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A rat model of otitis media with effusion caused by eustachian tube obstruction with and without Streptococcus pneumoniae infection: Methods and disease course
A rat model of otitis media with effusion caused by eustachian tube obstruction with and without Streptococcus pneumoniae infection: Methods and disease course OTAVIO B. PILTCHER, MD, PHD, J. DOUGLAS SWARTS, PHD, KARIN MAGNUSON, MD, PHD, CUNEYT M. ALPER, MD, WILLIAM J. DOYLE, PHD, and PATRICIA A. HEBDA, PHD, Pittsburgh, Pennsylvania
OBJECTIVE: To describe the clinical and histopathologic progression of a rat model of otitis media with effusion caused by eustachian tube obstruction (ETO) with and without Streptococcus pneumoniae infection. METHODS: In 164 rats, the left, bony eustachian tube was approached via a ventral incision and obstructed with dental material. Then 108 rats were infected via an intrabullar injection with S pneumoniae. At 48 hours, the infected rats were treated for 5 days with ampicillin. All ears were evaluated by weekly otomicroscopy. On each of days 1, 2, 7, 21, 35, 56, and 112, four rats were killed for histologic study. All effusions were cultured for bacteria. RESULTS: Fourteen rats died of surgical complications; effusion resolved by 2 weeks in 9 rats. During the first few days, infected ears with ETO had bulging tympanic membranes, followed by tympanic membrane retraction, purulent effusion, and otorrhea (50%) over the next few weeks, whereas uninfected ears with ETO developed retraction and serous effusion during the same time frame. At later times, all ears with ETO presented with retraction and serous or serous-mucoid effusion. S pneumoniFrom the Department of Otolaryngology, Children’s Hospital of Pittsburgh and the University of Pittsburgh School of Medicine. This work was supported in part by grants from the National Institutes of Health (DC 01260) and from CAPES (Coordenaçao de Aperfeiçoamento de Pessoal de Nível Superior, Brazil). Reprint requests: Patricia A. Hebda, Department of Pediatric Otolaryngology, Children’s Hospital of Pittsburgh, 3460 Fifth Ave, Pittsburgh, PA 15213; e-mail, [email protected]. Copyright © 2002 by the American Academy of Otolaryngology–Head and Neck Surgery Foundation, Inc. 0194-5998/2002/$35.00 + 0 23/1/124935 doi:10.1067/mhn.2002.124935
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ae was recovered only from the infected ears with ETO (days 1 and 2), with some colonization by nonpathogenic microorganisms observed equally in both groups of ears. Histology showed a typical acute inflammatory reaction in the challenged ears with ETO through day 14 and then a chronic inflammation for all ears with ETO. CONCLUSION: The experimental methods provoked reproducible pathologic signs similar to those for otitis media with effusion. Given the availability of rat-specific reagents, this model is well suited for studies of cytokine elaboration during disease pathogenesis. (Otolaryngol Head Neck Surg 2002;126:490-98.)
O
titis media with effusion (OME) is a common pediatric disease that remains one of the most frequent causes of nonwell medical visits, antibiotic prescriptions, and surgical procedures worldwide.1-3 The term OME describes the persistence of an inflammatory effusion within the middle ear (ME) space in the absence of clinical signs and symptoms of an acute infection and is synonymous with secretory or serous otitis media. To date, OME remains refractory to most medical treatments, and surgical interventions have relatively low efficacies. The cause of OME is recognized as multifactorial, with viral upper respiratory tract infection, nasal allergy, and/or bacterial infection of the ME mucosa precipitating the onset of OME and either intrinsic or acquired eustachian tube (ET) obstruction (ETO) playing a major role in disease persistence.3,4 Since early in the 20th century, animal models of OME were developed and used to study the pathogenesis and treatment of the disease.5-13 Animal species chosen for these models included
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the dog, cat, ferret, monkey, gerbil, chinchilla, and rat. The choice of the particular species for use in a given model depended on a variety of considerations, including the pathophysiologic mechanism to be studied, the susceptibility of the species to infection with specific pathogens, and the availability of commercial reagents for assay of biochemical mediators of inflammation. In this report, a model is described in the rat of OME caused by ETO without or with concurrent infection by the human pathogen Streptococcus pneumoniae. The surgical methods for initiating disease pathogenesis included modifications to those previously published6-10 and appear to better control for the insult and thereby avoid some of the criticisms of previously described models. The etiology of OME in this model is described on the basis of longitudinal results for otomicroscopy, microbiology, effusion type, effusion cytology, and mucosal histopathology. The large number of animals in this study serves to establish the consistency, reliability, and stability of this model of OME. MATERIALS AND METHODS Animals One hundred sixty-four specific pathogen-free male rats (Hilltop Laboratory Animals, Inc, Scottdale, PA) weighing between 150 and 200 g were used. The rats were anesthetized with a mixture of ketamine (60 mg/kg) and acepromazine (0.6 mg/kg) administered intramuscularly. The ears were examined by otomicroscopy to document a disease-free ME bilaterally, and then unilateral (left) ETO was created surgically in all rats. Unilateral ETO was used because this procedure was tolerated better than bilateral obstruction. Although not included in this report, a pilot study showed that animals with bilateral ETO exhibited markedly increased morbidity and mortality rates. Surgical Procedure The ventral neck from the limit of the jaw to the middle of the sternum was shaved and cleaned with a 10% povidone-iodine solution. Under enhanced visualization with an operating microscope (Storz; URBAN, St Louis, MO), a vertical midline incision was made to include the skin, superficial fascia, platisma, and deep fascia. Left
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Fig 1. Coronal cross section of a rat head for the 56-day timepoint (Hemotoxylin and eosin staining, dissection microscope original magnification ×15). Both middle ears (ME) are identified, with the left being filled with effusion. Arrows point to the eustachian tube, showing the presence of the dark staining gutta percha within the lumen of the left eustachian tube. The nasopharynx is also identified.
