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Nasal mucus transportability in children with cleft palate
International Journal of Pediatric Otorhinolaryngology (2008) 72, 581—585
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Nasal mucus transportability in children with cleft palate§ ˆngela Macchione a, Sergio Henrique Kiemle Trindade a,b,*, Maria ˜es a, Inge Elly Kiemle Trindade d, Eliane Tigre Guimara ´rio Nascimento Saldiva a, Geraldo Lorenzi-Filho a,c Paulo Hila a
˜o Paulo, Laboratory of Experimental Air Pollution, School of Medicine, University of Sa ˜o Paulo, SP, Brazil Sa b Division of Clinical Otorhinolaryngology, Department of Otorhinolaryngology and Ophthalmology, ˜o Paulo, SP, Brazil Medical School, University of Sao Paulo, Sa c ˜o Paulo, Sa ˜o Paulo, SP, Brazil Pulmonary Division, Medical School, University of Sa d Laboratory of Physiology, Hospital for Rehabilitation of Craniofacial Anomalies, ˜o Paulo, Bauru, SP, Brazil School of Dentistry, University of Sa Received 9 August 2007; received in revised form 2 January 2008; accepted 5 January 2008 Available online 5 March 2008
KEYWORDS Cleft palate; Children; Mucociliary transport; Mucus physical properties
Summary Objectives: Children with cleft palate (CP) have a high prevalence of sinusitis. Considering that nasal mucus properties play a pivotal role in the upper airway defense mechanism, the aim of the study was to evaluate nasal mucus transportability and physical properties from children with CP. Setting: Hospital for Rehabilitation of Craniofacial Anomalies, School of Dentistry, University of Sa ˜o Paulo, Bauru, SP, Brazil and Laboratory of Experimental Air Pollution, School of Medicine, University of Sa ˜o Paulo, Sa ˜o Paulo, SP, Brazil. Methods: Nasal mucus samples were collected by nasal aspiration from children with CP and without CP (non-CP). Sneeze clearance (SC) was evaluated by the simulated sneeze machine. In vitro mucus transportability (MCT) by cilia was evaluated by the frog palate preparation. Mucus physical surface properties were assessed by measuring the contact angle (CA). Mucus rheology was determined by means of a magnetic rheometer, and the results were expressed as log G* (vectorial sum of viscosity and elasticity) and tan d (relationship between viscosity and elasticity) measured at 1 and 100 rad/s. Results: Mucus samples from children with CP had a higher SC than non-CP children (67 30 and 41 24 mm, respectively, p < 0.05). Mucus samples from children
§ Presented at the Rhinology 2005-ISIAN, on the panel ‘‘Mucociliary Clearance and Mucus Hypersecretion’’, held in Sa ˜o Paulo, Brazil, 20—23 April 2005. * Corresponding author at: Rua Jure ´ia 394, apto 192, 04140-110 Sa ˜o Paulo, SP, Brazil. Tel.: +55 11 5572 3163. E-mail address: [email protected] (S.H.K. Trindade).
0165-5876/$ — see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2008.01.003
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S.H.K. Trindade et al. with CP had a lower CA (24 168 and 35 118, p < 0.05) and a higher tan d 100 (0.79 0.24 and 0.51 0.12, p < 0.05) than non-CP children. There were no significant differences in mucus MCT, log G* 1, tan d 1 and log G* 100 obtained for CP and non-CP children. Conclusions: Nasal mucus physical properties from children with CP are associated with higher sneeze transportability. The high prevalence of sinusitis in children with CP cannot be explained by changes in mucus physical properties and transportability. # 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cleft palate (CP) associated or not with cleft lip is a congenital malformation with a high incidence in humans, which occurs isolated or associated to other anomalies [1]. Disorders of deglutition, speech, facial growth, middle ear diseases, recurrent upper airways infections and high prevalence of nasal sinusitis are among the problems frequently related to CP, especially in the early infancy [2—4]. Several mechanisms have been implicated in the genesis of the increased upper airway infections in children with CP [3]. Ishikawa et al. found in two consecutive studies, a decreased mucociliary transport evaluated by the saccharin transport time test and a lower ciliary beating frequency evaluated by a photoelectric method in patients with cleft lip and palate [5,6]. These findings suggest that mucociliary clearance impairment may be a mechanism involved in the genesis of the increased upper airway infections associated with CP. Since mucociliary transport depends not only on the mechanical input provided by the cilia, but also on mucus physical properties, the further step is to investigate nasal mucus physical properties. Therefore, the aim of the present study was to evaluate nasal mucus transportability (MCT) and physical properties of samples collected from children with CP.
