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Effect of intranasal histamine challenge on Eustachian tube function
International Journal of Pediatric Otorhinolaryngology 63 (2002) 189– 198 www.elsevier.com/locate/ijporl
Effect of intranasal histamine challenge on Eustachian tube function Charles S. Ebert Jr. *, Hoke W. Pollock, Marc G. Dubin, Scott S. Scharer, Jiri Prazma, Chapman T. McQueen, Harold C. Pillsbury III Department of Otolaryngology – Head and Neck Surgery, CBc 7070, Burnett – Womack Clinical Science Building, Uni6ersity of North Carolina School of Medicine, Chapel Hill, NC 27599 -7070, USA Received 11 October 2001; accepted 14 December 2001
Abstract Objecti6e: To show a relationship between intranasal histamine challenge, the development of middle ear effusion and Eustachian tube (ET) dysfunction in a rat model. Methods: Non-allergic Sprague– Dawley rats weighing between 450–600 g were randomly assigned to receive an intranasal infusion of 16 ml of 10% histamine or normal saline. ET function was assessed by using the forced-response test to measure passive and active opening and closing pressures at time intervals of 6, 10, 14, 18, 22, and 26 min and 24 h post-infusion. Mucociliary clearance times (MCCTs) of the tubotympanum at 18 min post-infusion were measured by timing the transit of dye from the middle ear to the nasopharynx. Outcome measures were ET dysfunction and evidence of clinical effusion. Results: Intranasal histamine caused acute ET dysfunction when introduced into the nasopharynx demonstrated by significant elevations in passive and active opening and closing pressures (P 00.001) compared to controls. The largest difference was seen at 26 min post-infusion. Furthermore, MCCTs were 2.4 times longer after infusing intranasal histamine than after saline infusion. No clinically significant effusions were evident in either group at any time interval. Conclusion: These data demonstrate a successful development of an intranasal histamine rat model, in addition to a relationship between intranasal histamine challenge and development of acute ET dysfunction. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Histamine; Allergy; Otitis media; Intranasal; Eustachian tube dysfunction
1. Introduction
Presented at 2001 American Society of Pediatric Otolaryngology, Annual Meeting: May 9–12, 2001, Scottsdale, Arizona. * Corresponding author. Tel.: +1-919-955-3254; fax: + 1919-966-7656. E-mail address: [email protected] (C.S. Ebert, Jr.).
Otitis media (OM) and otitis media with effusion (OME) represent significant health problems for children in the United States today. Together they represent the most frequent primary diagnoses at visits to US physician offices for children younger than 15 years of age. OM predominantly
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affects infants and preschoolers, and accounts for nearly $4 billion in health-care expenditures annually [1]. Studies report that over 80% of all children will have at least one episode of OME by age three, and 40% will have three or more [2]. The sequelae of repeated exposures of OME range from few permanent deficits to significant impairments in receptive language skills, balance, coordination, and school readiness [2]. OME in children has been linked to various risk factors such as: first degree relative with allergy, attendance to day care, exposure to cigarette smoke, recurrent upper respiratory infection, short duration of breast feeding, and seasonal changes [3]. However, even with advances in the understanding of the pathogenesis and pathophysiology of OME, the incidence has remained the same over the last 20 years [4]. Since the Eustachian tube (ET) is situated in a way that allows for communication between the nasopharynx and middle ear, its position may permit changes in the middle ear due to reaction in the nose [4]. The major functions of the ET with respect to the middle ear involve ventilation with atmospheric pressure, drainage of secretions into the nasopharynx, and protection from nasopharyngeal secretions and sound pressure [5]. Obstruction of the ET, whether functional or mechanical, alters the partial pressure of middle ear gases, resulting in negative middle ear pressure, improper ventilation, and a supportive milieu for bacteria reproduction [1,2]. Children are further disadvantaged because the pediatric ET is shorter and situated in a more horizontal orientation compared to adults, which inherently impairs its protective function [6,7]. The etiology of OME involves many factors, and allergic inflammation around the nasopharyngeal portion of the ET may additionally lead to the breakdown of these protective functions resulting in increased risk for the development of OM [2]. Histamine is a well-recognized mediator of the early-phase allergic response that initiates edema formation by promoting exudation of fluid and proteins from mucosal vasculature into the extravascular space [8]. In the allergic response, edema caused by mediators of inflammation in the nasopharynx blocks the ET orifice, resulting
in improper ventilation and drainage [2]. Doyle et al. implicated nasal allergy in generating significantly impaired active ET function by intranasally challenging sensitized rhesus monkeys with pollen [9]. The production of ET obstruction by intranasal challenge of histamine has been reported; thereby supporting a role for allergy in the production of OME [10]. However, previous attempts using aerosolized histamine to develop an intranasal allergy model in anesthetized rats have been unsuccessful [11]. Recent evidence suggests that nasal allergy predisposes patients to develop OME [3]. Epidemiologic studies have shown that as many as 50% of all patients with OM have nasal allergy and 21% of patients with nasal allergy have OM [12]. In children, the incidence of OME in those with allergy has been reported to be two times that of those without allergy [13]. However, controversy regarding the exact role allergy plays in OME still exists is evident by published incidences of allergy in OME varying from 0 to 95% [10]. An improved understanding of the role of allergy in ETD is essential to enhance treatment options for OM and OME. The purpose of this study is to dynamically evaluate the relationship between intranasal histamine challenge and the development of ET dysfunction with or without middle ear effusion in a rat model. To do this, we placed a small catheter through the nasal bone in to the nasopharynx and injected 10% histamine or phosphate buffered saline (PBS) and tested various parameters of ET function.
