis and pathophysiology of the human inner ear at a molecular level since the early 1990s when the antigen retrieval (AR) technique was developed. This review article focuses on several critical technical issues of AR technique based predominantly on our experiences and suggestions concerning further development and standardization of AR-IHC for IHC study of human temporal bone section, as well as other tissues embedded in celloidin. Examples of using AR-IHC in human temporal bone sections collected include our unpublished data in order to indicate the potential utility of this novel method. Suggestions of further development of AR techniques are proposed for references and comments.     

The shortened cochlea: its classification and histopathologic features

INTRODUCTION: The term 'Mondini dysplasia' has been used to describe virtually any congenital abnormality of the osseous labyrinth resulting in confusion and seemingly contradictory observations and conclusions about this type of deformity. The purpose of this study is to histopathologically classify and describe temporal bones whose cochleas have less than 2.5 turns. METHODS: Of the 1800 temporal bones in our collection, 21 from 12 cases were found to have cochleas with less than 2.5 cochlear turns. Ages ranged from stillborn to 50 years. Temporal bones were harvested at autopsy, processed and embedded in celloidin. Sections were cut at a thickness of 20 microm and every 10th section stained with hematoxylin-eosin and examined using light microscopy. The number of turns, length of cochlea, integrity of cochlear base, length of modiolus, abnormalities of the semicircular canals and vestibule, enlargement of the vestibular aqueduct and middle ears were documented. Twenty-one temporal bones from age-matched patients without cochlear deformities were used as controls for modiolar length measurements. RESULTS: Malformation of the shortened cochlea was histopathologically classified into three groups as follows: (1) Common cavity, cochlear dysplasia (one ear)--severe dysplasia of the cochlea without a complete basal turn; (2) Mondini dysplasia (11 ears)--1.5 cochlear turns, a complete basal turn, an incomplete or absent interscalar septum and a complete bone at the base of the modiolus; and (3) Mondini-like dysplasia type A (five ears)--2 turns to the cochlea including a complete basal turn and complete bone at the base of the modiolus; and type B (four ears)--1.5-2 turns to the cochlea, hypoplasia of or a missing bone at the base of the modiolus (either with or without a communication between the internal auditory canal and the cochlea) and a complete basal turn. CONCLUSION: The range of congenital malformations in short cochlea is highly variable. Fundamental to the accurate evaluation of a labyrinthine anomaly, malformations of the inner ear should be classified according to the findings in the labyrinth. We suggest the use of common cavity cochlear dysplasia, Mondini dysplasia and Mondini-like dysplasia to describe these variable anomalies.