Otology & Neurotology 35:e178Ye186 Ó 2014, Otology & Neurotology, Inc.
Bacterial Invasion of the Inner Ear in Association With Pneumococcal Meningitis *Martin Nue MLller, †Christian Brandt, ‡Christian Kstergaard, and *§Per Caye-Thomasen *Department of Oto-Rhino-Laryngology, Head and Neck Surgery, University Hospital, Rigshospitalet/ Gentofte, Copenhagen; ÞDepartment of Infectious Diseases, Copenhagen University Hospital Hvidovre; þDepartment of Clinical Microbiology, Copenhagen University Hospital Hvidovre, Hvidovre; and §The Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen Denmark
Objective: To examine the pathways of bacterial invasion and subsequent spreading in the inner ear during pneumococcal meningitis. Study Design: A well-established adult rat model of Streptococcus pneumoniae meningitis was used. Methods: Thirty rats were inoculated intrathecally with S. pneumoniae serotype 1, 3 or 9 V and received no additional treatment. The rats were sacrificed when reaching terminal illness or on Day 7 and then prepared for serial sectioning and PAS-Alcian blue staining for light microscopy. Results: During the first few days after inoculation, bacteria invade the inner ear through the cochlear aqueduct, into the scala tympani of the cochlea (perilymphatic space). From here, bacteria spreads apically toward the helicotrema and subsequently basally through the scala vestibuli, toward the vestibule and the vestibular system. When the bacteria after 5 to 6 days had reached scala vestibuli of the basal turn of the cochlea, hematogenous spreading occurred to
the spiral ligament and into the cochlear endolymph, subsequently to the vestibular endolymph. We found no evidence of alternative routes for bacterial invasion in the inner ear. Several internal barriers to bacterial spreading were found within the inner ear. Bacterial elimination was evidenced by engulfment by macrophages within the inner ear. Conclusion: From the meninges, pneumococci invade the inner ear through the cochlear aqueduct during the first days of infection, whereas hematogenous invasion via the spiral ligament capillary bed occur at later stages. Although internal barriers exist within the inner ear, the spreading of bacteria occurs via the natural pathways of the fluid compartments. Bacterial elimination occurs by local macrophage engulfment. Key Words: Bacterial meningitisVInner earVLabyrinthitisVStreptococcus pneumoniaeVTemporal bone pathology. Otol Neurotol 35:e178Ye186, 2014.
The most common long-term sequela after pneumococcal meningitis is inner ear dysfunction.Thus, sensorineural hearing loss affects up to 54% of survivors (1Y3) and is often accompanied by vestibular dysfunction, leading to concurring vertigo and/or balance problems, which affects up to 15% (2,4,5). The cause of postmeningitic inner ear dysfunction is supposed to be bacterial toxins and/or inflammation (1,6Y9). Inner ear filtration of inflammatory cells seems in itself to be a major cause of meningitis-associated hearing loss, as it induces damage to the organ of Corti and spiral ganglion, conceivably through production of cytotoxic mediators (1,6,7,10Y12). This is substantiated by an increase of cochlear damage and hearing loss when the inflammatory response is boosted (11) and conversely by a reduction
in hearing loss by anti-inflammatory therapy with corticosteroids (13). As direct evidence of bacterial invasion of the inner ear during meningitis is lacking, it is unclear whether inner ear inflammation occurs secondary to direct invasion of bacteria from the meninges or the bloodstream or as a bystander phenomenon secondary to adjacent meningeal inflammation. During meningitis, inflammatory cell infiltration of the inner ear occurs from the meninges to the perilymphatic space of the cochlea through the cochlear aqueduct or the internal auditory canal, the route seemingly depending somewhat on species (6Y10). Subsequently, the endolymphatic compartment is infiltrated by diapedesis of inflammatory cells from the vascular bed of the spiral ligament in the cochlea (9). Assuming that this process is preceded by inner ear invasion of bacteria, the routes and temporary pattern are likely to mirror this pattern. This report presents direct evidence of bacterial invasion of the inner ear during meningitis. Using a well-established experimental model of pneumococcal meningitis, the routes
Address correspondence and reprint requests to Martin Nue MLller, M.D., Department of Oto-Rhino-Laryngology, Head and Neck Surgery, University Hospital, Rigshospitalet/Gentofte, 2100 Copenhagen, Denmark; E-mail: [email protected] Disclosure: The authors report no conflicts of interest.
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INNER EAR AND BACTERIAL MENINGITIS of bacterial invasion and the pathways and temporal pattern of bacterial spreading and elimination within the inner ear are documented. MATERIALS AND METHODS The experimental protocol was approved by the Danish Animal Inspectorate (Dyreforsoegstilsynet) and based on a well-established model of pneumococcal meningitis (14,15).
Adult Rat Model of Meningitis A total of 36 adult rats were included in the study. The adult rat model was chosen for the study because the histopathologic brain damage observed in this model resembles the findings in human meningitis more than other models of meningitis. Different serotypes were used, to determine potential serotype-related differences in inner ear invasion. Before inoculation, all strains were passed several times through CSF, to obtain maximum virulence of the pathogen. A frozen stock of the bacteria was thawed and grown on 5% blood agar plates for 18 hours, suspended in beef broth, and grown to midYlog-phase. The bacteria were washed, centrifuged (3,509 ÅÈ g, 4-C for 10 min) and diluted in cold saline to a final concentration of 0.9j2 105 CFU/ml, as confirmed by quantitative cultures. On the day of infection, young adult male Wistar rats (È200 g) were anesthetized subcutaneously with midazolam (1.88 mg/kg; Dormicum; Roche AG) and a combination of fentanyl and fluanisone (0.12 mg/kg; Hypnorm; Janssen Pharmaceutica NV) and infected with one of the serotypes (serotype 1, n = 10, (strain 277/99, Statens Serum Institute, Copenhagen, Denmark [SSI]); serotype 3, n = 10, (strain 68034, SSI); or serotype 9 V, n = 10, SSI), by intrathecal inoculation of 30 Kl of the bacterial suspension through the cisterna magna. After the bacterial inoculation, the rats were evaluated clinically 3 times daily and given 2.5 ml of isotonic saline during periods of severe illness, to prevent dehydration. The clinical score was a modification of the method of Leib et al. (16) and was graded as follows: 0, normal activity; 1, minimal ambulatory activity (turns upright in G5 s); 2,turns upright within 30 s; 3, does not turn upright; 4, spontaneously lying on the back or side; and 5, terminal illness (cyanosis, apnea, coma, and/or ophistotonus/ seizures). When terminal illness occurred, a CSF sample was obtained, and rats were killed with an intravenous injection of pentobarbital (200 mg/ml) and perfused with 1.5% paraformaldehyde (PFA) through the left ventricle of the heart. Rather than studying the pathology at predefined intervals, this approach was chosen to mimic the clinical setting and allow comparability to human temporal bones harvested from patients succumbing to meningitis. Surviving rats and sham-inoculated rats were observed for a total of 166 h, then killed with an injection of pentobarbital after CSF samples were obtained, then perfused with 1.5% PFA, as described previously. CSF was analyzed for bacterial concentration and white blood cell (WBC) count, to confirm meningitis.