2. Materials and methods
2.1. Animals All animals were handled according to the standards and protocols of ‘The Principles of Laboratory Animal Care’ as stated in the National Society for Medical Research, Institutional Animal Care and Use Committee, and the ‘Guide for the Care and Use of Laboratory Animals’ published by the National Institute of Health. Forty non-allergic, male Sprague–Dawley rats weighing
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between 350–600 g were randomly assigned to receive a 16 ml intranasal infusion of either 10% solution of histamine (H-7250; Sigma Chemical Co., St. Louis, MO) or PBS over a 3 min interval. The rats receiving a 10% histamine solution (n = 24) were divided into three groups to assess passive opening and closing pressures (POP/PCP) (n = 12), active clearance of positive and negative pressure (ACPP/ACNP) (n = 7), and mucociliary clearance times (MCCTs) of the tubotympanum (n = 5). Five animals in the treatment group (n = 2 POP/PCP, n=2 ACCP/ACNP, and n = 1 MCCT) were not infused secondary to dehiscence of catheters. The rats receiving PBS (n = 16) were separated in a similar manner with the groups containing 5, 4,and 4, respectively (POP/PCP: n =5, ACCP/ACNP: n = 4, MCCT: n = 4). Three animals in the control group (n = 2 POP/PCP, and n= 1 ACCP/ACNP) were not infused secondary to dehiscence of catheters.
2.2. Surgical procedures and peri-operati6e management Each rat was anesthetized with 0.1 ml/100 gm of a 1:1 mixture of xylazine (4 mg/ml) and ketamine hydrochloride (100 mg/ml). The depth of anesthesia was tested by applying pressure to joints. All animals were given 80 ml/min/kg of 300 mOsm Krebs Ringer solution (140 mmol/l Na+, 120 mmol/l Cl−, 5.2 mmol/l K+, 25 mmol/l HCO3 − , 1.1 mmol/l Ca2 + , 1.2 mmol/l Mg2 + , 0.4 mmol/l HPO2 − , and 5.6 mmol/l glucose) subcutaneously to maintain blood pressure and renal perfusion during the experiment. To maintain body temperature at 37 °C a thermistor-controlled heating pad was used each time anesthesia was administered. Once anesthetized all tympanic membranes were assessed using an operating microscope to exclude obvious middle ear pathology. The skin overlying the nasal bones and the nucal region were then shaved and injected with 1% xylocaine with 1:100 000 epinephrine for local anesthesia and hemostasis. Incisions were made (1 cm) over the skin of the animal’s shoulders and (2–3 cm) over the midline to expose the nasal bones. With a small hand drill, a hole (2 mm diameter) was
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bored 1 mm from the midline in the left nasal bone through to the nasopharynx. A PE-10 polyethylene tube (c 7401, Clay Adams, Parsippany, NJ) with one end heat-formed to have a 12 mm bend was then threaded through the drilled hole (12 mm) into the nasopharynx of each rat. The average distance from the catheter tip to the ET orifice was 0.5 cm. The free end of the catheter was passed through a subcutaneous tunnel exiting between the animal’s shoulders. The skin overlying the exposed nasal bones was sutured and the tube was secured in its position via subcutaneous and external sutures in the skin overlying the shoulders. After allowing all animals to repose for 7–10 days after catheter placement procedure, they were anesthetized with a xylazine/ketamine mixture and a solution of 10% histamine (experimental group) or PBS (control group) was infused into the nasopharynx through the implanted catheter. The infusion was 3 min at a rate of 5.33 ml/min. via a Razen microinfusion pump to deliver 16 ml of solution in to the nasopharynx. Larger volumes of intranasal histamine has been previously determined to cause aspiration and swallowing, compromising ET function tests [11]. During infusion all animals were stimulated to swallow a minimum of 10 times (4 times the first minute and 3 times the second and third minutes) by direct stimulation of the epiglottis with polyethylene tubing. Swallowing ventilates the ET and allows for contents of the nasopharynx to reflux through the tube and into the middle ear [5]. The dynamic function of the ET was assessed prior to surgery, before and after infusion, and 24 h post-infusion as described below.
2.3. Assessment of ET function Rats were anesthetized with a xylazine/ketamine mixture and placed in the recumbent position. Once the tympanic membrane was visualized through an operating microscope, incisions were made with a small myringotomy knife in the posteroinferior and anteroinferior quadrants. The myringotomies functioned as a port and vent for the application of positive and negative pressure to the middle ear. Baseline opening and closing
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pressures were measured employing the forced-response test analogous to that first described by Flisberg et al. [14]. A pressure measurement apparatus was created by inserting the end of a 6 French latex pediatric foley catheter with a 3 cc balloon (Medline, Mundelein, IL) under direct visualization into the external auditory canal and inflating the balloon to create an airtight seal (Fig. 1). The drainage port of the catheter was connected to a water manometer, thereby creating a closed system, which allowed for the application of positive pressure via a Harvard infusion pump (20 ml/min). Slow uniform pressure was then infused until the ET spontaneously opened, marked by the meniscus of the water column changing from convex to concave. This ‘breaking point’ was considered the passive opening pressure (POP). The meniscus of the water column then descended, and the point at which the column stopped descending corresponded to the
spontaneous closure of the ET. This point was considered the passive closing pressure (PCP). For those rats in the experimental and control groups assigned to have POP/PCP assessed, baseline pressures were made prior to catheter placement. In addition, before infusion (time 0 min), and at minutes 6, 10,14,18, 26, and 24 h post-infusion POP/PCP were measured. At each time interval each animal was assessed for evidence of effusion via an operating microscope. To assess the ability of the ET to actively clear middle ear pressure rats in the experimental and control groups were anesthetized as previously described and placed in the recumbent position. Once the 3 cc catheter balloon of the pressure measurement apparatus was placed and inflated in the external auditory canal, 15 cm H2O of positive pressure was infused. Rats were then stimulated to swallow with polyethylene tubing. Swallowing caused the tensor veli palatini to actively open the
Fig. 1. An infusion withdrawal pump was used to apply positive or negative pressure to the rat’s middle ear by infusing or withdrawing air into a closed system. A 6 French Foley catheter created a seal in the animal’s external auditory canal. A myringotomy created communication between the middle ear and the infusion/withdrawal pump so that changes in pressure could be recorded in cm H2O using a water manometer. A PE-10 catheter was inserted through the left nasal bone in order to deliver either a 10% histamine solution or PBS.
