Morten Friis

Gene expression of the endolymphatic sac.

Abstract Conclusion: The endolymphatic sac is part of the membranous inner ear and is thought to play a role in the fluid homeostasis and immune defense of the inner ear; however, the exact function of the endolymphatic sac is not fully known. Many of the detected mRNAs in this study suggest that the endolymphatic sac has multiple and diverse functions in the inner ear. Objectives:The objective of this study was to provide a comprehensive review of the genes expressed in the endolymphatic sac in the rat and perform a functional characterization based on measured mRNA abundance. Methods:Microarray technology was used to investigate the gene expression of the endolymphatic sac with the surrounding dura. Characteristic and novel endolymphatic sac genes were determined by comparing with expressions in pure dura. Results: In all, 463 genes were identified specific for the endolymphatic sac. Functional annotation clustering revealed 29 functional clusters.

Bacterial adherence in otitis media: determination of N-acetylgalactosamine (GalNAc) residues in the submucosal glands and surface epithelium of the normal and diseased Eustachian tube.

BACKGROUND Acute otitis media (AOM) is the most common childhood infection caused by bacteria. The pathogenesis of AOM implicates initial adherence of a pathogen to the nasopharyngeal epithelium, which is followed by bacterial colonization of the middle ear cavity through the Eustachian tube. N-acetylgalactosamine (GalNAc) is an important constituent of mucins and GalNAc containing sugar residues seem to be essential for initial adherence of respiratory bacteria to the surface of epithelial cells. OBJECTIVE To explore the localization of GalNAc residues, we incubated Eustachian tube sections from Streptococcus pneumoniae infected and normal control rats with seven biotinylated, GalNAc recognizing lectins: Bauhinia purpurea lectin (BPA), Psophocarpus tetragonolobus lectin (PTA), Helix aspersa lectin (HAA), Helix pomatia lectin (HPA), Phaseolus lunatus lectin (PLA), Sophora japonica lectin (SJA) and Vicia Villosa isolectin B4 (VVA-B4). RESULTS The mucin producing epithelium and submucosal glands of the normal Eustachian tube contained GalNAc residues, as evidenced by binding of several of the lectins. Lectin binding specificity and intensity changed following acute middle ear infection. BPA was the only lectin that exclusively stained the surface epithelium and the serous acini of the submucosal glands in the infected animals, whereas no binding was detected in the normal controls. HPA, HAA, PTA and VVA-B4 binding to surface epithelial cells increased after infection, indicating an active secretion of GalNAc containing glycans. Quantitative analysis of submucosal gland staining intensity showed significantly more GalNAc residues in the normal Eustachian tube, compared to infected animals. CONCLUSION We conclude that the mucous producing elements of the normal rat Eustachian tube contain GalNAc residues essential for respiratory pathogen adherence. In addition, the GalNAc residue specificity and reacting intensity change in relation to acute infection, which may be important in relation to subsequent development of secretory otitis media or formation of a bacterial biofilm in the middle ear. The results show that GalNAc residues increased in both the submucosal serous glands and in the surface epithelium of the Eustachian tube after middle ear infection with S. pneumoniae.

A Potential Portal Flow in the Inner Ear

Objectives/Hypothesis: The aim of the present study was to visualize the flow direction of blood in the extraosseous part of the vein of the vestibular aqueduct (VVA) and to explore the effect of an induced obstruction in the distal part of the VVA before it merges with the sigmoid sinus. The endolymphatic sac has been implicated as a potential endocrine gland, which venules drain to the VVA. A reversal of the direction of flow in the VVA toward the inner ear could, through vestibular arteriovenous anastomosis, cause portal circulation in the inner ear.

Objective measurement of the human endolymphatic sac dimensions in Ménière's disease.

Hypothesis: To identify structural differences between the endolymphatic sac of temporal bones with and without Ménières disease by applying design-based stereology. Background: The dimension of the human endolymphatic sac has previously been studied by extrapolating 2-dimensional irregularities on the sectional level into 3-dimensional quantities via unfolding algorithms. These computer renderings require certain geometrical assumptions of the object studied. The term design-based used in the present study signifies that the methods and sampling schemes are defined a priori, in such a manner that consideration of the size, shape, spatial orientation, and distribution of the investigated structures are eliminated. Materials and Methods: Archival materials of 15 human temporal bones with Ménières disease and 15 control specimens were investigated by design-based stereology. Results: The total surface area of the endolymphatic sac was significantly lower in the Ménières disease group (24.8 mm2) compared with the control group (47.0 mm2), p = 0.006. The volume fraction of the homogenous substance was significantly higher in the Ménières disease group (17.5%) compared with the control group (5.7%), p = 0.031. No significant differences were found between the volumes of the homogenous substance, the volumes of the endolymphatic sac, and the surface-to-volume ratio in the 2 groups. The study revealed a large biological variation. Conclusion: Design-based stereology is a robust, unbiased and efficient tool to quantify 3-dimensional structures derived from 2-dimensional histologic sections. A 2-fold reduction in the surface area of the endolymphatic sac and a 3-fold increase in the volume fraction of homogenous substance in temporal bones with Ménières disease may, at least in part, be involved in the dysfunction of endolymph fluid homeostasis and the development of endolymphatic hydrops.