and right subplatismal flaps were elevated and kept open using self-retractors. The triangular area between the sternomastoideus, sternohyoideus, and omohyoideus muscles was explored, and arteries found running medially in a superoinferior direction were protected by keeping the dissection more lateral and inferior. The dissection was extended more deeply to expose the ventral surface of the ME bulla, and that surface was denuded using a half-moon in a medial and superior direction until the bony part of the ET was identified. A hole was drilled midway between the beginning and the end of the bony ET at a 75-degree angle with the bone. The membranous cover under the bone was opened with the help of a sharp dissector ear instrument, and then small pieces of an inert dental material, gutta percha (points size “fine”; American Dental Cooperative, St Louis, MO), were placed into the defect. After each piece was fitted, it was melted using electrocautery and molded to conform to the ET lumen. Experimental Design After completion of the obstruction, 108 (66%) rats were randomized to the infected (I) group; they each received a transbullar inoculation in the
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Fig 2. Number of effusions recovered from the middle ear space of ears with eustachian tube obstruction in the uninfected (left) and infected (right) groups, visually typed as purulent (filled), serous (open), and seromucoid (shaded) on each of the sampling days (D, days; W, weeks). The results show an early disparity between uninfected and infected ears, with infected ears exhibiting signs of acute inflammation. Later in the time course (after 3 weeks), the 2 groups are similar, reflecting the persistent changes in the chronic phase of otitis media with effusion.
same ME of 15 µL phosphate-buffered saline (PBS) containing 1 × 108 colony-forming units (cfu) of an ampicillin-sensitive strain of Streptococcus pneumoniae, type 6A. The skin was sutured with nylon 4-0, and the animal recovered from the anesthesia. The average time for the surgical procedure was 20 minutes. The remaining 56 animals were placed into the uninfected (UI) group. Beginning 48 hours after the surgery, all animals challenged with S pneumoniae were treated twice daily with 100 mg/kg per day IM of ampicillin for 5 days. For all animals, bilateral otomicroscopy was performed weekly, and results were recorded for tympanic membrane position (retracted, normal, or bulging), presence or absence of effusion, and effusion type (serous, serousmucoid, or purulent). Twenty-eight rats (14 in group I infected with S pneumoniae, 14 in group UI) were assigned for histologic study, and 4 rats (2 in group I, 2 in group UI) were killed by exsanguination under deep anesthesia on each of days 0, 1, 2, 7, 21, 56, and
112 days with an overdose of ketamine-acepromazine (100 and 1 mg/kg IM). The animals were perfused with PBS and then with 4% paraformaldehyde in PBS for fixation, as previously described.14 The whole heads were decalcified in 10% formic acid. After complete decalcification, the specimens were divided into anterior and posterior bullar halves, and each part was processed for light microscopy, embedded in paraffin blocks, sliced coronally (8-µm thin sections) from posterior to anterior, and stained with hematoxylin and eosin (H&E). Sections from the anterior block were examined for evidence of ETO. Sections from the posterior block were examined for descriptive histopathology, and the thickness of the mucosa at the epitympanum, promontorium, and hypotympanum was measured using the Metamorph Imaging System (Universal Imaging Corporation, West Chester, PA). The remaining 136 animals were followed by weekly otoscopy, and the results are included in this report. Six animals in each group were killed by exsanguination under deep anesthesia on days
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Fig 3. Mean log number of cells within the effusion at the different sample times for ears with eustachian tube obstruction with (■) and without () prior Streptococcus pneumoniae infection. Horizontal bars designate the bounds of a 95% confidence interval about the mean for the ears with eustachian tube obstruction with S pneumoniae infection. Cellularity of the effusions shows early differences with infection (higher cellularity) that resolved with time and disease course; at later stages, there were relatively few cells in the effusions.
1, 2, 7, 21, 35, 56, and 112 and were perfused with saline and decapitated. ME effusions and mucosa were harvested and used for biochemical and molecular analyses reported elsewhere.15 The syringe used to collect the effusion was washed with 15 µL of sterile tryptic soy broth (Fisher Scientific Co, Pittsburgh, PA), and the washes were transferred onto chocolate agar plates (Fisher), incubated at 37°C for 48 hours in a 5% CO2 environment, and examined for bacterial recovery. This experimental protocol was reviewed and approved by the Children’s Hospital of Pittsburgh Animal Research and Care Committee (Animal Welfare Assurance No. A3617-01). The institution is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). Data Analysis Quantitative data, including bacterial colony counts, mucosal thickness, and bullar thickness, are reported as the means and 95% confidence intervals. Nonoverlapping confidence intervals
indicate that 2 values are significantly different (P < 0.05) (see Figs 3 and 4). RESULTS Of the 164 rats entered into the study, 14 (8.4%) died due to the surgical procedure, anesthesia, or infection. Of the 150 remaining animals, the effusion in 9 experimental ears (6%) was resorbed within 2 weeks. Those animals were killed, and the ET was shown to be patent by direct dissection. Microscopic examination of selected sections from the experimental ear of all animals documented at least one section in which the ET was observed to be filled with the dental material (Fig 1). Moreover, the region of obstruction was limited to the middle of the ET, whereas the pharyngeal and tympanic portions were spared and had no evidence of surgical trauma. The contralateral control ear exhibited no evidence of ETO, inflammation, or surgical trauma. Examination of the tympanic membranes of the ears with ETO in the UI group documented the progressive development of a retraction during the
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A
B Fig 4. Mean mucosal thickness (A) and bone thickness (B) for ears with eustachian tube obstruction with infection (2 per timepoint, ■) and without infection (2 per timepoint, ) and for the contralateral nonobstructed ears (control group, 4 per timepoint, ). Vertical bars designate the bounds of a 95% confidence interval about the mean values of the contralateral control ears. The results indicate that mucosal thickening (A) occurred transiently with eustachian tube obstruction, with the greatest change during the early phase of the disease and resolution during the chronic phase. In contrast, bony thickening (B) occurred more gradually but permanently, and this change was more pronounced in the infected group.