Preparation for Light Microscopy After decapitation, the cranium was stored in paraformaldehyde 1.5% for at least 1 week, followed by right temporal bone dissection and decalcification for 4 weeks in ethylenediaminetetraacetic acid (EDTA) 10%. The inner ear was subsequently split in 2 parts by an intended midmodiolar transection of the cochlea. The transection was performed by aligning the scalpel along the Eustachian tube, through the tympanic orifice and the cochlear apex, and cutting toward the center of the cochlear base in one careful movement. This was followed by paraffin embedding, serial
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5- to 10-Km sectioning, and periodic acid Schiff (PAS)-alcian blue staining. PAS-alcian blue staining is well documented for identification of bacteria with a capsule containing acid mucopolysaccharides, such as Streptococcus pneumoniae. The stained parts appear blue to bluish-green (17). Light microscopy and photodocumentation A Zeiss Axio Imager M1 microscope was used for light microscopy and a Zeiss Axiocam HRc camera for photodocumentation, using Zeiss Axiovision software for digital image processing.
RESULTS In all but the 6 sham-inoculated animals, meningitis was verified by clinical score, as well as CSF pleocytosis and a positive CSF culture for pneumococci. According to the occurrence of terminal illness, serotype 1 inoculated rats (N = 10) were sacrificed after 57, 59 (n = 2), 64, 67 (n = 2), 75, 77 and 166 (n = 2) hours, respectively. Serotype 3 inoculated rats (n = 10) were sacrificed after 27, 28, 30, 38, 43 (n = 2), 46 (n = 2), 67 and 91 hours, respectively, and serotype 9 V inoculated rats (n = 10) were sacrificed after 57, 66, 90 and 138 (n = 7) hours, respectively. Routes of Bacterial Invasion of the Inner Ear From the meninges, bacteria may invade the inner ear through the naturally occurring openings or soft tissue connections between the meninges and the inner ear, that is, the cochlear aqueduct, the internal auditory canal, and the endolymphatic sac. Alternatively, invasion may occur through the bony partitions between the posterior or medial cranial fossa and the inner ear. Finally, bacterial invasion of the inner ear may occur through hematogenous spread. Between the 3 pneumococcal serotypes, no qualitative differences were observed regarding the route of inner ear invasion or subsequent pathways of spreading within the inner ear. After intrathecal inoculation, the pneumococci were invading the inner ear though the cochlear aqueduct in all cases (Fig. 1). The bacteria were subsequently observed in scala tympani of the basal turn of the cochlea (the perilymphatic space) (Fig. 1). Bacteria did not invade the inner ear through the internal auditory canal, through the endolymphatic sac/duct or through bony partitions. Thus, in the sections from the first hours and days postinoculation, no bacteria were seen in the internal auditory canal (not shown) or in the vestibular end organs (Fig. 3). However, clear evidence of hematogenous spread was seen on Days 5 to 6, subsequent to invasion of both scala tympani and vestibuli (perilymphatic space), as endolymphatic invasion of bacteria from the spiral ligament capillary bed, spreading from dilated or trombotic vessels and into the scala media (endolymphatic space) (Figs. 1 and 4). In the modiolus, no bacterial extravasation, thrombosis, or vessel dilation was seen (Fig. 2). Pathways and Temporal Pattern of Bacterial Spread Within the Inner Ear As noted previously, bacterial invasion of the inner ear occurred through the cochlear aqueduct and into the scala tympani of the cochlea (perilymphatic space) on Days 1 to Otology & Neurotology, Vol. 35, No. 5, 2014
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FIG. 1. Overview of the primary routes of bacterial invasion from the meninges to the inner ear during experimental meningitis in the rat. A, Days 1 to 3 postinoculation. Bacterial invasion occur primarily from the meninges (MIN) and through the cochlear aqueduct (arrow) to the scala tympani (ST), in which the pneumococci accumulates in the basal turn of the cochlea. SM: scala media. RM: round window membrane. Bar: 0.2 mm. Insert right is a high power micrograph of the framed area in the cochlea aqueduct, showing abundant bacterial invasion and accumulation. Bar: 10 Km. Insert left is a higher magnification of the framed area in scala tympani, showing massive accumulation of bacteria. Bar: 20 Km. B, Days 5 to 7: Evidence of hematogenous spread. The arrow indicates bacteria adhering to the wall of a modiolar blood vessel. MOD: modiolus. Bar: 20 Km (C): Days 5 to 7: Shows direct spread of bacteria (arrows) from the scala tympani (ST) and into the spiral ganglion (SG) and Rosenthal’s canal (RC) of the modiolus. Bar: 40 Km.
2, regardless of serotype (Fig. 1). From there, the pneumococci spread rapidly within the scala tympani toward the medial cochlear turn and further to the helicotrema at the cochlear apex on Days 3 to 4, then descending basally within the scala vestibuli, eventually reaching the vestibule and the vestibular end organs on Days 5 to 6 (Figs. 3 and 5). Thus, bacteria in the vestibular system were observed first in the vestibule, invading the saccule and the utricle perilymphatic space in a clear continuation of the invasion of scala vestibuli of the basal turn of the cochlea (perilymphatic space) (Figs. 3 and 5). Somewhat later, invasion followed in the endolymphatic space of the vestibular system. This occurred subsequent to endolymphatic invasion from the spiral ligament capillary in the scala media of the cochlea (Fig. 4), as evidence of hematogenous spread of bacteria to the inner ear. In animals surviving more than 5 days, bacterial invasion reached the semicircular canals, in the perilymphatic
and endolymphatic space (Figs. 3 and 5). The lateral semicircular canal was infiltrated first in all cases and thus displayed a more pronounced bacterial accumulation at sacrifice (not shown). As the cochlea aqueduct was the main route of invasion, the accumulation of invading bacteria displayed a basal to apical gradient, with the basal part being the most heavily invaded (Figs. 3 and 5). The bacteria were more abundant in the perilymphatic space compared with the endolymphatic space, although this was less evident in the vestibular system, as perilymphatic and endolymphatic infiltration in the vestibular system occurred at roughly the same time (Figs. 3 and 5). As the pneumococci rarely and in only very severe cases (with massive bacterial invasion), infiltrated the endolymphatic space directly from the perilymphatic space through breach of the basilar or Reissner’s membrane (scala tympani or vestibuli respectively), massive bacterial
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FIG. 3. Overview of the cochlea and the vestibular system during pneumococcal meningitis. A, Days 3 to 5 postinoculation: Multiple image alignment showing the cochlea, the vestibule with the utricule (UT) and the semicircular canals (SCC). Note the beginning bacterial invasion of the perilymphatic part of the vestibular system, without concurrent endolymphatic involvement. In the cochlea, the bacterial invasion and inflammatory infiltration display a basal to apical gradient. The basal turn is the most heavily invaded and infiltrated, compared with that of the helicotrema (HT). MOD: modiolus. Bar: 200 Km. B, Semicircular canal Days 1 to 3 postinoculation. No bacterial invasion or inflammation occurs in the endolymphatic and perilymphatic space. Bar: 20 Km. C, Semicircular Days 5 to 7 postinoculation. Endolymphatic invasion of bacteria and inflammation is observed at this point (arrow). Bar: 20 Km.
