Apoptosis in meatal skin, cholesteatoma and squamous cell carcinoma of the ear.

Apoptosis in normal meatal skin, cholesteatoma and squamous cell carcinoma (SCC) of the ear was investigated by using morphological analysis and in-situ specific labelling of fragmented DNA. In meatal skin, apoptotic cells were localized mainly in the granular layers, but were not so restricted in cholesteatoma, while in SCC they were even more dispersed. The apoptotic index (AI) was low (1.59 +/- 0.10 SEM) in normal skin. It was increased in cholesteatoma (2.09 +/- 0.11), and was intermediate in SCC (1.72 +/- 0.14). By contrast, the mitotic index (MI) increased from 0.19 +/- 0.02 in normal skin, to 0.25 +/- 0.01 in cholesteatoma and to 0.25 +/- 0.02 in SCC. Our findings indicate that apoptosis is involved in the epithelial homeostasis of meatal skin, cholesteatoma and SCC of the ear. The hyperproliferation of epithelial cells in cholesteatoma is counteracted by an increased apoptosis rate, while in SCC the increased cell proliferation without a compensatory increase in apoptosis may be associated with the malignant transformation.

Lectin expression during wound healing of the rabbit sinus mucosa: a study of regenerating epithelium and early polyp formation.

Lectin expression during wound healing of the rabbit sinus mucosa was examined. Positive UEA-I staining was evident on squamous or cuboidal as well as columnar regenerating epithelial cells (RE cells). PNA staining of columnar RE cells first became evident after neuraminidase treatment, while squamous or cuboidal RE cells stained positively with PNA alone. Fucosylation within RE cells thus occurred from a relatively early period, and sialylation followed at later stages. Ingrowing epithelial cells of early polyp formation stained negatively with UEA-I, indicating that unfucosylated RE cells may represent aberrant cellular behavior. We concluded that these patterns of lectin staining indicate a functional maturation as well as an integration of regenerating mucosa.

Edema formation in the rat larynx.

In the rat larynx, plasma exudation and edema formation were studied by light and electron microscopy after i.v. injections of the mast cell activator compound 48/80, substance P, and capsaicin. The morphological effects of substance P and capsaicin on connective tissue mast cells in vivo were also examined. Of the drugs tested, only compound 48/80 degranulated the connective tissue mast cells. All drugs induced a subepithelial plasma exudation in the subglottic region, with edema in the lamina propria and widened intraepithelial intercellular spaces, though the tight junction regions seemed intact. In the epiglottis, 10 min after compound 48/80 injection, there was edema in the lamina propria on the lingual side, with an intact and tight epithelial lining. No morphological sign of edema was found in the epiglottis after injection of substance P or capsaicin. The pronounced effect found in the epiglottic region after compound 48/80 injection was due to the release of mediators such as histamine and 5-hydroxytryptamine from the connective tissue mast cells. This study supports the belief that substance P in vivo mediates an increased vascular permeability by a direct effect on the blood vessels - a mechanism distinct from mast cell degranulation.

Changes in glycoconjugate expression of the sinus mucosa during experimental sinusitis: a lectin histochemical study of the epithelium and goblet cell development.

Lectin expression in both normal and inflamed sinus mucosa in rabbit was investigated histochemically. A different staining pattern was observed in the inflamed mucosa, i.e. the degree of staining reactivity with Canavalia ensiformis (ConA) decreased, whereas staining with Ulex europaeus agglutinin-I (UEA-I), Arachis hypoga (PNA) with neuraminidase pretreatment and Triticum vulgaris (WGA) intensified, indicating an enhanced fucosylation as well as sialylation. Goblet cells were stained with UEA-I, WGA, PNA and PNA with neuraminidase pretreatment, but scarcely with ConA. Positive PNA staining of basal cells and epithelial secretory granules was observed in the inflamed mucosa, especially close to areas where goblet cell development was prolific. It was therefore assumed that basal cells can differentiate into goblet cells-through epithelial secretory cells accompanied by sialylation and/or sulphonation of their mucins. It is concluded that the changes in glycoconjugate expression as well as goblet cell development in the inflamed mucosa are of importance for both local host resistance and defence mechanisms against microorganisms during the early stages of the inflammatory process.

