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Cryopreservation of human cilia: A simplified technique of storing and transporting nasal biopsy samples
Otolaryngology Head and Neck Surgery Scientific Sessions- - Tuesday
Volume 115 Number 2
plied to the maxillary sinus membrane 20 minutes before the topical application of 4 lag PAF, and the dye transport time was measured. At the end of the procedure, mucosae were harvested for histopathologic study using a light microscope. Results: The dye transport times of groups 1 through VIII were 60, 70, 81, 155, 225, 302, more than 600, and 66 seconds, respectively. The transport times among group I through III showed no statistically significant differences. The transport times of groups IV through VII showed statistically significant differences compared with that of group II (70 seconds; p < 0.05). The transport time in group VIII did not show a statistically significant difference compared with that of group II, which was significantly different compared with that of group V. The light microscopy demonstrated exfoliations of the ciliated cells of the maxillary sinus in groups III through VII. Conclusions: PAF appears to impair the mucociliary function of the maxillary sinus in dose-dependent manner, and PAF antagonist seems to prevent the mucociliary dysfunction induced by PAF. This study suggests that PAF plays an important role in inducing the mucociliary dysfunction of the maxillary sinus. Poster 25
The Role of Prostaglandin E2 in the Regulation of Ciliary Beat Frequency SCOYF M. GAYNER, MD (presenter), and THOMAS V. McCAFFREY, MD, PhD, Rochester, Minn.
Prostaglandin E: (PGE2) is a known modulator in upper airway ciliary activity and may be involved in the transduction of the muscarinic acetylcholine receptor signal. We studied the in vitro effects of cholinergic and ~2-adrenergic ciliostimulation on ciliary beat frequency (CBF) and PGE 2 production in human adenoid explants before and after application of a cyclooxygenase inhibitor. CBF was determined with phase-contrast microscopy and PGE 2 production by radioimmunoassay. Methacholine, a muscarinic agonist; terbutaline, a ]]2-adrenergic agonist; and diclofenac, a cyclooxygenase inhibitor, were used at a concentration of 10-~ mol/L. Methacholine alone increased CBF by 21.5% _+ 4% (p < 0.0001). This effect was completely blocked by diclofenac application. Terbutaline alone increased CBF by 27.4% _+ 12% (p < 0.01). This effect was not abolished by diclofenac. The average PGE 2 production induced by methacholine before and after diclofenac application was 1.93 • 0.4 pg/lag protein and 0.66 + 0.15 pg/lag protein (p < 0.01), whereas for terbutaline the average PGE2 production was 0.50 • 0.11 pg/lag protein before diclofenac and 0.73 _+ 0.28 pg/lag protein after diclofenac (p > 0.05). These data show that endogenous PGE 2 production is necessary for methacholine-induced ciliostimulation while terbutaline ciliostimulation is not PGEz dependent. Understanding these pathways of cilioregulation may permit the selection of pharmacologic agents to enhance mucociliary clearance in pathologic states.
P 131
Poster 26
Cryopreservation of Human Cilia: A Simplified Technique of Storing and Transporting Nasal Biopsy Samples BIN YANG, MD (presenter), THOMAS V. MCCAFFREY, MD, PhD, and RODNEY J. SCHLOSSER, [kS, Rochester, Minn.
