Ewing sarcoma with myxoid stroma: Case report of an unusual histological variant.

We describe the case of a Ewing sarcoma with prominent myxoid stroma of the temporal bone in a 26-year-old female. Histologically, the tumor exhibited a fascicular growth pattern of round to spindled cells with a minimal amount of pale eosinophilic to clear cytoplasm and oval or spindled nuclei with finely dispersed chromatin and small nucleoli. Myxoid changes were prominent (>50%), with reticular or pseudoacinar growth of the loosely cohesive cells. The tumor showed strong expression of CD99, FLI1, and CD56 and was positive for the EWSR1-FLI1 fusion transcript. The diagnosis of Ewing sarcoma with myxoid stroma (myxoid variant) is particularly problematic owing to the large number of potential mimics. The tumor extends the morphologic spectrum of Ewing sarcoma beyond the previously described histological variants, and broadens the differential diagnosis. For any round/spindle cell sarcoma, prominent myxoid stroma and CD99 immunoreactivity should prompt consideration for molecular studies that include analysis of both EWSR1 and FLI1.

Tympanic membrane surface motions in forward and reverse middle ear transmissions.

Characterization of Tympanic Membrane (TM) surface motions with forward and reverse stimulation is important to understanding how the TM transduces acoustical and mechanical energy in both directions. In this paper, stroboscopic opto-electronic holography is used to quantify motions of the entire TM surface induced by forward sound and reverse mechanical stimulation in human cadaveric ears from 0.25 to 18.4 kHz. The forward sound stimulus was coupled to an anatomically realistic artificial ear canal that allowed optical access to the entire TM surface, and the reverse mechanical stimulus was applied to the body of the incus by a piezo-electric stimulator. The results show clear differences in TM surface motions evoked by the two stimuli. In the forward case, TM motion is dominated by standing-wave-like modal motions that are consistent with a relatively uniform sound-pressure load over the entire TM surface. With reverse mechanical stimulation, the TM surface shows more traveling waves, consistent with a localized mechanical drive applied to the manubrium embedded in the TM. With both stimuli, the manubrium moves less than the rest of the TM, consistent with the TM acting like a compliant membrane rather than a stiff diaphragm, and also consistent with catenary behavior due to the TM's curved shape.

Temporal bone thickness and texture are major determinants of the high rate of insonation failures of transcranial Doppler in Amerindians (the Atahualpa Project).

PURPOSE: To assess the role of temporal bone characteristics in transcranial Doppler (TCD) insonation failures in Amerindians living in rural Ecuador. METHODS: We evaluated thickness and texture of temporal bones in community-dwelling Amerindians ≥65 years old undergoing TCD. Using receiver operator characteristics curve analysis and generalized estimating equations, we investigated factors associated with insonation failures. RESULTS: Of 65 participants (mean age 74.7 ± 6.7 years, 60% women), 32 (49%) had uni- or bilateral insonation failure through temporal windows. Considering temporal bones independently, 57 of 130 (44%) had poor insonation. Mean thickness was higher (4.7 ± 1.2 versus 2.7 ± 0.9, p < 0.0001), and texture more often heterogeneous (93% versus 22%, p < 0.0001) in bones with poor acoustic windows. Thickness, better predicting poor insonation, was ≥3.6 mm if used alone, and ≥2.7 mm if used together with heterogeneous texture. For every millimeter of increase in thickness, subjects were 2.9 times more likely to have insonation failures. Per se, heterogeneous texture increased by 3.2 times the odds for poor insonation. In all models, being woman increased the odds for poor insonation by six to nine times. CONCLUSIONS: Temporal bone thickness and texture are independent predictors of TCD insonation failure in Amerindians.

Cochlear neuropathy in human presbycusis: Confocal analysis of hidden hearing loss in post-mortem tissue.

