Intrathecal Application of a Fluorescent Dye for the Identification of Cerebrospinal Fluid Leaks in Cochlear Malformation.

In cases of cerebrospinal fluid (CSF) leaks, reliable detection of their origins is needed to seal the leak sufficiently and prevent complications, such as meningitis. A method is presented here using intrathecal administered fluorescein in a clinical case of bilateral congenital ear malformation. A fluorescent dye is administered intrathecally to achieve intraoperative visualization of CSF leaks. The dye is applied 20 min before surgery, and concentration of 5% is used. Per every 10 kg of body weight, 0.1 mL of the fluid is applied intrathecally. The fluorescein is visualized using a fully digital microscope. The origin of the fluid leak is identified in the stapes footplate. During primary surgery, it is sealed, and cochlea implantation is performed for hearing restoration. In this specific case, 6 weeks later, the implant was explanted due to acute meningitis, and the electrode array was left as a spacer. Postoperatively, in the aural smear, ß-transferrin was detected. During a revision mastoidectomy, dislocated coverage of the leak was found. The stapes was removed and oval window sealed. Five days after revision surgery, no ß-transferrin was detected in the aural smear. During the revision of cochlea implantation 6 months later, intact coverage of the oval niche was observed. Thus, intrathecal fluorescein application proves to be a reliable tool for the detection of CSF leaks. It facilitates the orientation in malformations and complicated or unknown surgical situs. In the literature, its use is described for CSF fistulas in endonasal surgery but is rarely described in skull base and mastoid surgeries. The method has been used successfully in several cases with CSF leaks, and the results confirm the feasibility of safely accessing the origin of the leak.

High-resolution imaging of the human incudostapedial joint using synchrotron-radiation phase-contrast imaging.

The incudostapedial joint (ISJ) of the middle ear is important for proper transmission of sound energy to the cochlea. Recently, the biomechanics of the ISJ have been investigated using finite-element (FE) modelling, using simplified geometry. The objective of the present study was to investigate the feasibility of synchrotron-radiation phase-contrast imaging (SR-PCI) in visualising the ISJ ultrastructure. Three human cadaveric ISJs were dissected and scanned using SR-PCI at 0.9 µm isotropic voxel size. One of the samples was previously scanned at 9 µm voxel size. The images were visually compared and contrast-to-noise ratios (CNRs) were calculated (of both bone and soft tissues) for quantitative comparisons. The ISJ ultrastructure as well as adjacent bone and soft tissues were clearly visible in images with a 0.9 µm voxel size. The CNRs of the 0.9 µm images were relatively lower than those of the 9 µm scans, while the ratio of bone to soft tissue CNRs were higher, indicating better discernibility of bone from soft tissue in the 0.9 µm scans. This study was the first known attempt to image the ISJ ultrastructure using an SR-PCI scanner at submicron voxel size and results suggest that this method was successful. Future studies are needed to optimise the contrast and test the feasibility of imaging the ISJ in situ. LAY DESCRIPTION: The human middle ear consists of the eardrum, three small bones (the malleus, incus and stapes) and two joints connecting the bones (the incudostapedial joint and the incudomallear joint). The role of the middle ear is to amplify and transfer sound energy to the cochlea, the end organ of hearing. The incudostapedial joint (ISJ) of the middle ear is a synovial joint which is important for proper transmission of sound energy to the cochlea. Similar to other synovial joints it consists of meniscus, fluid and articulating surfaces. Recently, the biomechanics of the ISJ have been investigated using computational models, using grossly simplified geometry. Synchrotron radiation phase contrast imaging (SR-PCI) is a high-resolution imaging technique used to visualise small structures in three dimensions. The objective of the present study was to investigate the feasibility of using SR-PCI in visualising the ISJ ultrastructure. Three human cadaveric ISJs were dissected and scanned using SR-PCI at 0.9 µm isotropic voxel size. One of the samples was previously scanned at 9 µm voxel size. The images were both qualitatively and quantitatively compared. This study was the first known attempt to image the ISJ ultrastructure using an SR-PCI scanner at submicron voxel size and results suggest that this method was successful. Future studies are needed to optimise the contrast and feasibility of imaging the ISJ in situ.

