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Apoptosis and cell proliferation in the tympanic membrane and middle ear cholesteatoma
P148
Otolaryngology Head and Neck Surgery August 1995
Scientific Sessions- - Tuesday
previously shown that this upregulation of mitochondrial enzymes is accompanied by a rapid increase in mitochondrial volume within deafferented neurons. Inhibition of mitochondriat biogenesis with use of chloramphenicol significantly increased the amount of neuronal death to as high as 80%. Increases in intracellular calcium levels ([Ca2§ have been shown to trigger increases in mitochondrial function. Larger increases in [Ca2*]iactivate degradative pathways, which leads to cell death. Recently, Zirpel and colleagues showed that deafferentation of NM neurons results in an increase in [Ca~+]i within hours after cochlea removal (Zirpel et al., 1995). Our present set of experiments is designed to test the hypothesis that the deafferentation-induced increase in [Ca2§ triggers the metabolic upregulation in NM neurons and ultimately plays a role in the demise of deafferentation NM neurons. We used flunarizine, a calcium antagonist that crosses the blood-brain barrier, to block the [Ca z+] increase after deafferentation and then studied the effects on neuronal survival. When flunarizine (20 mg/kg) was administered to El8 chicks before unilateral cochlea removal, there was no significant increase in [Ca2+]i in deafferented NM neurons after 3 hours. In a separate set of experiments, flunarizine was administered to 10-day-old chicks before unilateral CR. There was no significant loss of NM neurons observed after deafferentation (p >0.6). We also did not find any significant neuronal loss in animals treated with chloramphenicol, flunarizine, and unilateral cochlea removal. Thus, it appears that [Ca2+]i increases are involved in the death of auditory neurons after deafferentation. Calcium antagonists such as flunarizine may prove useful in preserving central auditory pathways following sensorineural hearing loss. Poster 64 Apoptosis and Cell Proliferation in the Tympanic Membrane and Middle Ear Cholesteatoma REIKO KOBA, MD, (presenter), RYUICHI OSANAI, MD, and ISUZU KAWABATA, MD, Saitama, Japan
Introduction: The purpose of this study was to provide detailed data on cell proliferation in the tympanic membrane and in cholesteatoma. In addition, an attempt was made to study apoptosis. Materials and methods: Specimens of human tympanic membrane, meatal skin, retroauricular skin, and cholesteatoma were obtained during ear surgery. The specimens in the first group were incubated in FC43 emulsion (Green Cross Corp., Osaka, Japan) containing bromodeoxyuridine, and detection of bromodeoxyuridine-labeled cells was performed with use of a celt proliferation kit (Amersham International Ltd., Little Chalfont, United Kingdom). The other specimens were processed for in situ visualization of apoptosis using an apop tag kit (Oncor, Gaithersburg, Md.). Results: Statistical analysis of counts of bromo-
deoxyuridine-labeledcells within a distance of 200 ktm along the basal cell layer showed a significant difference between the annulus and other parts. Also, the count at the malleus handle was significantly different from those in other parts. In contrast, statistical analysis of counts of apoptotic cells within a distance of 500 ~tm did not show any significant difference. Discussion: The present study revealed the existence of proliferation centers of epidermal cells at the annulus and the malleus handle. We hypothesize that insertion of newly prolifereated cells at the proliferation center and random cell death cause cell migration over the tympanic membrane and the ear canal. In addition, no proliferation center was seen in the cholesteatoma epidermis, suggesting disturbance of epidermal cell migration in cholesteatoma. Poster 65 The Sheep: An Excellent Animal Model for Macro- and Micromorphologic Studies on the Eustachian Tube MARIEI-rA LINNARZ, MD (presenter), JORGEN U. G. HOPF, MD, HANS-JOACHIM MERKER, MD, and VIRAVUTH PRAPAVAT, DIPL.ING, Berlin, Germany
The introduction of transnasal microendoscopy of the eustachian tube and the middle ear into clinical practice is the precondition for every kind of minimal invasive therapy (MIT) in this delicate anatomical area. The application of fiber delivered laser systems via the working channel of small fiberscopes to remove obstructive soft tissue inside of the lumen of the eustachian tube is a possible procedure. However, before this new method can be applied in human patients, it has to be investigated in an animal model that is morphologically comparable with the human anatomy and physiology. Special considerations have to be given to the dimensions of the eustachian tube in the species chosen to serve as an adequate animal model. After comparative dissections of this region in different species and morpologic comparisons in literature, we found the sheep to be the best animal model for these investigations. Its gross shape and dimensions are very similar to the human eustachian tube. It allows the mother-baby-scope technique for transnasal tubo-tympanoscopy to be performed and laser fibers to be introduced under visual control to remove some soft tissue. This procedure would be exactly the same as it is therapeutically planned. Introducing the animal model "sheep" into middle ear and eustachian tube research, we present our profound analysis of macro- and micromorpologic findings in more than 200 tubes. Comparative histologic studies concerning kind, distribution, and constituents of the epithelium of the pharyngeal, isthmic, and tympanic section have been done by light microscopy, scanning microscopy, and transmission electron microscopy. The occurrence of cilia, microvilli, secretory and goblet cells, multilamellar bodies and lysosomes, the distribution of lysozyme and acid phosphatase has been evaluated critically. The cultivation of eustachian tube ciliated epithelium for in vitro experiments is presented.
