bromochloroacetic-acid and Tympanic-Membrane-Perforation

Most tympanic membrane (TM) perforations heal spontaneously, but approximately 10-20% remain open as chronic TM perforations. Chronic perforations can lead to an impaired hearing ability and recurrent middle ear infections. Traditionally, these perforations must be surgically closed, which is costly and time consuming. Therefore, there is a need for simpler therapeutic strategies. Previous studies by us have shown that plasminogen (plg) is a potent pro-inflammatory regulator that accelerates cutaneous wound healing in mice. We have also shown that the healing of TM perforations is completely arrested in plg-deficient (plg(-/-)) mice and that these mice develop chronic TM perforations. In the present study, we investigated the therapeutic potential of local plg injection in acute and chronic TM perforation mice models.. Plg(-/-) mice and wild-type mice were subjected to standardized TM perforations followed by local injection of plg into the soft tissue surrounding the TM. TM perforations with chronic characteristics were induced by leaving TM perforations in plg(-/-) mice untreated for 9 days before treatment. The healing process was observed through otomicroscope and finally confirmed by immunostaining. The quality of TM healing was evaluated based on the morphology of the TM.. Daily local injections of plg into the soft tissue surrounding the TM restored the ability to heal TM perforations in plg-/- mice in a dose-dependent manner, and potentiated the healing rate and quality in wild-type mice. A single local injection of plg initiated the healing of the chronic-like TM perforations in these mice, resulting in a closed TM with a continuous but rather thick outer keratinocyte layer. However, three plg injections led to a completely healed TM with a thin keratinizing squamous epithelium covering a connective tissue layer.. Our data suggests that plg is a promising drug candidate for the treatment of chronic TM perforations in humans.

Topics: Animals; Chronic Disease; Dose-Response Relationship, Drug; Immunohistochemistry; Injections, Intraperitoneal; Injections, Subcutaneous; Keratins; Mice; Mice, Inbred C57BL; Plasminogen; Tympanic Membrane Perforation; Wound Healing

Mice deficient in plasminogen, the precursor of plasmin, show completely arrested healing of tympanic membrane (TM) perforations, indicating that plasmin plays an essential role in TM healing. The activation of plasminogen to plasmin is performed by two plasminogen activators (PAs), urokinase-type PA (uPA) and tissue-type PA (tPA). To elucidate the functional roles of PAs in the healing of TM perforations, we investigated the phenotypes of single gene-deficient mice lacking uPA (uPA(-/-)) or tPA (tPA(-/-)) after TM perforation. Delayed healing of TM perforations was observed in uPA(-/-) mice but not tPA(-/-) mice. The migration of keratinocytes was clearly delayed and seemed to be misoriented in uPA(-/-) mice. Furthermore, fibrin deposition and the inflammatory response were persistent in these mice. Our findings demonstrate that uPA plays a role in the healing of TM perforations. The observed phenotypes in uPA(-/-) mice are most likely due to the reduced generation of plasmin.

Topics: Animals; Cell Movement; Fibrinolysin; Immunohistochemistry; Keratinocytes; Keratins; Mice; Mice, Knockout; Otoscopy; Phenotype; Tissue Plasminogen Activator; Tympanic Membrane Perforation; Urokinase-Type Plasminogen Activator; Wound Healing

Plasminogen has been proposed to play an important role in different tissue remodeling processes such as wound healing and tissue regeneration after injuries. The healing of tympanic membrane perforations is a well-organized chain of inflammatory events, with an initial invasion of inflammatory cells followed by reparative and restoration phases. Here we show that the healing of tympanic membrane perforations is completely arrested in plasminogen-deficient mice, with no signs of any healing even 143 days after perforation. Inflammatory cells were recruited to the wounded area, but there were no signs of tissue debridement. In addition, removal of fibrin, keratinocyte migration and in-growth of connective tissue were impaired. This contrasts with skin wound healing, where studies have shown that, although the healing process is delayed, it reaches completion in all plasminogen-deficient mice. Our finding that keratinocyte migration and re-epithelialization were completely arrested in plasminogen-deficient mice indicates that plasminogen/plasmin plays a more profound role in the healing of tympanic membrane perforations than in the healing of other epithelial wounds.

Topics: Animals; Cell Proliferation; Fibrin; Fibrinolysin; Keratinocytes; Keratins; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Knockout; Neutrophil Infiltration; Neutrophils; Plasminogen; Time Factors; Tympanic Membrane Perforation; Wound Healing

The origin and behaviour of keratinizing stratified squamous epithelium, an essential component of cholesteatoma occurring in the middle-ear cavity, has puzzled otologists for decades. In this experimental study in 16 cats, central (n = 23) and peripheral (n = 9) tympanic membrane perforations were observed for up to 63 days before sacrifice. The tympanic membranes with bony rim were excised, decalcified and embedded in Epon 812. Sections were stained with toluidine blue and examined using a light microscope. The perforation had been sealed by meatal epithelium exhibiting pronounced hyperplasia and keratin formation, lying on a bed of granulation tissue. Subtotal central perforations healed within 14 days, forming a bowl-shaped tympanic membrane and leaving parts of the handle of the malleus (with meatal epithelium) protruding freely into the middle-ear cavity. Stratified squamous epithelium, morphologically identical with that of external ear canal epidermis, could be observed on the malleus even 63 days after operation. This meatal epithelium was non-keratinizing, non-invasive, and showed no destructive properties typical of acquired cholesteatoma. During certain circumstances, the cell cycle of hyperplastic epidermal epithelium within the middle-ear cavity can evidently be arrested and inactivated by a local defence mechanism.

