Effects of fixative and embedding medium on morphology and immunostaining of the cochlea.

The localization of proteins by immunostaining is a powerful method to investigate otologic disorders. However, the use of fixatives and embedding media (necessary for the preservation of morphology) can obscure antigens, making it difficult to perform immunoassays. We performed a systematic investigation of the effects of fixative and embedding medium on morphology and immunostaining of the mouse cochlea. Three different fixative solutions [4% formaldehyde (F), 4% formaldehyde + 1% acetic acid (FA), and 4% formaldehyde + 1% acetic acid + 0.1% glutaraldehyde (FGA)] and 3 different embedding media (paraffin, polyester wax, and celloidin) were used. Morphology was assessed using light microscopy. Immunostaining was studied using a panel of 6 antibodies (to prostaglandin D synthase, aquaporin 1, connective tissue growth factor, 200-kDa neurofilament, tubulin and Na(+),K(+)-ATPase). Preservation of morphology was suboptimal with paraffin, adequate with polyester wax and superb with celloidin. Immunostaining was successful using all 6 antibodies in all 3 fixatives and all 3 embedding media. While there were differences in strength of signal and localization of antigen between the 3 fixatives, overall, FA and FGA gave the most uniform results. For a given fixative and antibody, there was surprisingly little difference in the quality of immunostaining between celloidin and paraffin, while results in polyester wax were not as good in some cases. These results suggest that celloidin may be the embedding medium of choice for both morphological and pathological studies, including immunostaining when morphology must be optimized.

Technical report: laser microdissection of cochlear structures from celloidin embedded human temporal bone tissues and detection of the mitochondrial DNA common deletion using real time PCR.

Laser microdissection (LMD) has been used to isolate groups of cells and single cells from numerous tissues. In this study, we describe a technique for isolating cochlear structures and individual spiral ganglion cells from archival celloidin embedded human temporal bone sections. The specimens isolated are suitable for quantifying the mitochondrial DNA (mtDNA) common deletion (CD) within these tissues using a real time polymerase chain reaction (PCR) assay. The results presented in this manuscript demonstrate the feasibility of using this LMD technique to study the accumulation of mtDNA deletions in diseases of the ear. To our knowledge, this approach to analyzing archival human temporal bone tissues has not been previously reported.

The effect of collagen reinforcement in the behaviour of the temporomandibular joint disc.

In this paper, the influence of collagen fibres in the behaviour of the temporomandibular joint disc is studied. A three-dimensional finite element model of the joint is developed from a set of medical images. The model comprises the mandible, part of the cranium and both temporomandibular joints. Joints have been considered to be composed of the articular discs and the temporomandibular ligaments. A fibre-reinforced porohyperelastic model was used to study the response under clenching of the fibrocartilage that composes the articular disc. This was divided in an intermediate zone, and two bands, an anterior and other posterior, in order to define the orientation of collagen fibres. The study demonstrates that the introduction of collagen fibres in the biphasic behaviour of the articular disc implies for a prescribed displacement not only an increase of the pressurization in the tissue, but also higher stresses in the anterior and posterior bands, as well as in the lateral zone of the disc. Thus, modelling the disc as an isotropic solid matrix leads in this case to an overestimation of the stresses in the intermediate zone, an underestimation of the pore pressure in this area, and an underestimation of the stresses in the rest of the disc.

Structure and Size-selective Permeability of the Synovial Membrane of the Temporomandibular Joint of the Mouse Measured by MR Imaging at 7T.

PURPOSE: We analyzed the anatomical structure of the temporomandibular joint (TMJ) and molecular weight dependency of synovial membrane permeability in mice using 7-tesla magnetic resonance (MR) imaging. METHODS: We obtained 3-dimensional (3D) T1-weighted gradient echo (3D-T1W) and 3D T2-weighted rapid acquisition with relaxation enhancement (3D-T2W RARE) MR images of the TMJ of male C57BL6 mice with voxel resolution of 65 µm. Two-dimensional (2D) T1w images were measured every 45 s before and after bolus intravenous (IV) injection of contrast reagents: gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA; 0.5 kDa); oligomer-based contrast agent (CH3-DTPA-Gd; 2.1 kDa); gadolinium-labeled polylysine (Gd-polylysine; 10 kDa); and gadolinium-labeled albumin (Gd-albumin; 74 kDa). RESULTS: T1W images depicted the temporal bone and mandibular condyle as regions with lower signal intensity and the disc as a region of intermediate intensity. In the Gd-DTPA-enhanced T1W and T2W images, the articular disc could be identified as a region with lower signal intensity than that of the upper and lower joint cavities. After IV injection of Gd-DTPA or CH3-DTPA-Gd, the signal intensity of the joint cavities increased within 10 min, but this increase was not shown with Gd-polylysine and Gd-albumin. CONCLUSION: The structural findings obtained by MR imaging agreed with those obtained by hematoxylin-eosin staining under light microscopy. Contrast-enhanced MR imaging suggested that smaller (<2.1 kDa) but not larger (>10 kDa) molecules can permeate the synovial membrane. Our results suggest the utility of MR imaging for analyzing the structure of the TMJ as well as permeability of the synovial membrane.