2. Patients and methods The study included 19 children with CP (12 male and 7 female) and 16 children non-CP (14 male and 2 female) for control purposes. Mean age was 4.3 2.9 years for the CP group, ranging from 6 months to 12 years, and 2.8 2.5 years for the control group, ranging from 6 months to 10 years. Types of clefts were the following: five cases of bilateral cleft lip and palate (BCLP), eight of isolated CP and six of unilateral cleft lip and palate (UCLP). Children with CP were admitted to primary or secondary surgeries of the lip and/or palate and non-CP children were recruited when submitted to elective surgeries not related to craniofacial
anomalies (inguinal herniorraphy and postectomy). Isolated cleft lip, age under 6 months and above 12 years and history of acute respiratory diseases within 4 weeks before surgery were exclusion criteria. The study was reviewed and approved by the institutional research oversight committee. Samples were collected at the operating room by nasal aspiration, immediately after anesthesia induction and oral intubation. A polyethylene catheter was inserted into the nasal cavity and the samples were obtained, while the patient was under general anesthesia, immediately before surgical procedures began, by applying negative pressure sufficient to bring the mucus just inside the distal end of the catheter, which was then withdrawn and cut off. The mucus was extracted from the catheter with the aid of a needle and immediately immersed in mineral oil to prevent dehydration. The suction conditions were kept to a minimum to reduce the degree of shear thinning and incorporation of air bubbles [7]. Samples were stored at 70 8C ( 94 8F) in sealed plastic containers for further analysis by the methods described below.
2.1. In vitro mucociliary transport by cilia In vitro mucociliary transportability (MCT) was evaluated by the frog palate preparation [8—10]. The frog palate is covered with a pseudo-stratified ciliated epithelium similar to that present in the mammals airways. For testing how a nasal mucus sample reacts when transported by the cilia, a small nasal mucus drop was positioned on the beginning of the surface of the palate, and the time elapsed for the displacement of the sample in a pre-determined distance was measured, with the aid of a sterioscopic microscope equipped with a reticulated eyepiece. Briefly, the velocity of the tested nasal mucus sample was compared to the velocity of autologous frog mucus, and the results were expressed in terms of relative speed (patient mucus/frog mucus). In this experimental setting, the most important variables that influences mucus transport are the rheological and physical properties of the tested mucus [11,12].
Nasal mucus transportability in children
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2.2. In vitro sneeze clearance (SC)
2.5. Statistical analysis
In vitro sneeze clearance experiment was determined by the ‘‘simulated sneeze machine’’. This device mimics flow rates and time pattern close to those occurring when turbulent flow interacts and displaces mucus in conditions such as cough and sneeze [13]. SC was determined by observing the displacement of the mucus samples and expressed in millimeters. Gas release delivered at a pressure of 40 psi (pounds per square inch) was controlled by a solenoid valve to the outflow part of the gas reservoir. After a simulated sneeze, mucus dislocation was measured with the aid of a ruler.
After testing for normality and equal variance, all variables of the two groups were compared with the Student t-test. Values of p < 0.05 were considered statistically significant.
2.3. Contact angle (CA) Contact angle is the angle between the tangent of the liquid-air interface and a horizontal line [14,15]. Using an eyepiece goniometer with an angular scale of 0—1808, the angle was measured between a mucus drop and the surface of a glass plate treated with sulfochromic acid solution to remove electrostatic charge. CA characterizes the wettability of the mucus sample; in other words, it corresponds to the ability of a biological fluid to spread when deposited in a solid planar surface [16].
2.4. Mucus rheologic properties The magnetic microrheometer, as described by King and MacKlem [17], and modified by Silveira et al. [18], was used to measure rheologic properties of mucus. A small steel ball was inserted into the mucus sample, and its displacement, under the influence of a sinusoidal oscillating magnetic field, was measured. This ball acts as a rheologic probe, because viscous and elastic forces oppose its movement. Measurements were made at two different frequencies: 1 rad/s, simulating ciliary movement, and 100 rad/s, simulating the stimulus provided by the sneeze. Two parameters were obtained: log G*, representing the overall impedance of the mucus, that is, the vectorial sum of elasticity and viscosity or rigidity factor, and tan d, representing the ratio between viscosity and elasticity or recoil factor.