Early stages in the development of goblet cells in the paranasal sinuses: a multimethodological study in the rabbit.

To evaluate the pattern of goblet cell differentiation in sinus mucosa in response to external stimuli, New Zealand White rabbits were subjected to either experimental sinusitis or topical capsalcin application. Sinus mucosa was examined by light microscopy after serial sectioning, whole-mount preparation or immunohistochemistry. The mucosa was also examined by electron microscopy after perfusion fixation or high-pressure freezing. While goblet cells were normally very scarce in the healthy rabbit maxillary sinus mucosa, such cells were frequent after experimental sinusitis or topical capsaicin application. The process of goblet cell differentiation seems to follow a sequential path where serous secretory cells start to produce an increasing amount of mucous granules which appear electron lucent after conventional fixation. Parallel to this shift in secretion production, the cell assumes a bulging appearance after conventional fixation. It is concluded that newly formed goblet cells are recruited from intermediate secretory cell stages rather than from ciliated cells.

Recurrent primary pleomorphic adenomas of salivary gland origin: intrasurgical rupture, histopathologic features, and pseudopodia.

BACKGROUND: The rate of tumor recurrence after surgery for benign salivary gland pleomorphic adenoma varies considerably in different clinical settings and seems to depend to a great extent on the surgical technique used. The importance of tumor spillage for subsequent recurrence has recently been questioned. The current follow-up study was undertaken to ascertain whether intrasurgical rupture, tumor spillage, or any histopathologic feature might have had an impact on the rate of recurrence. METHODS: The medical records of all 255 patients operated on for benign salivary gland pleomorphic adenoma between the years 1974 and 1993 at the Department of Otorhinolaryngology, Huddinge University Hospital, were reviewed. All patients alive in April 1995 (n = 230) were sent a simple questionnaire. Two hundred thirteen of these patients received follow-up. All cases of tumor recurrence after surgery or intrasurgical rupture of the tumor capsule were reviewed histopathologically. RESULTS: Two (7.1%) of the 28 patients who had macroscopic capsule rupture during surgery experienced recurrence at a later stage. This was not a statistically higher rate than the 4.1% recurrence rate for the rest of the material. As many as 5 of the 9 primary tumors that subsequently recurred (56%) sent fingerlike tumor extensions or pseudopodia outside the pseudocapsule. The rate of occurrence of such structures was statistically higher than that of the tumors that ruptured during surgery (25%) and the examined uncomplicated cases (8%). CONCLUSIONS: Occurrence of pseudopodia--microscopic fingerlike formations of tumor tissue that extend beyond the main lump of the tumor--is a significant risk factor for local recurrence.

Effects of experimental Mycoplasma pulmonis infection on sensory neuropeptides and airway mucosa in the rat.

The effect of airway infection on neurogenic inflammation is not known. The present study examines the effect of Mycoplasma pulmonis infection on the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) in the trigeminal ganglion and in the mucosa of the nose and trachea in rats. We compared germ-free (GF), conventionally raised (CV) and specific pathogen-free (SPF) rats. The concentrations of SP and CGRP in the nasal mucosa were assessed with immunohistochemistry, and their prohormonal transcripts in the trigeminal ganglion were assessed with Northern blot. Mucosa was also processed for light microscopy and electron microscopy. SP-like immunoreactivity was greater in the nasal mucosa of infected animals than in uninfected controls. CGRP-like immunoreactivity was greater in the nasal septum, but not in the nasal turbinate, of infected than uninfected animals. In contrast, no change was evident in the expression levels of the prohormonal transcripts in the trigeminal ganglion. Infected nasal and tracheal mucosa was oedematous and locally infiltrated with inflammatory cells. In the nose of uninfected GF rats, subepithelial lymphoid aggregations were scarce and appeared inactive. We conclude that Mycoplasma pulmonis infection results in increased immunoreactivity of substance P, probably within nerves. There was no clear evidence of increased synthesis of the precursors of substance P and calcitonin gene-related peptide.

Expression of transforming growth factor-alpha and epidermal growth factor receptor in middle ear cholesteatoma.