accumulation appeared adjacent these structures (Figs. 2 and 4). In few cases, bacteria were invading the peripheral parts of the modiolus from the perilymphatic space on Days 5 to 6 (Fig. 1). As noted previously, no qualitative differences of inner ear invasion were seen between the serotypes. However, a marked serotype-related difference was seen concerning the degree of bacterial invasion and accumulation over time. Serotype 3 seemed to have a more aggressive course of disease overall, but a less pronounced pneumococcal invasion of the inner ear, in timely comparison with serotype 1 and 9 V. No animals inoculated with serotype 3 survived the full observational period of 7 days, and endolymphatic and vestibular infiltration were observed only scarcely. Elimination of Bacteria From the Inner Ear Clear evidence of bacterial elimination was found in sections from animals surviving more than a few days, increasing numbers of macrophages engulfing bacteria were accumulating in especially scala tympani (perilymphatic
space), primarily at the localities were the bacteria were most abundant, that is, adjacent the basilar membrane (Fig. 2).
DISCUSSION In the present study, a visual display of the routes, pathways, and dynamics of Streptococcus pneumoniae invasion of and spreading within the inner ear during meningitis is provided for the first time, using a well-proven rat model. In addition, partial elimination by macrophage migration to the inner ear and subsequent bacterial engulfment is documented. During the first few days after inoculation, the bacteria invade the inner ear through the cochlear aqueduct and into the scala tympani of the cochlea (perilymphatic space). Through scala tympani, spreading progresses apically toward the helicotrema and into the apical part of scala vestibuli (perilymphatic space). From there, further spreading occurs basally through the scala vestibuli toward the vestibule and further into the vestibular system. At the time when the bacteria reach scala vestibuli of the basal turn of the cochleaVafter Otology & Neurotology, Vol. 35, No. 5, 2014
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FIG. 4. Days 5 to 7: Hematogenous spread and bacterial invasion of the endolymphatic space. A, Overview of the scala media (SM), scala vestibuli (SV) and the spiral ligament (SL). Bar: 40 Km. B, Higher magnification of the framed area in A showing a Reissner’s membrane (arrows) resistant to penetration and breakthrough of massively accumulating bacteria in scala vestibuli (SV). Bar: 20 Km. C, Higher magnification of the framed area in A showing bacteria (horizontal arrows) spreading from the thrombosed and dilated spiral ligament vessels and into the endolymphatic lumen of the scala media (SM). Bar: 20 Km.
5 to 6 daysVhematogenous spread occurs to the spiral ligament of the scala media and from there into the cochlear endolymph, subsequently to the endolymph of the vestibular end organs. In the rat model, we found no evidence of bacterial invasion of the inner ear through the internal auditory canal and modiolus, through the endolymphatic sac and duct, or through bony partitions between the intracranial space and the inner ear. Our findings indicate that the internal auditory canal and the modiolus is an unlikely route of bacterial invasion in rats, which is also the case in rabbits (6,10), although this pathway has been implicated in studies on human temporal bones (7). Differences in species anatomy may explain this, as the human cochlear aqueduct is long and narrow compared with the rodents. Furthermore, the cochlea aqueduct is always patent in rodents, which may not be the case in humans (15,18,19). However, the human cochlear aqueduct has been suggested in a recent study as potential route of infection in patients with pneumococcal meningitis after cochlear implantation. This is supported by experimental animal studies, although the implant in itself seems to cause
susceptibility to bacterial meningitis, regardless of the route of infection (20). In the present study, modiolar invasion of bacteria from scala tympani (perilymphatic space) was seen scarcely and only in the peripheral parts. Given the method of perfusion fixation, one cannot expect bacteria to be present within modiolar blood vessels, despite potential hematogenous spread. However, in a few cases, we did observe bacteria adhering to the wall of modiolar vessels, coinciding temporally with hematogenous spread to the spiral ligament and scala media. Previous reports of hematogenous spread into scala media are conflicting, as perineural invasion through the organ of Corti and migration through the basal membrane have been proposed, although in different species (6,7,10,21). As noted, the observations in the present study indicate that hematogenous spread of bacteria to the scala media endolymph occurs in animals surviving more than a few days. Although a single study has demonstrated bacteria in the endolymphatic space in absence of concurrent inflammation (10), the sequence of events after bacterial invasion of the inner ear implicates subsequent inflammation
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FIG. 2. Overview of the natural, internal barriers to bacterial spreading within the inner ear, as well as bacterial elimination. Days 3 to 5 postinoculation. A, Micrograph of the basal turn of the cochlea. SV: scala vestibuli. SM: scala media. Bar: 40 Km. B, Higher magnification of the framed area in (A). Note the clear distinction between a pronounced bacterial invasion and accumulation in the scala vestibuli and the absence of bacteria in scala media (SM), indicating a Reissner’s membrane (arrows) resistant to bacterial penetration and breakthrough. Bar: 10 Km. C, Micrograph from the basal part of the modiolus (MOD), showing leukocyte infiltration (arrow) but no bacteria. Bar: 40 Km. D, Micrograph of the middle turn of the cochlea. ST: scala tympani. SG: spiral ganglion. Bar: 20 Km. E, Higher magnification of the framed area in D, showing multiple macrophages engulfing bacteria. Bar: 10 Km.
and induction of a local production of cytokines and proinflammatory mediators by attracted leukocytes, leading to further inflammation, disruption of the blood-labyrinth barrier, damage to the organ of Corti, spiral ganglion neuron loss, and a concurrent hearing loss (9,11,14). Inflammation and cochlear damage is potentially augmented by chemokine release from spiral ligament fibrocytes, which have been shown to occur in response to S. pneumoniae (22).