Differential gene expression in the otic capsule and the middle ear--an annotation of bone-related signaling genes.

Hypothesis A number of bone-related genes may be responsible for the unique suppression of perilabyrinthine bone remodeling. Background Bone remodeling is highly inhibited around the inner ear space most likely because of osteoprotegerin (OPG), which is a well-known potent inhibitor of osteoclast formation and function. However, other signaling molecules may also be responsible for the inhibition of bone remodeling within the otic capsule. Methods Microarray technology was used to determine bone-related genes differentially expressed between the lining tissues of the otic capsule (spiral ligament and stria vascularis) and the lining tissues from the middle ear of the rat. Data was analyzed with statistical bioinformatics tools. Gene expression levels of selected genes were validated using quantitative polymerase chain reaction. Results A total of 413 genes were identified when young inner bulla (growing) were compared with young otic capsule and 358 genes were identified when adult inner bulla (quiescent) were compared with adult otic capsule. Fourteen genes were involved in bone metabolism of which four genes have been previously discussed in the literature of perilabyrinthine bone biology. Conclusion The gene expression of the otic capsule was significantly different from that of the middle ear. This study identified a number of differentially expressed bone-related mRNAs of potential significance and confirmed the OPG/receptor activator of nuclear factor kappa-B (RANK)/RANK ligand (RANKL) pathway as the key signaling system for the unique behavior of bone cells within the otic capsule. No differentially expressed up- or downstream messengers in the OPG/RANK/RANKL pathway were found.

Bone Signaling in Middle Ear Development: A Genome‐Wide Differential Expression Analysis

Common middle ear diseases may affect bone behavior in the middle ear air cell system. To understand this pathologic pneumatization, the normal development of bone in the middle ear should be investigated. The objective of this study was to analyze gene expression of bone‐related signaling factors and gene sets in the developing middle ear. Microarray technology was used to identify bone‐related genes and gene sets, which were differentially expressed between the lining tissue of adult (quiescent) bulla and young (resorbing/forming) bulla. Data were analyzed using tools of bioinformatics and expression levels of selected genes were validated using quantitative polymerase chain reaction. The candidate gene products were compared with previously published data on middle ear bone metabolism. No differentially expressed genes were found on the outer surface of bulla. On the inner lining a total of 260 genes were identified of which 22 genes were involved in bone metabolism. Gene set analysis revealed five enriched bone‐related gene sets. The identified differentially expressed bone‐related mRNAs and gene sets are of potential significance in the normally developing bulla. These factors and gene sets may also play important roles during pathologic pneumatization of the middle ear air cell system in common middle ear diseases. In addition, this study suggests that the control of growth rate and wall thickness from resorptive as well as formative signals all originate from the inner lining cells of the bulla wall. Anat Rec, 297:2349–2355, 2014.

Experimental Hyperactivity of the Endolymphatic Sac

Injury to the endolymphatic sac may play an important role in the pathogenesis of Ménière’s disease, an inner ear disorder characterized by hearing loss, tinnitus and attacks of vertigo. Isoimmunization of 16 inbred Lewis rats with a crude endolymphatic sac extract and complete Freund’s adjuvant induced hyperactivity of the endolymphatic sac. One group of rats was immunized by a single dose whereas a second group was immunized twice. Control animals were injected with Freund’s adjuvant in saline only. Serum was collected from all rats by the end of the study and harvested autoantibodies were tested by immunohistochemistry. The endolymphatic sacs were investigated by transmission electron microscopy. Endolymphatic sac stimulation was observed in all immunized rats. Based on detailed ultrastructural observations, the degree of reactivity seemed proportional to the number of injections and the extent of immunization. Moreover, the ribosome-rich cells seemed hyperactive with an extravagant content of intracellular components: numerous rough endoplasmic reticulum and free ribosomes, morphological signs of extensive endo- and exocytosis, vesicles of material with a density similar to the homogeneous substance of which many were observed to fuse with primary lysozymes. Basolateral foldings were numerous and in the subepithelial capillaries formation of multiple and apposing fenestrations were observed. No endolymphatic sac stimulation was observed in the control animals. Specific ribosome-rich cell alterations identical to those present in the endolymphatic sac of Ménière’s disease were observed 21 days after the first immunization. The observations suggest that either an autoantigen or a trophic factor, capable of inducing a hyperactivity of the ribosome-rich cells and an imbalance of the homogeneous substance metabolism, exists in the endolymphatic sac of the rat.

