Free radical production by antibiotic-killed bacteria in the guinea pig middle ear

OBJECTIVES: Oxygen free radicals are implicated in the pathogenesis of otitis media Recent investigations with animal models have demonstrated that free radical-mediated damage of the middle ear mucosa, measured as lipid hydroperoxide, occurs when the middle ear cavity is inoculated with Streptococcus pneumoniae. The present study was conducted to examine the effect of antibiotics on free radical-mediated damage in pneumococcal acute otitis media. STUDY DESIGN: Animal model of acute otitis media. METHODS: Seventy-eight guinea pigs underwent bilateral middle ear inoculation with 100 microl of 1) sterile saline as a control, 2) 50 microg/mL amoxicillin, 3) 10(7) colony forming units (CFU)/mL Streptococcus pneumoniae killed with 50 microg/mL amoxicillin, or 4) 10(7) CFU/mL S. pneumoniae. Animals were killed on postoperative day 1 or 5, and the middle ear mucosa was examined for lipid peroxidation as evidence of free radical damage. RESULTS: Mucosal lipid hydroperoxide was significantly elevated compared with control subjects on day 1 in both the antibiotic-killed S. pneumoniae group and the S. pneumoniae-infected group. On day 5, the S. pneumoniae-infected mucosa had significantly higher lipid hydroperoxide levels compared with the antibiotic-killed group and the control subjects. Histological studies confirmed mucosal edema and the presence of inflammatory cells in the infected groups. CONCLUSIONS: Antibiotic-killed bacteria seem to produce free radical-mediated damage to the middle ear mucosa in the early phase of acute otitis media. The clinical implication of this study is that free radical damage to the middle ear mucosa may occur in otitis media despite appropriate antibiotic therapy.     

Oral vaccine therapy for pneumococcal otitis media in an animal model

We investigated whether mucosal IgA response in the middle ear cavity against Streptococcus pneumoniae type 19F is enhanced by use of enteric capsules, and whether the resulting mucosal immunity can prevent pneumococcal otitis media. Adult Hartley guinea pigs were employed. With intratympanic inoculation of 10(5) and 10(6) live S pneumoniae, the occurrence of pneumococcal otitis media significantly decreased in guinea pigs that received intraduodenal and intragastric immunization by enteric capsules. In these guinea pigs, the values of salivary IgA antibody titers against S pneumoniae were significantly greater, and histologic changes of the middle ear mucosa were also slighter than those of control guinea pigs. These findings indicate that oral vaccination by enteric capsules elicits mucosal IgA responses, as well as intraduodenal immunization, to prevent pneumococcal otitis media. These results suggest the possibility of clinical application of oral vaccination by enteric capsules for the prevention of middle ear infection.     

Role of interleukin-1beta in a murine model of otitis media with effusion.

To clarify the role of interleukin-1beta (IL-1beta) in the pathogenesis of otitis media with effusion (OME), we developed and investigated a murine model of this disease. Specific pathogen-free male BALB/c mice received intratympanic injections of 20 microg of endotoxin derived from nontypeable Haemophilus influenzae. Three days after injection, middle ear effusions were observed through the eardrum. Similar pathological changes were observed after inoculation with 100 ng of recombinant IL-1beta. Anti-IL-1 receptor antibodies inhibited the pathological changes induced by the endotoxin. In situ hybridization showed expression of IL-1beta messenger RNA in the epithelium of the middle ear mucosa. These results suggest that IL-1beta might be associated with endotoxin-induced inflammation in the middle ear and might play an important role in the induction of otitis media with effusion.     

First forty-eight hours of developing otitis media: an experimental study

The early inflammatory changes in the tympanic membrane were explored in 2 rat models. Acute otitis media was induced by instillation of Streptococcus pneumoniae type 3 into the middle ear cavity, and otitis media with effusion was induced by blockage of the eustachian tube. Otomicroscopic examination was performed before the rats were painlessly sacrificed at 3, 6, 9, 12, 18, 24, or 48 hours after initiation of the otitis media conditions. The tympanic membrane was studied by light and electron microscopy. Both acute otitis media and otitis media with effusion caused early inflammatory changes of the tympanic membrane, and the pars flaccida was the portion that reacted first. The inflammatory alterations were most pronounced in the acute otitis media model. The course of inflammation showed a bimodal pattern with an early deposition of a filamentous material with a band pattern, typical of fibrin. Despite a fluid-filled middle ear cavity, the inflammatory changes in the otitis media with effusion model were moderate, as was consistent with the clinical appearance of the tympanic membrane.     