Disruption of the blood-labyrinth barrier has been demonstrated after pneumococcal meningitis, and interestingly, the spiral ligament was assessed as being a potential route of bacterial invasion into the scala media and the endolymphatic space (14). It should be noted though that a direct cytotoxic effect of the bacterial invasion and associated release of toxins, most likely pneumolysin, may cause or exasperate cochlea damage (8). This may be supported by Otology & Neurotology, Vol. 35, No. 5, 2014
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FIG. 5. Pathways and chronology of bacterial spread within the cochlea. The pneumococci spread via the scala tympani of the perilymphatic space toward the helicotrema and from here basally through the scala vestibuli, eventually reaching the vestibule and the vestibular system. A, Low power micrograph Days 1 to 3. Bar: 400 Km. a: Higher magnification of the framed area in (A), showing accumulating bacteria (long arrow) in scala tympani (ST) and beginning bacterial invasion (short arrow) of the apical part of scala vestibuli (SV). Note the absence of bacteria and inflammation in the endolymphatic space (asterisk). Bar: 50 um. B, Low power micrograph representing Days 3 to 5. Bar: 400 Km. b: Higher magnification of the framed area in (B), showing abundant bacteria (long arrow) in scala tympani and bacterial invasion (short arrow) of the scala vestibuli. Bacteria and inflammation are still absent from the endolymphatic space (asterisk). Bar: 50 Km. C, Low power micrograph from Days 5 to 7. Bar: 400 Km. c: Higher magnification of the framed area in (C), showing abundant bacteria (long arrow) in the perilymphatic space of the vestibule (VE). At this point, bacteria are also observed in the endolymphatic space (asterisk). Bar: 50 Km.
an observed correlation between CSF bacterial count and hearing loss in a clinical study (23). Regarding the pathways of bacterial spreading within the inner ear and invasion of the vestibular end organs, a perilymphatic barrier in the transition zone between the basal turn of scala tympani and the vestibule may exist along the periphery of ductus Reunions (which connects the base of the cochlea to the vestibulum directly) because the bacteria in all cases traveled the significantly longer distance through
the scala tympani perilymphatic space toward the cochlear apex and back to the cochlear base via the scala vestibuli before reaching the vestibule. This is in agreement with the fact that no bacteria occurred in the vestibular end organs in the first few days after inoculation. In animals surviving more than 5 days, the bacteria were found in both the perilymphatic and endolymphatic space of the vestibular system. Thus, the ductus Reuniens mirrors Reissner’s membrane with respect to the remarkable resistance to bacterial penetration
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INNER EAR AND BACTERIAL MENINGITIS and breakthrough. The findings indicate that the inner ear has several natural, internal barriers to bacterial spreading, primarily protecting invasion of the endolymphatic space and the vestibular system. Considering the previously mentioned route of meningeal infection after cochlear implantation, it may be possible that the internal barriers dictate the route of infection through the cochlear aqueduct. This could explain the low bacterial threshold needed to induce meningitis through inner ear inoculation and the increased susceptibility to infection after cochlear implantation (20). The route and delay of invasion into the vestibular end organs and the accumulating bacteria in the lateral semicircular canal are in agreement with clinical observations of delayed ossification and vestibular deficits after meningitis (2,4,24Y27). One of these studies indicated that lateral semicircular canal ossification may predict subsequent cochlea ossification (26). The present observations of the routes and pathways of bacterial invasion and spreading may support this hypothesis, as bacterial invasion and inflammation of the lateral semicircular canal are preceded by cochlear invasion and inflammation, suggesting that fibrosis and subsequent ossification are induced in the same order that the invasion appears to occur. As the inflammation is more massive, the process of ossification is likely to be delayed, although this is speculative. In the present study, we found no qualitative differences concerning the pattern of inner ear invasion between the 3 serotypes included. However, a relative absence of bacterial invasion in rats inoculated with serotype 3 pneumococci was noted, as was the fact that these animals did not survive the entire observational period. Unlike the quantitative pattern for serotype 1 and 9 V, serotype 3 pneumococci were not accumulating massively in the cochlea and did only scarcely invade the vestibule and vestibular end organs. Interestingly though, massive cochlear inflammation was noted as the serotype 3 animals succumbed to advanced disease, indicating that extensive inner ear inflammation may also occur subsequent to only sparse or even no bacterial invasion. From the observed differences, it may be speculated that the degree of cochlear damage and delayed cochlear ossification may depend on bacterial serotype, which is supported by a recent clinical study on hearing loss after pneumococcal meningitis (13). The routes and pathways of bacterial invasion of the inner ear and the subsequent inflammation reinforce the need for and relevance of the development of local pharmacologic intervention strategies, by which a far higher inner ear drug concentration and a prevention of hearing loss may be achieved. As suggested in earlier studies (28Y30), this could be accomplished by intratympanic administration of antibiotics and/or anti-inflammatory drugs. A possible synergistic effect could be obtained by cochlear microperfusion (31).
CONCLUSION From the meninges, pneumococci invade the inner ear through the cochlear aqueduct during the first days of infection, whereas hematogenous invasion via the spiral ligament
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capillary bed occur at later stages. Although internal barriers exist within the inner ear, the spreading of bacteria occurs via the natural pathways of the fluid compartments. Bacterial elimination occurs by local macrophage engulfment. REFERENCES 1. Dodge PR, Davis H, Feigin RD, et al. Prospective evaluation of hearing impairment as a sequelae of acute bacterial meningitis. N Engl J Med 1984;311:869Y74. 2. Rasmussen N, Johnsen NJ, Bohr VA. Otologic sequelae after pneumococcal meningitis: a survey of 164 consecutive cases with a follow-up of 94 survivors. Laryngoscope 1991;101:876Y82. 3. WorsLe L, Caye´-Thomasen P, Brandt CT, Thomsen J, Kstergaard C. Factors associated with the occurrence of hearing loss after pneumococcal meningitis. Clin Infect Dis 2010;51:917Y24. 4. Cushing SL, Papsin BC, Rutka JA, James AL, Blaser SL, Gordon KA. Vestibular end-organ and balance deficits after meningitis and cochlear implantation in children correlate poorly with functional outcome. Otol Neurotol 2009;30:488Y95. 5. Zingler VC, Weintz E, Jahn K, et al. Causative factors, epidemiology, and follow-up of bilateral vestibulopathy. Ann N Y Acad Sci 2009; 1164:505Y8. 6. Bhatt S, Halpin C, Hsu W, et al. Hearing loss and pneumococcal meningitis: an animal model. Laryngoscope 1991;101:1285Y92. 7. Merchant SN, Gopen Q. A human temporal bone study of acute bacterial meningogenic labyrinthitis. Am J Otol 1996;17:375Y85. 8. Winter AJ, Comis SD, Osborne MP, et al. A role for pneumolysin but not neuraminidase in the hearing loss and cochlear damage induced by experimental pneumococcal meningitis in guinea pigs. Infect Immun 1997;65:4411Y8. 9. Caye´-Thomasen P, Worsøe L, Brandt CT, et al. Routes, dynamics, and correlates of cochlear inflammation in terminal and recovering experimental meningitis. Laryngoscope 2009;119:1560Y70. 