first week with the subsequent development of serous and, later, seromucoid effusion. In contrast, the ears with ETO in the I group initially presented with a bulging tympanic membrane, which progressed to retraction after 7 to 14 days. Those ears initially had a purulent effusion that changed by 2 weeks to a seromucoid effusion. None of the contralateral control ears had evidence of disease on otomicroscopic examination.
Figure 2 shows the distribution of effusion types based on macroscopic examination at the time of death for the ears with ETO in the I and UI groups. These results are similar to those made on the basis of in vivo otomicroscopic observations and confirm the development of purulent effusion only in the ears with ETO of the I group. The results show an early disparity between uninfected and infected ears, with infected ears exhibiting signs of acute
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Fig 5. Coronal cross sections (H&E staining, original magnification ×40) of an uninfected ear with eustachian tube obstruction (ETO) (A), an infected ear with ETO (B), and a contralateral control ear (C) at days 2 and 56 (D to F). Designators are provided for the middle ear (ME), epithelial layer (E), submucosa (SM), and bone (B). Effusion-mucosa separation artifact in B was corrected with computerized imaging software.
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inflammation. Later in the time course (after 3 weeks), the 2 groups are similar, reflecting the persistent changes in the chronic phase of otitis media with effusion. Effusion was not recovered from the contralateral control ears of any study animal. S pneumoniae was recovered from all ears with ETO in the I group (n = 11) on days 1 and 2 but not at any time later. That organism was not recovered at any time from the ears with ETO in the UI group or from the contralateral control ears. At all timepoints, gram-negative rods, α and β Streptococcus hemolyticus, Streptococcus pyogenes, Pseudomonas aeruginosa, and other bacteria grew on the chocolate agar plates swabbed with the wash fluids from the aspiration needle, but in the absence of concomitant S pneumoniae infection, none of the source ears showed any signs of acute infection, and the total cfu was very low in the UI group relative to the I group. Histologic study of the ears with ETO in the 2 groups showed early differences that disappeared between study days 7 and 21. For example, during the first week, the cell density within the effusion was greater for the ears with ETO in the I group than for those in the UI group, but by day 21 and subsequently, the 2 groups were indistinguishable for that variable (Fig 3). In both groups, the ME mucosa in ears with ETO progressively thickened through day 21 vis-a-vis the contralateral control ear with some resolution during the later timepoints (Fig 4A). Compared with the control sides, there was a slow but progressive formation of new bone under the ME mucosa in both groups with ETO, and this process continued throughout the period of follow-up, with the I group showing greater bony thickening at the end of study period (16 weeks) (Fig 4B). The epithelium initially developed a hyperplasia that was more extensive in the ears with ETO of the I group. In both groups, there was a progressive reduction in the mucosal area, characterized as a respiratory epithelium with an increase in cuboidal and squamous cell types, which reflected an increasing degree of metaplasia. The submucosa showed early vascular and lymphatic dilatations with inflammatory cell infiltration mainly during the first 21 days, and this was followed by fibroplasia and collagen deposition. In none of the specimens was there evidence
of giant cells, which are indicative of a foreign body reaction. These changes were not seen in the contralateral control ears (Fig 5). DISCUSSION There are many publications describing experimental models for the study of OME. For example, mucosal inflammation and effusion were observed after mechanical or functional block of the ET, after exposure of the ME to infectious or inflammatory agents (bacteria, virus, and inflammatory mediators), or after inducement of systemic inflammatory reactions. Also, a variety of animal species have been used, with the choice dictated by the specific etiology being modeled and by such factors as cost, ease of approach, physiology of the ET dilatory mechanism, anatomic similarities to the human ME, susceptibility to infection with pathogens, and the availability of specific analytical reagents. Indeed, there is no single ideal nonhuman species for mimicking disease pathogenesis by all suspected OME etiologies and for all model applications (eg, treatment, pathogenesis, etc). The animal model described in this report was developed for study of the temporal relationship between cytokine elaboration and mucosal inflammation in ears with OME caused by ETO with and without concurrent bacterial infection. Such a model would be useful for preclinical evaluation of the efficacy with respect to disease resolution of interventions that upregulate, downregulate, or otherwise modify the expression of specific cytokines elaborated during disease pathogenesis. Of the animal models commonly used for study of OME (including different experimental manipulations of the monkey, chinchilla, gerbil, or rat), the specific requirements of the proposed application are most compatible with the choice of the rat as the experimental species. For example, monkeys are discounted because they are resistant to infection with pathogens common to OME in humans, whereas chinchilla- and gerbil-specific reagents for assay of cytokine protein and mRNA are not commercially available.16 On the other hand, rats can be infected with human pathogens, surgical techniques have been described for blocking the rat ET, and assay reagents are readily available.8-11,17 Also, the general anatomy of the rat ME and ET is similar to