Role of phagocytes in antimicrobial defence of the middle ear.

The role of phagocytes in the antimicrobial defence of the middle ear was investigated in this experiment, using Hartley strain guinea pigs with an experimental otitis media. Otitis media was induced with an inoculation of Streptococcus pneumoniae into the tympanic cavity through the ear drum. For depletion of peripheral blood phagocyte population such as monocytes and polymorphonuclear neutrophils (PMNs), whole body irradiation (250 rad or 500 rad) was carried out on guinea pigs three days before S. pneumoniae inoculation into the middle ears. Carrageenan was also used for selective depletion of mononuclear cells, to distinguish their role from polymorphonuclear neutrophils. In control animals, otitis media was induced reproducibly with middle ear inoculation of more than 10(6) S. pneumoniae. In irradiated animals, which underwent 10(2) or 10(4) S. pneumoniae inoculation, the incidence of otitis media because of S. pneumoniae infection became higher in accordance with the dosage of irradiation. However, no significant difference was seen in the occurrence of otitis media and the number of viable bacteria recovered from bulla washings between controls and carrageenan-treated animals. These results suggest that phagocytes, particularly neutrophils, are essential for antimicrobial defense at the early phase of the middle ear infection with S. pneumoniae.     

Effects of active immunization with a pneumococcal surface protein (PspA) and of locally applied antibodies in experimental otitis media.

An experimental rat model for Streptococcus pneumoniae otitis media was used to investigate passive protection by anti-type 3 capsular antibodies and effects of immunization with pneumococcal surface protein A (PspA). Anti-type 3 antibodies instilled into the middle ear reduced purulent otitis media as compared to control animals (p = 0.015). Secondly, after immunization with PspA, the right middle ear was inoculated with S. pneumoniae type 6A in a dose calibrated to induce purulent otitis media. There was an anti-PspA antibody response in all rats immunized and a reduction in signs of purulent otitis media as compared to control animals (p = 0.026). Thus, purulent (acute) otitis media can be reduced by local application of antibodies in the middle ear and also by immunization with a non-type-specific pneumococcal protein, PspA.     

Carcinoembryonic antigen in chronic otitis media: an immunohistochemical study of the middle ear mucosa

Carcinoembryonic antigen (CEA) has an important role as a tumor marker and also as a cell adhesion molecule. The expression and the role of CEA in chronic inflammation of middle ear mucosa has not been studied previously. Immunohistochemical analysis with monoclonal antibodies against human CEA was performed in order to detect CEA in formalin fixed and paraffin embedded biopsy specimens of middle ear mucosa in chronic otitis media. In this study 118 specimens of middle ear mucosa from different regions of the middle ear cleft were analysed. All specimens were taken during surgery of otitis media with and without cholesteatoma. The results showed that CEA expression was present in mucosal samples in chronic otitis media with cholesteatoma, predominantly in squamous epithelium, as well as in the subepithelial layer and among the connective tissue, especially on collagen fibres of fibroproliferative stroma. CEA was seen on endothelial cells of blood vessels. Occasionally it was present on the surface of columnar epithelium of the middle ear mucosa in chronic otitis media without cholesteatoma. The expression patterns of CEA in chronic inflammation of middle ear mucosa suggest potential functional activities similar to adhesion molecules and signal regulatory proteins which will be the subject of further study.     