10. Osborne MP, Comis SD, Tarlow MJ, Stephen J. The cochlear lesion in experimental bacterial meningitis of the rabbit. Int J Exp Pathol 1995;76:317Y30. 11. Brandt CT, Caye´-Thomasen P, Lund SP, et al. Hearing loss and cochlear damage in experimental pneumococcal meningitis, with special reference to the role of neutrophil granulocytes. Neurobiol Dis 2006;23:300Y11. Epub 2006 Jun 22. 12. Demel C, Hoegen T, Giese A, et al. Reduced spiral ganglion neuronal loss by adjunctive neurotrophin-3 in experimental pneumococcal meningitis. J Neuroinflammation 2011;8:7. 13. WorsLe L, Brandt CT, Lund SP, Kstergaard C, Thomsen J, Caye´Thomasen P. Systemic steroid reduces long-term hearing loss in experimental pneumococcal meningitis. Laryngoscopoe 2010;120:1872Y9. 14. Kastenbauer S, Klein M, Koedel U, Pfister HW. Reactive nitrogen species contribute to bloodlabyrinth barrier disruption in suppurative labyrinthitis complicating experimental pneumococcal meningitis in the rat. Brain Res 2001;904:208Y17. 15. Brandt CT, Lundgren JD, Lund SP, et al. Attenuation of the bacterial load in blood by pretreatment with granulocyte-colony-stimulating factor protects rats from fatal outcome and brain damage during Streptococcus pneumoniae meningitis. Infect Immun 2004;72:4647Y53. 16. Leib SL, Kim YSM, Ferriero DM, Tauber MG. Neuroprotective effect of excitatory amino acid antagonist kynurenic acid in experimental bacterial meningitis. J Infect Dis 1996;173:166Y71. 17. Fusaro RM, Goltz RW. A comparative study of the periodic acidschiff and alcian blue stains. Investig Dermatol 1960;35:305Y7. 18. Palva T, Dammert K. Human cochlea aqueduct. Acta Otolaryngol 1969:246:1Y58. 19. Rask-Andersen H, Stahle J, Wilbrand H. Human cochlea aqueduct and its accessory canals. Ann Otol Rhinol Laryngol 1977;86:1Y16. 20. Wei BP, Shepherd RK, Robins-Browne RM, Clark GM, O’Leary SJ. Pneumococcal meningitis post-cochlear implantation: potential routes of infection and pathophysiology. Otolaryngol Head Neck Surg 2010;143:S15YS23. 21. Rappaport JM, Bhatt SM, Kimura RS, Lauretano AM, Levine RA. Electron microscopic temporal bone histopathology in experimental Otology & Neurotology, Vol. 35, No. 5, 2014
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27. Brodie HA, Thompson TC, Vassilian L, Lee BN. Induction of labyrinthitis ossificans after pneumococcal meningitis: an animal model. Otolaryngol Head Neck Surg 1998;118:15Y21. 28. Bird PA, Begg EJ, Zhang M, Keast AT, Murray DP, Balkany TJ. Intratympanic versus intravenous delivery of methylprednisolone to cochlear perilymph. Otol Neurotol 2007;28:1124Y30. 29. WorsLe L, Brandt CT, Lund SP, Ostergaard C, Thomsen J, Caye´Thomasen P. Intratympanic steroid prevents long-term spiral ganglion neuron loss in experimental meningitis. Otol Neurotol 2010;31: 394Y403. 30. Salt AN, Hartsock J, Plontke S, LeBel C, Piu F. Distribution of dexamethasone and preservation of inner ear function following intratympanic delivery of a gel-based formulation. Audiol Neurootol 2011;16:323Y35. doi: 10.1159/000322504. Epub 2010 Dec 19. 31. Barkdull GC, Vu C, Keithley EM, Harris JP. Cochlear microperfusion: experimental evaluation of a potential new therapy for severe hearing loss caused by inflammation. Otol Neurotol 2005;26:19Y26.
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Bacterial Invasion of the Inner Ear in Association With Pneumococcal Meningitis *Martin Nue MLller, †Christian Brandt, ‡Christian Kstergaard, and *§Per Caye-Thomasen *Department of Oto-Rhino-Laryngology, Head and Neck Surgery, University Hospital, Rigshospitalet/ Gentofte, Copenhagen; ÞDepartment of Infectious Diseases, Copenhagen University Hospital Hvidovre; þDepartment of Clinical Microbiology, Copenhagen University Hospital Hvidovre, Hvidovre; and §The Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen Denmark
Objective: To examine the pathways of bacterial invasion and subsequent spreading in the inner ear during pneumococcal meningitis. Study Design: A well-established adult rat model of Streptococcus pneumoniae meningitis was used. Methods: Thirty rats were inoculated intrathecally with S. pneumoniae serotype 1, 3 or 9 V and received no additional treatment. The rats were sacrificed when reaching terminal illness or on Day 7 and then prepared for serial sectioning and PAS-Alcian blue staining for light microscopy. Results: During the first few days after inoculation, bacteria invade the inner ear through the cochlear aqueduct, into the scala tympani of the cochlea (perilymphatic space). From here, bacteria spreads apically toward the helicotrema and subsequently basally through the scala vestibuli, toward the vestibule and the vestibular system. When the bacteria after 5 to 6 days had reached scala vestibuli of the basal turn of the cochlea, hematogenous spreading occurred to
the spiral ligament and into the cochlear endolymph, subsequently to the vestibular endolymph. We found no evidence of alternative routes for bacterial invasion in the inner ear. Several internal barriers to bacterial spreading were found within the inner ear. Bacterial elimination was evidenced by engulfment by macrophages within the inner ear. Conclusion: From the meninges, pneumococci invade the inner ear through the cochlear aqueduct during the first days of infection, whereas hematogenous invasion via the spiral ligament capillary bed occur at later stages. Although internal barriers exist within the inner ear, the spreading of bacteria occurs via the natural pathways of the fluid compartments. Bacterial elimination occurs by local macrophage engulfment. Key Words: Bacterial meningitisVInner earVLabyrinthitisVStreptococcus pneumoniaeVTemporal bone pathology. Otol Neurotol 35:e178Ye186, 2014.
The most common long-term sequela after pneumococcal meningitis is inner ear dysfunction.Thus, sensorineural hearing loss affects up to 54% of survivors (1Y3) and is often accompanied by vestibular dysfunction, leading to concurring vertigo and/or balance problems, which affects up to 15% (2,4,5). The cause of postmeningitic inner ear dysfunction is supposed to be bacterial toxins and/or inflammation (1,6Y9). Inner ear filtration of inflammatory cells seems in itself to be a major cause of meningitis-associated hearing loss, as it induces damage to the organ of Corti and spiral ganglion, conceivably through production of cytotoxic mediators (1,6,7,10Y12). This is substantiated by an increase of cochlear damage and hearing loss when the inflammatory response is boosted (11) and conversely by a reduction
in hearing loss by anti-inflammatory therapy with corticosteroids (13). As direct evidence of bacterial invasion of the inner ear during meningitis is lacking, it is unclear whether inner ear inflammation occurs secondary to direct invasion of bacteria from the meninges or the bloodstream or as a bystander phenomenon secondary to adjacent meningeal inflammation. During meningitis, inflammatory cell infiltration of the inner ear occurs from the meninges to the perilymphatic space of the cochlea through the cochlear aqueduct or the internal auditory canal, the route seemingly depending somewhat on species (6Y10). Subsequently, the endolymphatic compartment is infiltrated by diapedesis of inflammatory cells from the vascular bed of the spiral ligament in the cochlea (9). Assuming that this process is preceded by inner ear invasion of bacteria, the routes and temporary pattern are likely to mirror this pattern. This report presents direct evidence of bacterial invasion of the inner ear during meningitis. Using a well-established experimental model of pneumococcal meningitis, the routes
Address correspondence and reprint requests to Martin Nue MLller, M.D., Department of Oto-Rhino-Laryngology, Head and Neck Surgery, University Hospital, Rigshospitalet/Gentofte, 2100 Copenhagen, Denmark; E-mail: [email protected] Disclosure: The authors report no conflicts of interest.