Forward mandibular positioning up-regulates SOX9 and type II collagen expression in the glenoid fossa.

Regulatory factors governing the formation of bone in the glenoid fossa in response to functional appliance therapy have not been identified. Therefore, the purpose of this study was to investigate the temporal pattern of expression of two key chondrogenesis markers-SOX9 and its target gene, type II collagen-in the glenoid fossa by immunostaining in a 35-day-old Sprague Dawley rat model during both natural growth and forward mandibular positioning. The expression of both factors was up-regulated when the mandible was positioned forward, indicating an enhancement of chondrocyte differentiation and chondroid matrix formation. Our results indicate that chondroid bone formation in the glenoid fossa in response to forward mandibular positioning is regulated by molecular markers indicative of endochondral ossification.

Amplification of mitochondrial DNA from archival temporal bone specimens.

Archival temporal bone collections are an invaluable resource for studying the molecular genetics of many types of otopathology. The irreplaceable nature of temporal bone sections makes efficiency of DNA extraction of paramount importance. Several protocols are available for extracting DNA from fresh and preserved tissue. To establish the best protocol for reliably extracting DNA from celloidin-embedded temporal bone sections, a variety of DNA extraction techniques were tested. Using the optimum protocol, mitochondrial DNA fragments ranging in size from less than 100 base pairs to more than 400 base pairs were amplified, and the authenticity of polymerase chain reaction (PCR) products was confirmed through comparative sequence analysis.

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

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

Direct adenovirus-mediated gene delivery to the temporomandibular joint in guinea-pigs.

Adenovirus vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, recombinant adenovirus vectors harbouring beta-galactosidase gene (LacZ) was injected directly into the temporomandibular joints of Hartley guinea-pigs to clarify the in vivo transfer availability of the adenovirus vectors. Specifically, recombinant adenovirus harbouring LacZ gene (AxlCALacZ) was injected into the upper joint cavities of both mandibular joints of four male 6-week-old Hartley guinea-pigs. Either the same amount of recombinant adenovirus without LacZ gene (Axlw) suspension (placebo) or the same amount of phosphate-buffered saline solution (control) were injected into the upper joint cavities of both joints of another four male guinea-pigs. At 1, 2, 3 and 4 weeks after injection, the joints were dissected and the expression of delivered LacZ was examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining and reverse transcriptase-polymerase chain reaction (RT-PCR). To investigate the expression of transferred gene in other organs, total RNA was extracted from liver, kidney, heart and brain and the expression of LacZ mRNA and 18 S ribosomal RNA were analysed by RT-PCR. Clear expression of LacZ was observed in the articular surfaces of the temporal tubercle, articular disc and synovium of the temporomandibular joints even 4 weeks after injection in the AxlCALacZ-injected group, while no expression was detected in placebo and control groups. Histological examination confirmed that LacZ activity was clearly detected in a few cell layers of the articular surface tissues, which is much more efficient than in a previously study of the knee joint. In the other organs, expression of the delivered transgene was not observed. Based on these findings, direct gene delivery into the articular surface of the temporomandibular joint using the adenovirus vector is feasible as an effective in vivo method.

An immunohistochemical study of the effects of surgical induction of anterior disc displacement in the rabbit craniomandibular joint on type I and type II collagens.

The right craniomandibular joint (CMJ) was exposed surgically and all the discal attachments severed except for the posterior one. The disc was then repositioned anteriorly and sutured to the zygomatic arch. The left joint served as a sham-operated control; 10 other joints were used as non-operated controls. Deeply anaesthetized rabbits were perfused with 2% buffered formalin 2 weeks (10 rabbits) or 6 weeks (10 rabbits) after the induction of the anterior disc displacement (ADD). The articular disc, bilaminar zone, mandibular condyle and articular eminence were excised. The condyles and the articular eminences were demineralized in EDTA. All tissues were then sectioned at 10 microns in a cryostat. Sections were incubated with polyclonal antibodies directed against type I or type II collagens. Following incubation in the appropriate fluorescein isothiocyanate-labelled secondary antibodies, these specimens were studied under the fluorescence microscope. At 2 weeks there was a reduction in type II collagen immunostaining; some areas of the experimental condylar cartilage showed a switch from type II to type I collagen. However, at 6 weeks there was an increase in type II collagen immunostaining and a decrease in type I compared to the 2-week group. It is concluded that surgical induction of ADD in the rabbit CMJ leads to alteration in the condylar cartilage collagen phenotype similar to that reported for osteoarthritic cartilage of other synovial joints.