Otolaryngology Head and Neck Surgery August 1995
Scientific Sessions- - Tuesday
previously shown that this upregulation of mitochondrial enzymes is accompanied by a rapid increase in mitochondrial volume within deafferented neurons. Inhibition of mitochondriat biogenesis with use of chloramphenicol significantly increased the amount of neuronal death to as high as 80%. Increases in intracellular calcium levels ([Ca2§ have been shown to trigger increases in mitochondrial function. Larger increases in [Ca2*]iactivate degradative pathways, which leads to cell death. Recently, Zirpel and colleagues showed that deafferentation of NM neurons results in an increase in [Ca~+]i within hours after cochlea removal (Zirpel et al., 1995). Our present set of experiments is designed to test the hypothesis that the deafferentation-induced increase in [Ca2§ triggers the metabolic upregulation in NM neurons and ultimately plays a role in the demise of deafferentation NM neurons. We used flunarizine, a calcium antagonist that crosses the blood-brain barrier, to block the [Ca z+] increase after deafferentation and then studied the effects on neuronal survival. When flunarizine (20 mg/kg) was administered to El8 chicks before unilateral cochlea removal, there was no significant increase in [Ca2+]i in deafferented NM neurons after 3 hours. In a separate set of experiments, flunarizine was administered to 10-day-old chicks before unilateral CR. There was no significant loss of NM neurons observed after deafferentation (p >0.6). We also did not find any significant neuronal loss in animals treated with chloramphenicol, flunarizine, and unilateral cochlea removal. Thus, it appears that [Ca2+]i increases are involved in the death of auditory neurons after deafferentation. Calcium antagonists such as flunarizine may prove useful in preserving central auditory pathways following sensorineural hearing loss. Poster 64 Apoptosis and Cell Proliferation in the Tympanic Membrane and Middle Ear Cholesteatoma REIKO KOBA, MD, (presenter), RYUICHI OSANAI, MD, and ISUZU KAWABATA, MD, Saitama, Japan
Introduction: The purpose of this study was to provide detailed data on cell proliferation in the tympanic membrane and in cholesteatoma. In addition, an attempt was made to study apoptosis. Materials and methods: Specimens of human tympanic membrane, meatal skin, retroauricular skin, and cholesteatoma were obtained during ear surgery. The specimens in the first group were incubated in FC43 emulsion (Green Cross Corp., Osaka, Japan) containing bromodeoxyuridine, and detection of bromodeoxyuridine-labeled cells was performed with use of a celt proliferation kit (Amersham International Ltd., Little Chalfont, United Kingdom). The other specimens were processed for in situ visualization of apoptosis using an apop tag kit (Oncor, Gaithersburg, Md.). Results: Statistical analysis of counts of bromo-
deoxyuridine-labeledcells within a distance of 200 ktm along the basal cell layer showed a significant difference between the annulus and other parts. Also, the count at the malleus handle was significantly different from those in other parts. In contrast, statistical analysis of counts of apoptotic cells within a distance of 500 ~tm did not show any significant difference. Discussion: The present study revealed the existence of proliferation centers of epidermal cells at the annulus and the malleus handle. We hypothesize that insertion of newly prolifereated cells at the proliferation center and random cell death cause cell migration over the tympanic membrane and the ear canal. In addition, no proliferation center was seen in the cholesteatoma epidermis, suggesting disturbance of epidermal cell migration in cholesteatoma. Poster 65 The Sheep: An Excellent Animal Model for Macro- and Micromorphologic Studies on the Eustachian Tube MARIEI-rA LINNARZ, MD (presenter), JORGEN U. G. HOPF, MD, HANS-JOACHIM MERKER, MD, and VIRAVUTH PRAPAVAT, DIPL.ING, Berlin, Germany
The introduction of transnasal microendoscopy of the eustachian tube and the middle ear into clinical practice is the precondition for every kind of minimal invasive therapy (MIT) in this delicate anatomical area. The application of fiber delivered laser systems via the working channel of small fiberscopes to remove obstructive soft tissue inside of the lumen of the eustachian tube is a possible procedure. However, before this new method can be applied in human patients, it has to be investigated in an animal model that is morphologically comparable with the human anatomy and physiology. Special considerations have to be given to the dimensions of the eustachian tube in the species chosen to serve as an adequate animal model. After comparative dissections of this region in different species and morpologic comparisons in literature, we found the sheep to be the best animal model for these investigations. Its gross shape and dimensions are very similar to the human eustachian tube. It allows the mother-baby-scope technique for transnasal tubo-tympanoscopy to be performed and laser fibers to be introduced under visual control to remove some soft tissue. This procedure would be exactly the same as it is therapeutically planned. Introducing the animal model "sheep" into middle ear and eustachian tube research, we present our profound analysis of macro- and micromorpologic findings in more than 200 tubes. Comparative histologic studies concerning kind, distribution, and constituents of the epithelium of the pharyngeal, isthmic, and tympanic section have been done by light microscopy, scanning microscopy, and transmission electron microscopy. The occurrence of cilia, microvilli, secretory and goblet cells, multilamellar bodies and lysosomes, the distribution of lysozyme and acid phosphatase has been evaluated critically. The cultivation of eustachian tube ciliated epithelium for in vitro experiments is presented.