Recent animal work has suggested that cochlear synapses are more vulnerable than hair cells in both noise-induced and age-related hearing loss. This synaptopathy is invisible in conventional histopathological analysis, because cochlear nerve cell bodies in the spiral ganglion survive for years, and synaptic analysis requires special immunostaining or serial-section electron microscopy. Here, we show that the same quadruple-immunostaining protocols that allow synaptic counts, hair cell counts, neuronal counts and differentiation of afferent and efferent fibers in mouse can be applied to human temporal bones, when harvested within 9 h post-mortem and prepared as dissected whole mounts of the sensory epithelium and osseous spiral lamina. Quantitative analysis of five "normal" ears, aged 54-89 yrs, without any history of otologic disease, suggests that cochlear synaptopathy and the degeneration of cochlear nerve peripheral axons, despite a near-normal hair cell population, may be an important component of human presbycusis. Although primary cochlear nerve degeneration is not expected to affect audiometric thresholds, it may be key to problems with hearing in noise that are characteristic of declining hearing abilities in the aging ear.

Protuberant fibro-osseous lesions of the temporal bone: two additional case reports.

The most commonly encountered fibro-osseous lesions of the skull bone are fibrous dysplasia and ossifying fibroma. Two cases of a unique "protuberant fibro-osseous lesion of the temporal bone" were first described by Selesnick and colleagues in 1999, and 2 further cases were reported in 2010 under the name "Bullough lesion". We recently found 2 new cases of this rare entity. Two Korean female patients aged 70 and 54 years presented with slow growing postauricular masses without pain or tenderness for 6 and 7 years, respectively. Computed tomography revealed a 2.9 cm calcified mass in the temporal bone of the first patient, and a 5.5 cm enhancing mass with internal cartilaginous matrix in the temporal bone of the second patient. Intramedullary or intracranial extension was not found in either case, and en bloc removals were performed. Microscopically, multiple round to oval osseous islands were scattered throughout the bland fibrous stroma in both cases. The osseous islands varied in size and were lamellar or woven, without osteoblastic rimming, and surrounded by fibroblastic bands. Neither patient has shown evidence of postoperative recurrence for 18 months. The location, histology, and clinical course of these 2 cases were identical to the 4 cases previously reported, although age and sex varied. The lesions were tested for the R201H mutation in the GNAS gene, which is present in fibrous dysplasia. No mutations were found, suggesting a different genetic background for these lesions.

Endolymphatic sac tumor with von Hippel-Lindau disease: report of a case with atypical pathology of endolymphatic sac tumor.

The authors described a case of a patient with co-existing endolymphatic sac tumor (ELST) and hemangioblastoma in the posterior cranial fossa, which belonged to a subtype of Von Hippel-Lindau (VHL) disease confirmed by the test of VHL-gene. The signs in this 42-year-old female included intermittent headache and dizziness. Imaging revealed a giant mass in the right cerebellopontine angle (CPA) region and another lesion in the left cerebellar hemisphere. The results of biopsy after two operations confirmed the diagnosis respectively. Both of the tumors were resected totally. Nevertheless, we had to confess the misdiagnosis as vascular tumor instead of ELST at the initial diagnosis because of the rarity of ELST associated with atypical histological characteristics. The purposes we reported this case were to describe the atypical pathological feature of ELST and the mutation of germline VHL not mentioned in previously literature, furthermore, to foster understanding of ELSTs with the avoidance of the similar misdiagnosis as far as possible in future.

Sporadic endolymphatic sac tumor--a diagnostic and therapeutic challenge.

Endolymphatic sac tumor (ELST) is a rare low-grade locally aggressive neoplasm of the inner ear that may occur sporadically or in the setting of von Hippel-Lindau syndrome. We herein present a case of sporadic ELST in a 39-year-old man, treated using an interdisciplinary approach (surgery+radiotherapy), with a 10-year follow-up. The patient presented with hearing loss of sudden onset. The treatment of choice for ELST is radical tumor resection, which is associated with a good long-term prognosis. Remission may last for years, but there may be local recurrences, probably as a result of incomplete resection. Adjuvant radiotherapy is an option in case of recurrence and could be discussed after incomplete resection. The purpose of this report is to call attention to ELSTs, which are difficult to diagnose due to their rarity and variety of presentations.

Citrate content of bone for time since death estimation: results from burials with different physical characteristics and known PMI.