Giant cell reparative granuloma of the temporal bone: neuroradiological and immunohistochemical findings.

A 38-year-old man presented with a giant cell reparative granuloma (GCRG) of the left temporal bone. Computed tomography showed a osteolytic middle cranial mass lesion. Magnetic resonance (MR) imaging showed the lesion as low intensity with heterogeneous enhancement by gadolinium on the T1-weighted images, and extremely low intensity on the T2-weighted images. Angiography showed the lesion as highly vascular and fed by branches of the left external carotid artery. After preoperative embolization, gross total removal of the tumor was performed. The postoperative course was uneventful and no evidence of recurrence has been found for more than 4 years. Histological examination revealed GCRG with multinucleated giant cells in the fibrous background, abundant collagen bundles, hemosiderin deposits, and trabeculae of reactive bone. Some of the mononuclear stromal cells and almost all of the giant cells were positive for CD68, suggesting histiocytic differentiation. These histological features reflect the marked decrease in signal intensity on T2-weighted MR images.

Fetal and early post-natal mineralization of the tympanic bulla in fin whales may reveal a Hitherto undiscovered evolutionary trait.

The evolution of the cetacean skeleton followed a path that differentiated this group from other terrestrial mammals about 50 million years ago [1], and debate is still going on about the relationships between Cetacea and Artiodactyla [2], [3], [4]. Some skeletal traits of the basilosaurids (the more advanced forms of Archaeocetes), such as the expansion of the peribullary air sinuses, dental modification and vertebral size uniformity [5] are maintained and further emphasized also in contemporary odontocetes and mysticetes. Using Dual-Energy X-Ray Absorptiometry here we report that the deposition of bone mineral in fetal and newborn specimens of the fin whale Balaenoptera physalus is remarkably higher in the bulla tympanica than in the adjacent basal skull or in the rest of the skeleton. Ossification of the tympanic bulla in fetal Artiodactyla (bovine, hippopotamus) is minimal, becomes sensible after birth and then progresses during growth, contrarily to the precocious mineralization that we observed in fin whales. Given the importance of the ear bones for the precise identification of phylogenetic relationship in therian evolution [6], this feature may indicate a specific evolutionary trait of fin whales and possibly other cetacean species or families. Early mineralization of the tympanic bulla allows immediate sound conduction in the aquatic medium and consequently holds potential importance for mother-calf relationship and postnatal survival.

Strategies for drug delivery to the human inner ear by multifunctional nanoparticles.

UNLABELLED: Hearing loss is a very significant health problem. The methods currently available for inner ear drug delivery are limited and a noninvasive cell-specific drug delivery strategy needs to be found. AIM: In this study we investigated the ability of polymersomes, lipid core nanocapsules and hyperbranched poly-L-lysine to cross the round window membrane. MATERIALS & METHODS: Nanoparticles (NPs) used in this study have different size and chemical compositions. Freshly frozen human temporal bones were used for this investigation. Intact human round window membrane within the freshly frozen human temporal bone served as an excellent model to test the membrane permeation and distribution within the tissues. RESULTS: In this investigation we were able to visualize the NPs across the round window membrane. The NPs were subsequently found to be distributed in the sensory hair cells, nerve fibers and to other cells of the cochlea. CONCLUSION: This finding raises hope in terms of future multifunctional NP-based drug delivery strategy to the human inner ear.

A histochemical study on condylar cartilage and glenoid fossa during mandibular advancement.