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ET and thus, ventilating the middle ear causing the water column to fall. Rats were stimulated to swallow until positive pressure could no longer be cleared represented by the point at which the water column would no longer fall after swallowing. The difference in pressures between these two points were divided by 15 cm H2O to quantify the percent active clearance of positive pressure (ACPP). To measure the active clearance of negative pressure (ACNP) the infusion/withdrawal pump was used to remove 10 cm H2O from the system. Again, rats were stimulated to swallow ventilating the ET allowing for clearance of negative pressure. When repeated swallowing no longer caused the water column to rise, the differences pressures between the two points were divided by 10 cm H2O to signify the percent ACNP. ACCP and ACNP measurements were only taken at intervals of 0, 6, and 24 min post-infusion due to the duration of time for adequate swallowing to clear pressure. To assess the mucociliary clearance function of the ET, rats in the experimental and control groups were deeply anesthetized to prevent swallowing and active opening of the ET. After infusion of either 10% histamine or PBS the rats were placed in the supine position, and a modified standard nasal speculum was used as a headholder device to aid in the visualization of the soft palate. A small midline incision was made in the soft palate posterior to the posterior border of the hard palate. Hemostasis and anesthesia were maintained by swabbing the incision with 1% xylocaine with 1:100 000 epinephrine. A miniature hand mirror was inserted through the incision to visualize the ET orifice under the operating microscope. Rats were then placed in the right lateral recumbent position and incisions in the anteroinferior and posteroinferior quadrants of the tympanic membrane were made with a small myringotomy knife. Eighteen minutes after infusion 4 ml of blue dye (Coumassie Brilliant Blue, B-0149; Sigma Chemical Co.) were injected into the middle ear using a micropipette. A stopwatch was started and the rats were returned to the supine position. The nasopharyngeal orifice of the ET was again visualized under the operating mi-
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croscope. The time at which dye appeared at the orifice was measured. This time signified the MCCT of the tubotympanum.
2.4. Statistical analysis All statistical analyses were done using SigmaStat Statistical software Version 2.03 (SPSS Inc.). Pressures were analyzed by one-way analysis of variance with pair wise comparisons of means evaluated by paired t-tests. ACPP/ACNP was converted to a percentage of total possible pressure for clearance and then analyzed. Mean mucociliary times were analyzed using paired t-tests. Statistical significance was determined prior to the experiment for P values of B0.05. Graphs were constructed using means and the standard error of the mean (SEM).
3. Results
3.1. Eustachian tube function The intranasal administration of histamine significantly increased the pressure required to open (Fig. 2) and close (Fig. 3) the ET in response to the infusion of positive pressure compared to controls, respectively. The increase was progressive, with the largest difference occurring at 26 min after infusion. Twenty-four hours post-infusion the mean POP and PCP returned to baseline. Figs. 2 and 3 depict time versus pressure in cm H2O and illustrate the notable differences of POP and PCP at various intervals post-infusion between the treatment group and controls. Significant differences are marked by asterisks. There were no significant differences between the treatment and control groups at times pre-catheter placement, 0 min, and 24 h post-infusion for both POP and PCP. According to the analysis of variance, the differences in the means among and within the treatment groups for both POP and PCP are greater than would have been expected by chance (P 00.001). In addition, no clinically evident effusions were noted at any time interval. Intranasal infusion of histamine also decreased the ability to clear positive and negative pressure
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Fig. 2. Mean POPs for experimental (histamine, black) and control (PBS, gray) in cm H2O at intervals prior to catheter placement, 0, 6, 10, 14, 18, 22, and 26 min as well as 24 h post-infusion of either 10% histamine or PBS. (Note: y-axis begins at 30 cm H2O.) Values are significantly greater for the histamine infused group at intervals 6, 10, 14, 18, 22, and 26 min after infusion. Significant differences are marked by asterisks. The maximum difference between the experimental group and controls were observed at 26 min. There was no significant differences between the means of the groups at times prior to surgery, 0 min, and 24 h. Error bars represent SEM.
Fig. 3. Mean PCPs for experimental (histamine, black) and control (PBS, gray) in cm H2O at intervals prior to catheter placement, 0, 6, 10, 14, 18, 22, and 26 min as well as 24 h post-infusion of either 10% histamine or PBS. Values are significantly greater for the histamine infused group at intervals 6, 10, 14, 18, 22, and 26 min after infusion. Significant differences are marked by asterisks. The maximum difference between the experimental group and controls were observed at 26 min. There was no significant differences between the means of the groups at times prior to surgery, 0 min, and 24 h. Error bars represent SEM.
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Fig. 4. Mean ACPPs for experimental (histamine, black) and control (PBS, gray) in cm H2O at intervals prior to catheter placement, 6, and 24 min post-infusion of either 10% histamine or PBS. Values are significantly greater for the histamine infused group at only 24 min after infusion. Significant differences are marked by asterisks. The maximum amount of possible percent clearance was 100%. There was no significant differences between the means of the groups at times prior to surgery, 0, and 6 min. Error bars represent SEM.
Fig. 5. Mean ACNPs for experimental (histamine, black) and control (PBS, gray) in cm H2O at intervals prior to catheter placement, 6, and 24 min post-infusion of either 10% histamine or PBS. Values are significantly greater for the histamine infused group at 6 and 24 min after infusion. Significant differences are marked by asterisks. The maximum amount of possible percent clearance was 100%. There was no significant differences between the means of the groups at times before surgery and at 0 min. Error bars represent SEM.