3. Results A total of 19 mucus samples from children with CP and 16 from children non-CP patients were obtained. The volume of mucus obtained from each patient was small. Because it is common to loose sample fractions during the different tests, it was not possible to submit all samples to all methods. Despite this fact, the number of samples evaluated by the different methods provided adequate statistical power. Table 1 shows the results of the mucociliary transport, sneeze clearance, contact angle and the number of samples evaluated in each test. SC was higher and CA was lower in children with CP as compared to children non-CP. No significant differences between groups were observed for MCT. Table 2 shows the results of the rheological properties of seven mucus samples of children with CP and nine of non-CP children. The mean value of tan d 100 was found to be significantly higher in the CP group. The other parameters did not differ between the two groups.
4. Discussion The present study was developed under the hypothesis that the nasal mucus of children with CP has a central role in the genesis of the recurrent upper airway infections usually seen in this population. Jaffe and DeBlanc reported, for instance, a high incidence of sinusitis in subjects with CP [3]. Analyzing the mucociliary clearance of the nasal cavity by the saccharin test, Ishikawa et al. [5,6] found a higher saccharin time in CP patients compared to non-CP patients and a lower ciliary beating frequency measured by a photoelectric method. These
Table 1 Mean values of sneeze clearance (SC), in vitro mucociliary transport by cilia (MCT) and contact angle (CA) of children with cleft palate (CP) and children without cleft palate (non-CP) (*p < 0.05), and numbers of samples evaluated in the different tests (n) Variable
Non-CP
CP
SC (mm) MCT (relative speed) CA (8)
41 24 (n = 19) 0.87 0.29 (n = 16) 35 11 (n = 15)
67 30* (n = 16) 0.81 0.34 (n = 16) 24 16* (n = 16)
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S.H.K. Trindade et al.
Table 2 Mean values of rheologic parameters of children with CP and without CP (non-CP), where log G* 1 and log G* 100 represent the rigidity factor, measured, respectively, at 1 and 100 rad/s, and tan d 1 and tan d 100 represent the recoil factor, measured, respectively, at 1 and 100 rad/s (*p < 0.05) Variable
Non-CP
CP
tan d 1 tan d 100 log G* 1 (dyne/cm2) log G* 100 (dyne/cm2)
0.42 0.10 0.51 0.12 1.43 0.40 1.52 0.28
0.52 0.28 0.79 0.24 * 1.66 0.76 1.63 0.58
results led them to conclude that the higher prevalence of sinusitis in their patients with CP was caused by mucociliary impairment. Since mucociliary transport is influenced not only by ciliary beating frequency, but also by mucus physical properties, this was the focus of the present study. Mucus transportability by cilia, evaluated by the frog palate method, showed no differences between CP and non-CP children. In contrast, mucus samples of children with CP showed a higher transportability by sneeze, when evaluated by the sneeze clearance machine. These results are consistent with the measurements of physical properties as explained below. Contact angle represents the wettability of the mucus, which corresponds to the ability of the mucus to spread on a glass surface. A mucus sample with a low-contact angle presents a low wettability. There is a negative relation between the mucus wettability and its transportability by cough as demonstrated by Albers et al. [19]. In other words, a mucus with a low wettability is supposed to be better transported by cough and sneeze and this is in agreement with the findings in the CP group, i.e., children with CP were shown to have a lower wettability and a higher transportability by sneeze. The same authors did not find relationship between the wettability and the transport by cilia. Accordingly, our results showed no differences in the mucociliary transport between the two groups, as previously mentioned. The results obtained in the rheological analysis further support our findings of sneeze clearance and transportability by cilia. Previous studies showed that the rheologic analysis of mucus samples in low frequencies (1 rad/s) reflects the way the mucus will react when transported by cilia and the rheologic analysis at high frequencies (100 rad/s) reflects the way the mucus will be transported by cough and sneeze [12,13]. The absence of a significant difference in the mucus transportability by cilia between the two groups was confirmed by the absence of a significant difference in the log G* at 1 rad/s. In contrast, the tan del measured at 100 rad/s was significantly higher in the CP children