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that of humans, and the ME mucosa of the rat and human has similar histologic features with respect to cell type and ciliary clearance tracts.18-21 Reported limitations to the use of the rat in modeling OME include the reported high frequency of natural infection of the ME with bacteria, which may confound an ETO model that does not include infection; the small size of the ME, which significantly limits the volumes of effusion and mucosa that can be collected from a single animal; and the difficulty of the surgical approach to block the ET.11,12,18 The actual incidence of natural ME infections among rats is not known, and the use of pathogenfree animals should significantly reduce this background frequency. In this large sample, no rat had evidence of naturally occurring ME disease. In previous studies, the small volume of recovered effusion required dilution before biochemical assay, and that for the mucosa was accommodated by pooling samples across ears.14,17 Although these procedures limit the threshold sensitivity for biochemical assays and increase the number of animals required to achieve a designated sample size for the mucosal analyses, these drawbacks can be managed. Finally, surgical procedures to access the bony ET using a ventral neck incision and careful dissection of the area between specific muscles, nerves and blood vessels were previously described.8-10 Although they are difficult and require approximately 30 minutes, those procedures and the modifications described here (substitution of gutta percha for dental wax and a vascular clip) have a low mortality rate (9% in this study) and a high success rate (94%) and reliably produce a persistent OME that lasts for >6 months. Finally, the intersample consistency reported here for the otomicroscopic, microbiologic, and histologic observations within the different groups shows that this model is useful for studying disease evolution along the OME continuum. Obstruction and changes in the ET were localized to tissue in the immediate region, with no histologic evidence of inflammation at the nasopharyngeal or tympanic orifices of the ET. Thus, it is unlikely that the effusion and observed mucosal changes were caused by a foreign body reaction to the dental material or by inflammation
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secondary to infection. Of some concern was the recovery of nonpathogenic bacteria from all MEs with ETO. Although not clearly associated with signs of infection or inflammation, this will be studied in the future to determine if those bacteria are also recovered from the contralateral control MEs. The S pneumoniae culture results were consistent with the expectations that the organism would be recovered only from ears challenged with the bacteria and only at the early time points. That S pneumoniae was not recovered on or after day 7 suggests that the antimicrobial therapy instituted at 48 hours effectively sterilized the ME cleft. The histopathologic changes observed in response to ETO and infection reproduced the descriptions provided in earlier studies using similar models.7,13 In general, ETO and concomitant S pneumoniae infection elicited a more robust inflammatory response than ETO alone. However, after eradication of the pathogen with antibiotics, by day 21 the disease course during the chronic phase was quite similar in both groups. These results suggest that the chronic mucosal changes documented in the ears of these animals result from the persistent ETO and that reversal of that condition should be one goal of therapeutic intervention. The investigators thank Chia-Yee Lo, MS, and Julianne Banks, BS, for their able assistance with the microbiologic, surgical, and technical aspects of this study. Consultation for histopathologic analysis was graciously provided by Edwin C. Klein, VMD, veterinarian and veterinary pathologist at the University of Pittsburgh.
REFERENCES
1. Schappert SM. Office Visits for Otitis Media: United States 1975-1990. Bethesda, MD: US Dept of Health and Human Services, US Public Health Service, Centers for Disease Control and Prevention, National Center for Health Statistics; 1991. 2. Haggard MP. Otitis media outcomes and impact in families. In: Adam D, Ehrlich GD, eds. International Clinical Practice Series, Otitis Media: Prospects for Management, Tunbridge Wells, England: Wells Medical Ltd; 1996. p. 29-35. 3. Bluestone CD, Klein JO. Otitis media in infants and children. Philadelphia: WB Saunders; 1998. 4. Paparella MM. Middle ear effusions: definitions and terminology. Ann Otol Rhinol Laryngol 1976;85:8-11;
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6. 7. 8. 9. 10. 11.
12. 13.
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Modified report of the Ad Hoc Committee on Definition and Classification of Otitis Media. Ann Otol Rhinol Laryngol 1980;89:(Suppl68):3-4. Haynemann L. Arch Ohrenheilk 1912;267; Ibid. 1913; 92;1; Ibid. 1914;93:1; Ibid. 1914;95:99., quoted in Freidmann I. The comparative pathology of otitis media: experimental and human. I. Experimental otitis of the guinea pig. J Laryngol Otol 1955;69:27-50. Proud GO, Odoi H. Effects of eustachian tube ligation. Ann Otol Rhinol Laryngol 1970;79:30-2. Kuijpers W, van der Beek JMH, Willart ECT. The effect of experimental tubal obstruction on middle ear. Acta Otolaryngol 1979;345-52. Stenfors LE, Carlsoo B, Winbald B. Structure and healing of the tympanic membrane after eustachian tube occlusion. Acta Otolaryngol (Stockh) 1981;75-84. Russell JD, Giles SJ. A persistent otitis media with effusion: a new experimental model. Laryngoscope 1998; 108:1181-4. Jewett BS, Prazma JP, Hunter SE, et al. Systemic reactivation of otitis media with effusion in a rat model. Otolaryngol Head Neck Surg 1999;121:7-12. Giebink GS, Payene EE, Mills EL, et al. Experimental otitis media due to Streptococcus pneumoniae: immunopathologic response in the chinchilla. J Infect Dis 1976;134:595-604. Kuijpers W, van der Beek JMH. The role of microorganisms in experimental eustachian tube obstruction. Acta Otolaryngol (Stockh) 1984;(suppl 414):58-66. Kaneko Y, Paparella MM. Middle ear epithelium in the
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presence of experimental effusion. Acta Otolaryngol 1971;72:206-14. 14. Hebda PA, Alper CM, Doyle WJ, et al. Upregulation of messenger RNA for inflammatory cytokines in middle ear mucosa in a rat model of acute otitis media. Ann Otol Rhinol Laryngol 1998;107:501-7. 15. Hebda PA, Piltcher OB, Swarts JD, et al. Cytokine profiles in a rat model of otitis media with effusion caused by eustachian tube obstruction with and without S.penumoniae infection. Laryngoscope 2002 (in press). 16. Doyle WJ. Animal models of otitis media: other pathogens. Pediatr Infect Dis J 1989;8:S45-S48. 17. Zeevi A, Alper CM, Balaban CD, et al. Early inflammatory events in a rat model of OM caused by infection with S pneumoniae. Proceedings of the Sixth International Symposium on Recent Advances in Otitis Media, Ft Lauderdale, FL; 1995. p. 258-61. 18. Hal DJ III, Fulghum RS, Brinn JE, et al. Comparative anatomy of eustachian tube and middle ear cavity in animal models for otitis media. Ann Otol Rhinol Laryngol 1982;91:82-9. 19. Hellstrom S, Salen B, Stenfors LE. Anatomy of the rat middle ear. A study under the dissection microscope. Acta Anat 1982;112:346-52. 20. Albiin N, Hellstrom S, Salen B, et al. The anatomy of the eustachian tube in the rat. Anat Rec 1983;207:51321. 21. Albiin N, Hellstrom S, Stenfors LE, et al. Middle ear mucosa in rats and humans. Ann Otol Rhinol Laryngol 1986;(suppl 126)95:1-15.