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Fig. 6. Mean MCCTs for experimental (histamine, black) and control (PBS, gray) groups versus time (min). MCCT was significantly greater for the histamine infused groups compared to the controls. Significant differences are marked by asterisks. Error bars represent SEM.
from the middle ear compared to controls (Figs. 4 and 5). Fig. 4 shows the mean ACPP in cm H2O over various time intervals. Significant differences are marked by asterisks. At 24 min the ACPP for the histamine infused group was appreciably impaired compared to baseline at 0 min. Similarly, the ACNP was impaired by the infusion of histamine. Fig. 5 illustrates mean ACNP in cm H2O over various time intervals with the ACNP most impaired at 24 min. Fig. 6 showing treatment group versus time demonstrates that the mean MCCT was prolonged by histamine when compared to controls. The average time for dye to be transmitted through the ET was 534 s for the treatment group compared to 220 s for controls. Thus, MCCT was on average 2.4 times longer for the histamine group than the PBS group.
4. Discussion The infusion of a 10% histamine solution caused acute ET dysfunction when introduced into the nasopharynx in rats. Histamine produced notable impairment of the ET’s ventilation functions as evident by increased mean POP/PCP and a notable reduction in the ability to actively clear both positive and negative pressure. Furthermore,
the ciliary function of the ET was impaired by histamine when compared to controls as evidenced by the prolongation of the MCCT of the tubotympanum. These results demonstrate a relationship between histamine challenge and the development of ET dysfunction. Examining the ventilatory parameters assessed in this experiment (POP, PCP, ACPP, and ACNP), all showed a maximal impairment of function at approximately 24 min post-infusion with initial significant differences observed as early as 6 min. By 24 h, the effect had disappeared. Particularly important are the decreased percent ACNP since the equalization of negative pressure within the middle ear by deglutition is considered the most significant and accurate parameter of tubal function by Miller and others [15,16]. Additionally, ciliary function was impaired 18 min post-infusion. These data are consistent with previous work conducted by Doyle with rhesus monkeys where aerosolized histamine and other allergens were administered into the nasal cavity, which resulted in similar ETD [9,17]. While few would argue that either intrinsic or extrinsic obstruction of the ET results in improper ventilation/pressure regulation and effusion, there remains some debate whether the middle epithelium functions in a manner parallel to other respiratory mucosa. In a recent series of studies,
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Hurst showed that the middle ear epithelium of persons with atopy had all the tools needed to respond in a manner like that of the rest of the upper respiratory system, and was able to have an allergic immune response [18]. Supporting this observation, the middle ear mucosa develops from the same ectoderm as the other respiratory epithelium, and animal studies have shown that it responds immunologically to antigens as do the epithelium of the nasopharynx, sinuses, and bronchi [18,19]. In addition, White showed that dye injected into the nasopharynx of rats can be refluxed into the middle ear and that gastric reflux into the nasopharynx caused significant ETD using analogous parameters of testing ET function [15]. Although this study was not designed to further elucidate the exact site of action of the allergic response, the effects of histamine most likely acted on both the middle ear mucosa due to reflux as seen by delayed MCCT and by blocking the nasopharyngeal orifice resulting in significantly decreased ACPP/ACNP as well as increased POP/PCP. Our study builds on previous work examining the role of histamine and ETD, and represents the development of a model to continue investigation in this area [11,17,20]. Future study will be directed at dynamically evaluating the dose-response relationship of histamine infused intranasally with ETD. Also, we plan to use this model to examine intranasal allergen delivery on ET function and test employment of immunomodulators on response the to allergen. It is hoped that our work will continue to build on the understanding of the pathogenesis and pathophysiology of OM and OME, specifically that related to allergy. An improved understanding of the role of allergy in ETD is vital for the enhancement of treatment options for OM and OME.
Acknowledgements The authors would like to thank Doug C. Fitzpatrick for critical review without which this paper would not have been completed.
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References [1] K.A. Daly, et al., Chronic otitis media with effusion, Pediatr. Rev. 20 (3) (1999) 85 – 94. [2] J.M. Berstein, Role of allergy in Eustachian tube blockage and otitis media with effusion: a review, Otolaryngol. Head Neck Surg. 114 (4) (1996) 562 – 568. [3] O.P. Alho, H. Oja, M. Koivu, M. Sorri, Risk factors for chronic otitis media with effusion in infancy. Each acute otitis media episode inducesa high but transient risk, Arch. Otolaryngol. Head Neck Surg. 121 (8) (1995) 839 – 843. [4] P. Fireman, Otitis media and eustachian tube dysfunction: connection to allergic rhinitis, J. Allergy Immunol. 99 (2) (1997) 787 – 797. [5] C.D. Bluestone, W.J. Doyle, Anatomy and physiology of eustachian tube and middle ear related otitis media, J. Allergy Clin. Immunol. 81 (1988) 997 – 1003. [6] C.D. Bluestone, Pathogenesis of otitis media: role of eustachian tube, Pediatr. Infect. Dis. J. 15 (1996) 281 – 291. [7] B. Procter, Embryology and anatomy of the eustachian tube, Arch. Otolaryngol. 86 (1967) 503 – 514. [8] D.D. Metcalfe, D. Baram, Y.A. Mekori, Mast cells, Physiol. Rev. 77 (1997) 1033 – 1079. [9] W.J. Doyle, R. Friedman, P. Fireman, C.D. Bluestone, Eustachian tube obstruction after provocative nasal antigen challenge, Arch. Otolaryngol. 110 (8) (1984) 508 – 511. [10] M.J. Derebery, et al., Allergy for the otologist external canal to inner ear, Otolaryngol. Clin. North Amer. 31 (1) (1998) 157 – 173. [11] B.W. Downs, H.F. Butehorn III, J. Prazma, A.S. Rose, J.C. Stamat, H.C. Pillsbury III, Action of histamine on Eustachian tube function, Otolaryngol. Head Neck Surg. 124 (4) (2001) 414 – 420. [12] AAFP Clinical Recommendation: Otitis Media with Effusion in Young Children. Available from http:// www.aafp.org/policy/camp/otitis – c.html. [13] W.L. Draper, Secretory otitis media in children: a study of 540 children, Laryngoscope 77 (1967) 636. [14] K. Flisberg, S. Ingelstedt, U. O8 rtegren, Controlled ear aspiration of air: a physiological test of the tubal function, Acta Otolaryngol. (Stockh) S182 (1963) 35 – 38. [15] D.R. White, S.B. Heavner, S.M. Hardy, J. Prazma, Gastroesophageal reflux and Eustachian tube dysfunction in an animal model. Presented at the Southern Section of the Triological Society Meeting, San Marco Island, Florida, January 2001. [16] G.F. Miller, Eustachian tubal function in normal and diseased ears, Arch. Otolaryngol. 81 (1965) 41 – 48. [17] W.J. Doyle, A.S. Ingraham, P. Fireman, The effects of intranasal histamine challenge on Eustachian tube function, J. Allergy Clin. Immunol. 76 (1985) 551 – 556. [18] D.S. Hurst, The Middle Ear: The Inflammatory Response in Children with Otitis Media with Effusion and the Impact of Atopy, Dissertation Faculty of Medicine Uppsala University, 2000, pp. 7 – 51.
