Experimental Study on Cochlear Damage Induced by an Endotoxin-like Substance

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1 Otol Jpn 1 (1): 22-27, 1991 Experimental Study on Cochlear Damage Induced by an Endotoxin-like Substance Takehiko Harada, Tatsuya Yamasoba, Masashi Sugasawa Department Otolaryngology, University Tokyo School Medicine The pathogenesis sensorineural hearing loss which accompanies various forms otitis media is attracting increasing interest. Endotoxin frequently found in middle ear effusion is considered to be one substances which might penetrate round window membrane from middle ear exhibit ototoxicity in inner ear. In this paper, possible pathogenesis sensorineural hearing loss by a similar process was investigated by using endotoxin-like substance (lipoteichoic acid from Staphylococcus aureus). In Group A, middle ear bulla 31 guinea pigs was opened 5 mg lipoteichoic acid was placed on round window membrane. In 12 guinea pigs Group B, a very small amount lipoteichoic acid solution (10 mg/ml) was injected into scala tympani through round window membrane. After various time intervals, animals were sacrificed, temporal bones removed, histologic sections prepared. The cochlea was examined under a light microscope for possible pathology. No definite pathology was observed eir in Group A or in control group. In 5 out 12 guinea pigs Group B, severe cochlear pathology was observed. The pathology consisted various combinations fibrosis in scala tympani scala vestibuli, atrophy cystic stria vascularis, cyst-like formation Reissner's membrane, organ Corti with loss hair cells, disappearance tectorial membrane, inflammatory cell infiltration endolymphatic space. The results study did not prove that an endotoxin-like substance in middle ear could cause cochlear damage, but y suggest possibility that, once introduced into inner ear, it could potentially cause cochlear pathology. Key words: Guinea pig, Lipoteichoic acid, Endotoxin, Cochlea, Round window membrane Introduction The mechanism by which this sensorineural hearing loss develops is not known, but it has been Sensorineural hearing loss sometimes accompanies chronic otitis media. Chronic suppurative otitis media has long been known to produce sensorineural hearing loss 1) `5), but recently otitis media with effusion has been shown to have same effect. Arnold et al 6) reported that significant sensorineural hearing loss was observed in about 20% children with otitis media with effusion. Aviel Ostfeld 7) reported on 3 patients suffering from otitis media with effusion in whom irreversible sensorineural hearing loss developed. variously hyposized. One hyposes is that sensorineural hearing loss may be result infiltration infectious process from middle ear into inner ear through round window membrane. 4), 7) The presence inflammatory elements in chronic otits media raises possibility that sensorineural hearing loss is result penetration microorganism or its toxins through round window membrane into inner ear. In support this hyposis is fact that endotoxin is frequently found in middle ear

2 Harada, et al. Cochlear Damage by Endotoxin-like Substance 23 effusion children suffering from otitis media with effusion 8) 1), that endotoxin in middle ear can pass through round window membrane enter inner ear." The purpose this study is to investigate experimentally wher same hyposis could be applied to a similar bacterial product. For this purpose, an endotoxin-like substance was eir placed on round window membrane or injected into inner ear guinea pig, which was n examined for any cochlear pathology induced by this substance. in solution 2% glutaraldehyde 2%paraformaldehyde. Some bones were postfixed in 1% osmium tetroxide without decalcification, dehydrated in alcohol, embedded in Epon 812. They were n cut into blocks representing each cochlear turn, sections 1ƒÊm thick were cut from each block. The rest bones were decalcified in 10%EDTA, postfixed in 1% osmium tetroxide, dehydrated in alcohol, embedded in resin. Relatively large sections 2ƒÊm thick were cut at planes which included modiolus /or round window membrane. The sections were n mounted on glass slides stained with toluidine Materials methods blue, cochlear pathology was examined under a light microscope. The experiments were conducted on 43 healthy young guinea pigs (body weight g), lipoteichoic acid extracted from Staphylococcus aureus (Sigma Chemical Company, St. Louis, Mo) was selected as endotoxin-like substance. The guinea pigs were divided into two groups. In both groups, under general anessia with sodium pentobarbital administered intraperitoneally, a postauricular incision was made, middle ear bulla was opened so as to expose round window membrane directly. In Group A, which consisted 31 guinea pigs, 5 mg lipoteichoic acid was administered by absorbing substance with a small piece Gelfoam (R) soaked in distilled water applying it directly on surface round window membrane. In Group B, which consisted 12 guinea pigs, a very small amount lipoteichoic acid dissolved in distilled water (10 mg /ml) was injected directly into scala tympani through round window membrane by a 27 gauge needle. After injection, round window membrane was immediately covered with a small piece Gelfoam (R) soaked in same solution. In both groups, bulla was closed by suturing skin incision. After various intervals time (Group A: 1, 3, The control study was conducted on 14 guinea pigs. In control study for Group A, a small piece Gelfoam (R) was soaked in normal saline placed on round window membrane 8 guinea pigs. In control study for Group B, a very small amount normal saline was injected directly into scala tympani 6 guinea pigs. After various time intervals (Control Group A: 1 5 days; Control Group B: 7 14 days after experiments), guinea pigs were sacrificed temporal bones were removed immediately. They were n processed examined by methods described above. Results The results for Group A are summarized in Table 1. The intervals between experiments sacrifice varied from 1 day to 30 days, but compared with findings control group, no definite cochlear pathology was observed in this group. The results for Group B are summarized in Table 2. Table 1 Results for Group A, in which lipoteichoic acid was placed on round window membrane 7, 14, 30 days; Group B: 7 days after experiments), guinea pigs were sacrificed, temporal bones were removed fixed immediately