Topics: Animals; Cats; Cholesteatoma, Middle Ear; Epithelium; Female; Keratins; Male; Tympanic Membrane; Tympanic Membrane Perforation; Wound Healing

Topics: Epithelial Cells; Hearing Loss, Sensorineural; Humans; Keratins; Skin; Tympanic Membrane Perforation

The presence of keratin in the middle ear cavity is usually associated with the diagnosis of cholesteatoma or epidermoid metaplasia. Analysis of a series of 18 cases suggests that it may correspond to a specific entity developing in the course of severe or long-lasting opened chronic otitis. This condition, we called mallear epidermosis, is characterized by: i) a perforation of the tympanic membrane lining the handle of the malleus and the umbo; ii) a proliferation of keratin surrounding the handle of the malleus and diffusing into the mesotympanum on the internal side of the tympanic membrane, without matrix; and iii) a mild inflammation of the middle ear epithelium. The process is usually limited to the mesotympanum and does not extend towards the attic and the posterior cavities. Epidemiological, clinical, pathophysiological, and histological features allow this entity to be distinguished from cholesteatoma and epidermoid metaplasia. Management is either medical consisting of local treatment and microaspiration, or surgical including resection of the umbo, removal of the tympanic membrane invaded by the adherent hyperkeratotic layers and repair by conventional underlay myringoplasty. This report emphasizes the need for a clear identification of the various types of chronic otitis media presenting with keratin in the middle ear, as they do not share the same course and do not require the same therapeutic management.

Topics: Adolescent; Adult; Aged; Cholesteatoma, Middle Ear; Chronic Disease; Diagnosis, Differential; Ear, Middle; Epithelium; Female; Humans; Keratins; Male; Malleus; Metaplasia; Middle Aged; Myringoplasty; Otitis Media; Suction; Tympanic Membrane; Tympanic Membrane Perforation

To evaluate the type of differentiation of keratinocytes of acquired cholesteatoma and its significance for cholesteatoma invasiveness.. Forty acquired cholesteatomas and 10 tympanic membranes with persisting perforations were snap frozen and processed for immunohistochemical studies. Cytokeratin antibodies that represented all subgroups and antibodies that were directed against collagen components of the basal lamina were applied. Expression of these constituents was scored by using light microscopy.. The phenotype of the matrix was generally characterized by an extension of expression of basal cell cytokeratin 14 and hyperproliferation-associated cytokeratins 6, 16, and 17 into the suprabasal cell layers, while the expression of keratinization marker cytokeratin 10 was down-regulated. These features varied greatly at different sites of the matrix and were most marked at the advancing front of the cholesteatoma. A comparable expression pattern, but less pronounced, was observed at the epidermal front of the mucocutaneous junction of the tympanic membrane perforations. This phenomenon was invariably associated with a mononuclear cell infiltrate in the dermis at both junctions. The basal lamina was always intact.. Acquired cholesteatomas show hyperproliferative features. There is a striking similarity between the pronounced expression of this phenotype and the associated inflammation at the mucocutaneous junctions of cholesteatomas and tympanic membrane perforations and those that are observed after epidermal injury. This indicates that epidermis and middle ear epithelium do not form stable junctions and the front can be considered to be a persisting epidermal defect. This involves the permanent presence of "activated keratinocytes" in the junction area that will lead to proliferation and migration, when additional triggers are present.

Topics: Cell Division; Cholesteatoma, Middle Ear; Ear, Middle; Epidermis; Epithelium; Humans; Immunohistochemistry; Keratinocytes; Keratins; Tympanic Membrane; Tympanic Membrane Perforation

The effects of keeping rat tympanic membranes with an artificially made pars tensa perforation in tissue culture were observed under a scanning electron microscope. After one day and onwards, spreading and thickening of the keratinizing, outer squamous epithelium (OE) was noted. In addition, ballooning of the innermost cells of the outer epithelium apposing the inner tympanic epithelium (IE) was seen. No appreciable reaction was noted in the connective tissue layer of the drum. The inner tympanic epithelium appeared to be swollen, containing spherical structures in the cytoplasm, especially close to the area of contact with the outer meatal epithelium. No complete cover of the drum defect was seen after 14 days of tissue culture. Hyperplasia and spreading of the keratinizing, outer squamous epithelium of the drum is not sufficient to achieve covering of a drum perforation and complete healing cannot take place unless supported by granulation tissue formation.

Topics: Animals; Culture Techniques; Epithelium; Keratins; Male; Microscopy, Electron, Scanning; Rats; Rats, Sprague-Dawley; Time Factors; Tympanic Membrane; Tympanic Membrane Perforation; Wound Healing