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Effect of platelet-activating factor on the mucocillary transport of the maxillary sinus in rabbits
P130
Otolaryngology Head and Neck Surgery August 1996
Scientific Sessions- - Tuesday
Poster 21 Vestibular Function in the Fetus PATRICK J. ANTONELLI, MD (presenter), ROBERTM, ABRAMS, PhD, and KENNETH J. GERHARDT, PhD, Gainesville, Fla,
Auditory stimulation (hearing) is critical for normal brain development. Deprivation of auditory function in the fetal sheep results in profound depression of central nervous system metabolism. We hypothesized that vestibular function is similarly important in early (fetal) central nervous system functional maturation. The purpose of this preliminary study was to evaluate the fetal vestibular response to caloric stimulation. Late gestational fetal sheep (n = 4) were instrumented through a midline hysterotomy using general anesthesia and aseptic techniques. Copper caloric probes were inserted into the right bulla and beneath the left facial skin. Screw electrodes were placed in the skull for monitoring electrooculography and electrocortical activity. The caloric probes were irrigated with water (100 ml/minute) at body temperature, 50 ~ C, and 4 ~ C at various stages in the fetal "sleep" cycle. Response to ice-water infusion of the bulla consistently produced well recognized slow-phase deviations followed by saccades directed contralaterally, consistent with vestibular nystagmus. The direction of the response was reversed with warm-water irrigation. The response was absent with irrigation at body temperature. Electrooculography responses to cold water irrigation were best observed during fetal quiet "sleep." Only random eye movements were observed in response to caloric stimulation of the facial skin. These results demonstrate that the vestibular system is functional prenatally. Prenatal vestibular function may be important for normal fetal brain maturation.
Poster 22 Leukotrlenes C 4 and D4 Increase the Ciliary Beat Frequency in Human Upper Airway Mucosa In Vitro CARLOS B. CYRUS (presenter), THOMAS V. McCAFFREY, MD, PhD, and BIN JULIA YANG, MD, Hershey, Pa., Rochester, Minn,, and Baltimore, Md.
It has been suggested that leukotrienes C 4 ( L T C 4 ) and D 4 (LTD 4) released from upper respiratory mucosa affect mucociliary transport during allergic reactions. We studied the in vitro effects of leukotrienes C 4 and D 4 o n the ciliary beat frequency (CBF) of human adenoid explants over a 5hour period. Tissue explants were cultured at 35 ~ C and covered with 1.0 ml of minimum essential medium Eagle (MEM). CBF was measured using phase-contrast microscopy and microphotometry. Measurements of CBF were recorded in medium alone and in 1.0 ml MEM containing LTC 4 or LTD 4 at concentrations of 10 -6 and 10 -8 mol/L. LTC 4 or LTD 4 increased CBF in a dose-dependent manner with mean maximum increases of 20.51% _+ 2.69% and 29.84% _+ 4.06%, respectively. To determine the specificity of LTC 4 and LTD 4 effects, the leukotriene antagonist, LY-171,883
was added before administration of LTC 4 or LTD 4. LY171,883 (10 -~ mol/L) significantly inhibited the ciliostimulatory effects of both leukotrienes. Our findings indicate that LTC 4 and LTD 4 increase CBF in vitro. Poster 23 The Differential Effect of Isotonic and Isometric Exercise on Nasal Blood Flow as Measured by Laser Doppler Analysis R.W. CLARKE (presenter), Sheffield, England
Aim: Isotonic and isometric exercise have different cardiovascular effects. Although isotonic exercise causes a fall in nasal resistance, this may be due to flaring of the nostrils. As nasal blood flow can now be reliably measured by laser Doppler analysis, this technique was applied to study the change in blood flow after exercise. Method: Nasal blood flow was measured at rest and after vigorous isotonic exercise on a bicycle ergometer. Readings were repeated for isometric exercise of equivalent work on a hand dynamometer. Analysis by Wilcoxon Signed Rank test showed a significant decrease in nasal blood flow with isotonic exercise but no change with isometric (Z = -3.4, two-tailed p = 0.006). Conclusion: The fall in nasal resistance with isotonic exercise is caused at least partly by reduced blood flow and not by reflex alar dilation. Poster 24 Effect of Platelet-actlvatlng Factor on the Mucoclliary Transport of the Maxillary Sinus In Rabbits PIL-SEOB JEONG (presenter), CHUNG-KU RHEE,SANGYOUNG JEONG, and HAN-GYUN KIM, Cheonan Chungnam Do, Korea
Objective: Platelet-activating factor (PAF) is a biologically active phospholipid and a potent inflammatory mediator. Recent reports have indicated that PAF may impair the mucociliary function of ciliated mucosa. The aims of this study are to investigate the effect of PAF on the mucociliary function of the maxillary sinus mucosa in rabbits and to test whether PAF antagonist can prevent the mucociliary dysfunction induced by PAF. Methods: The average mucociliary transport time was measured with india ink from the floor of the maxillary sinus to the natural ostium of rabbits. Rabbits were divided into the following eight groups. In group I the dye was applied to the floor of the maxillary sinus, and normal transport time was measured. In group II (control group) normal saline solution was topically applied to the maxillary sinus membrane, and the dye transport time was measured 15 minutes after the application of normal saline solution. In groups III through VII, 1, 2, 4, 8, and 16 !ag/ml, respectively, of PAF was topically applied on the maxillary sinus membrane and the dye transport time was measured 15 minutes after the application of PAF. In group VIII, PAF antagonist (WEB-2170, 3 mg/ml) was topically ap-
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
Otolaryngology Head and Neck Surgery August 1996
Scientific Sessions- - Tuesday
Poster 21 Vestibular Function in the Fetus PATRICK J. ANTONELLI, MD (presenter), ROBERTM, ABRAMS, PhD, and KENNETH J. GERHARDT, PhD, Gainesville, Fla,
Auditory stimulation (hearing) is critical for normal brain development. Deprivation of auditory function in the fetal sheep results in profound depression of central nervous system metabolism. We hypothesized that vestibular function is similarly important in early (fetal) central nervous system functional maturation. The purpose of this preliminary study was to evaluate the fetal vestibular response to caloric stimulation. Late gestational fetal sheep (n = 4) were instrumented through a midline hysterotomy using general anesthesia and aseptic techniques. Copper caloric probes were inserted into the right bulla and beneath the left facial skin. Screw electrodes were placed in the skull for monitoring electrooculography and electrocortical activity. The caloric probes were irrigated with water (100 ml/minute) at body temperature, 50 ~ C, and 4 ~ C at various stages in the fetal "sleep" cycle. Response to ice-water infusion of the bulla consistently produced well recognized slow-phase deviations followed by saccades directed contralaterally, consistent with vestibular nystagmus. The direction of the response was reversed with warm-water irrigation. The response was absent with irrigation at body temperature. Electrooculography responses to cold water irrigation were best observed during fetal quiet "sleep." Only random eye movements were observed in response to caloric stimulation of the facial skin. These results demonstrate that the vestibular system is functional prenatally. Prenatal vestibular function may be important for normal fetal brain maturation.