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INNER EAR AND BACTERIAL MENINGITIS of bacterial invasion and the pathways and temporal pattern of bacterial spreading and elimination within the inner ear are documented. MATERIALS AND METHODS The experimental protocol was approved by the Danish Animal Inspectorate (Dyreforsoegstilsynet) and based on a well-established model of pneumococcal meningitis (14,15).
Adult Rat Model of Meningitis A total of 36 adult rats were included in the study. The adult rat model was chosen for the study because the histopathologic brain damage observed in this model resembles the findings in human meningitis more than other models of meningitis. Different serotypes were used, to determine potential serotype-related differences in inner ear invasion. Before inoculation, all strains were passed several times through CSF, to obtain maximum virulence of the pathogen. A frozen stock of the bacteria was thawed and grown on 5% blood agar plates for 18 hours, suspended in beef broth, and grown to midYlog-phase. The bacteria were washed, centrifuged (3,509 ÅÈ g, 4-C for 10 min) and diluted in cold saline to a final concentration of 0.9j2 105 CFU/ml, as confirmed by quantitative cultures. On the day of infection, young adult male Wistar rats (È200 g) were anesthetized subcutaneously with midazolam (1.88 mg/kg; Dormicum; Roche AG) and a combination of fentanyl and fluanisone (0.12 mg/kg; Hypnorm; Janssen Pharmaceutica NV) and infected with one of the serotypes (serotype 1, n = 10, (strain 277/99, Statens Serum Institute, Copenhagen, Denmark [SSI]); serotype 3, n = 10, (strain 68034, SSI); or serotype 9 V, n = 10, SSI), by intrathecal inoculation of 30 Kl of the bacterial suspension through the cisterna magna. After the bacterial inoculation, the rats were evaluated clinically 3 times daily and given 2.5 ml of isotonic saline during periods of severe illness, to prevent dehydration. The clinical score was a modification of the method of Leib et al. (16) and was graded as follows: 0, normal activity; 1, minimal ambulatory activity (turns upright in G5 s); 2,turns upright within 30 s; 3, does not turn upright; 4, spontaneously lying on the back or side; and 5, terminal illness (cyanosis, apnea, coma, and/or ophistotonus/ seizures). When terminal illness occurred, a CSF sample was obtained, and rats were killed with an intravenous injection of pentobarbital (200 mg/ml) and perfused with 1.5% paraformaldehyde (PFA) through the left ventricle of the heart. Rather than studying the pathology at predefined intervals, this approach was chosen to mimic the clinical setting and allow comparability to human temporal bones harvested from patients succumbing to meningitis. Surviving rats and sham-inoculated rats were observed for a total of 166 h, then killed with an injection of pentobarbital after CSF samples were obtained, then perfused with 1.5% PFA, as described previously. CSF was analyzed for bacterial concentration and white blood cell (WBC) count, to confirm meningitis.
Preparation for Light Microscopy After decapitation, the cranium was stored in paraformaldehyde 1.5% for at least 1 week, followed by right temporal bone dissection and decalcification for 4 weeks in ethylenediaminetetraacetic acid (EDTA) 10%. The inner ear was subsequently split in 2 parts by an intended midmodiolar transection of the cochlea. The transection was performed by aligning the scalpel along the Eustachian tube, through the tympanic orifice and the cochlear apex, and cutting toward the center of the cochlear base in one careful movement. This was followed by paraffin embedding, serial
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5- to 10-Km sectioning, and periodic acid Schiff (PAS)-alcian blue staining. PAS-alcian blue staining is well documented for identification of bacteria with a capsule containing acid mucopolysaccharides, such as Streptococcus pneumoniae. The stained parts appear blue to bluish-green (17). Light microscopy and photodocumentation A Zeiss Axio Imager M1 microscope was used for light microscopy and a Zeiss Axiocam HRc camera for photodocumentation, using Zeiss Axiovision software for digital image processing.
RESULTS In all but the 6 sham-inoculated animals, meningitis was verified by clinical score, as well as CSF pleocytosis and a positive CSF culture for pneumococci. According to the occurrence of terminal illness, serotype 1 inoculated rats (N = 10) were sacrificed after 57, 59 (n = 2), 64, 67 (n = 2), 75, 77 and 166 (n = 2) hours, respectively. Serotype 3 inoculated rats (n = 10) were sacrificed after 27, 28, 30, 38, 43 (n = 2), 46 (n = 2), 67 and 91 hours, respectively, and serotype 9 V inoculated rats (n = 10) were sacrificed after 57, 66, 90 and 138 (n = 7) hours, respectively. Routes of Bacterial Invasion of the Inner Ear From the meninges, bacteria may invade the inner ear through the naturally occurring openings or soft tissue connections between the meninges and the inner ear, that is, the cochlear aqueduct, the internal auditory canal, and the endolymphatic sac. Alternatively, invasion may occur through the bony partitions between the posterior or medial cranial fossa and the inner ear. Finally, bacterial invasion of the inner ear may occur through hematogenous spread. Between the 3 pneumococcal serotypes, no qualitative differences were observed regarding the route of inner ear invasion or subsequent pathways of spreading within the inner ear. After intrathecal inoculation, the pneumococci were invading the inner ear though the cochlear aqueduct in all cases (Fig. 1). The bacteria were subsequently observed in scala tympani of the basal turn of the cochlea (the perilymphatic space) (Fig. 1). Bacteria did not invade the inner ear through the internal auditory canal, through the endolymphatic sac/duct or through bony partitions. Thus, in the sections from the first hours and days postinoculation, no bacteria were seen in the internal auditory canal (not shown) or in the vestibular end organs (Fig. 3). However, clear evidence of hematogenous spread was seen on Days 5 to 6, subsequent to invasion of both scala tympani and vestibuli (perilymphatic space), as endolymphatic invasion of bacteria from the spiral ligament capillary bed, spreading from dilated or trombotic vessels and into the scala media (endolymphatic space) (Figs. 1 and 4). In the modiolus, no bacterial extravasation, thrombosis, or vessel dilation was seen (Fig. 2). Pathways and Temporal Pattern of Bacterial Spread Within the Inner Ear As noted previously, bacterial invasion of the inner ear occurred through the cochlear aqueduct and into the scala tympani of the cochlea (perilymphatic space) on Days 1 to Otology & Neurotology, Vol. 35, No. 5, 2014
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FIG. 1. Overview of the primary routes of bacterial invasion from the meninges to the inner ear during experimental meningitis in the rat. A, Days 1 to 3 postinoculation. Bacterial invasion occur primarily from the meninges (MIN) and through the cochlear aqueduct (arrow) to the scala tympani (ST), in which the pneumococci accumulates in the basal turn of the cochlea. SM: scala media. RM: round window membrane. Bar: 0.2 mm. Insert right is a high power micrograph of the framed area in the cochlea aqueduct, showing abundant bacterial invasion and accumulation. Bar: 10 Km. Insert left is a higher magnification of the framed area in scala tympani, showing massive accumulation of bacteria. Bar: 20 Km. B, Days 5 to 7: Evidence of hematogenous spread. The arrow indicates bacteria adhering to the wall of a modiolar blood vessel. MOD: modiolus. Bar: 20 Km (C): Days 5 to 7: Shows direct spread of bacteria (arrows) from the scala tympani (ST) and into the spiral ganglion (SG) and Rosenthal’s canal (RC) of the modiolus. Bar: 40 Km.