A Biochemical Analysis of the Stapes.

BACKGROUND Otosclerosis is a primary disease of the bony labyrinth. In the course of otosclerosis, abnormal resorption and recalcification of the endochondral layer of the temporal bone is observed. The otosclerotic process most commonly develops in the anterior part of the oval window. MATERIAL AND METHODS We analyzed stapes superstructures from 4 patients undergoing surgery for otosclerosis. The first step involved tissue assessment under a scanning electron microscope. The resulting images were analyzed in terms of morphological changes. The stapes superstructure was then divided into small "ossicles", including fragments from the closest vicinity of the stapes footplate and a fragment of the head of the stapes. This material was examined using a scanning electron microscope with a unit for chemical analysis in microareas. RESULTS Chemical analysis confirms the appearance of considerable quantities of the following elements: carbon, oxygen, potassium, and calcium, and the appearance of small quantities of sodium and magnesium. Based on a detailed analysis of the chemical composition, these fragments could represent a calcium phosphate compound from the following system: CaO-P2O5-H2O. Fragments of the superstructure from the region closest to the base of the stapes demonstrated a considerably larger presence of carbon, oxygen, and nitrogen, which most likely suggests an increased metabolic process in this region. CONCLUSIONS Our analysis revealed an increased metabolic activity in the closest vicinity of the otosclerotic focus, the fissula ante fenestram. The increased metabolism correlated with the bone tissue changes seen on scanning electron microscopy.

Macroscopic description of the external and middle ear of paca (Cuniculus paca Linnaeus, 1766) / Descrição macroscópica da orelha externa e media da paca (Cuniculus paca Linnaeus, 1766)

Paca (Cuniculus paca), one of the largest rodents of the Brazilian fauna, has inherent characteristics of its species which can conribute as a new option for animal experimantation. As there is a growing demand for suitable experimental models in audiologic and otologic surgical research, the gross anatomy and ultrastructural ear of this rodent have been analyzed and described in detail. Fifteen adult pacas from the Wild Animals Sector herd of Faculdade de Ciências Agrárias e Veterinárias, Unesp-Jaboticabal, were used in this study. After anesthesia and euthanasia, we evaluated the entire composition of the external ear, registering and ddescribing the details; the temporal region was often dissected for a better view and detailing of the tympanic bulla which was removed and opened to expose the ear structures analyzed mascroscopically and ultrastructurally. The ear pinna has a triangular and concave shape with irregular ridges and sharp apex. The external auditory canal is winding in its path to the tympanic mebrane. The tympanic bulla is is on the back-bottom of the skull. The middle ear is formed by a cavity region filled with bone and membranous structures bounded by the tympanic membrane and the oval and round windows. The tympanic membrane is flat and seals the ear canal. The anatomy of the paca ear is similar to the guinea pig and from the viewpoint of experimental model has major advantages compared with the mouse ear.