A recently introduced method to determine the postmortem interval (PMI) based on quantification of the citrate content in bone was applied on the temporal bones and femora of 20 individuals buried in wooden coffins (WO) and body bags (BB), respectively. Concerning known vs. calculated PMI, a significant difference between the temporal and the femur bone samples of the same individuals was observed in the BB group (p = 0.012). In contrast, differences were insignificant for the WO group (p = 0.400). Moreover, similar levels of underestimation of PMIs resulted from the analysis of the femora for both burial groups (p = 0.247). Also, there was consistently less citrate preserved in the flat temporal bones as compared to the femora, indicating that the cortical layer of the long bones should be preferentially employed for citrate-based PMI estimations. The results call for additional research on subsurface-buried and surface-deposited remains to enhance the accuracy of the published PMI equation.

Osteogenetic changes in elongated styloid processes of Eagle syndrome patients.

Abnormal elongation of the styloid process, or Eagle syndrome, can be painful, and is associated with differential diagnoses including cranio-facial malformations and vasculo-neurological disturbances. The precise molecular mechanism leading to styloid process elongation is unknown. In this study, elongated styloid processes with periosteal fibrous ligament tissue were obtained from three patients with Eagle syndrome and examined by immunohistochemical methods using different antisera. In all cases, marked bony deposition was found at the apex of the styloid process. The osteogenetic proteins, such as osteonectin, osteocalcin, BMP-2, BMP-4, and RANKL were strongly positive by immunohistochemistry in both the ligament fibers and the periosteal membrane attached to the styloid process apex. Staining for protective proteins, HO-1, HSP-70, and HSP-90 was also positive. These results suggest that styloid process elongation is related to increased expression of osteogenetic and protective proteins. Therefore, we propose that Eagle syndrome results from a protective response to increased tensile stress in the ligament attached to the styloid process, which could also signal osteogenetic protein expression in the periosteal fibrous tissue.

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.

Quantitative analysis of mRNA in human temporal bones.

CONCLUSION: Well-preserved mRNA could be extracted from frozen human inner ears. Therefore, this study demonstrates that analysis of mRNA could be performed to study the molecular mechanisms of inner ear disorders using human specimens. OBJECTIVES: Analysis of RNA as well DNA is requisite to study the molecular mechanisms of inner ear disorders. Methods of isolating RNA from experimental animals have been established, while isolation of RNA from human inner ears is much more challenging. In the present study, we demonstrate a method by which messenger RNA (mRNA) was extracted from human inner ears and quantitatively analyzed. MATERIALS AND METHODS: COCH mRNA as well as GAPDH mRNA was extracted from membranous labyrinths dissected from three formalin-fixed and three frozen human temporal bones, removed at autopsy. The length of COCH mRNA and quantity of GAPDH mRNA was compared between the two groups by quantitative RT-PCR. RESULTS: COCH mRNA could be amplified as much as 976 bp in all three frozen specimens. By contrast, it was amplified to 249 bp in two of the three formalin-fixed specimens, with no amplification observed in the remaining. The quantity of amplifiable GAPDH mRNA in the formalin specimens was only 1% of that of the frozen specimens.

Detection of mitochondrial DNA from human inner ear using real-time polymerase chain reaction and laser microdissection.

CONCLUSIONS: In this study we were able to amplify and analyze extremely small amounts of template DNA from only a few individually dissected cells. We anticipate that this approach will facilitate the detection and analysis of mitochondrial (mt) DNA mutations in specific cell types in the inner ear, which should shed new light on genetic disorders leading to hearing loss. OBJECTIVE: To isolate mtDNA from selected tissues in the inner ear. Although several methods for extracting DNA from formalin-fixed, celloidin-embedded, archival human temporal bones have been reported, the isolation of DNA from the inner ear by means of laser microdissection has not been previously demonstrated. MATERIAL AND METHODS: This was a retrospective study. Temporal bones were obtained from subjects with no known otological history at autopsy. The combined method of laser microdissection and real-time polymerase chain reaction was used to isolate mtDNA from selected tissues in the inner ear. RESULTS: mtDNA could be isolated from the stria vascularis, spiral ligament, spiral ganglion cells and organ of Corti.

Osteoprotegerin in the inner ear may inhibit bone remodeling in the otic capsule.