Nitric oxide contributes to control of effusion in experimental otitis media

OBJECTIVES/HYPOTHESIS: Nitric oxide (NO) is a small, short-lived free radical involved in cellular signaling and known to play a role in inflammation. It is generated on demand by the enzyme nitric oxide synthase (NOS) on arginine. We have previously found that mRNA encoding NOS is produced in the middle ear during otitis media. The role of NO was therefore explored in an experimental model of immune-mediated otitis media. STUDY DESIGN AND METHODS: Guinea pigs were systemically immunized and later challenged in the middle ear with the same antigen. One ear of each animal was challenged with antigen alone. In the opposite ear, antigen was combined with a potent inhibitor of NOS, N(G)-amino-L-arginine (L-NAA). After survival for 24, 48, or 72 hours, the middle ears were evaluated for otitis media. RESULTS: Inhibition of NOS resulted in significantly increased middle ear effusion at all three time periods. This increase was blocked by the addition of excess 1-arginine, which bypasses the inhibitory effects of L-NAA. The infiltration of cells into the middle ear lumen and the hyperplasia of the middle ear mucosa were unaffected by L-NAA administration. CONCLUSIONS: The results suggest that NO is involved in regulating the permeability of the middle ear vascular, the transudation of serum into the middle ear mucosa, and/or the movement of extracellular fluid across the middle ear mucosal epithelium.     

Induction of mucous cell metaplasia in the middle ear of rats using a three-step method: an improved model for otitis media with mucoid effusion

Otitis media with mucoid effusion, characterized by mucous cell metaplasia in the middle ear cleft and thick fluid accumulation in the middle ear cavity, is a common otological disease that frequently affects young children. Multiple factors are involved in the development of this disease, especially middle ear infection and Eustachian tube dysfunction. In this study, in order to induce otitis media with effusion in rats, we introduced a three-step method, namely inoculation of Streptococcus pneumoniae at 10(7) colony-forming units (CFU)/ear or Haemophilus influenzae at 5 x 10(7) CFU/ear into the middle ear cavity twice at 2-week intervals, followed by Eustachian tube obstruction (ETO) for 4 and 8 weeks. Animals inoculated with phosphate-buffered saline (PBS) twice in the same manner followed by ETO served as controls. Middle ear effusion and mucosa were harvested for evaluation of carbohydrate concentrations and mucous cell density, respectively. We found that rats inoculated with S. pneumoniae twice, followed by ETO at 8 weeks, yielded the highest carbohydrate concentration in middle ear effusion and the highest goblet cell density in the middle ear cavity compared to the H. influenzae and PBS groups. It is tentatively concluded that inoculation of S. pneumoniae at 10(7) CFU/ear into the middle ear cavity of rats twice at 2-week intervals, followed by ETO for 8 weeks, is a promising animal model for otitis media with mucoid effusion which may be valuable for studying the human counterpart.     

Pathophysiology of Streptococcus pneumoniae otitis media: kinetics of the middle ear biochemical and cytologic host responses.

Streptococcus pneumoniae is an important bacterial pathogen in the pathophysiology of otitis media. To elucidate the inflammatory responses that occur during pneumococcal otitis media, the kinetics of the biochemical and cytologic middle ear responses to heat-killed encapsulated and nonencapsulated pneumococci were studied in the chinchilla model. Inoculation of the middle ear cavity with at least 10(6) S pneumoniae cells induced an early, brief vascular response with leakage of small (albumin) followed by larger (alpha 2-macroglobulin) proteins, followed by sustained influx of acute inflammatory cells and lysozyme. The threshold for a sustained lysozyme response was 1,000 times lower for nonencapsulated than for encapsulated pneumococci. These results indicate that nonviable S pneumoniae organisms with an intact envelope initiate the middle ear inflammatory response. Therefore, interventions that enhance the clearance of pneumococcal cells from the middle ear may reduce the inflammatory response and prevent chronic middle ear inflammation.     