To study the possible autocrine growth stimulation of cholesteatoma epithelium, the expression of transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) in middle ear cholesteatoma was investigated by immunohistochemical staining techniques. Twenty cholesteatoma samples obtained at operation and six normal skin specimens collected from the external ear canal were used in the study. Immunostaining for TGF-alpha showed a diffuse cytoplasmic staining pattern. It was weakly expressed in the basal layer of the normal epidermal epithelium but was more strongly expressed in all cholesteatoma specimens. EGFR showed a dot-like/diffuse cytoplasmic and cell membrane staining pattern. EGFR-positive cells were seen in the basal layer of normal epidermis. In the cholesteatoma specimens, expression of EGFR was not only confined to the basal layer but persisted into the upper layers of the epithelium. Our findings indicate that an autocrine growth stimulation by TGF-alpha and EGFR may contribute to the unrestrained growth of cholesteatoma epithelium in the middle ear cavity.

Zenker's diverticulum--a historical review and trends in therapy.

More than 200 years have passed since the pulsion diverticulum of hypopharynx was first described. This paper reviews different opinions on its etiology over the centuries. The German pathologist F.A. von Zenker, who successfully pursued research on a variety of topics, is often associated with this diverticulum through his classical work from 1867 "Krankheiten des Oesophagus", which deals with the pathogenesis and clinical presentations of this herniation of the posterior mucosal wall. Numerous surgical techniques, which have been practised mainly during this century, are also reviewed. Different options of endoscopic surgery, which is presently the preferred approach at many medical centers, are described and discussed. Our own experience of endoscopic surgery in patients with Zenker's diverticulum is also presented.

Experimentally induced polyps in the sinus mucosa: a structural analysis of the initial stages.

To document polyp formation in the sinus mucosa, the authors of this study subjected New Zealand white rabbits to different modes of manipulation intended to induce inflammation of the maxillary sinus. These manipulations included a combination of bacterial infection and mechanical trauma, the deposition of agarose into the sinus cavity, and the deposition of N-formyl-methionyl-leucyl-phenylalanine, a chemotactic peptide, into the sinus cavity. A majority of animals developed polyps, which were examined by light and electron microscopy. Polyp formation appears to involve epithelial disruption and the migration of immature branching epithelium. While part of the migrating epithelium eventually covers the mucosal defect, other branches spread into the underlying connective tissue, where intraepithelial microcavities with a differentiated, ciliated lining are formed. Fusing cavities separate the developing polyp body from the adjacent mucosa. With the described method, mucosal polyps can be induced with high reproducibility.

A comparison of morphological effects on the rabbit nasal and sinus mucosa after surgical denervation and topical capsaicin application.

In order to study morphological effects on the nasal and sinus mucosa, New Zealand White rabbits underwent either unselective, regional sectioning of sensory and parasympathetic nerve branches or topical treatment of the mucosa with capsaicin. Ten days after treatment, mucosal specimens were analyzed by light and electron microscopy. Immunohistochemistry was used to evaluate neuropeptides present, in particular substance P, calcitonin gene-related peptide, vasoactive intestinal peptide and neuropeptide Y. In surgically denervated rabbits, mucosal glands were found to be enlarged and contained an increased number of zymogen granules having a bipartite substructure. Topical capsaicin application caused localized epithelial changes in the sinus mucosa and maxilloturbinal region of the nose, including clotting of cilia and an increased number of goblet cells. Reduced amounts of all neuropeptides investigated were found in the surgically denervated animals, while topical capsaicin treatment had only marginal effects on the mucosal neuropeptide content. The morphological changes observed after surgical denervation suggest an imbalance between neural stimulation and secretory capacity of the mucosal glands. These findings could explain the difference in clinical effect noted between sectioning of the vidian nerve and topical treatment with capsaicin in patients with perennial rhinitis.

Antigen expression of epithelial markers, collagen IV and Ki67 in middle ear cholesteatoma. An immunohistochemical Study.