2, regardless of serotype (Fig. 1). From there, the pneumococci spread rapidly within the scala tympani toward the medial cochlear turn and further to the helicotrema at the cochlear apex on Days 3 to 4, then descending basally within the scala vestibuli, eventually reaching the vestibule and the vestibular end organs on Days 5 to 6 (Figs. 3 and 5). Thus, bacteria in the vestibular system were observed first in the vestibule, invading the saccule and the utricle perilymphatic space in a clear continuation of the invasion of scala vestibuli of the basal turn of the cochlea (perilymphatic space) (Figs. 3 and 5). Somewhat later, invasion followed in the endolymphatic space of the vestibular system. This occurred subsequent to endolymphatic invasion from the spiral ligament capillary in the scala media of the cochlea (Fig. 4), as evidence of hematogenous spread of bacteria to the inner ear. In animals surviving more than 5 days, bacterial invasion reached the semicircular canals, in the perilymphatic
and endolymphatic space (Figs. 3 and 5). The lateral semicircular canal was infiltrated first in all cases and thus displayed a more pronounced bacterial accumulation at sacrifice (not shown). As the cochlea aqueduct was the main route of invasion, the accumulation of invading bacteria displayed a basal to apical gradient, with the basal part being the most heavily invaded (Figs. 3 and 5). The bacteria were more abundant in the perilymphatic space compared with the endolymphatic space, although this was less evident in the vestibular system, as perilymphatic and endolymphatic infiltration in the vestibular system occurred at roughly the same time (Figs. 3 and 5). As the pneumococci rarely and in only very severe cases (with massive bacterial invasion), infiltrated the endolymphatic space directly from the perilymphatic space through breach of the basilar or Reissner’s membrane (scala tympani or vestibuli respectively), massive bacterial
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FIG. 3. Overview of the cochlea and the vestibular system during pneumococcal meningitis. A, Days 3 to 5 postinoculation: Multiple image alignment showing the cochlea, the vestibule with the utricule (UT) and the semicircular canals (SCC). Note the beginning bacterial invasion of the perilymphatic part of the vestibular system, without concurrent endolymphatic involvement. In the cochlea, the bacterial invasion and inflammatory infiltration display a basal to apical gradient. The basal turn is the most heavily invaded and infiltrated, compared with that of the helicotrema (HT). MOD: modiolus. Bar: 200 Km. B, Semicircular canal Days 1 to 3 postinoculation. No bacterial invasion or inflammation occurs in the endolymphatic and perilymphatic space. Bar: 20 Km. C, Semicircular Days 5 to 7 postinoculation. Endolymphatic invasion of bacteria and inflammation is observed at this point (arrow). Bar: 20 Km.
accumulation appeared adjacent these structures (Figs. 2 and 4). In few cases, bacteria were invading the peripheral parts of the modiolus from the perilymphatic space on Days 5 to 6 (Fig. 1). As noted previously, no qualitative differences of inner ear invasion were seen between the serotypes. However, a marked serotype-related difference was seen concerning the degree of bacterial invasion and accumulation over time. Serotype 3 seemed to have a more aggressive course of disease overall, but a less pronounced pneumococcal invasion of the inner ear, in timely comparison with serotype 1 and 9 V. No animals inoculated with serotype 3 survived the full observational period of 7 days, and endolymphatic and vestibular infiltration were observed only scarcely. Elimination of Bacteria From the Inner Ear Clear evidence of bacterial elimination was found in sections from animals surviving more than a few days, increasing numbers of macrophages engulfing bacteria were accumulating in especially scala tympani (perilymphatic
space), primarily at the localities were the bacteria were most abundant, that is, adjacent the basilar membrane (Fig. 2).
DISCUSSION In the present study, a visual display of the routes, pathways, and dynamics of Streptococcus pneumoniae invasion of and spreading within the inner ear during meningitis is provided for the first time, using a well-proven rat model. In addition, partial elimination by macrophage migration to the inner ear and subsequent bacterial engulfment is documented. During the first few days after inoculation, the bacteria invade the inner ear through the cochlear aqueduct and into the scala tympani of the cochlea (perilymphatic space). Through scala tympani, spreading progresses apically toward the helicotrema and into the apical part of scala vestibuli (perilymphatic space). From there, further spreading occurs basally through the scala vestibuli toward the vestibule and further into the vestibular system. At the time when the bacteria reach scala vestibuli of the basal turn of the cochleaVafter Otology & Neurotology, Vol. 35, No. 5, 2014
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FIG. 4. Days 5 to 7: Hematogenous spread and bacterial invasion of the endolymphatic space. A, Overview of the scala media (SM), scala vestibuli (SV) and the spiral ligament (SL). Bar: 40 Km. B, Higher magnification of the framed area in A showing a Reissner’s membrane (arrows) resistant to penetration and breakthrough of massively accumulating bacteria in scala vestibuli (SV). Bar: 20 Km. C, Higher magnification of the framed area in A showing bacteria (horizontal arrows) spreading from the thrombosed and dilated spiral ligament vessels and into the endolymphatic lumen of the scala media (SM). Bar: 20 Km.