OBJECTIVES: To elucidate factors that may be responsible for the inhibition of remodeling of bone within the otic capsule. METHODS: Expression of osteoprotegerin (OPG), receptor activator of nuclear factor kappa B (RANK), and RANK ligand (RANKL) were assayed in samples of bone obtained from the otic capsule, calvarium, and femur, and from the soft tissue within the cochlea using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) in mice. Immunostaining was used for histologic localization of the gene products. An enzyme-linked immunosorbent assay (ELISA) was used to quantify the amount of OPG within perilymph, serum, and cerebrospinal fluid. The micro-anatomy of the interface between the otic capsule and the fluid spaces of the cochlea was investigated by brightfield and phase-contrast microscopy and by three-dimensional reconstruction in the mouse and human. RESULTS: OPG, a powerful inhibitor of bone remodeling, was expressed at extremely high levels within the soft tissue of the cochlea and was present in the perilymph at very high concentrations. The OPG produced within the inner ear may diffuse into the surrounding otic capsule, where it may be responsible for inhibition of bone turnover. Our anatomic studies revealed an extensive system of interconnected canaliculi within the otic capsule that had direct openings into the fluid spaces of the inner ear, thus providing a possible anatomic route for the diffusion of OPG from the inner ear into the surrounding bone. CONCLUSION: OPG, a potent inhibitor of osteoclast formation and function, is expressed at high levels within the inner ear and is secreted into the perilymph and the surrounding bone and may serve to inhibit active bone remodeling within the otic capsule, especially immediately adjacent to the cochlea. By this means, the cochlear soft tissue may control the nature of the surrounding petrous bone.

Giant cell granuloma of the temporal bone: a case report with immunohistochemical, enzyme histochemical, and in vitro studies.

A case of giant cell granuloma (GCG) that occurred in the right temporal bone is reported. The lesion showed histologic features identical to GCG. The multinuclear giant cells (MGCs) in the lesion showed strong reactivity with CD68, but patchy staining for myeloid/histiocyte antigen, alpha-1-antitrypsin, alpha-1-antichymotrypsine, and lysozyme. Activity of tartrate-resistant acid phosphatase was also consistently detected in the MGCs. Some of the mononuclear cells of the lesion exhibited similar immunocytochemical and histochemical reactivity as the MGCs. Ki-67 staining, however, was only detected in the mononuclear cells. The MGCs isolated from the lesion presented characteristic morphology of osteoclasts and possessed the ability to excavate bone in vitro. Thus, the MGCs in GCG appeared to express both macrophage- and osteoclast-associated phenotypes. The mononuclear cells were the major proliferative elements in the lesion and a subpopulation of these cells may represent precursors of the MGCs.

Temporal bone histopathological and quantitative analysis of mitochondrial DNA in MELAS.

OBJECTIVES/HYPOTHESIS: Although hearing loss is common in MELAS (syndrome of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes), the histopathology of the temporal bone has not been reported. The majority of cases of MELAS are linked to a mitochondrial DNA (mtDNA) mutation at nucleotide 3243. In MELAS, normal mtDNA and mutant mtDNA coexist in a heteroplasmic manner. The purpose of the study was to report the otopathological findings from two patients with MELAS and quantitative mtDNA analysis in the inner ear of one of these patients. STUDY DESIGN: Basic scientific histopathological examination and quantitative mtDNA analysis of the temporal bone. METHODS: Temporal bones were embedded in celloidin and sectioned for light microscopic study. Graphic reconstruction of the cochlea was performed using the method described by Schuknecht. For quantitative mtDNA analysis, total DNA from the membranous part of the inner ear was collected, amplified by polymerase chain reaction, and digested with the restriction enzyme. The percentage of mutant/total mtDNA was measured by the ratio of fluorescence intensity. RESULTS: Histopathological examination revealed severe degeneration of the stria vascularis and degenerative change of spiral ganglion cells in both patients. The quantitative DNA studies showed that the proportion of mutant to wild-type mtDNA was similar in both histologically affected and histologically unaffected tissues within the inner ear. CONCLUSION: Dysfunction of the stria vascularis and spiral ganglion cells causes sensorineural hearing loss in MELAS.