A paca (Cuniculus paca), um dos maiores roedores da fauna brasileira, possui características inerentes à sua espécie que podem contribuir como uma nova opção de animal experimental; assim, considerando-se que há crescente busca por modelos experimentais apropriados para pesquisas audiológicas e otológica cirúrgicas foram analisados e descritos em detalhes a anatomia macroscópica e ultraestrutural da orelha desse roedor. Para o estudo, utilizaram-se 15 animais adultos provenientes do plantel do Setor de Animais Silvestres da Faculdade de Ciências Agrárias e Veterinárias, Unesp-Jaboticabal, Jaboticabal/SP. Após anestesia e eutanásia, avaliou-se toda a composição da orelha externa, registrando-se e descrevendo-se os detalhes, também se dissecou a região temporal para melhor visibilização e detalhamento da bula timpânica e estas foram removidas e abertas a fim de expor as estruturas da orelha, as quais foram analisadas, macroscopicamente e ultraestruturalmente. O pavilhão auricular apresenta forma triangular e côncava com cristas irregulares e ápice pontiagudo; o conduto auditivo externo é sinuoso em seu trajeto até a membrana timpânica; a bula timpânica encontra-se na parte posterior-inferior do crânio; a orelha média é formada por uma região cavitária preenchida por estruturas ósseas e membranosas. É delimitada pela membrana timpânica e as janelas redonda e oval, sendo a membrana timpânica de forma plana e que veda todo o conduto auditivo. A anatomia da orelha da paca é semelhante à da cobaia e do ponto de vista de modelo experimental apresenta grandes vantagens em comparação com a orelha do rato.

Histochemical localization of the extracellular matrix components in the annular ligament of rat stapediovestibular joint with special reference to fibrillin, 36-kDa microfibril-associated glycoprotein (MAGP-36), and hyaluronic acid.

The annular ligament across the stapediovestibular joint connects the stapes footplate and the vestibular window and plays an important role in the sound conductive system of the ear. In this study, we investigated the distribution of extracellular matrix components in the ligament by histochemical methods at light and electron microscopic levels. As results, light microscopic immunohistochemistry of fibrillin and 36-kDa microfibril-associated glycoprotein (MAGP-36) showed intense immunoreactivities in the annular ligament between the stapes footplate and vestibular window. In addition, the histochemical localization of hyaluronic acid by using biotinylated hyaluronic acid-binding protein (HABP) clarifi ed the presence of hyaluronic acid in the annular ligament. At the electron microscopic level, the immunogold labeling of fibrillin showed intense labeling on the periphery of the electron-dense mantle. Furthermore, the labeling of fibrillin was preferentially seen on the fibrous components among the electronlucent amorphous substance. The immunogold labeling of MAGP-36 was seen on the electron-dense mantle and scattered on the electron-lucent amorphous substance. The gold labeling with biotinylated HABP clearly showed a distribution of hyaluronic acid throughout the amorphous space in the ligament. The present results provide a histochemical profile of the annular ligament of the rat stapediovestibular joint that may provide clues to elucidation of pathological changes in the ligaments and conductive hearing loss in humans.

Three-dimensional regular arrangement of the annular ligament of the rat stapediovestibular joint.

The stapes footplate articulates with the vestibular window through the annular ligament. This articulation is known as the stapediovestibular joint (SVJ). We investigated the ultrastructure of adult rat SVJ and report here on the characteristic ultrastructure of the corresponding annular ligament. Transmission electron microscopy showed that this annular ligament comprises thick ligament fibers consisting of a peripheral mantle of microfibrils and an electron-lucent central amorphous substance that is regularly arranged in a linear fashion, forming laminated structures parallel to the horizontal plane of the SVJ. Scanning electron microscopy revealed that transverse microfibrils cross the thick ligament fibers, showing a lattice-like structure. The annular ligament was vividly stained with elastica van Gieson's stain and the Verhoeff's iron hematoxylin method. Staining of the electron-lucent central amorphous substance of the thick ligament fibers by the tannate-metal salt method revealed an intense electron density. These results indicate that the annular ligament of the SVJ is mainly composed of mature elastic fibers.