OBJECTIVE: To describe the clinical and histopathologic progression of a rat model of otitis media with effusion caused by eustachian tube obstruction (ETO) with and without Streptococcus pneumoniae infection. METHODS: In 164 rats, the left, bony eustachian tube was approached via a ventral incision and obstructed with dental material. Then 108 rats were infected via an intrabullar injection with S pneumoniae. At 48 hours, the infected rats were treated for 5 days with ampicillin. All ears were evaluated by weekly otomicroscopy. On each of days 1, 2, 7, 21, 35, 56, and 112, four rats were killed for histologic study. All effusions were cultured for bacteria. RESULTS: Fourteen rats died of surgical complications; effusion resolved by 2 weeks in 9 rats. During the first few days, infected ears with ETO had bulging tympanic membranes, followed by tympanic membrane retraction, purulent effusion, and otorrhea (50%) over the next few weeks, whereas uninfected ears with ETO developed retraction and serous effusion during the same time frame. At later times, all ears with ETO presented with retraction and serous or serous-mucoid effusion. S pneumoniFrom the Department of Otolaryngology, Children’s Hospital of Pittsburgh and the University of Pittsburgh School of Medicine. This work was supported in part by grants from the National Institutes of Health (DC 01260) and from CAPES (Coordenaçao de Aperfeiçoamento de Pessoal de Nível Superior, Brazil). Reprint requests: Patricia A. Hebda, Department of Pediatric Otolaryngology, Children’s Hospital of Pittsburgh, 3460 Fifth Ave, Pittsburgh, PA 15213; e-mail, [email protected]. Copyright © 2002 by the American Academy of Otolaryngology–Head and Neck Surgery Foundation, Inc. 0194-5998/2002/$35.00 + 0 23/1/124935 doi:10.1067/mhn.2002.124935
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ae was recovered only from the infected ears with ETO (days 1 and 2), with some colonization by nonpathogenic microorganisms observed equally in both groups of ears. Histology showed a typical acute inflammatory reaction in the challenged ears with ETO through day 14 and then a chronic inflammation for all ears with ETO. CONCLUSION: The experimental methods provoked reproducible pathologic signs similar to those for otitis media with effusion. Given the availability of rat-specific reagents, this model is well suited for studies of cytokine elaboration during disease pathogenesis. (Otolaryngol Head Neck Surg 2002;126:490-98.)
O
titis media with effusion (OME) is a common pediatric disease that remains one of the most frequent causes of nonwell medical visits, antibiotic prescriptions, and surgical procedures worldwide.1-3 The term OME describes the persistence of an inflammatory effusion within the middle ear (ME) space in the absence of clinical signs and symptoms of an acute infection and is synonymous with secretory or serous otitis media. To date, OME remains refractory to most medical treatments, and surgical interventions have relatively low efficacies. The cause of OME is recognized as multifactorial, with viral upper respiratory tract infection, nasal allergy, and/or bacterial infection of the ME mucosa precipitating the onset of OME and either intrinsic or acquired eustachian tube (ET) obstruction (ETO) playing a major role in disease persistence.3,4 Since early in the 20th century, animal models of OME were developed and used to study the pathogenesis and treatment of the disease.5-13 Animal species chosen for these models included
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the dog, cat, ferret, monkey, gerbil, chinchilla, and rat. The choice of the particular species for use in a given model depended on a variety of considerations, including the pathophysiologic mechanism to be studied, the susceptibility of the species to infection with specific pathogens, and the availability of commercial reagents for assay of biochemical mediators of inflammation. In this report, a model is described in the rat of OME caused by ETO without or with concurrent infection by the human pathogen Streptococcus pneumoniae. The surgical methods for initiating disease pathogenesis included modifications to those previously published6-10 and appear to better control for the insult and thereby avoid some of the criticisms of previously described models. The etiology of OME in this model is described on the basis of longitudinal results for otomicroscopy, microbiology, effusion type, effusion cytology, and mucosal histopathology. The large number of animals in this study serves to establish the consistency, reliability, and stability of this model of OME. MATERIALS AND METHODS Animals One hundred sixty-four specific pathogen-free male rats (Hilltop Laboratory Animals, Inc, Scottdale, PA) weighing between 150 and 200 g were used. The rats were anesthetized with a mixture of ketamine (60 mg/kg) and acepromazine (0.6 mg/kg) administered intramuscularly. The ears were examined by otomicroscopy to document a disease-free ME bilaterally, and then unilateral (left) ETO was created surgically in all rats. Unilateral ETO was used because this procedure was tolerated better than bilateral obstruction. Although not included in this report, a pilot study showed that animals with bilateral ETO exhibited markedly increased morbidity and mortality rates. Surgical Procedure The ventral neck from the limit of the jaw to the middle of the sternum was shaved and cleaned with a 10% povidone-iodine solution. Under enhanced visualization with an operating microscope (Storz; URBAN, St Louis, MO), a vertical midline incision was made to include the skin, superficial fascia, platisma, and deep fascia. Left
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Fig 1. Coronal cross section of a rat head for the 56-day timepoint (Hemotoxylin and eosin staining, dissection microscope original magnification ×15). Both middle ears (ME) are identified, with the left being filled with effusion. Arrows point to the eustachian tube, showing the presence of the dark staining gutta percha within the lumen of the left eustachian tube. The nasopharynx is also identified.