group. This result is consistent because, as previously stated, there is a positive correlation between the tan del measured at 100 rad/s and the transportability by cough and sneeze [12]. Children with chronic or recurrent airway infections present important nasal mucosal changes, such as disruption of the tight junctions, ultrastructural ciliary defects, increase in the number of the goblet cells and submucosal glands, and alterations in the basal membrane of the respiratory epithelium [20— 22]. In a first moment, as an adaptative response, young patients under chronic aggression to the respiratory epithelium react with an improvement in the mucociliary transport, by enhancing physical and transport properties of the respiratory mucus. However it has been shown that the persistent exposure to chronic aggression can determine the loss of this protective effect [23]. The changes observed in mucus physical and transport properties in children with CP can represent this adaptative mechanism in the response to the chronic aggression represented by recurrent infections. In the same manner, children with CP in a first moment react improving physical and transport properties of the nasal mucus to compensate for the ciliary impairment. To our knowledge, this is the first study to investigate nasal mucus physical properties and transportability from children with CP. In summary, we found a lower CA and a higher tan d 100 in children with CP when compared to controls. Mucus of children with CP presented a higher transportability by sneeze due to physical alterations in the mucus, without alterations in the transportability by the cilia.
5. Conclusion Based on the present findings, we conclude that the cause of the recurrent upper airway infections in children with CP cannot be explained by alterations of the nasal mucus. Contrary to our initial hypothesis, the nasal mucus of children with CP does not seem to present any difficulty to be transported.
Acknowledgements Financial support: FAPESP, CNPq, LIM-05-FMUSP, Brazil.
References [1] M.M. Cohen Jr., Etiology and pathogenesis of orofacial clefting, Oral Max. Surg. Clin. North Am. 12 (2000) 379—397.
Nasal mucus transportability in children [2] K.R. Bzoch, Communicative Disorders Related to Cleft Lip and Palate, Pro-ed, Austin, 1997. [3] B.F. Jaffe, C.B. DeBlanc, Sinusitis in children with clef lip and palate, Arch. Otolaryngol. 93 (1971) 479—482. [4] T. Koppe, C. Weigel, M. Ba ¨renklau, W. Kaduk, T. Bayerklein, T. Gedrange, Maxilary sinus pneumatization of an adult skull with an untreated bilateral cleft palate, J. Craniomaxillofac. Surg. 34 (Suppl. 2) (2006) 91—95. [5] Y. Ishikawa, M. Kawano, I. Honjo, R. Amitani, The cause of nasal sinusitis in subjects with cleft palate, Arch. Otolaryngol. Head Neck Surg. 115 (1989) 442—446. [6] Y. Ishikawa, R. Amitani, Nasal and paranasal sinus disease in patients with congenital velopharyngeal insufficiency, Arch. Otolaryngol. Head and Neck Surg. 120 (8) (1994) 861—865. [7] N.K. Nakagawa, M. Macchione, M.S. Petrolino, E.T. Guimaraes, M. King, P.H. Saldiva, et al., Effects of a heat and moisture exchanger and heated humidifier on respiratory mucus in patients undergoing mechanical ventilation, Crit. Care Med. 28 (2000) 312—317. [8] B.K. Rubin, O. Ramirez, M. King, Mucus-depleted frog palate as a model for the study of the mucociliary clearance, J. Appl. Physiol. 69 (1990) 424—429. [9] E. Puchelle, J.M. Zahm, P. Sadoul, Mucociliary frequency of frog palate epithelium, Am. J. Physiol. 242 (1982) C31—C35. [10] M. Macchione, M. King, G. Lorenzi Filho, E.T. Guimaraes, W.A. Zin, G.M. Bohm, et al., Rheological determinants of mucociliary transport in the nose of the rat, Respir. Physiol. 99 (1995) 165—172. [11] M. King, Interrelation between mechanical properties of mucus and mucociliary transport: effect of pharmacologic interventions, Biorheology 16 (1979) 57—68. [12] G. Lorenzi, G.M. Bo ¨hm, E.T. Guimara ˜es, M.A. Vaz, M. King, P.H. Saldiva, Correlation between rheological properties and in vitro ciliary transport of rat nasal mucus, Biorheology 29 (1992) 433—440. [13] M. King, G. Brock, C. Lundell, Clearance of mucus by stimulated cough, J. Appl. Physiol. 58 (1985) 1776—1782.