¿Qué sabemos de otoesclerosis? aspectos anatomopatológicos / What do we know about otosclerosis? anatomo-pathological aspects

La otoesclerosis es una enfermedad que produce hipoacusia de conducción por un proceso de remodelación ósea denominado otoespongiosis. Se han estudiado aspectos anatomopatológicos para evaluar el foco de inicio, y las características de este proceso llamado otoespongiosis. Nos motivó a realizar este estudio el deseo de conocer qué pasaba en nuestra población, clasificar las observaciones anatomopatológicas, relacionar estos aspectos microscópicos con aspectos clínicos como: tiempo de evolución clínica y aspecto macroscópico de la platina. Para esto se realizó un estudio prospectivo tomando todos los pacientes operados de estapedostomía en el Hospital Clínico de la Universidad de Chile desde octubre 2003 a octubre 2004. Fueron un total de 22 pacientes que se agruparon en 4 subgrupos según su aspecto anatomopatológico. Este estudio mostró que la otoesclerosis es más frecuente en mujeres en la quinta década de vida, en el oído derecho y presenta un tiempo de evolución clínica promedio de 7 años, que no varía con el grado de otoespongiosis de la supraestructura del estribo. Se observó un 45 por ciento de otoespongiosis en los huesecillos estudiados, aspecto no descrito en la bibliografía. No se observó correlación entre el aspecto microscópico y macroscópico de la platina, lo que sugiere otros focos de otoespongiosis o un avance rápido desde la fissula antefenestra a las cruras del estribo. Se continuará este estudio aumentando la muestra y realizando un seguimiento a los pacientes para relacionar el resultado anatomopatológico y el resultado auditivo post-estapedostomía.

[Experimental joint incus-stapedial injuries in scanning electron microscopy]. / Doswiadczalne uszkodzenia stawu kowadelkowo-strzemiaczkowego w skaningowej mikroskopii elektronowej.

Because all machines and vibration devices also produce noise, the combined activities of both factors are usually examined together. The opinion dominates that vibration exerts only a weak, additionally traumatic influence on the hearing organ. The aim of our investigation was to determine the influence of long-term, whole-body vibration on the incudo-stapedial joint which integrity is indispensable for the protection of the inner ear from the effect of noise. To realize the experimental conditions, sinusoidal vertical shaking (10 Hz, 5 mm, 1.4 g rms), an own noiseless apparatus was consisted. The experiment was carried out on 30 young, healthy, colored guinea pigs. They were subjected to vibration over 1, 3, and 6 months (132, 396, and 792 hours). The investigation was based on examination of the structures of the incudo-stapedial joint in the scanning electron microscopy. Among experimental animals two kinds of changes were ascertained which can be attributed to the activity of vibration. One was an extensive damage to the surfaces of the incudo-stapedial joint itself. The other was an often observed thickening of the bursa of the joint. The frequencies of occurrence and stages of advancement of both were in direct relationship to the duration of the experiment. The evaluation of the observed changes permits an explanation of the mechanism of the damage to hearing of persons subjected to noise and vibration. Whole-body vibration damages the incudo-stapedial joint, making its separation difficult. This may, than, facilitate the transfer of noise and its injurious influence to the inner ear.

Imaging of a congenital perilymphatic fistula.

A 7-year-old boy with a history of purulent meningitis and watery rhinorrhea was studied using computed tomography (CT) and magnetic resonance imaging (MRI). He had a common cavity in the left inner ear. With high-resolution heavily T2-weighted MRI, leakage of the inner ear fluid into the middle ear at the oval window area through a congenital perilymphatic fistula could be visualized. Surgery to close the fistula showed a perforation in the stapes footplate.

[Otosclerosis and Van der Hoeven's syndrome: a contribution]. / Aportación al estudio de la otosclerosis y del síndrome de Van der Hoeve.

Morphological and microchemical changes that effect to the otosclerotic stape in the Van der Hoeve's syndrome were examined with a scanning electron microscope equipped with an energy dispersive X-ray fluorescence. Using the Ca/P ratio as criterion--measured by the characteristic x-ray fluorescence--it was shown that the Van der Hoeve stape had a higher Ca/P ratio (2.6:1) as compared to the normal stape (2:1). The Van der Hoeve's syndrome lesions as poorly mineralized, with low calcium salt and apparent increase of phosphates. This finding indicates a possible change from hydroxyapatite (or apatite) to brushite, which imply an acidification of bone.