中耳细菌感染诱导的急性期HSP-70相关反应的免疫组织化学研究

探讨中耳细菌感染急性期时 ,哺乳动物中、内耳热休克反应的部位 ,以及中耳细菌感染诱生的热休克蛋白是否可能引发内耳自身免疫损伤。运用中耳注射肺炎克雷伯杆菌制成豚鼠中耳急性感染动物模型 ,分别于接种后第 1、3、5、7天处死动物取材。应用免疫组化技术 ,研究了中耳粘膜和耳蜗表达热休克蛋白 70 (HSP- 70 )的部位。结果表明 :正常状态下 ,中耳粘膜表层的上皮细胞和内耳膜迷路血管纹、螺旋韧带、Corti氏器均有弱的阳性反应 ,感染应激后 ,上述同样部位均有强的阳性显色。不同取材时段显示的阳性位置无差异。说明在中耳急性细菌感染期 ,中耳粘膜和内耳组织均表达了同源 HSP- 70蛋白分子 ,这些同源 HSP- 70为引发内耳自身免疫损伤提供了物质基础。     

Course of IL-1beta, IL-6, IL-8, and TNF-alpha in the middle ear fluid of the guinea pig otitis media model induced by nonviable Haemophilus influenzae.

To characterize the local response in acute otitis media, courses of interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF)alpha in middle ear fluid (MEF) of the guinea pig otitis media model induced by nonviable Haemophilus influenzae were investigated with enzyme-linked immunosorbent assay (ELISA) kits. The IL-1beta concentration in H. influenzae-inoculated ears peaked 24 hours after inoculation. The IL-8 concentration was significantly higher in H. influenzae-inoculated ears than in controls 48 and 96 hours after inoculation. The TNF-alpha concentration in H. influenzae-inoculated ears had an initial peak 6 hours after inoculation and had significant late increases 48 and 96 hours after inoculation. The results suggest that IL-1beta and TNF-alpha were produced by middle ear mucosa in the early stage of the experiment by stimulation of bacterial inoculation, which caused subsequent inflammatory cell accumulation, and that IL-8 and TNF-alpha were produced in the late stage by accumulating inflammatory cells.     

Effect of 10 pharmacologic probes on mRNA levels of inducible nitric oxide synthetase and selected inflammatory cytokines in a rat model of acute otitis media

Ten drugs were screened for their ability to decrease inflammatory mediator (IL-6, inducible nitric oxide synthetase [iNOS], IL-1beta and monocyte chemotactic protein [MCP-1]) expression in a rat model of acute otitis media caused by Streptococcus pneumoniae. Six adult rats were randomly assigned to each of 12 groups corresponding to uninfected controls and treatments with saline, aminoguanidine, anisomycin, dexamethasone, ketorolac, L-N(G)-nitroarginine methylester, methylprednisolone, mycophenolic acid, pentoxiphylline, tacrolimus or WEB2086. Forty-eight h after the start of treatment, the ears of the animals in the 11 treatment groups were challenged with S. pneumoniae. Forty-eight h later, all animals were killed and middle ear mucosa was harvested and assayed for RNA message. Messages for IL-6, iNOS and MCP-1 were significantly increased as a result of infection. Most treatments decreased MCP-1 and four decreased IL-6 and iNOS. Tacrolimus and dexamethasone decreased IL-6, iNOS and MCP-1. These results show that pharmacological agents can modify the expression of inflammatory mediators in this model and may have clinically relevant effects.     

Inadequate opening capacity of the eustachian tube in Meniere's disease

Ten drugs were screened for their ability to decrease inflammatory mediator (IL-6, inducible nitric oxide synthetase [iNOS], IL-1 &;#103 and monocyte chemotactic protein [MCP-1]) expression in a rat model of acute otitis media caused by Streptococcus pneumoniae. Six adult rats were randomly assigned to each of 12 groups corresponding to uninfected controls and treatments with saline, aminoguanidine, anisomycin, dexamethasone, ketorolac, L-N G -nitroarginine methylester, methylprednisolone, mycophenolic acid, pentoxiphylline, tacrolimus or WEB2086. Forty-eight h after the start of treatment, the ears of the animals in the 11 treatment groups were challenged with S. pneumoniae. Forty-eight h later, all animals were killed and middle ear mucosa was harvested and assayed for RNA message. Messages for IL-6, iNOS and MCP-1 were significantly increased as a result of infection. Most treatments decreased MCP-1 and four decreased IL-6 and iNOS. Tacrolimus and dexamethasone decreased IL-6, iNOS and MCP-1. These results show that pharmacological agents can modify the expression of inflammatory mediators in this model and may have clinically relevant effects.