585 [14] B.A. Hills, Gastric mucosal barrier: stabilization of hydrophobic lining to the stomach by mucus, Am. J. Physiol. 249 (1985) G342—G349. [15] E. Puchelle, J.M. Zahm, J. Jacquot, C. Plotkwski, C. Duvivier, A simple technique for measuring adhesion properties of human bronchial secretions, Eur. J. Respir. Dis. 71 (1987) 281—282. [16] M. Macchione, E.T. Guimara ˜es, P.H. Saldiva, G. LorenziFilho, Methods for studying respiratory mucus and mucus clearance, Braz. J. Med. Biol. Res. 28 (1995) 1347—1355. [17] M. King, P.T. MacKlem, Rheological properties of microliter quantities of normal mucus, J. Appl. Physiol. 42 (1977) 797— 802. [18] P.S. Silveira, G.M. Bo ¨hm, H.M. Yang, C.L. Wen, E.T. Guimaraes, M.A. Parada, et al., Computer-assisted rheological evaluation of microsamples of mucus, Comput. Methods Programs Biomed. 39 (1992) 51—60. [19] G.M. Albers, R.P. Tomkiewicz, M.K. May, O.E. Ramirez, B.K. Rubin, Ring distraction technique for measuring surface tension of sputum: relationship to sputum clearability, J. Appl. Physiol. 81 (1996) 2690—2695. [20] B. Petruson, H.A. Hansson, Nasal mucosal changes in children with frequent infections, Arch. Otolaryngol. Head Neck Surg. 113 (1987) 1294—1300. [21] E.G. Barlocco, E.A. Valleta, M. Canciani, G. Lungarella, C. Gardi, M. De Santi, et al., Ultrastructural ciliary defects in children with recurrent infections of the lower respiratory tract, Pediatr. Pulmol. 10 (1991) 11—17. [22] J.U. Ponikau, D.A. Sherris, G.M. Kephart, E.B. Kern, T.A. Gaffey, J.E. Tarara, et al., Features of airway remodeling and eosinophilic inflammation in chronic rhinosinusitis: is the histopathology similar to asthma? J. Allergy Clin. Immunol. 112 (2003) 877—882. [23] B.K. Rubin, O. Ramirez, G. Zayas, B. Finegan, M. King, Respiratory mucus from asymptomatic smokers is better hydrated and more easily cleared by mucociliary action, Am. Rev. Respir. Dis. 145 (1992) 545—547.
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Nasal mucus transportability in children with cleft palate§ ˆngela Macchione a, Sergio Henrique Kiemle Trindade a,b,*, Maria ˜es a, Inge Elly Kiemle Trindade d, Eliane Tigre Guimara ´rio Nascimento Saldiva a, Geraldo Lorenzi-Filho a,c Paulo Hila a
˜o Paulo, Laboratory of Experimental Air Pollution, School of Medicine, University of Sa ˜o Paulo, SP, Brazil Sa b Division of Clinical Otorhinolaryngology, Department of Otorhinolaryngology and Ophthalmology, ˜o Paulo, SP, Brazil Medical School, University of Sao Paulo, Sa c ˜o Paulo, Sa ˜o Paulo, SP, Brazil Pulmonary Division, Medical School, University of Sa d Laboratory of Physiology, Hospital for Rehabilitation of Craniofacial Anomalies, ˜o Paulo, Bauru, SP, Brazil School of Dentistry, University of Sa Received 9 August 2007; received in revised form 2 January 2008; accepted 5 January 2008 Available online 5 March 2008
KEYWORDS Cleft palate; Children; Mucociliary transport; Mucus physical properties
Summary Objectives: Children with cleft palate (CP) have a high prevalence of sinusitis. Considering that nasal mucus properties play a pivotal role in the upper airway defense mechanism, the aim of the study was to evaluate nasal mucus transportability and physical properties from children with CP. Setting: Hospital for Rehabilitation of Craniofacial Anomalies, School of Dentistry, University of Sa ˜o Paulo, Bauru, SP, Brazil and Laboratory of Experimental Air Pollution, School of Medicine, University of Sa ˜o Paulo, Sa ˜o Paulo, SP, Brazil. Methods: Nasal mucus samples were collected by nasal aspiration from children with CP and without CP (non-CP). Sneeze clearance (SC) was evaluated by the simulated sneeze machine. In vitro mucus transportability (MCT) by cilia was evaluated by the frog palate preparation. Mucus physical surface properties were assessed by measuring the contact angle (CA). Mucus rheology was determined by means of a magnetic rheometer, and the results were expressed as log G* (vectorial sum of viscosity and elasticity) and tan d (relationship between viscosity and elasticity) measured at 1 and 100 rad/s. Results: Mucus samples from children with CP had a higher SC than non-CP children (67 30 and 41 24 mm, respectively, p < 0.05). Mucus samples from children