The expression of epithelial markers (cytokeratins, Filaggrin, BerEp4 and EMA), collagen IV and Ki67 was studied immunohistochemically in cholesteatoma and compared with that in epidermis of meatal skin, squamous epithelium of eardrum and simple epithelium of middle ear mucosa. MNF116 (cytokeratin 10, 17, 18) stained the full layer of normal epithelium and all cholesteatoma specimens. CK10 and Filaggrin were expressed in the upper layer of epidermis but more diffusely in cholesteatoma. BerEp4 was found in the basal layer of normal epithelium but was detected in most epithelial cells in cholesteatoma matrix. Variability was observed in EMA and CK14 immunostaining. Collagen IV was localized in the basement membrane of normal epithelium with a continuously staining pattern, an observation also made in the cholesteatomas studied. However, in one of these small areas the basement membrane was not stained with collagen IV. Ki67 was expressed in nuclei of the cells in the basal layer of normal epithelium but extended to epithelial cells in the upper layers of cholesteatoma matrix. The results of the present study indicate that the expression pattern of epithelial markers in cholesteatoma corresponds to that in normal epidermis. The increasing expression of BerEp4 and Ki67 confirms the hyperproliferative nature of cholesteatoma. Whether or not the lack of expression of collagen IV in one of the cholesteatomas reflects a true degradation of the basement membrane needs further investigation in extended materials.

Formation of mucosal polyps in the nasal and maxillary sinus cavities by infection.

Unilateral maxillary sinusitis was experimentally induced in New Zealand White rabbits with Streptococcus pneumoniae serotype 3, Bacteroides fragilis NCTC 9343, and Staphylococcus aureus V8. In another group of rabbits, sinusitis was induced by blocking of the sinus ostium only. Bacteriologic and light microscopic analysis was performed after 5 days to 1 month. Granulation-like polyps developed after deep mucosal inflammatory trauma initiating fibroblast proliferation, angiogenesis, and epithelial migration to cover the polyp. In regions of a more superficial trauma-characterized by epithelial desquamation and fibroblast growth-proliferation and differentiation of basal cells resulted in the formation of microcavities dissecting off edematous polyps. Polyps could be found in all sinusitis groups, irrespective of inducing agent. The cellular events of polyp formation appear to be the result of a continuous inflammatory reaction and are not directly related to the presence of a certain microorganism. Instead, the potential of any microorganism to induce a deep mucosal trauma or epithelial desquamation seems essential for its ability to initiate polyp formation.

Mucosal fine structure in experimental sinusitis.

Rabbit maxillary sinuses were inoculated with Streptococcus pneumoniae and Bacteroides fragilis, and the histologic response in the sinus mucosa was observed over a 12-week period. An increased height of the cylindric cells and hyperplasia of the basal cells were frequent findings irrespective of the pathogen inoculated. The disease was found to influence the character of the secretory product from epithelial secretory cells and to degranulate the subepithelial glands. Ciliary loss was a transitional finding. A reduction in the number of mitochondria, the occurrence of deformed short microvilli, and cytoplasmic blebbing were seen in the cells devoid of normal cilia. It is inferred from this study that pneumococcal sinusitis in rabbits is a self-limiting process, and the mucosal sequelae of the acute infection are persisting goblet cells, slight focal fibrosis, and edema. Inoculation with B fragilis produces a chronic inflammatory process, with infiltration of mononuclear cells, luminal dilatation of the glands exhibiting zymogen granule depletion, and an increased thickness of the whole mucosal layer.

Regeneration of maxillary sinus mucosa following surgical removal. Experimental study in rabbits.

A rapid regeneration of the epithelium takes place in the maxillary sinus in rabbits after experimental operative removal of the mucosa. Two weeks postoperatively the previously denuded areas have reepithelialized. The subepithelial glands, however, do not seem to regenerate. The normal sinus mucosa contains numerous serous glands in the lamina propria, but in the regenerated mucosa these glands are replaced by dense connective tissue. Atypical glands and polyp formations are sometimes encountered, but goblet cells are sparse. Furthermore, the sinus cavity on the operated side is reduced in size compared with the nonoperated side because of fibrosis and periosteal reactions including bone degradation and neogenesis. This study indicates that although the mucosa is reepithelialized within 2 weeks, the regeneration of the lamina propria is incomplete, and reactive cellular processes such as bone remodeling, fibroblast proliferation, and formation of polyps and "atypical glands" are characteristic of regenerating mucosa.