Mucociliary transport affected by the cilia beat frequency (CBF) is believed to be an important defense mechanism for preventing infection and eliminating foreign debris from the upper airway. Because ciliary activity deteriorates rapidly and only a limited number of centers are equipped for routine ciliary function analysis, a simplified and reliable method of storing and transporting of nasal mucosal biopsy samples is desirable. We evaluated several methods of cryopreservation of airway ciliated mucosa for this purpose. The ciliated samples were frozen by a slow or fast freezing method. All samples were stored in liquid nitrogen (-196 ~ C) for 1 week. The frozen samples were thawed by two methods: (1) (rapid thawing) 37 ~ C water bath for 3 to 4 minutes, and (2) (slow thawing) room temperature for 15 minutes. Prefreeze and postthaw CBF were measured by microphotometry. The slow freezing and fast thawing method (SFFT) resulted in the best viability. The ciliated samples processed by SFFI" method were examined after being frozen in liquid nitrogen (-196 ~ C) for 1 week, 2 weeks, and 1 month. There was no significant decrease of baseline CBF after cryopreservation for at least 2 weeks (p > 0.05). Postthaw CBF decreased 7.25% -+ 0.87% after freezing for 1 month (p < 0.05). There was no statistical difference between fresh and cryopreserved samples exposed to terbutaline, a ~,-adrenergic agonist, suggesting that nasal cilia retain their physiologic activity after cryopreservation. The electron microscopy also showed that there was no ultrastructure change in the ciliated samples after freezing for 2 weeks. We believe that SFFI" cryopreservation is a soluble method for preserving viable airway cilia for at least 2 weeks.
Poster 27
Aldosterone Regulation of No, K-ATPase Activity in the Mammalian Olfactory Mucosa KAREN J. FONG, MD (presenter), ROBERTC. KERN, MD, JAMES FOSTER,PhD, and DIMITRI Z. PITOVSKI, MD, Chicago, II1,, and Nashville, Tenn.
Na, K-ATPase maintains the ionic gradients necessary for resting and odorant-induced ion flux across the olfactory mucosa (OM). In addition, this enzyme is likely necessary for secretion and modification of olfactory mucus from Bowman's glands. In many other tissues Na, K-ATPase activity is modulated by corticosteroid hormones. In this study the effect of alteration of serum aldosterone levels on olfactory Na, K-ATPase activity in the guinea pig was investigated. A high-sodiunl/low-potassium diet offered ad libiturn for 5 days was used to decrease serum aldosterone significantly in male Hartley guinea pigs compared with controis. An injection of aldosterone (10 lag/100 body wt) 21 hours before sacrifice resulted in significant elevation of
Volume 115 Number 2
plied to the maxillary sinus membrane 20 minutes before the topical application of 4 lag PAF, and the dye transport time was measured. At the end of the procedure, mucosae were harvested for histopathologic study using a light microscope. Results: The dye transport times of groups 1 through VIII were 60, 70, 81, 155, 225, 302, more than 600, and 66 seconds, respectively. The transport times among group I through III showed no statistically significant differences. The transport times of groups IV through VII showed statistically significant differences compared with that of group II (70 seconds; p < 0.05). The transport time in group VIII did not show a statistically significant difference compared with that of group II, which was significantly different compared with that of group V. The light microscopy demonstrated exfoliations of the ciliated cells of the maxillary sinus in groups III through VII. Conclusions: PAF appears to impair the mucociliary function of the maxillary sinus in dose-dependent manner, and PAF antagonist seems to prevent the mucociliary dysfunction induced by PAF. This study suggests that PAF plays an important role in inducing the mucociliary dysfunction of the maxillary sinus. Poster 25
The Role of Prostaglandin E2 in the Regulation of Ciliary Beat Frequency SCOYF M. GAYNER, MD (presenter), and THOMAS V. McCAFFREY, MD, PhD, Rochester, Minn.