§ Presented at the Rhinology 2005-ISIAN, on the panel ‘‘Mucociliary Clearance and Mucus Hypersecretion’’, held in Sa ˜o Paulo, Brazil, 20—23 April 2005. * Corresponding author at: Rua Jure ´ia 394, apto 192, 04140-110 Sa ˜o Paulo, SP, Brazil. Tel.: +55 11 5572 3163. E-mail address: [email protected] (S.H.K. Trindade).
0165-5876/$ — see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2008.01.003
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S.H.K. Trindade et al. with CP had a lower CA (24 168 and 35 118, p < 0.05) and a higher tan d 100 (0.79 0.24 and 0.51 0.12, p < 0.05) than non-CP children. There were no significant differences in mucus MCT, log G* 1, tan d 1 and log G* 100 obtained for CP and non-CP children. Conclusions: Nasal mucus physical properties from children with CP are associated with higher sneeze transportability. The high prevalence of sinusitis in children with CP cannot be explained by changes in mucus physical properties and transportability. # 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cleft palate (CP) associated or not with cleft lip is a congenital malformation with a high incidence in humans, which occurs isolated or associated to other anomalies [1]. Disorders of deglutition, speech, facial growth, middle ear diseases, recurrent upper airways infections and high prevalence of nasal sinusitis are among the problems frequently related to CP, especially in the early infancy [2—4]. Several mechanisms have been implicated in the genesis of the increased upper airway infections in children with CP [3]. Ishikawa et al. found in two consecutive studies, a decreased mucociliary transport evaluated by the saccharin transport time test and a lower ciliary beating frequency evaluated by a photoelectric method in patients with cleft lip and palate [5,6]. These findings suggest that mucociliary clearance impairment may be a mechanism involved in the genesis of the increased upper airway infections associated with CP. Since mucociliary transport depends not only on the mechanical input provided by the cilia, but also on mucus physical properties, the further step is to investigate nasal mucus physical properties. Therefore, the aim of the present study was to evaluate nasal mucus transportability (MCT) and physical properties of samples collected from children with CP.
2. Patients and methods The study included 19 children with CP (12 male and 7 female) and 16 children non-CP (14 male and 2 female) for control purposes. Mean age was 4.3 2.9 years for the CP group, ranging from 6 months to 12 years, and 2.8 2.5 years for the control group, ranging from 6 months to 10 years. Types of clefts were the following: five cases of bilateral cleft lip and palate (BCLP), eight of isolated CP and six of unilateral cleft lip and palate (UCLP). Children with CP were admitted to primary or secondary surgeries of the lip and/or palate and non-CP children were recruited when submitted to elective surgeries not related to craniofacial
anomalies (inguinal herniorraphy and postectomy). Isolated cleft lip, age under 6 months and above 12 years and history of acute respiratory diseases within 4 weeks before surgery were exclusion criteria. The study was reviewed and approved by the institutional research oversight committee. Samples were collected at the operating room by nasal aspiration, immediately after anesthesia induction and oral intubation. A polyethylene catheter was inserted into the nasal cavity and the samples were obtained, while the patient was under general anesthesia, immediately before surgical procedures began, by applying negative pressure sufficient to bring the mucus just inside the distal end of the catheter, which was then withdrawn and cut off. The mucus was extracted from the catheter with the aid of a needle and immediately immersed in mineral oil to prevent dehydration. The suction conditions were kept to a minimum to reduce the degree of shear thinning and incorporation of air bubbles [7]. Samples were stored at 70 8C ( 94 8F) in sealed plastic containers for further analysis by the methods described below.