and right subplatismal flaps were elevated and kept open using self-retractors. The triangular area between the sternomastoideus, sternohyoideus, and omohyoideus muscles was explored, and arteries found running medially in a superoinferior direction were protected by keeping the dissection more lateral and inferior. The dissection was extended more deeply to expose the ventral surface of the ME bulla, and that surface was denuded using a half-moon in a medial and superior direction until the bony part of the ET was identified. A hole was drilled midway between the beginning and the end of the bony ET at a 75-degree angle with the bone. The membranous cover under the bone was opened with the help of a sharp dissector ear instrument, and then small pieces of an inert dental material, gutta percha (points size “fine”; American Dental Cooperative, St Louis, MO), were placed into the defect. After each piece was fitted, it was melted using electrocautery and molded to conform to the ET lumen. Experimental Design After completion of the obstruction, 108 (66%) rats were randomized to the infected (I) group; they each received a transbullar inoculation in the
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Fig 2. Number of effusions recovered from the middle ear space of ears with eustachian tube obstruction in the uninfected (left) and infected (right) groups, visually typed as purulent (filled), serous (open), and seromucoid (shaded) on each of the sampling days (D, days; W, weeks). The results show an early disparity between uninfected and infected ears, with infected ears exhibiting signs of acute inflammation. Later in the time course (after 3 weeks), the 2 groups are similar, reflecting the persistent changes in the chronic phase of otitis media with effusion.
same ME of 15 µL phosphate-buffered saline (PBS) containing 1 × 108 colony-forming units (cfu) of an ampicillin-sensitive strain of Streptococcus pneumoniae, type 6A. The skin was sutured with nylon 4-0, and the animal recovered from the anesthesia. The average time for the surgical procedure was 20 minutes. The remaining 56 animals were placed into the uninfected (UI) group. Beginning 48 hours after the surgery, all animals challenged with S pneumoniae were treated twice daily with 100 mg/kg per day IM of ampicillin for 5 days. For all animals, bilateral otomicroscopy was performed weekly, and results were recorded for tympanic membrane position (retracted, normal, or bulging), presence or absence of effusion, and effusion type (serous, serousmucoid, or purulent). Twenty-eight rats (14 in group I infected with S pneumoniae, 14 in group UI) were assigned for histologic study, and 4 rats (2 in group I, 2 in group UI) were killed by exsanguination under deep anesthesia on each of days 0, 1, 2, 7, 21, 56, and
112 days with an overdose of ketamine-acepromazine (100 and 1 mg/kg IM). The animals were perfused with PBS and then with 4% paraformaldehyde in PBS for fixation, as previously described.14 The whole heads were decalcified in 10% formic acid. After complete decalcification, the specimens were divided into anterior and posterior bullar halves, and each part was processed for light microscopy, embedded in paraffin blocks, sliced coronally (8-µm thin sections) from posterior to anterior, and stained with hematoxylin and eosin (H&E). Sections from the anterior block were examined for evidence of ETO. Sections from the posterior block were examined for descriptive histopathology, and the thickness of the mucosa at the epitympanum, promontorium, and hypotympanum was measured using the Metamorph Imaging System (Universal Imaging Corporation, West Chester, PA). The remaining 136 animals were followed by weekly otoscopy, and the results are included in this report. Six animals in each group were killed by exsanguination under deep anesthesia on days
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Fig 3. Mean log number of cells within the effusion at the different sample times for ears with eustachian tube obstruction with (■) and without () prior Streptococcus pneumoniae infection. Horizontal bars designate the bounds of a 95% confidence interval about the mean for the ears with eustachian tube obstruction with S pneumoniae infection. Cellularity of the effusions shows early differences with infection (higher cellularity) that resolved with time and disease course; at later stages, there were relatively few cells in the effusions.