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[19] P. Bikhazi, A.F. Ryan, Expression of immunoregulatory cytokines during acute and chronic middle ear immune response, Laryngoscope 105 (1995) 629 –634. [20] J.T. Seroky, C.M. Alper, R. Tabari, W.J. Doyle, Effects
of intranasal challenge with histamine, bradykinin, and prostaglandin on middle ear pressure and blood flow in cynomolgus monkeys, Acta Otolaryngol. (Stockh) 115 (1995) 83 – 87.
Effect of intranasal histamine challenge on Eustachian tube function Charles S. Ebert Jr. *, Hoke W. Pollock, Marc G. Dubin, Scott S. Scharer, Jiri Prazma, Chapman T. McQueen, Harold C. Pillsbury III Department of Otolaryngology – Head and Neck Surgery, CBc 7070, Burnett – Womack Clinical Science Building, Uni6ersity of North Carolina School of Medicine, Chapel Hill, NC 27599 -7070, USA Received 11 October 2001; accepted 14 December 2001
Abstract Objecti6e: To show a relationship between intranasal histamine challenge, the development of middle ear effusion and Eustachian tube (ET) dysfunction in a rat model. Methods: Non-allergic Sprague– Dawley rats weighing between 450–600 g were randomly assigned to receive an intranasal infusion of 16 ml of 10% histamine or normal saline. ET function was assessed by using the forced-response test to measure passive and active opening and closing pressures at time intervals of 6, 10, 14, 18, 22, and 26 min and 24 h post-infusion. Mucociliary clearance times (MCCTs) of the tubotympanum at 18 min post-infusion were measured by timing the transit of dye from the middle ear to the nasopharynx. Outcome measures were ET dysfunction and evidence of clinical effusion. Results: Intranasal histamine caused acute ET dysfunction when introduced into the nasopharynx demonstrated by significant elevations in passive and active opening and closing pressures (P 00.001) compared to controls. The largest difference was seen at 26 min post-infusion. Furthermore, MCCTs were 2.4 times longer after infusing intranasal histamine than after saline infusion. No clinically significant effusions were evident in either group at any time interval. Conclusion: These data demonstrate a successful development of an intranasal histamine rat model, in addition to a relationship between intranasal histamine challenge and development of acute ET dysfunction. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Histamine; Allergy; Otitis media; Intranasal; Eustachian tube dysfunction
1. Introduction
Presented at 2001 American Society of Pediatric Otolaryngology, Annual Meeting: May 9–12, 2001, Scottsdale, Arizona. * Corresponding author. Tel.: +1-919-955-3254; fax: + 1919-966-7656. E-mail address: [email protected] (C.S. Ebert, Jr.).