2.1. In vitro mucociliary transport by cilia In vitro mucociliary transportability (MCT) was evaluated by the frog palate preparation [8—10]. The frog palate is covered with a pseudo-stratified ciliated epithelium similar to that present in the mammals airways. For testing how a nasal mucus sample reacts when transported by the cilia, a small nasal mucus drop was positioned on the beginning of the surface of the palate, and the time elapsed for the displacement of the sample in a pre-determined distance was measured, with the aid of a sterioscopic microscope equipped with a reticulated eyepiece. Briefly, the velocity of the tested nasal mucus sample was compared to the velocity of autologous frog mucus, and the results were expressed in terms of relative speed (patient mucus/frog mucus). In this experimental setting, the most important variables that influences mucus transport are the rheological and physical properties of the tested mucus [11,12].
Nasal mucus transportability in children
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2.2. In vitro sneeze clearance (SC)
2.5. Statistical analysis
In vitro sneeze clearance experiment was determined by the ‘‘simulated sneeze machine’’. This device mimics flow rates and time pattern close to those occurring when turbulent flow interacts and displaces mucus in conditions such as cough and sneeze [13]. SC was determined by observing the displacement of the mucus samples and expressed in millimeters. Gas release delivered at a pressure of 40 psi (pounds per square inch) was controlled by a solenoid valve to the outflow part of the gas reservoir. After a simulated sneeze, mucus dislocation was measured with the aid of a ruler.
After testing for normality and equal variance, all variables of the two groups were compared with the Student t-test. Values of p < 0.05 were considered statistically significant.
2.3. Contact angle (CA) Contact angle is the angle between the tangent of the liquid-air interface and a horizontal line [14,15]. Using an eyepiece goniometer with an angular scale of 0—1808, the angle was measured between a mucus drop and the surface of a glass plate treated with sulfochromic acid solution to remove electrostatic charge. CA characterizes the wettability of the mucus sample; in other words, it corresponds to the ability of a biological fluid to spread when deposited in a solid planar surface [16].
2.4. Mucus rheologic properties The magnetic microrheometer, as described by King and MacKlem [17], and modified by Silveira et al. [18], was used to measure rheologic properties of mucus. A small steel ball was inserted into the mucus sample, and its displacement, under the influence of a sinusoidal oscillating magnetic field, was measured. This ball acts as a rheologic probe, because viscous and elastic forces oppose its movement. Measurements were made at two different frequencies: 1 rad/s, simulating ciliary movement, and 100 rad/s, simulating the stimulus provided by the sneeze. Two parameters were obtained: log G*, representing the overall impedance of the mucus, that is, the vectorial sum of elasticity and viscosity or rigidity factor, and tan d, representing the ratio between viscosity and elasticity or recoil factor.
3. Results A total of 19 mucus samples from children with CP and 16 from children non-CP patients were obtained. The volume of mucus obtained from each patient was small. Because it is common to loose sample fractions during the different tests, it was not possible to submit all samples to all methods. Despite this fact, the number of samples evaluated by the different methods provided adequate statistical power. Table 1 shows the results of the mucociliary transport, sneeze clearance, contact angle and the number of samples evaluated in each test. SC was higher and CA was lower in children with CP as compared to children non-CP. No significant differences between groups were observed for MCT. Table 2 shows the results of the rheological properties of seven mucus samples of children with CP and nine of non-CP children. The mean value of tan d 100 was found to be significantly higher in the CP group. The other parameters did not differ between the two groups.