5 to 6 daysVhematogenous spread occurs to the spiral ligament of the scala media and from there into the cochlear endolymph, subsequently to the endolymph of the vestibular end organs. In the rat model, we found no evidence of bacterial invasion of the inner ear through the internal auditory canal and modiolus, through the endolymphatic sac and duct, or through bony partitions between the intracranial space and the inner ear. Our findings indicate that the internal auditory canal and the modiolus is an unlikely route of bacterial invasion in rats, which is also the case in rabbits (6,10), although this pathway has been implicated in studies on human temporal bones (7). Differences in species anatomy may explain this, as the human cochlear aqueduct is long and narrow compared with the rodents. Furthermore, the cochlea aqueduct is always patent in rodents, which may not be the case in humans (15,18,19). However, the human cochlear aqueduct has been suggested in a recent study as potential route of infection in patients with pneumococcal meningitis after cochlear implantation. This is supported by experimental animal studies, although the implant in itself seems to cause
susceptibility to bacterial meningitis, regardless of the route of infection (20). In the present study, modiolar invasion of bacteria from scala tympani (perilymphatic space) was seen scarcely and only in the peripheral parts. Given the method of perfusion fixation, one cannot expect bacteria to be present within modiolar blood vessels, despite potential hematogenous spread. However, in a few cases, we did observe bacteria adhering to the wall of modiolar vessels, coinciding temporally with hematogenous spread to the spiral ligament and scala media. Previous reports of hematogenous spread into scala media are conflicting, as perineural invasion through the organ of Corti and migration through the basal membrane have been proposed, although in different species (6,7,10,21). As noted, the observations in the present study indicate that hematogenous spread of bacteria to the scala media endolymph occurs in animals surviving more than a few days. Although a single study has demonstrated bacteria in the endolymphatic space in absence of concurrent inflammation (10), the sequence of events after bacterial invasion of the inner ear implicates subsequent inflammation
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FIG. 2. Overview of the natural, internal barriers to bacterial spreading within the inner ear, as well as bacterial elimination. Days 3 to 5 postinoculation. A, Micrograph of the basal turn of the cochlea. SV: scala vestibuli. SM: scala media. Bar: 40 Km. B, Higher magnification of the framed area in (A). Note the clear distinction between a pronounced bacterial invasion and accumulation in the scala vestibuli and the absence of bacteria in scala media (SM), indicating a Reissner’s membrane (arrows) resistant to bacterial penetration and breakthrough. Bar: 10 Km. C, Micrograph from the basal part of the modiolus (MOD), showing leukocyte infiltration (arrow) but no bacteria. Bar: 40 Km. D, Micrograph of the middle turn of the cochlea. ST: scala tympani. SG: spiral ganglion. Bar: 20 Km. E, Higher magnification of the framed area in D, showing multiple macrophages engulfing bacteria. Bar: 10 Km.
and induction of a local production of cytokines and proinflammatory mediators by attracted leukocytes, leading to further inflammation, disruption of the blood-labyrinth barrier, damage to the organ of Corti, spiral ganglion neuron loss, and a concurrent hearing loss (9,11,14). Inflammation and cochlear damage is potentially augmented by chemokine release from spiral ligament fibrocytes, which have been shown to occur in response to S. pneumoniae (22).
Disruption of the blood-labyrinth barrier has been demonstrated after pneumococcal meningitis, and interestingly, the spiral ligament was assessed as being a potential route of bacterial invasion into the scala media and the endolymphatic space (14). It should be noted though that a direct cytotoxic effect of the bacterial invasion and associated release of toxins, most likely pneumolysin, may cause or exasperate cochlea damage (8). This may be supported by Otology & Neurotology, Vol. 35, No. 5, 2014
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FIG. 5. Pathways and chronology of bacterial spread within the cochlea. The pneumococci spread via the scala tympani of the perilymphatic space toward the helicotrema and from here basally through the scala vestibuli, eventually reaching the vestibule and the vestibular system. A, Low power micrograph Days 1 to 3. Bar: 400 Km. a: Higher magnification of the framed area in (A), showing accumulating bacteria (long arrow) in scala tympani (ST) and beginning bacterial invasion (short arrow) of the apical part of scala vestibuli (SV). Note the absence of bacteria and inflammation in the endolymphatic space (asterisk). Bar: 50 um. B, Low power micrograph representing Days 3 to 5. Bar: 400 Km. b: Higher magnification of the framed area in (B), showing abundant bacteria (long arrow) in scala tympani and bacterial invasion (short arrow) of the scala vestibuli. Bacteria and inflammation are still absent from the endolymphatic space (asterisk). Bar: 50 Km. C, Low power micrograph from Days 5 to 7. Bar: 400 Km. c: Higher magnification of the framed area in (C), showing abundant bacteria (long arrow) in the perilymphatic space of the vestibule (VE). At this point, bacteria are also observed in the endolymphatic space (asterisk). Bar: 50 Km.
an observed correlation between CSF bacterial count and hearing loss in a clinical study (23). Regarding the pathways of bacterial spreading within the inner ear and invasion of the vestibular end organs, a perilymphatic barrier in the transition zone between the basal turn of scala tympani and the vestibule may exist along the periphery of ductus Reunions (which connects the base of the cochlea to the vestibulum directly) because the bacteria in all cases traveled the significantly longer distance through
the scala tympani perilymphatic space toward the cochlear apex and back to the cochlear base via the scala vestibuli before reaching the vestibule. This is in agreement with the fact that no bacteria occurred in the vestibular end organs in the first few days after inoculation. In animals surviving more than 5 days, the bacteria were found in both the perilymphatic and endolymphatic space of the vestibular system. Thus, the ductus Reuniens mirrors Reissner’s membrane with respect to the remarkable resistance to bacterial penetration
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INNER EAR AND BACTERIAL MENINGITIS and breakthrough. The findings indicate that the inner ear has several natural, internal barriers to bacterial spreading, primarily protecting invasion of the endolymphatic space and the vestibular system. Considering the previously mentioned route of meningeal infection after cochlear implantation, it may be possible that the internal barriers dictate the route of infection through the cochlear aqueduct. This could explain the low bacterial threshold needed to induce meningitis through inner ear inoculation and the increased susceptibility to infection after cochlear implantation (20). The route and delay of invasion into the vestibular end organs and the accumulating bacteria in the lateral semicircular canal are in agreement with clinical observations of delayed ossification and vestibular deficits after meningitis (2,4,24Y27). One of these studies indicated that lateral semicircular canal ossification may predict subsequent cochlea ossification (26). The present observations of the routes and pathways of bacterial invasion and spreading may support this hypothesis, as bacterial invasion and inflammation of the lateral semicircular canal are preceded by cochlear invasion and inflammation, suggesting that fibrosis and subsequent ossification are induced in the same order that the invasion appears to occur. As the inflammation is more massive, the process of ossification is likely to be delayed, although this is speculative. In the present study, we found no qualitative differences concerning the pattern of inner ear invasion between the 3 serotypes included. However, a relative absence of bacterial invasion in rats inoculated with serotype 3 pneumococci was noted, as was the fact that these animals did not survive the entire observational period. Unlike the quantitative pattern for serotype 1 and 9 V, serotype 3 pneumococci were not accumulating massively in the cochlea and did only scarcely invade the vestibule and vestibular end organs. Interestingly though, massive cochlear inflammation was noted as the serotype 3 animals succumbed to advanced disease, indicating that extensive inner ear inflammation may also occur subsequent to only sparse or even no bacterial invasion. From the observed differences, it may be speculated that the degree of cochlear damage and delayed cochlear ossification may depend on bacterial serotype, which is supported by a recent clinical study on hearing loss after pneumococcal meningitis (13). The routes and pathways of bacterial invasion of the inner ear and the subsequent inflammation reinforce the need for and relevance of the development of local pharmacologic intervention strategies, by which a far higher inner ear drug concentration and a prevention of hearing loss may be achieved. As suggested in earlier studies (28Y30), this could be accomplished by intratympanic administration of antibiotics and/or anti-inflammatory drugs. A possible synergistic effect could be obtained by cochlear microperfusion (31).
CONCLUSION From the meninges, pneumococci invade the inner ear through the cochlear aqueduct during the first days of infection, whereas hematogenous invasion via the spiral ligament
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