OBJECTIVE: To evaluate cellular hypertrophic activities in the mandibular condylar cartilage (MCC) and the glenoid fossa (GF) during mandibular advancement in the temporomandibular joint (TMJ) of Sprague-Dawley rats, as evidenced by fibroblast growth factor 8 (FGF8). METHODS AND MATERIALS: Fifty-five female 24-day-old Sprague-Dawley rats were randomly divided into four experimental and control groups, with a mandibular advancement appliance on the experimental rats' lower incisors. The rats were euthanized on days 3, 14, 21, and 30 of the study, and their TMJ was prepared for a immunohistochemical staining procedure to detect FGF8. RESULTS: FGF8 expression was significantly higher among the experimental rats (P  =  .002). Patterns of ascension and descension of FGF8 expression were similar in experimental and control samples. The results show an overall enhanced osteogenic transition occurring in both the MCC and the GF in experimental rats in comparison with controls. The level of cellular changes in the MCC is remarkably higher than in the GF. CONCLUSION: In the MCC and the GF, cellular morphologic and hypertrophic differentiations increase significantly during mandibular advancement. It is also concluded that endochondral ossification in the MCC and intramembranous ossification in the GF occur during adaptive remodeling.

The biological mechanisms of PCNA and BMP in TMJ adaptive remodeling.

OBJECTIVES: To histologically and immunohistochemically assess the pattern of expression of bone morphogenic proteins 2 and 4 (BMP2/4) and proliferating cell nuclear antigen (PCNA) in response to bite jumping appliances in the condylar cartilage and the glenoid fossa. MATERIALS AND METHODS: Fifty-five 4-week-old female Sprague-Dawley rats were randomly divided into four experimental and four control groups. Bite-jumping appliances were fitted to the experimental animals. The rats were sacrificed at 3, 14, 21, and 30 days, and the temporomandibular structures were analyzed histologically and immunohistochemically. RESULTS: The expression of BMP2/4 in response to bite-jumping appliances was statistically significant in the condylar cartilage and the glenoid fossa. Cell proliferation was not significant. CONCLUSION: BMP2/4 plays an important role in bone formation in response to mandibular advancement by accelerating and enhancing the differentiation of mesenchymal cells into bone-forming cells.

Recombinant adeno-associated virus serotype 2 (rAAV2)-An efficient vector for gene delivery in condylar cartilage, glenoid fossa and TMJ disc in an experimental study in vivo.

OBJECTIVE: To elaborate whether rAAV2 can be used for future TMJ gene therapy, we examined the infection efficiencies of rAAV2 in vitro, and the transgene expression pattern mediated by rAAV2 in glenoid fossa, TMJ disc and condylar cartilage in vivo. MATERIALS AND METHODS: Different dosages of rAAV2-eGFP (MOI: 5 x 10(4), 1 x 10(4), 5 x 10(3)) were applied to primary cultured condylar chondrocytes of rats. Infection efficiencies were analysed by FACSCalitur at different time points. Vastatin, a molecule not naturally expressed in TMJ, was used as a reporter for detection of rAAV2 mediated transgene expression in vivo. Thirty SD rats were injected with either rAAV2-sec-Vastatin (experimental group) or rAAV2-eGFP (control group) into both sides of TMJ. They were sacrificed at the indicated time (7, 14, 21, 30 and 60 days of injection) and the TMJ samples were collected for RT-PCR and immunostaining analysis. RESULTS: High dosage (MOI 5 x 10(4)) of rAAV2-eGFP can achieve desirable transduction efficiencies in vitro after 5 days. Transgene expression of rAAV-sec-Vastatin persisted for about 21 days in glenoid fossa, around 7 days in TMJ disc and at least 60 days in condylar cartilage in vivo. In condylar cartilage, transgene expression was found in the proliferative layer and chondroblast layer (day 7), chondrocyte layer (day 14), pre-hypertrophic and hypertrophic layer (day 21), hypertrophic layer and deep hypertrophic layer (day 30 and 60). CONCLUSION: Recombinant AAV2 could be considered as a promising vector for gene therapy in TMJ which can mediate therapeutic gene expression in glenoid fossa, articular disc and condylar cartilage in vivo.

Shox2-deficiency leads to dysplasia and ankylosis of the temporomandibular joint in mice.

The temporomandibular joint (TMJ) is a unique synovial joint whose development differs from the formation of other synovial joints. Mutations have been associated with the developmental defects of the TMJ only in a few genes. In this study, we report the expression of the homeobox gene Shox2 in the cranial neural crest derived mesenchymal cells of the maxilla-mandibular junction and later in the progenitor cells and undifferentiated chondrocytes of the condyle as well as the glenoid fossa of the developing TMJ. A conditional inactivation of Shox2 in the cranial neural crest-derived cells causes developmental abnormalities in the TMJ, including dysplasia of the condyle and glenoid fossa. The articulating disc forms but fuses with the fibrous layers of the condyle and glenoid fossa, clinically known as TMJ ankylosis. Histological examination indicates a delay in development in the mutant TMJ, accompanied by a significantly reduced rate of cell proliferation. In situ hybridization further demonstrates an altered expression of several key osteogenic genes and a delayed expression of the osteogenic differentiation markers. Shox2 appears to regulate the expression of osteogenic genes and is essential for the development and function of the TMJ. The Shox2 conditional mutant thus provides a unique animal model of TMJ ankylosis.