Otitis media (OM) and otitis media with effusion (OME) represent significant health problems for children in the United States today. Together they represent the most frequent primary diagnoses at visits to US physician offices for children younger than 15 years of age. OM predominantly
0165-5876/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 5 8 7 6 ( 0 2 ) 0 0 0 0 7 - 1
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affects infants and preschoolers, and accounts for nearly $4 billion in health-care expenditures annually [1]. Studies report that over 80% of all children will have at least one episode of OME by age three, and 40% will have three or more [2]. The sequelae of repeated exposures of OME range from few permanent deficits to significant impairments in receptive language skills, balance, coordination, and school readiness [2]. OME in children has been linked to various risk factors such as: first degree relative with allergy, attendance to day care, exposure to cigarette smoke, recurrent upper respiratory infection, short duration of breast feeding, and seasonal changes [3]. However, even with advances in the understanding of the pathogenesis and pathophysiology of OME, the incidence has remained the same over the last 20 years [4]. Since the Eustachian tube (ET) is situated in a way that allows for communication between the nasopharynx and middle ear, its position may permit changes in the middle ear due to reaction in the nose [4]. The major functions of the ET with respect to the middle ear involve ventilation with atmospheric pressure, drainage of secretions into the nasopharynx, and protection from nasopharyngeal secretions and sound pressure [5]. Obstruction of the ET, whether functional or mechanical, alters the partial pressure of middle ear gases, resulting in negative middle ear pressure, improper ventilation, and a supportive milieu for bacteria reproduction [1,2]. Children are further disadvantaged because the pediatric ET is shorter and situated in a more horizontal orientation compared to adults, which inherently impairs its protective function [6,7]. The etiology of OME involves many factors, and allergic inflammation around the nasopharyngeal portion of the ET may additionally lead to the breakdown of these protective functions resulting in increased risk for the development of OM [2]. Histamine is a well-recognized mediator of the early-phase allergic response that initiates edema formation by promoting exudation of fluid and proteins from mucosal vasculature into the extravascular space [8]. In the allergic response, edema caused by mediators of inflammation in the nasopharynx blocks the ET orifice, resulting
in improper ventilation and drainage [2]. Doyle et al. implicated nasal allergy in generating significantly impaired active ET function by intranasally challenging sensitized rhesus monkeys with pollen [9]. The production of ET obstruction by intranasal challenge of histamine has been reported; thereby supporting a role for allergy in the production of OME [10]. However, previous attempts using aerosolized histamine to develop an intranasal allergy model in anesthetized rats have been unsuccessful [11]. Recent evidence suggests that nasal allergy predisposes patients to develop OME [3]. Epidemiologic studies have shown that as many as 50% of all patients with OM have nasal allergy and 21% of patients with nasal allergy have OM [12]. In children, the incidence of OME in those with allergy has been reported to be two times that of those without allergy [13]. However, controversy regarding the exact role allergy plays in OME still exists is evident by published incidences of allergy in OME varying from 0 to 95% [10]. An improved understanding of the role of allergy in ETD is essential to enhance treatment options for OM and OME. The purpose of this study is to dynamically evaluate the relationship between intranasal histamine challenge and the development of ET dysfunction with or without middle ear effusion in a rat model. To do this, we placed a small catheter through the nasal bone in to the nasopharynx and injected 10% histamine or phosphate buffered saline (PBS) and tested various parameters of ET function.
2. Materials and methods
2.1. Animals All animals were handled according to the standards and protocols of ‘The Principles of Laboratory Animal Care’ as stated in the National Society for Medical Research, Institutional Animal Care and Use Committee, and the ‘Guide for the Care and Use of Laboratory Animals’ published by the National Institute of Health. Forty non-allergic, male Sprague–Dawley rats weighing
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between 350–600 g were randomly assigned to receive a 16 ml intranasal infusion of either 10% solution of histamine (H-7250; Sigma Chemical Co., St. Louis, MO) or PBS over a 3 min interval. The rats receiving a 10% histamine solution (n = 24) were divided into three groups to assess passive opening and closing pressures (POP/PCP) (n = 12), active clearance of positive and negative pressure (ACPP/ACNP) (n = 7), and mucociliary clearance times (MCCTs) of the tubotympanum (n = 5). Five animals in the treatment group (n = 2 POP/PCP, n=2 ACCP/ACNP, and n = 1 MCCT) were not infused secondary to dehiscence of catheters. The rats receiving PBS (n = 16) were separated in a similar manner with the groups containing 5, 4,and 4, respectively (POP/PCP: n =5, ACCP/ACNP: n = 4, MCCT: n = 4). Three animals in the control group (n = 2 POP/PCP, and n= 1 ACCP/ACNP) were not infused secondary to dehiscence of catheters.
2.2. Surgical procedures and peri-operati6e management Each rat was anesthetized with 0.1 ml/100 gm of a 1:1 mixture of xylazine (4 mg/ml) and ketamine hydrochloride (100 mg/ml). The depth of anesthesia was tested by applying pressure to joints. All animals were given 80 ml/min/kg of 300 mOsm Krebs Ringer solution (140 mmol/l Na+, 120 mmol/l Cl−, 5.2 mmol/l K+, 25 mmol/l HCO3 − , 1.1 mmol/l Ca2 + , 1.2 mmol/l Mg2 + , 0.4 mmol/l HPO2 − , and 5.6 mmol/l glucose) subcutaneously to maintain blood pressure and renal perfusion during the experiment. To maintain body temperature at 37 °C a thermistor-controlled heating pad was used each time anesthesia was administered. Once anesthetized all tympanic membranes were assessed using an operating microscope to exclude obvious middle ear pathology. The skin overlying the nasal bones and the nucal region were then shaved and injected with 1% xylocaine with 1:100 000 epinephrine for local anesthesia and hemostasis. Incisions were made (1 cm) over the skin of the animal’s shoulders and (2–3 cm) over the midline to expose the nasal bones. With a small hand drill, a hole (2 mm diameter) was
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bored 1 mm from the midline in the left nasal bone through to the nasopharynx. A PE-10 polyethylene tube (c 7401, Clay Adams, Parsippany, NJ) with one end heat-formed to have a 12 mm bend was then threaded through the drilled hole (12 mm) into the nasopharynx of each rat. The average distance from the catheter tip to the ET orifice was 0.5 cm. The free end of the catheter was passed through a subcutaneous tunnel exiting between the animal’s shoulders. The skin overlying the exposed nasal bones was sutured and the tube was secured in its position via subcutaneous and external sutures in the skin overlying the shoulders. After allowing all animals to repose for 7–10 days after catheter placement procedure, they were anesthetized with a xylazine/ketamine mixture and a solution of 10% histamine (experimental group) or PBS (control group) was infused into the nasopharynx through the implanted catheter. The infusion was 3 min at a rate of 5.33 ml/min. via a Razen microinfusion pump to deliver 16 ml of solution in to the nasopharynx. Larger volumes of intranasal histamine has been previously determined to cause aspiration and swallowing, compromising ET function tests [11]. During infusion all animals were stimulated to swallow a minimum of 10 times (4 times the first minute and 3 times the second and third minutes) by direct stimulation of the epiglottis with polyethylene tubing. Swallowing ventilates the ET and allows for contents of the nasopharynx to reflux through the tube and into the middle ear [5]. The dynamic function of the ET was assessed prior to surgery, before and after infusion, and 24 h post-infusion as described below.