1, 2, 7, 21, 35, 56, and 112 and were perfused with saline and decapitated. ME effusions and mucosa were harvested and used for biochemical and molecular analyses reported elsewhere.15 The syringe used to collect the effusion was washed with 15 µL of sterile tryptic soy broth (Fisher Scientific Co, Pittsburgh, PA), and the washes were transferred onto chocolate agar plates (Fisher), incubated at 37°C for 48 hours in a 5% CO2 environment, and examined for bacterial recovery. This experimental protocol was reviewed and approved by the Children’s Hospital of Pittsburgh Animal Research and Care Committee (Animal Welfare Assurance No. A3617-01). The institution is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). Data Analysis Quantitative data, including bacterial colony counts, mucosal thickness, and bullar thickness, are reported as the means and 95% confidence intervals. Nonoverlapping confidence intervals
indicate that 2 values are significantly different (P < 0.05) (see Figs 3 and 4). RESULTS Of the 164 rats entered into the study, 14 (8.4%) died due to the surgical procedure, anesthesia, or infection. Of the 150 remaining animals, the effusion in 9 experimental ears (6%) was resorbed within 2 weeks. Those animals were killed, and the ET was shown to be patent by direct dissection. Microscopic examination of selected sections from the experimental ear of all animals documented at least one section in which the ET was observed to be filled with the dental material (Fig 1). Moreover, the region of obstruction was limited to the middle of the ET, whereas the pharyngeal and tympanic portions were spared and had no evidence of surgical trauma. The contralateral control ear exhibited no evidence of ETO, inflammation, or surgical trauma. Examination of the tympanic membranes of the ears with ETO in the UI group documented the progressive development of a retraction during the
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A
B Fig 4. Mean mucosal thickness (A) and bone thickness (B) for ears with eustachian tube obstruction with infection (2 per timepoint, ■) and without infection (2 per timepoint, ) and for the contralateral nonobstructed ears (control group, 4 per timepoint, ). Vertical bars designate the bounds of a 95% confidence interval about the mean values of the contralateral control ears. The results indicate that mucosal thickening (A) occurred transiently with eustachian tube obstruction, with the greatest change during the early phase of the disease and resolution during the chronic phase. In contrast, bony thickening (B) occurred more gradually but permanently, and this change was more pronounced in the infected group.
first week with the subsequent development of serous and, later, seromucoid effusion. In contrast, the ears with ETO in the I group initially presented with a bulging tympanic membrane, which progressed to retraction after 7 to 14 days. Those ears initially had a purulent effusion that changed by 2 weeks to a seromucoid effusion. None of the contralateral control ears had evidence of disease on otomicroscopic examination.
Figure 2 shows the distribution of effusion types based on macroscopic examination at the time of death for the ears with ETO in the I and UI groups. These results are similar to those made on the basis of in vivo otomicroscopic observations and confirm the development of purulent effusion only in the ears with ETO of the I group. The results show an early disparity between uninfected and infected ears, with infected ears exhibiting signs of acute
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Fig 5. Coronal cross sections (H&E staining, original magnification ×40) of an uninfected ear with eustachian tube obstruction (ETO) (A), an infected ear with ETO (B), and a contralateral control ear (C) at days 2 and 56 (D to F). Designators are provided for the middle ear (ME), epithelial layer (E), submucosa (SM), and bone (B). Effusion-mucosa separation artifact in B was corrected with computerized imaging software.
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inflammation. Later in the time course (after 3 weeks), the 2 groups are similar, reflecting the persistent changes in the chronic phase of otitis media with effusion. Effusion was not recovered from the contralateral control ears of any study animal. S pneumoniae was recovered from all ears with ETO in the I group (n = 11) on days 1 and 2 but not at any time later. That organism was not recovered at any time from the ears with ETO in the UI group or from the contralateral control ears. At all timepoints, gram-negative rods, α and β Streptococcus hemolyticus, Streptococcus pyogenes, Pseudomonas aeruginosa, and other bacteria grew on the chocolate agar plates swabbed with the wash fluids from the aspiration needle, but in the absence of concomitant S pneumoniae infection, none of the source ears showed any signs of acute infection, and the total cfu was very low in the UI group relative to the I group. Histologic study of the ears with ETO in the 2 groups showed early differences that disappeared between study days 7 and 21. For example, during the first week, the cell density within the effusion was greater for the ears with ETO in the I group than for those in the UI group, but by day 21 and subsequently, the 2 groups were indistinguishable for that variable (Fig 3). In both groups, the ME mucosa in ears with ETO progressively thickened through day 21 vis-a-vis the contralateral control ear with some resolution during the later timepoints (Fig 4A). Compared with the control sides, there was a slow but progressive formation of new bone under the ME mucosa in both groups with ETO, and this process continued throughout the period of follow-up, with the I group showing greater bony thickening at the end of study period (16 weeks) (Fig 4B). The epithelium initially developed a hyperplasia that was more extensive in the ears with ETO of the I group. In both groups, there was a progressive reduction in the mucosal area, characterized as a respiratory epithelium with an increase in cuboidal and squamous cell types, which reflected an increasing degree of metaplasia. The submucosa showed early vascular and lymphatic dilatations with inflammatory cell infiltration mainly during the first 21 days, and this was followed by fibroplasia and collagen deposition. In none of the specimens was there evidence