Scanning electron microscopy images and energy-dispersive X-ray microanalysis of the stapes in otosclerosis and Van der Hoeve syndrome.

OBJECTIVE/HYPOTHESIS: The objective of this study was to evaluate the morphological and microchemical changes that affect sclerotic stapes in otospongiosis and van der Hoeve syndrome. METHODS: A scanning electron microscope equipped with an energy-dispersive x-ray analyzer was used in the experiments. RESULTS: In otosclerosis, focal lesions are poorly mineralized, with low calcium salt and reduced calcium-to-phosphorus (Ca/P) ratio (1.9:1). This finding correlates with a spongiotic type of lesion and indicates unstable mineralization with possible change from hydroxyapatite to calcium triphosphate. In van der Hoeve syndrome the presence of magnesium in stapes suggests osteoclastic function stimulation. The osteoclasts secrete many protons, causing an acidified microenvironment. Brushite is formed, and Ca/P ratio decreases in comparison with that of control patients (2.0:1 vs. 2.6:1).

Effects of pulsed laser systems on stapes footplate.

BACKGROUND AND OBJECTIVE: The aim of the present study was to investigate the tissue ablation capacity of various pulsed lasers at the stapes footplate. STUDY DESIGN/MATERIALS AND METHODS: Isolated human stapes and bovine compact-bone platelets were used to determine the effective laser parameters and appropriate application technique for achieving a perforation measuring 500-600 microns in diameter. Of interest were also the shape and quality of the perforations, the reproducibility of the perforation effect, and the thermically altered marginal zones occurring at the footplate. Three pulsed laser systems were used: excimer, holmium:YAG (Ho:YAG), and erbium:YSGG (Er:YSGG) lasers. RESULTS: The tissue-ablating effect of pulsed laser systems permits a precise and controlled management of the stapes footplate through low and readily reproducible ablation rates. The extent of thermic side effects at the footplate is lower in comparison to the purely thermically acting cw and superpulse laser systems. The Er:YSGG laser exhibits the highest ablation rate at the stapes and is thus the most effective laser for perforation of the stapes footplate. Though somewhat less effective, the Ho:YAG laser also appears to be suitable for stapedotomy. On the other hand, we do not consider the applied excimer laser (308 nm) to be particularly appropriate at the stapes because of its low ablation rates. CONCLUSION: Thus, the erbium laser could represent an alternative to the argon, KTP 532, and CO2 lasers, already clinically successful in stapes surgery. However, further studies are necessary to examine the transmission of thermic energy into the vestibule and the acoustic stress to the inner ear during laser stapedotomy, to be able to make a definitive statement about the safest and most effective laser system for stapes surgery.

[Experimental laser stapedectomy. Scanning electron microscopy studies]. / Doswiadczalna stapedotomia laserowa. Badania w mikroskopie elektronowym.

The results of experimental tests performed upon 16 human stapes taken from cadavers were presented. The aim of the experiment was to define optimal parameters of laser stapedotomy. The less harmful range of argon laser energy for making the hole 0.6 mm diameter in the stape footplate was determined. There were two zones of tissue damage around the footplate's hole seen in scanning microscope. The internal area was made of carbonized tissue. The osseous tissue of the external zone had cavernous-cystic structure and lamellar stratification.

Oval and round window membrane changes in otitis media in the human. An ultrastructural study.

An ultrastructural study of oval and round window changes in otitis media in humans was done. Ten cases were evaluated. In this first ultrastructural study of oval and round windows in otitis media, done at different stages of the disease, the round window membrane changes were similar to those of the mucoperiostium. Morphologic evidence suggests that the round window membrane layers participate in absorption and secretion of substances to and from the inner ear, such that the entire membrane could play a role in a middle and inner ear "defense system." Although the middle ear side of the footplate of the stapes had histopathological changes, the vestibular side remained essentially unchanged.