VEGF and bone formation in the glenoid fossa during forward mandibular positioning.

This study was designed to identify the relationship between vascularization and bone formation in the glenoid fossa during natural growth and functional appliance therapy. The temporal pattern of vascular endothelial growth factor (VEGF) expression and bone formation in the glenoid fossa during natural growth was identified and compared with that during forward mandibular positioning. We randomly divided 150 female Sprague-Dawley rats, 35 days old, into 10 experimental and 10 control groups. Appliances were fitted to position the mandible forward in the experimental groups. The rats were then killed at different times. Sections were cut and stained with anti-VEGF antibodies to evaluate VEGF expression, and with periodic acid and Schiff's reagent to evaluate new bone formation. Both VEGF expression and newly formed bone were measured by a computer-assisted image analyzing system. The results showed that, during natural growth and forward mandibular positioning, VEGF expression and new bone formation were highest in the posterior region of the glenoid fossa. There were significant increases of VEGF and new bone formation in the experimental groups compared with the controls. The highest amount of VEGF expression occurred before the highest amount of bone formation was reached. Forward mandibular positioning causes significant increases in vascularization and new bone formation in the glenoid fossa. A close correlation exists between vascularization and bone formation.

Gallium-67 SPECT scintigraphy may be useful in diagnosis of temporal arteritis.

BACKGROUND: Temporal arteritis (TA) is a common syndrome in the elderly, consisting of persistent pain in the temporal area of the skull, jaw claudication, sudden visual loss, high erythrocyte sedimentation rate, and tenderness on palpation in the temporal area. The diagnosis of this condition is relatively straightforward when the typical symptoms and a positive temporal artery biopsy are present. However, only half of the patients have a positive temporal artery biopsy. Other diagnostic procedures, such as colour Doppler sonography or superficial carotid artery angiography which have been proved to be useful for the diagnosis of TA, do not discriminate between inflammatory and non-inflammatory temporal artery disease and may be helpful only in experienced hands. Gallium-67 ((67)Ga) planar scan was reported to be useful in the diagnosis of the disease. Quantitative (67)Ga single photon emission computed tomography (SPECT) may raise the accuracy of the diagnosis. OBJECTIVE: To investigate the effectiveness and usefulness of (67)Ga SPECT scintigraphy in the diagnosis of TA. METHODS: Nine patients (five male, four female) and six controls were included in the study. All of them received 8-10 mCi (67)Ga intravenously 48 hours before the scan.(67)Ga uptake ratios were calculated on transaxial and coronal slices. RESULTS: All patients showed increased uptake in the temporal area of the skull compared with controls. CONCLUSION: The data suggest that (67)Ga skull SPECT may be useful in the diagnosis of TA, especially if the uptake ratio in the area of interest is calculated. Further studies are needed to confirm these data.

Growth and type-II collagen expression in the glenoid fossa of the temporomandibular joint during altered loading: a study in the rat.

The aim of this study was to measure changes in growth of the glenoid fossa and its articular eminence after decreased loading. A further aim was to evaluate the role of mechanical forces in relation to the existence of a cartilage layer, by determining type-II collagen secretion. A total of 99 Wistar rats were used: 48 animals were fed whole pellets and 51 were fed ground pellets. At age 21 days, after weaning, the upper and lower incisors of the soft-diet group were shortened by cutting them, twice a week. Ten animals fed whole pellets and 10 fed ground pellets were injected i. p. with Alizarin red (200 mg/kg) at ages 22, 30 and 40 days, and killed at ages 30, 40 and 50 days respectively. The heads were freed from the soft tissue and the zygomatic process cut sagittally at the deepest point of the greatest transversal concavity of the eminence. Bone apposition was measured. The other animals were used for studies involving collagen II immunostaining. Bone growth decreased in the group fed ground pellets except in the anterior-most part of the glenoid fossa at 50 days. Immunohistochemical analysis revealed larger areas of anti-collagen II staining in the group fed whole pellets, most markedly in the posterior part of the glenoid fossa. Growth of the articulationg surface of the temporal component of the temporomandibular joint appears to depend on mechanical factors, such as the condyle. The underlying mechanics seem likely to be different. The presence of type-II collagen is obviously not regulated only by compressive forces but probably also by tension loading.