3 24 Table In 2 5 Results for Group injected into scala 12 pathology were guinea was observed control The B, in which pigs, severe, observed, in lipoteichoic acid was tympani definite while no remaining cochlear such 7 guinea changes pigs or in group. pathologic changes in se cochleas endolymphatic characteristically observed in perilymphatic spaces, gy was generally in lower more turns, severe both observed were in Fig. 1. Fig. Enlargement severe patholo- in upper turns as shown Fig. 2 changes mphatic apical both turn in Fig. 1, showing endolymphatic perily- spaces. than 2 is Fig. 3 Cystic loss hair #3 cells stria tectorial vascularis along membrane. (Guinea with pig in Table 3. basal turn) Fig. 1 Pathologic changes observed in cochlea a guinea pig. The pathology is most severe in apical turn. (Guinea pig#1 in Table 3) enlargement apical turn same cochlea, demonstrates shows fibrosis most in most scala part scala vestibuli. spiral ligament, degenerated In remaining stria specimen severe tympani stria for This cystic is most The in vascularis part. cystic prominent organ has most part stria vascularis, vascularis It The fibrosis is invading disappeared is noted. (Fig. 3). pathology. Fig. 4 Cyst-like Corti, formation scala Reissner' s membrane. vestibuli, disappearance are also noted.(guinea pig#3 tectorial in Table Fibrosis organ membrane 3, third turn) in anor Corti has eir completely disappeared (Fig. 2) with loss hair cells (Fig. 3). or degenerated The tectorial memb-

4 Harada, et al. Cochlear Damage by Endotoxin-like Substance 25 Discussion To our knowledge, re are two reports which describe inner ear disturbances induced by endotoxins which apparently penetrated round window membrane from middle ear into inner ear. Kawaguchi et al.12) instilled lipopolysaccaride extracted from Salmonella typhimurium into middle ear chinchilla observed marked Fig. 5 Inflammatory cell infiltration into endolymphatic space. Slight fibrosis in scala vestibuli organ Corti are also noted, along with disappearance tectorial membrane. (Guinea pig #5 in Table 3, basal turn) rane is missing. Fig. 4 is from still anor specimen, shows fibrosis in scala vestibuli invading spiral ligament. The stria vascularis tectorial membrane are missing, organ Corti shows loss hair cells. Here, prominent feature is a cyst-like formation Reissner's membrane. In Fig. 5, in addition to fibrosis in scala vestibuli, atrophic stria vascularis, loss hair cells, missing tectorial membrane, outsting feature is inflammatory cell infiltration into endolymphatic space. The observed pathology is summarized in Table 3. Table 3 Summary pathologic findings pathologic change inner ear. Spow et al.13) inoculated lipopolysaccharide from E. coli on round window niche rat demonstrated dysfunction inner ear by frequency-specific ABR technique, but failed to detect any morphological change inner ear. These investigations imply that lipopolysaccharides bacteria can pass through round window membrane enter inner ear, reby demonstrating ir ototoxicity eir electrophysiologically or morphologically. A lipopolysaccharide is endotoxin extracted from cell wall gram-negative bacteria, acts as inducer inflammation modulator immune response. While effects endotoxin on middle ear mucosa have been fairly well studied, little is known about its effect on inner ear. In above-cited studies using endotoxins, somewhat contradictory results were reported, this may be because difference in lipopolysaccharides used in se two studies. In view fact that se lipopolysaccharides are from bacteria which do not exist in middle ear under ordinary conditions, we found it necessary to repeat same types experiment using endotoxin or an endotoxinlike substance which can be found in middle ear. When culture probes are taken from middle ear effusions, most frequently isolated bacteria are Streptococcus pneumoniae Hemophilus influenzae.14) In this experiment, however, we used lipoteichoic acid extracted from Staphylococcus aureus since this was easily available on a commercial basis. Lipoteichoic acid is a component cell wall gram-positive bacteria provides wall's antigenic specificity. Thus, we considered lipoteichoic acid as endotoxin-like substance.