Poster 22 Leukotrlenes C 4 and D4 Increase the Ciliary Beat Frequency in Human Upper Airway Mucosa In Vitro CARLOS B. CYRUS (presenter), THOMAS V. McCAFFREY, MD, PhD, and BIN JULIA YANG, MD, Hershey, Pa., Rochester, Minn,, and Baltimore, Md.
It has been suggested that leukotrienes C 4 ( L T C 4 ) and D 4 (LTD 4) released from upper respiratory mucosa affect mucociliary transport during allergic reactions. We studied the in vitro effects of leukotrienes C 4 and D 4 o n the ciliary beat frequency (CBF) of human adenoid explants over a 5hour period. Tissue explants were cultured at 35 ~ C and covered with 1.0 ml of minimum essential medium Eagle (MEM). CBF was measured using phase-contrast microscopy and microphotometry. Measurements of CBF were recorded in medium alone and in 1.0 ml MEM containing LTC 4 or LTD 4 at concentrations of 10 -6 and 10 -8 mol/L. LTC 4 or LTD 4 increased CBF in a dose-dependent manner with mean maximum increases of 20.51% _+ 2.69% and 29.84% _+ 4.06%, respectively. To determine the specificity of LTC 4 and LTD 4 effects, the leukotriene antagonist, LY-171,883
was added before administration of LTC 4 or LTD 4. LY171,883 (10 -~ mol/L) significantly inhibited the ciliostimulatory effects of both leukotrienes. Our findings indicate that LTC 4 and LTD 4 increase CBF in vitro. Poster 23 The Differential Effect of Isotonic and Isometric Exercise on Nasal Blood Flow as Measured by Laser Doppler Analysis R.W. CLARKE (presenter), Sheffield, England
Aim: Isotonic and isometric exercise have different cardiovascular effects. Although isotonic exercise causes a fall in nasal resistance, this may be due to flaring of the nostrils. As nasal blood flow can now be reliably measured by laser Doppler analysis, this technique was applied to study the change in blood flow after exercise. Method: Nasal blood flow was measured at rest and after vigorous isotonic exercise on a bicycle ergometer. Readings were repeated for isometric exercise of equivalent work on a hand dynamometer. Analysis by Wilcoxon Signed Rank test showed a significant decrease in nasal blood flow with isotonic exercise but no change with isometric (Z = -3.4, two-tailed p = 0.006). Conclusion: The fall in nasal resistance with isotonic exercise is caused at least partly by reduced blood flow and not by reflex alar dilation. Poster 24 Effect of Platelet-actlvatlng Factor on the Mucoclliary Transport of the Maxillary Sinus In Rabbits PIL-SEOB JEONG (presenter), CHUNG-KU RHEE,SANGYOUNG JEONG, and HAN-GYUN KIM, Cheonan Chungnam Do, Korea
Objective: Platelet-activating factor (PAF) is a biologically active phospholipid and a potent inflammatory mediator. Recent reports have indicated that PAF may impair the mucociliary function of ciliated mucosa. The aims of this study are to investigate the effect of PAF on the mucociliary function of the maxillary sinus mucosa in rabbits and to test whether PAF antagonist can prevent the mucociliary dysfunction induced by PAF. Methods: The average mucociliary transport time was measured with india ink from the floor of the maxillary sinus to the natural ostium of rabbits. Rabbits were divided into the following eight groups. In group I the dye was applied to the floor of the maxillary sinus, and normal transport time was measured. In group II (control group) normal saline solution was topically applied to the maxillary sinus membrane, and the dye transport time was measured 15 minutes after the application of normal saline solution. In groups III through VII, 1, 2, 4, 8, and 16 !ag/ml, respectively, of PAF was topically applied on the maxillary sinus membrane and the dye transport time was measured 15 minutes after the application of PAF. In group VIII, PAF antagonist (WEB-2170, 3 mg/ml) was topically ap-
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