2.3. Assessment of ET function Rats were anesthetized with a xylazine/ketamine mixture and placed in the recumbent position. Once the tympanic membrane was visualized through an operating microscope, incisions were made with a small myringotomy knife in the posteroinferior and anteroinferior quadrants. The myringotomies functioned as a port and vent for the application of positive and negative pressure to the middle ear. Baseline opening and closing
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pressures were measured employing the forced-response test analogous to that first described by Flisberg et al. [14]. A pressure measurement apparatus was created by inserting the end of a 6 French latex pediatric foley catheter with a 3 cc balloon (Medline, Mundelein, IL) under direct visualization into the external auditory canal and inflating the balloon to create an airtight seal (Fig. 1). The drainage port of the catheter was connected to a water manometer, thereby creating a closed system, which allowed for the application of positive pressure via a Harvard infusion pump (20 ml/min). Slow uniform pressure was then infused until the ET spontaneously opened, marked by the meniscus of the water column changing from convex to concave. This ‘breaking point’ was considered the passive opening pressure (POP). The meniscus of the water column then descended, and the point at which the column stopped descending corresponded to the
spontaneous closure of the ET. This point was considered the passive closing pressure (PCP). For those rats in the experimental and control groups assigned to have POP/PCP assessed, baseline pressures were made prior to catheter placement. In addition, before infusion (time 0 min), and at minutes 6, 10,14,18, 26, and 24 h post-infusion POP/PCP were measured. At each time interval each animal was assessed for evidence of effusion via an operating microscope. To assess the ability of the ET to actively clear middle ear pressure rats in the experimental and control groups were anesthetized as previously described and placed in the recumbent position. Once the 3 cc catheter balloon of the pressure measurement apparatus was placed and inflated in the external auditory canal, 15 cm H2O of positive pressure was infused. Rats were then stimulated to swallow with polyethylene tubing. Swallowing caused the tensor veli palatini to actively open the
Fig. 1. An infusion withdrawal pump was used to apply positive or negative pressure to the rat’s middle ear by infusing or withdrawing air into a closed system. A 6 French Foley catheter created a seal in the animal’s external auditory canal. A myringotomy created communication between the middle ear and the infusion/withdrawal pump so that changes in pressure could be recorded in cm H2O using a water manometer. A PE-10 catheter was inserted through the left nasal bone in order to deliver either a 10% histamine solution or PBS.
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ET and thus, ventilating the middle ear causing the water column to fall. Rats were stimulated to swallow until positive pressure could no longer be cleared represented by the point at which the water column would no longer fall after swallowing. The difference in pressures between these two points were divided by 15 cm H2O to quantify the percent active clearance of positive pressure (ACPP). To measure the active clearance of negative pressure (ACNP) the infusion/withdrawal pump was used to remove 10 cm H2O from the system. Again, rats were stimulated to swallow ventilating the ET allowing for clearance of negative pressure. When repeated swallowing no longer caused the water column to rise, the differences pressures between the two points were divided by 10 cm H2O to signify the percent ACNP. ACCP and ACNP measurements were only taken at intervals of 0, 6, and 24 min post-infusion due to the duration of time for adequate swallowing to clear pressure. To assess the mucociliary clearance function of the ET, rats in the experimental and control groups were deeply anesthetized to prevent swallowing and active opening of the ET. After infusion of either 10% histamine or PBS the rats were placed in the supine position, and a modified standard nasal speculum was used as a headholder device to aid in the visualization of the soft palate. A small midline incision was made in the soft palate posterior to the posterior border of the hard palate. Hemostasis and anesthesia were maintained by swabbing the incision with 1% xylocaine with 1:100 000 epinephrine. A miniature hand mirror was inserted through the incision to visualize the ET orifice under the operating microscope. Rats were then placed in the right lateral recumbent position and incisions in the anteroinferior and posteroinferior quadrants of the tympanic membrane were made with a small myringotomy knife. Eighteen minutes after infusion 4 ml of blue dye (Coumassie Brilliant Blue, B-0149; Sigma Chemical Co.) were injected into the middle ear using a micropipette. A stopwatch was started and the rats were returned to the supine position. The nasopharyngeal orifice of the ET was again visualized under the operating mi-
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croscope. The time at which dye appeared at the orifice was measured. This time signified the MCCT of the tubotympanum.
2.4. Statistical analysis All statistical analyses were done using SigmaStat Statistical software Version 2.03 (SPSS Inc.). Pressures were analyzed by one-way analysis of variance with pair wise comparisons of means evaluated by paired t-tests. ACPP/ACNP was converted to a percentage of total possible pressure for clearance and then analyzed. Mean mucociliary times were analyzed using paired t-tests. Statistical significance was determined prior to the experiment for P values of B0.05. Graphs were constructed using means and the standard error of the mean (SEM).
3. Results
3.1. Eustachian tube function The intranasal administration of histamine significantly increased the pressure required to open (Fig. 2) and close (Fig. 3) the ET in response to the infusion of positive pressure compared to controls, respectively. The increase was progressive, with the largest difference occurring at 26 min after infusion. Twenty-four hours post-infusion the mean POP and PCP returned to baseline. Figs. 2 and 3 depict time versus pressure in cm H2O and illustrate the notable differences of POP and PCP at various intervals post-infusion between the treatment group and controls. Significant differences are marked by asterisks. There were no significant differences between the treatment and control groups at times pre-catheter placement, 0 min, and 24 h post-infusion for both POP and PCP. According to the analysis of variance, the differences in the means among and within the treatment groups for both POP and PCP are greater than would have been expected by chance (P 00.001). In addition, no clinically evident effusions were noted at any time interval. Intranasal infusion of histamine also decreased the ability to clear positive and negative pressure
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Fig. 2. Mean POPs for experimental (histamine, black) and control (PBS, gray) in cm H2O at intervals prior to catheter placement, 0, 6, 10, 14, 18, 22, and 26 min as well as 24 h post-infusion of either 10% histamine or PBS. (Note: y-axis begins at 30 cm H2O.) Values are significantly greater for the histamine infused group at intervals 6, 10, 14, 18, 22, and 26 min after infusion. Significant differences are marked by asterisks. The maximum difference between the experimental group and controls were observed at 26 min. There was no significant differences between the means of the groups at times prior to surgery, 0 min, and 24 h. Error bars represent SEM.