5 26 Considering that endotoxin can pass through round window membrane11) `13), it might be assumed that lipoteichoic acid behaves similarly enters inner ear from middle ear. In this study, however, no cochlear pathology was observed In order to elucidate mechanism, furr investigations in pursuit this kind hyposis are necessary along with those aimed at possibility or explanations, such as disturbance oxygen supply from middle ear to inner ear through round window membrane.6),5) in animals Group A, in which lipoteichoic acid was placed directly on round window membrane. Thus, our study failed to prove hyposis that an endotoxin-like substance in middle ear might express its ototoxicity after entering inner ear through round window membrane. In some animals Group B, on or h, severe cochlear pathology was observed after lipoteichoic acid was injected directly into scala tympani. Since no change occurred in animals control group, observed pathology is considered to be due to direct activity lipoteichoic acid in inner ear. The pathology may be summarized as inflammatory reaction inner ear, but it is somewhat different from that reported by Kawaguchi et al.12) Although y observed changes in both endolymphatic perilymphatic spaces, as in our study, our pathology In experiments is much more severe. with Group B, problem might be that this type invasive experiment is not physiological, or that concentration lipoteichoic acid in this condition will be extremely high. There may also be such questions as why severe changes occurred only in some animals while ors were spared pathology, why in some animals fibrosis was more severe in scala vestibuli while lipoteichoic or why pathology acid was injected into scala tympani, was more severe in upper turns. For moment, we don't have answers to se problems questions. Neverless, results may be interpreted as suggesting possibility that an endotoxin-like substance, once introduced into inner ear, could potentially cause cochlear pathology. We consider that our study would show no more than one possibilities to interpret Mechanism by which sensorineural hearing loss occurrs during course various types otitis media. References 1) Paparella MM, Brady DR, Hoel R: Sensorineural hearing loss in chronic otitis media mastoiditis. Trans Am Acad Ophthalmol Otolaryngol 74: , ) English GM, Norrn JL, Fria TJ: Chronic otitis media as a cause sensorineural hearing loss. Arch Otolaryngol 98: 18-22, ) Prado 5, Paparella MM: Sensorineural hearing loss secondary to bacterial infection. Otolaryngol Clin North Am 11: 35-41, ) Paparella MM, Goycoolea MV, Meyerhf WL: Inner ear pathology otitis media. A review. Ann Otol Rhinol Laryngol 89, Suppl 68: , ) Walby AP, Barrera A, Schuknecht HF: Cochlear pathology in chronic suppurative otitis media. Ann Otol Rhinol Laryngol 92, Suppl 103: 3-19, ) Arnold W, Ganzer U, Kleinmann H: Sensorineural hearing loss in mucous otitis. Arch Oto- Rhino-Laryngol 215: 91-93, ) Aviel A, Ostfeld E: Acquired irreversible sensorineural hearing loss associated with otitis media with effusion. Am J Otolaryngol 3: , ) Bernstein JM, Praino MD, Neter E: Detection endotoxin in ear specimens from patients with chronic otitis media by means Limulus amebocyte lysate test. Can J Microbiol 26: , ) DeMaria TF, Prior RB, Briggs BR, et al: Endotoxin in middle-ear effusions from patients with chronic otitis media with effusion. J Clin Microbiol 20: 15-17, ) Eno Y, Kaneko Y, Takasaka T: Endotoxin in middle ear effusions tested with Limulus assay.

6 Harada, et al. Cochlear Damage by Endotoxin-like Substance 27 Acta Otolaryngol 100: 42-50, ) Juhn SK, Hamaguchi Y, Goycoolea M: Review round window membrane permeability. Acta Otolaryngol, Suppl 457: 43-48, ) Kawaguchi H, DeMaria TF, Lim DJ: Endotoxin permeability through round window. Acta Otolaryngol, Suppl 457: , ) Spow O, Anniko M, Hellstrom S: Inner ear disturbances following inoculation endotoxin into middle ear. Acta Otolaryngol 107: 90-96, ) Liu YS, Lang R, Lim DJ, et al: Microorganisms in chronic otitis media with effusion. Ann Otol Rhinol Laryngol 85, Suppl 25: , ) Yamashita T, Sasa H, Okazaki N, et al: A cause sensorineural hearing loss in otitis media with effusion. ANL 12, Suppl I: S40-S42, Send reprint request to: Takehiko Harada, MD. Department Otolaryngology University Tokyo School Medicine Hongo 7-3-1, Bunkyo-ku, Tokyo 113, JAPAN