Labyrinthitis Ossificans: On the Mechanism of Perilabyrinthine Bone Remodeling.

Introduction: It has been suggested that remodeling of the otic capsule is highly suppressed by the action of anti-resorptive signals emanating from structures of the inner ear space. Labyrinthitis ossificans (LO) is a severe complication to bacterial meningitis and is characterized by destruction of inner ear structures by the formation of new bone. The aim of this study was to explore the impact of LO on bone remodeling of the otic capsule. Material and Methods: In 11 human temporal bones with extensive LO and 10 control specimens, the degree of bone remodeling was explored indirectly by estimating the viability of osteocytes in perilabyrinthine bone and the mastoid. Results: The viability of osteocytes was significantly lower in the perilabyrinthine bone compared to the mastoid in both groups. However, the loss of perilabyrinthine osteocytes was the same in the 2 groups, and the presence of cartilage remnants appeared to be the same. Conclusion: This study indicates that the factors affecting bone remodeling of the otic capsule and the degeneration of osteocytes are not altered by wholesale destruction of inner ear soft tissue and its replacement by bone. Therefore, alternative mechanisms may be implicated in the suppression of capsular bone remodeling.

The vein of the vestibular aqueduct with potential pathologic perspectives.

Hypothesis: Pathologic changes around the vein of the vestibular aqueduct (VVA) may cause obstruction to the flow of blood toward the sigmoid sinus. Furthermore, a distal obstruction of this vessel may be responsible for a development of a retrograde flow of blood with concomitant drainage of endolymphatic sac (ES) substances to the inner ear. Background: The VVA is responsible for the venous drainage of the vestibular apparatus and endolymphatic duct and ES. Previous studies have linked the VVA to Ménières disease. The aim of the present article was a 3-dimensional perspective study of the VVA with its adjacent anatomic structures. Methods: In 14 rats, the VVA was examined by 3-dimensional reconstruction of 2-&mgr;m serial sections, corrosion cast technique, and scanning electron microscopy. Results: From the external aperture of the vestibular aqueduct, the VVA is interposed between the ES and the operculum. Three to 4 collecting venules from the ES drain into the VVA. The VVA merges at an oblique angle with the sigmoid sinus. Conclusion: The VVA courses near the ES, operculum, and sigmoid sinus and is potentially vulnerable to expanding structures in the cranial posterior fossa. The possible role of the VVA for the function of the ES under normal and pathologic conditions is discussed.

Auto-inflammatory challenge of the endolymphatic sac--Cochlear damage measured by distortion product oto-acoustic emissions.

Abstract Conclusion: Twenty-five rats were challenged by an immunologic attack of the endolymphatic sac. After 6 months, distortion product oto-acoustic emissions (DPOAE) revealed a dysfunction of the outer hair cells and immunological active cells were observed in the endolymphatic sac. This information could contribute to the understanding of Ménière’s disease. Objectives: This study investigated if an autoimmune challenge of the endolymphatic sac could affect DPOAE output measurements in rats. Also, a potential autoimmune cell infiltration of the endolymphatic sac was investigated. Methods: Eighteen Lewis rats were immunized with a crude endolymphatic sac extract in complete Freund’s adjuvant. Seven control animals were injected with Freund’s adjuvant in saline. Cochlear damage was estimated by DPOAE dynamics 3 weeks and 6 months after the immunization. Infiltrative cells in the endolymphatic sac were investigated with transmission electron microscopy. Results: The hearing assessment 6 months after immunization revealed a reduction of the DPOAE, on the full range of frequencies (2–63 kHz) in an average of the mean, of 2 dB ± 1.1 in the immunized group compared to the controls (p < 0.05). The same test showed a 2.5 dB decrease from 2 to 5 kHz (p < 0.01). Immunological active cells were observed in the endolymphatic sac in most of the immunized rats.