Lysozyme and lactoferrin in human maxillary sinus mucosa during chronic sinusitis. An immunohistochemical study.

Immunohistochemistry was used to study the localization of lysozyme (LZ) and lactoferrin (LF) in the human sinus mucosa during recurrent and chronic sinusitis. Serous cells of submucosal mixed glands and polymorphonuclear leukocytes both displayed a strongly positive staining reaction to both LZ and LF in the normal mucosa. A positive though weak staining for LZ and LF could also be found occasionally within goblet cells. In the mucosa from patients with recurrent or chronic sinusitis, the staining reaction to LZ appeared to intensify in goblet cells. Furthermore, an increased immunoreactivity of glands vis-à-vis LZ and LF was also noted occasionally. Atypical glands were frequently found in mucosa from patients with chronic sinusitis. The epithelium of these latter glands often showed an intense staining reaction to LF, but a rather weak reaction to LZ. The results of the present study suggest that the observed increase in LZ and LF secreting activity of goblet cells, epithelial cells and newly formed atypical glands may play a part in the defense mechanism of the sinus mucosa during the course of chronic sinusitis.

Lactic acid isomers and fatty acids in sinus secretion: a longitudinal study of bacterial and leukocyte metabolism in experimental sinusitis.

Concentrations of the two optic isomers of lactate (D- and L-form) as well as glucose, succinate, acetate, butyrate, isovalerate and valerate were examined in purulent sinus secretions. The samples were obtained from rabbit maxillary sinuses, experimentally infected with Streptococcus pneumoniae or Bacteroides fragilis. More soluble acids such as acetate displayed relatively low levels in the secretion, despite a high microbial production. A substantial increase in D-lactate concentration was found in secretions only the first day after induction of pneumococcal sinusitis, and not in bacteroides sinusitis. L-lactate levels were particularly high in secretions of a marked purulent character, and this isomer can be considered as indicator of anaerobic glycolysis in the leukocytes. Less diffusible fatty acids such as butyrate and isovalerate accumulated in the secretion, in spite of a relatively lower production rate, and are thus more reliable indicators of bacterial metabolism.

Experimental maxillary sinusitis induced by Bacteroides fragilis. A bacteriological and histological study in rabbits.

Experimental anaerobic maxillary sinusitis was induced in New Zealand White rabbits by blocking the ostium and inoculating Bacteroides fragilis, strain NCTC 9343. The animals were examined histologically and bacteriologically after 5 days, and 2, 3 and 4 weeks. All the infected sinuses displayed signs of moderate or severe inflammation throughout the study period. Ciliary damage and desquamation, hyperplasia and metaplasia of the epithelium were characteristic features. Furthermore, heavy leukocyte- and, particularly, round cell-infiltration, fibrosis, periosteal hyperplasia and bone degradation and -formation were also frequently encountered. The secretory cell count in the epithelium increased, including the regeneration of goblet cells. After 4 weeks no obvious recovery could be seen, and the inducing microorganism was re-isolated in the majority of cases. In comparison with experimental pneumococcal sinusitis, the B. fragilis infection exerts a more prolonged and severe inflammation.

Cellular regeneration and recovery of the maxillary sinus mucosa. An experimental study in rabbits.

Unilateral maxillary sinusitis was induced in 30 New Zealand White rabbits with Streptococcus pneumoniae or Bacteroides fragilis. In another group of 15 rabbits without infection, the sinus mucosa was surgically removed in defined areas. In both series, the sinuses were serially sectioned for histological analysis of the cellular regenerative capacity. In maxillary sinusitis induced by Bacteroides fragilis, an inflammatory and also reparative process involving all mucosal layers including the underlying periosteum was seen. The more superficial trauma as found in pneumococcal sinusitis eventually led to restitution ad integrum. Following surgical removal, the denuded sinus-lining was reepithelized by a flattened ciliated epithelium on a lamina propria displaying fibrosis and lacking serous glands. The restoration of the rabbit maxillary sinus mucosa after surgical trauma thus leads to structural abnormalities of the epithelium as well as the lamina propria, and these changes are likely to interfere with the normal function of the sinus mucosa.