4. Discussion The present study was developed under the hypothesis that the nasal mucus of children with CP has a central role in the genesis of the recurrent upper airway infections usually seen in this population. Jaffe and DeBlanc reported, for instance, a high incidence of sinusitis in subjects with CP [3]. Analyzing the mucociliary clearance of the nasal cavity by the saccharin test, Ishikawa et al. [5,6] found a higher saccharin time in CP patients compared to non-CP patients and a lower ciliary beating frequency measured by a photoelectric method. These
Table 1 Mean values of sneeze clearance (SC), in vitro mucociliary transport by cilia (MCT) and contact angle (CA) of children with cleft palate (CP) and children without cleft palate (non-CP) (*p < 0.05), and numbers of samples evaluated in the different tests (n) Variable
Non-CP
CP
SC (mm) MCT (relative speed) CA (8)
41 24 (n = 19) 0.87 0.29 (n = 16) 35 11 (n = 15)
67 30* (n = 16) 0.81 0.34 (n = 16) 24 16* (n = 16)
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Table 2 Mean values of rheologic parameters of children with CP and without CP (non-CP), where log G* 1 and log G* 100 represent the rigidity factor, measured, respectively, at 1 and 100 rad/s, and tan d 1 and tan d 100 represent the recoil factor, measured, respectively, at 1 and 100 rad/s (*p < 0.05) Variable
Non-CP
CP
tan d 1 tan d 100 log G* 1 (dyne/cm2) log G* 100 (dyne/cm2)
0.42 0.10 0.51 0.12 1.43 0.40 1.52 0.28
0.52 0.28 0.79 0.24 * 1.66 0.76 1.63 0.58
results led them to conclude that the higher prevalence of sinusitis in their patients with CP was caused by mucociliary impairment. Since mucociliary transport is influenced not only by ciliary beating frequency, but also by mucus physical properties, this was the focus of the present study. Mucus transportability by cilia, evaluated by the frog palate method, showed no differences between CP and non-CP children. In contrast, mucus samples of children with CP showed a higher transportability by sneeze, when evaluated by the sneeze clearance machine. These results are consistent with the measurements of physical properties as explained below. Contact angle represents the wettability of the mucus, which corresponds to the ability of the mucus to spread on a glass surface. A mucus sample with a low-contact angle presents a low wettability. There is a negative relation between the mucus wettability and its transportability by cough as demonstrated by Albers et al. [19]. In other words, a mucus with a low wettability is supposed to be better transported by cough and sneeze and this is in agreement with the findings in the CP group, i.e., children with CP were shown to have a lower wettability and a higher transportability by sneeze. The same authors did not find relationship between the wettability and the transport by cilia. Accordingly, our results showed no differences in the mucociliary transport between the two groups, as previously mentioned. The results obtained in the rheological analysis further support our findings of sneeze clearance and transportability by cilia. Previous studies showed that the rheologic analysis of mucus samples in low frequencies (1 rad/s) reflects the way the mucus will react when transported by cilia and the rheologic analysis at high frequencies (100 rad/s) reflects the way the mucus will be transported by cough and sneeze [12,13]. The absence of a significant difference in the mucus transportability by cilia between the two groups was confirmed by the absence of a significant difference in the log G* at 1 rad/s. In contrast, the tan del measured at 100 rad/s was significantly higher in the CP children
group. This result is consistent because, as previously stated, there is a positive correlation between the tan del measured at 100 rad/s and the transportability by cough and sneeze [12]. Children with chronic or recurrent airway infections present important nasal mucosal changes, such as disruption of the tight junctions, ultrastructural ciliary defects, increase in the number of the goblet cells and submucosal glands, and alterations in the basal membrane of the respiratory epithelium [20— 22]. In a first moment, as an adaptative response, young patients under chronic aggression to the respiratory epithelium react with an improvement in the mucociliary transport, by enhancing physical and transport properties of the respiratory mucus. However it has been shown that the persistent exposure to chronic aggression can determine the loss of this protective effect [23]. The changes observed in mucus physical and transport properties in children with CP can represent this adaptative mechanism in the response to the chronic aggression represented by recurrent infections. In the same manner, children with CP in a first moment react improving physical and transport properties of the nasal mucus to compensate for the ciliary impairment. To our knowledge, this is the first study to investigate nasal mucus physical properties and transportability from children with CP. In summary, we found a lower CA and a higher tan d 100 in children with CP when compared to controls. Mucus of children with CP presented a higher transportability by sneeze due to physical alterations in the mucus, without alterations in the transportability by the cilia.
5. Conclusion Based on the present findings, we conclude that the cause of the recurrent upper airway infections in children with CP cannot be explained by alterations of the nasal mucus. Contrary to our initial hypothesis, the nasal mucus of children with CP does not seem to present any difficulty to be transported.
Acknowledgements Financial support: FAPESP, CNPq, LIM-05-FMUSP, Brazil.
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