Temporal bone dissection: a possible route for prion transmission?

The aim of this study was to determine whether neural tissue is present in the bone 'dust' given off during temporal bone drilling. Bone 'dust' from three temporal bone dissections was collected and examined. Evidence of neural tissue was present in two out of the three specimens. Neural tissue is present in the bone dust given off during temporal bone drilling. This poses the question as to the risk of prion transmission during such dissection.

Recognition of basic fuchsin prestained microfissures of intravital origin with fluorescence microscopy: validation of a shortcut.

For 70 years it has been suspected that not all microfissures in histological bone sections are artifacts, but that some are provoked in vivo through repetitive stress. The development of undecalcified bone techniques and of the bulk staining technique has established a method for demonstrating the existence of intravital cracks and enhanced the discrimination towards artifactual microfissures in the load-bearing skeleton. Recently the presence of intravital microfissures has also been ascertained in temporal bones by these techniques. Due to the fluorescent properties of basic fuchsin it is possible to use epifluorescence microscopy for analysis of microfissures after bulk staining with basic fuchsin. This provides a more steady microscopic background and a sharper delineation of surface level structures since no projection from lower levels interfere. Artifactual cracks, which in transmitted light microscopy may look like darkly stained intravital microfissures due to refraction phenomena, become invisible or colorless. Epifluorescence microscopy enhances the detection of both smaller and larger prestained intravital microfissures, and leaves only a minor part of the cracks without certain categorization. The epifluorescence mode of analysis has the further advantage of being independent of slice thickness, making feasible whole-specimen analysis by serial stepwise grinding. The present study shows that the number and the length of microfissures in the human otic capsule, counted and measured under the epifluorescence microscope, equals numerically the findings in light microscopy, enabling the routine use of this mode of analysis. This may prove to be of particular value in the research into the etiology and pathogenesis of otosclerosis as well as perilymphatic fistulae.

Destructive tophaceous calcium hydroxyapatite tumor of the infratemporal fossa. Case report and review of the literature.

Tophaceous pseudogout is one of the rarest forms of crystal deposition disease, typically presenting as a destructive and invasive mass involving the temporomandibular joint or the infratemporal fossa region in the absence of any other articular manifestations. Previous cases have been assumed to be caused by calcium pyrophosphate dihydrate (CPPD) crystal deposition, based on finding weakly birefringent crystals in the involved tissues. The authors present the unique case of a 65-year-old woman with a destructive and invasive facial mass extending to the middle cranial fossa with microscopic and clinical features consistent with tophaceous pseudogout. High-resolution x-ray crystallographic powder diffraction and Fourier transformed infrared spectroscopy subsequently revealed that the crystals were composed of calcium hydroxyapatite without CPPD. The patient was later found to have primary hyperparathyroidism and mild hypercalcemia. This case demonstrates that tissue deposits of calcium hydroxyapatite can cause a destructive and invasive mass containing weakly birefringent crystals and raises the question of whether previous cases attributed to tophaceous pseudogout resulting from CPPD actually were composed of birefringent calcium hydroxyapatite.

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.

Distribution of Eph-related molecules in the developing and mature cochlea.

Receptors and ligands of the Eph family have recently been shown to influence the development of a variety of tissues. In the present study, the temporal and spatial distribution of Eph receptors and ligands were investigated in the embryonic and postnatal cochlea using Northern blot and immunohistochemical analysis. The results of Northern blot experiments revealed that a large number of Eph family members were present in embryonic cochlear and vestibular ganglia. Immunohistochemical studies revealed that ligands and receptors of the GPI subclass were distributed in complementary patterns within the differentiating spiral limbus, inner sulcus and outer sulcus. The distribution of these molecules became more restricted beginning in the first postnatal week. In contrast, members of the transmembrane subclass of Eph ligands were largely associated with cochlear neurons and their target hair cells. Expression of these ligands appeared to increase during the second postnatal week, corresponding to the period of peripheral nerve fiber reorganization in the cochlea. Together, these studies suggest that multiple Eph family members play unique roles in formation of the cochlea.