Fig. 3. Mean PCPs for experimental (histamine, black) and control (PBS, gray) in cm H2O at intervals prior to catheter placement, 0, 6, 10, 14, 18, 22, and 26 min as well as 24 h post-infusion of either 10% histamine or PBS. Values are significantly greater for the histamine infused group at intervals 6, 10, 14, 18, 22, and 26 min after infusion. Significant differences are marked by asterisks. The maximum difference between the experimental group and controls were observed at 26 min. There was no significant differences between the means of the groups at times prior to surgery, 0 min, and 24 h. Error bars represent SEM.
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Fig. 4. Mean ACPPs for experimental (histamine, black) and control (PBS, gray) in cm H2O at intervals prior to catheter placement, 6, and 24 min post-infusion of either 10% histamine or PBS. Values are significantly greater for the histamine infused group at only 24 min after infusion. Significant differences are marked by asterisks. The maximum amount of possible percent clearance was 100%. There was no significant differences between the means of the groups at times prior to surgery, 0, and 6 min. Error bars represent SEM.
Fig. 5. Mean ACNPs for experimental (histamine, black) and control (PBS, gray) in cm H2O at intervals prior to catheter placement, 6, and 24 min post-infusion of either 10% histamine or PBS. Values are significantly greater for the histamine infused group at 6 and 24 min after infusion. Significant differences are marked by asterisks. The maximum amount of possible percent clearance was 100%. There was no significant differences between the means of the groups at times before surgery and at 0 min. Error bars represent SEM.
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Fig. 6. Mean MCCTs for experimental (histamine, black) and control (PBS, gray) groups versus time (min). MCCT was significantly greater for the histamine infused groups compared to the controls. Significant differences are marked by asterisks. Error bars represent SEM.
from the middle ear compared to controls (Figs. 4 and 5). Fig. 4 shows the mean ACPP in cm H2O over various time intervals. Significant differences are marked by asterisks. At 24 min the ACPP for the histamine infused group was appreciably impaired compared to baseline at 0 min. Similarly, the ACNP was impaired by the infusion of histamine. Fig. 5 illustrates mean ACNP in cm H2O over various time intervals with the ACNP most impaired at 24 min. Fig. 6 showing treatment group versus time demonstrates that the mean MCCT was prolonged by histamine when compared to controls. The average time for dye to be transmitted through the ET was 534 s for the treatment group compared to 220 s for controls. Thus, MCCT was on average 2.4 times longer for the histamine group than the PBS group.
4. Discussion The infusion of a 10% histamine solution caused acute ET dysfunction when introduced into the nasopharynx in rats. Histamine produced notable impairment of the ET’s ventilation functions as evident by increased mean POP/PCP and a notable reduction in the ability to actively clear both positive and negative pressure. Furthermore,
the ciliary function of the ET was impaired by histamine when compared to controls as evidenced by the prolongation of the MCCT of the tubotympanum. These results demonstrate a relationship between histamine challenge and the development of ET dysfunction. Examining the ventilatory parameters assessed in this experiment (POP, PCP, ACPP, and ACNP), all showed a maximal impairment of function at approximately 24 min post-infusion with initial significant differences observed as early as 6 min. By 24 h, the effect had disappeared. Particularly important are the decreased percent ACNP since the equalization of negative pressure within the middle ear by deglutition is considered the most significant and accurate parameter of tubal function by Miller and others [15,16]. Additionally, ciliary function was impaired 18 min post-infusion. These data are consistent with previous work conducted by Doyle with rhesus monkeys where aerosolized histamine and other allergens were administered into the nasal cavity, which resulted in similar ETD [9,17]. While few would argue that either intrinsic or extrinsic obstruction of the ET results in improper ventilation/pressure regulation and effusion, there remains some debate whether the middle epithelium functions in a manner parallel to other respiratory mucosa. In a recent series of studies,
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Hurst showed that the middle ear epithelium of persons with atopy had all the tools needed to respond in a manner like that of the rest of the upper respiratory system, and was able to have an allergic immune response [18]. Supporting this observation, the middle ear mucosa develops from the same ectoderm as the other respiratory epithelium, and animal studies have shown that it responds immunologically to antigens as do the epithelium of the nasopharynx, sinuses, and bronchi [18,19]. In addition, White showed that dye injected into the nasopharynx of rats can be refluxed into the middle ear and that gastric reflux into the nasopharynx caused significant ETD using analogous parameters of testing ET function [15]. Although this study was not designed to further elucidate the exact site of action of the allergic response, the effects of histamine most likely acted on both the middle ear mucosa due to reflux as seen by delayed MCCT and by blocking the nasopharyngeal orifice resulting in significantly decreased ACPP/ACNP as well as increased POP/PCP. Our study builds on previous work examining the role of histamine and ETD, and represents the development of a model to continue investigation in this area [11,17,20]. Future study will be directed at dynamically evaluating the dose-response relationship of histamine infused intranasally with ETD. Also, we plan to use this model to examine intranasal allergen delivery on ET function and test employment of immunomodulators on response the to allergen. It is hoped that our work will continue to build on the understanding of the pathogenesis and pathophysiology of OM and OME, specifically that related to allergy. An improved understanding of the role of allergy in ETD is vital for the enhancement of treatment options for OM and OME.
Acknowledgements The authors would like to thank Doug C. Fitzpatrick for critical review without which this paper would not have been completed.
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