Prostaglandin E: (PGE2) is a known modulator in upper airway ciliary activity and may be involved in the transduction of the muscarinic acetylcholine receptor signal. We studied the in vitro effects of cholinergic and ~2-adrenergic ciliostimulation on ciliary beat frequency (CBF) and PGE 2 production in human adenoid explants before and after application of a cyclooxygenase inhibitor. CBF was determined with phase-contrast microscopy and PGE 2 production by radioimmunoassay. Methacholine, a muscarinic agonist; terbutaline, a ]]2-adrenergic agonist; and diclofenac, a cyclooxygenase inhibitor, were used at a concentration of 10-~ mol/L. Methacholine alone increased CBF by 21.5% _+ 4% (p < 0.0001). This effect was completely blocked by diclofenac application. Terbutaline alone increased CBF by 27.4% _+ 12% (p < 0.01). This effect was not abolished by diclofenac. The average PGE 2 production induced by methacholine before and after diclofenac application was 1.93 • 0.4 pg/lag protein and 0.66 + 0.15 pg/lag protein (p < 0.01), whereas for terbutaline the average PGE2 production was 0.50 • 0.11 pg/lag protein before diclofenac and 0.73 _+ 0.28 pg/lag protein after diclofenac (p > 0.05). These data show that endogenous PGE 2 production is necessary for methacholine-induced ciliostimulation while terbutaline ciliostimulation is not PGEz dependent. Understanding these pathways of cilioregulation may permit the selection of pharmacologic agents to enhance mucociliary clearance in pathologic states.
P 131
Poster 26
Cryopreservation of Human Cilia: A Simplified Technique of Storing and Transporting Nasal Biopsy Samples BIN YANG, MD (presenter), THOMAS V. MCCAFFREY, MD, PhD, and RODNEY J. SCHLOSSER, [kS, Rochester, Minn.
Mucociliary transport affected by the cilia beat frequency (CBF) is believed to be an important defense mechanism for preventing infection and eliminating foreign debris from the upper airway. Because ciliary activity deteriorates rapidly and only a limited number of centers are equipped for routine ciliary function analysis, a simplified and reliable method of storing and transporting of nasal mucosal biopsy samples is desirable. We evaluated several methods of cryopreservation of airway ciliated mucosa for this purpose. The ciliated samples were frozen by a slow or fast freezing method. All samples were stored in liquid nitrogen (-196 ~ C) for 1 week. The frozen samples were thawed by two methods: (1) (rapid thawing) 37 ~ C water bath for 3 to 4 minutes, and (2) (slow thawing) room temperature for 15 minutes. Prefreeze and postthaw CBF were measured by microphotometry. The slow freezing and fast thawing method (SFFT) resulted in the best viability. The ciliated samples processed by SFFI" method were examined after being frozen in liquid nitrogen (-196 ~ C) for 1 week, 2 weeks, and 1 month. There was no significant decrease of baseline CBF after cryopreservation for at least 2 weeks (p > 0.05). Postthaw CBF decreased 7.25% -+ 0.87% after freezing for 1 month (p < 0.05). There was no statistical difference between fresh and cryopreserved samples exposed to terbutaline, a ~,-adrenergic agonist, suggesting that nasal cilia retain their physiologic activity after cryopreservation. The electron microscopy also showed that there was no ultrastructure change in the ciliated samples after freezing for 2 weeks. We believe that SFFI" cryopreservation is a soluble method for preserving viable airway cilia for at least 2 weeks.
Poster 27
Aldosterone Regulation of No, K-ATPase Activity in the Mammalian Olfactory Mucosa KAREN J. FONG, MD (presenter), ROBERTC. KERN, MD, JAMES FOSTER,PhD, and DIMITRI Z. PITOVSKI, MD, Chicago, II1,, and Nashville, Tenn.
Na, K-ATPase maintains the ionic gradients necessary for resting and odorant-induced ion flux across the olfactory mucosa (OM). In addition, this enzyme is likely necessary for secretion and modification of olfactory mucus from Bowman's glands. In many other tissues Na, K-ATPase activity is modulated by corticosteroid hormones. In this study the effect of alteration of serum aldosterone levels on olfactory Na, K-ATPase activity in the guinea pig was investigated. A high-sodiunl/low-potassium diet offered ad libiturn for 5 days was used to decrease serum aldosterone significantly in male Hartley guinea pigs compared with controis. An injection of aldosterone (10 lag/100 body wt) 21 hours before sacrifice resulted in significant elevation of