of giant cells, which are indicative of a foreign body reaction. These changes were not seen in the contralateral control ears (Fig 5). DISCUSSION There are many publications describing experimental models for the study of OME. For example, mucosal inflammation and effusion were observed after mechanical or functional block of the ET, after exposure of the ME to infectious or inflammatory agents (bacteria, virus, and inflammatory mediators), or after inducement of systemic inflammatory reactions. Also, a variety of animal species have been used, with the choice dictated by the specific etiology being modeled and by such factors as cost, ease of approach, physiology of the ET dilatory mechanism, anatomic similarities to the human ME, susceptibility to infection with pathogens, and the availability of specific analytical reagents. Indeed, there is no single ideal nonhuman species for mimicking disease pathogenesis by all suspected OME etiologies and for all model applications (eg, treatment, pathogenesis, etc). The animal model described in this report was developed for study of the temporal relationship between cytokine elaboration and mucosal inflammation in ears with OME caused by ETO with and without concurrent bacterial infection. Such a model would be useful for preclinical evaluation of the efficacy with respect to disease resolution of interventions that upregulate, downregulate, or otherwise modify the expression of specific cytokines elaborated during disease pathogenesis. Of the animal models commonly used for study of OME (including different experimental manipulations of the monkey, chinchilla, gerbil, or rat), the specific requirements of the proposed application are most compatible with the choice of the rat as the experimental species. For example, monkeys are discounted because they are resistant to infection with pathogens common to OME in humans, whereas chinchilla- and gerbil-specific reagents for assay of cytokine protein and mRNA are not commercially available.16 On the other hand, rats can be infected with human pathogens, surgical techniques have been described for blocking the rat ET, and assay reagents are readily available.8-11,17 Also, the general anatomy of the rat ME and ET is similar to
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that of humans, and the ME mucosa of the rat and human has similar histologic features with respect to cell type and ciliary clearance tracts.18-21 Reported limitations to the use of the rat in modeling OME include the reported high frequency of natural infection of the ME with bacteria, which may confound an ETO model that does not include infection; the small size of the ME, which significantly limits the volumes of effusion and mucosa that can be collected from a single animal; and the difficulty of the surgical approach to block the ET.11,12,18 The actual incidence of natural ME infections among rats is not known, and the use of pathogenfree animals should significantly reduce this background frequency. In this large sample, no rat had evidence of naturally occurring ME disease. In previous studies, the small volume of recovered effusion required dilution before biochemical assay, and that for the mucosa was accommodated by pooling samples across ears.14,17 Although these procedures limit the threshold sensitivity for biochemical assays and increase the number of animals required to achieve a designated sample size for the mucosal analyses, these drawbacks can be managed. Finally, surgical procedures to access the bony ET using a ventral neck incision and careful dissection of the area between specific muscles, nerves and blood vessels were previously described.8-10 Although they are difficult and require approximately 30 minutes, those procedures and the modifications described here (substitution of gutta percha for dental wax and a vascular clip) have a low mortality rate (9% in this study) and a high success rate (94%) and reliably produce a persistent OME that lasts for >6 months. Finally, the intersample consistency reported here for the otomicroscopic, microbiologic, and histologic observations within the different groups shows that this model is useful for studying disease evolution along the OME continuum. Obstruction and changes in the ET were localized to tissue in the immediate region, with no histologic evidence of inflammation at the nasopharyngeal or tympanic orifices of the ET. Thus, it is unlikely that the effusion and observed mucosal changes were caused by a foreign body reaction to the dental material or by inflammation
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secondary to infection. Of some concern was the recovery of nonpathogenic bacteria from all MEs with ETO. Although not clearly associated with signs of infection or inflammation, this will be studied in the future to determine if those bacteria are also recovered from the contralateral control MEs. The S pneumoniae culture results were consistent with the expectations that the organism would be recovered only from ears challenged with the bacteria and only at the early time points. That S pneumoniae was not recovered on or after day 7 suggests that the antimicrobial therapy instituted at 48 hours effectively sterilized the ME cleft. The histopathologic changes observed in response to ETO and infection reproduced the descriptions provided in earlier studies using similar models.7,13 In general, ETO and concomitant S pneumoniae infection elicited a more robust inflammatory response than ETO alone. However, after eradication of the pathogen with antibiotics, by day 21 the disease course during the chronic phase was quite similar in both groups. These results suggest that the chronic mucosal changes documented in the ears of these animals result from the persistent ETO and that reversal of that condition should be one goal of therapeutic intervention. The investigators thank Chia-Yee Lo, MS, and Julianne Banks, BS, for their able assistance with the microbiologic, surgical, and technical aspects of this study. Consultation for histopathologic analysis was graciously provided by Edwin C. Klein, VMD, veterinarian and veterinary pathologist at the University of Pittsburgh.
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presence of experimental effusion. Acta Otolaryngol 1971;72:206-14. 14. Hebda PA, Alper CM, Doyle WJ, et al. Upregulation of messenger RNA for inflammatory cytokines in middle ear mucosa in a rat model of acute otitis media. Ann Otol Rhinol Laryngol 1998;107:501-7. 15. Hebda PA, Piltcher OB, Swarts JD, et al. Cytokine profiles in a rat model of otitis media with effusion caused by eustachian tube obstruction with and without S.penumoniae infection. Laryngoscope 2002 (in press). 16. Doyle WJ. Animal models of otitis media: other pathogens. Pediatr Infect Dis J 1989;8:S45-S48. 17. Zeevi A, Alper CM, Balaban CD, et al. Early inflammatory events in a rat model of OM caused by infection with S pneumoniae. Proceedings of the Sixth International Symposium on Recent Advances in Otitis Media, Ft Lauderdale, FL; 1995. p. 258-61. 18. Hal DJ III, Fulghum RS, Brinn JE, et al. Comparative anatomy of eustachian tube and middle ear cavity in animal models for otitis media. Ann Otol Rhinol Laryngol 1982;91:82-9. 19. Hellstrom S, Salen B, Stenfors LE. Anatomy of the rat middle ear. A study under the dissection microscope. Acta Anat 1982;112:346-52. 20. Albiin N, Hellstrom S, Salen B, et al. The anatomy of the eustachian tube in the rat. Anat Rec 1983;207:51321. 21. Albiin N, Hellstrom S, Stenfors LE, et al. Middle ear mucosa in rats and humans. Ann Otol Rhinol Laryngol 1986;(suppl 126)95:1-15.