Significance of chondrification in the development of otosclerotic stapedial footplate.

Thirty-four otosclerotic stapes of active type were investigated histologically with hematoxylin-eosin (H-E), periodic acid-Schiff-alcine blue (PAS-AB), and/or toluidine blue stain. The results show that chondrification plays an important role in the development of otosclerosis. To date, this specific histopathologic change has not been well recognized. Based on the findings of this study, it is suggested that the active type of otosclerosis be subdivided into two phases, the osteolytic phase and spongy chondroid phase.

Diagnosis of the ossicular chain in the middle ear by high-resolution CT.

This study was conducted to assess the usefulness and limitations of high-resolution CT for diagnosing the ossicular chain in the middle ear. The CT images in this study were obtained in as much detail as possible and 2 direction images. Preoperative CT findings of the ossicular chain were compared with operative findings in 26 patients with ossicular defects. Preoperative detection of the complete defect of the malleus head and the body and long process of the incus by high-resolution CT was possible in all cases, while detection of the defect of the manubrium of the malleus and superstructure of the stapes could be made in 33.3 and 60%, respectively. The defect of the incudostapedial joint (1 case) and partial defect of the stapes crus (2 cases) could not be diagnosed correctly by preoperative estimation.

Effect of mechanical trauma on the stapedial footplate after stapedectomy. A scanning electron microscopic study.

The possible occurrence of inner-ear trauma linked to stapedectomy was studied by scanning electron microscopy of the medial (or labyrinthine) side of human stapedial footplates after performing a hole with different instruments. The anatomic variations induced experimentally by the different procedures are presented in detail and discussed. Manual instruments were shown to induce irregularities on the rim of the hole, whilst the utilization of either electric or pneumatic drills produced more regular margins. Finally, laser-produced holes were also examined. Rather neat rims were observed, but the thermal effect produced by this device has to be considered the major parameter involved in a possible inner-ear postoperative trauma.

Ultrastructural and immunohistochemical evidence of measles virus in active otosclerosis.

Because of the similarity between otosclerosis and Paget's disease of bone, and the mounting evidence of a viral cause in Paget's disease, we have investigated a possible viral cause for otosclerosis. Transmission electron microscopy of stapes footplate fragments with active otosclerosis has revealed structures morphologically identical with measles virus nucleocapsid in osteoblasts and preosteoblasts. Immunofluorescence and immunoperoxidase studies have confirmed the presence of measles nucleocapsid antigen in active lesions. Application of sera from patients with subacute sclerosing panencephalitis, a defective measles virus infection of the central nervous system, resulted in positive immunoreaction in areas of active otosclerosis.

Morphologic and immunohistochemical observation of otosclerotic stapes: a preliminary study.

Stapes with otosclerotic lesions obtained during stapedectomies were examined with light microscopy, histochemistry, immunochemistry, and electron microscopy to elucidate the cellular mechanism(s) involved in this disease process. Three types of lesions were identified: cellular (spongiotic), fibrotic, and sclerotic. The cellular type is characterized by monocyte, macrophage, osteoblast, and osteoclast recruitment and their activation. Macrophage recruitment is an early event of otosclerosis. The fibrotic type is characterized by extensive fibrosis of the bone, and the sclerotic type is characterized by a paucity of bone cells. Cytochemical results showed a large accumulation of granular substances positive for periodic acid-Schiff stain along the edge of the marrow spaces coinciding with ultrastructural calcospherite deposits, suggesting that glycosaminoglycans are involved in the mineralization process. Acid phosphatase is largely localized in the osteoclasts, but a sporadic diffusion of this enzyme was observed in the demineralizing front of the preotosclerotic lesion. Immunoglobulin G and complement C3 were colocalized in pericapillary tissue, suggesting deposit of immune complex in the spongiotic lesion. This finding indicates a possibility that immune mechanisms are involved in otosclerosis.