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The effects of superoxide dismutase in gerbils with bacterial meningitis
Otolaryngology– Head and Neck Surgery NOVEMBER 2004
VOLUME 131
NUMBER 5
AWARD WINNERS The effects of superoxide dismutase in gerbils with bacterial meningitis NORMAN N. GE,
MD,
SHAUNA A. BRODIE, STEVEN P. TINLING,
BACKGROUND: Inflammatory products, such as oxygen radicals generated during the course of bacterial meningitis, can damage nerve endings, hair cells, and/or supporting cells in the cochlea. Superoxide dismutase (SOD), an O2-scavenger, has been shown to play an important role in the protection against radical toxicity in various animal experiments. OBJECTIVE: To study the antioxidant effects of SOD on the inflammatory response of gerbils with bacterial meningitis. STUDY DESIGN: Meningitis was induced in three groups of 10 gerbils by intrathecal (IT) injection of Streptococcus pneumoniae into the cisterna magna. Group 1 received IT SOD, group 2 received intramuscular (IM) SOD, and group 3, the control group, received IM normal saline. Histologic data and auditory brainstem responses (ABR) were obtained from each gerbil. RESULTS: Fibrosis and/or neo-ossification were near absent in the IT SOD group and significantly less
From the Department of Otolaryngology–Head and Neck Surgery, University of California, Davis Medical Center, Davis, CA. Presented at the Annual Meeting of the American Academy of Otolaryngology–Head and Neck Surgery, San Diego, CA, September 22-25, 2002. Supported by the Resident Research Grant from the American Academy of Otolaryngology, Head and Neck Surgery Foundation. Winner of 1st place, 2002 Resident Research Award, Clinical Science. Reprint requests: Hilary A. Brodie, MD, PhD, 2521 Stockton Blvd, Suite 7200, Department of Otolaryngology–Head and Neck Surgery, UC Davis, Sacramento, CA 95817; e-mail, [email protected]. 0194-5998/$30.00 Copyright © 2004 by the American Academy of Otolaryngology–Head and Neck Surgery Foundation, Inc. doi:10.1016/j.otohns.2004.03.046
MA,
and HILARY A. BRODIE,
MD, PHD,
Davis, California
fibrosis occurred in the IM group (IT vs. IM: P ⴝ 0.010; IT vs. control group: P ⴝ 0.001). The amount of surviving spiral ganglion cells correlated inversely with the extent of fibrosis (r ⴝ -0.753, P < 0.00001). CONCLUSIONS: IT injection of SOD significantly reduced cochlear fibrosis and neo-ossification, reduced the spiral ganglion cell loss, and decreased damage of the cochlear components following bacterial meningitis. (Otolaryngol Head Neck Surg 2004;131:563-72.)
B
acterial meningitis is the most common cause of both acquired profound bilateral sensorineural hearing loss in childhood and labyrinthitis ossificans.1-3 The reported incidence of hearing loss following meningitis ranges from 6% to 37%, with an estimated 5% suffering from profound deafness.4-8 Inflammatory products, such as oxygen radicals generated during the course of bacterial meningitis, can damage nerve endings, hair cells, and/or supporting cells in the cochlea.9 However, the mechanism responsible for the development of hearing loss in hosts with meningitis has not been fully elucidated. The effects of antiinflammatory agents in humans and animals with bacterial meningitis are under investigation. Among many anti-inflammatory agents, superoxide dismutase (SOD) has been used in numerous animal studies for its scavenger effect on free oxygen radicals and anti-inflammatory properties. Intrathecal (IT) administration has been shown to decrease brain damage during acute ischemic events, but its protective effects in hosts with bacterial meningitis have not been reported. 563
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HEARING LOSS IN BACTERIAL MENINGITIS Historically the three most common organisms responsible for bacterial meningitis are Hemophilus influenza (64%), Streptococcus pneumoniae (16%), and Nisseria meningitides (10%).10-12 The relative frequencies of these bacterial infections are changing with the advent of newer vaccines. The incidence of hearing loss following meningitis is greatest with S. pneumoniae (31%) and lowest with H. influenza (6%). The mortality rate of S. pneumoniae meningitis (19% in children; 20%-30% in adults) is also the highest of the three infecting organisms.13-14 As high as 80% of patients with profound postmeningitic deafness have been noted to have some degree of labyrinthine ossification.8 Bacterial meningitis and the associated inflammatory process may damage various components of the auditory pathway, (i.e., from the cochlea to the auditory cortex). Temporal bone histopathologic studies15-19 have indicated that the likely cause of such sensorineural hearing loss (SNHL) is serous or suppurative labyrinthitis. PATHOLOGY OF LABYRINTHITIS OSSIFICANS Following the onset of labyrinthitis, the inflammatory process proceeds through three characteristic stages: acute, fibrous, and ossification.20-21 In the acute stage, the perilymphatic spaces are infiltrated by bacteria, polymorphonuclear leukocytes (PMN), and a serofibrinous exudate. However, some experimental evidence suggests that the acute inflammatory process may begin before bacteria are present within the inner ear.22 The fibrous stage, which follows the formation of granulation tissue with associated angiogenesis, is composed of hypertrophic fibroblasts and collagen deposition. The ossification stage is characterized by the formation of osteoid and subsequent mineralization. This last stage progresses with bone formation and remodeling, which obliterates the perilymphatic and endolymphatic spaces. The temporal progression of bone formation in labyrinthitis ossificans has not been fully delineated. Ossification in humans has been noted to occur within a year after meningitis even though the hearing loss resulting from meningitis may occur as early as 48 hours after infection.12,23 A cellular infiltrate via the cochlear aqueduct has been noted to develop between several hours and 3 days following the onset of infection in animal models.24,25 INFLAMMATORY PROCESS In bacterial meningitis, a vigorous inflammatory response occurs as a result of the triggering of local host defenses by components of the bacterial cell
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wall.23,26,27 One of the factors that results in higher morbidity and mortality of S. pneumoniae as compared with other organisms is that lipoteichoic acids of the S. pneumoniae cell wall are potent activators of the alternative complement pathway.28,29 Additional pathogenic mechanisms include: 1) cytotoxic bacterial cell wall components; 2) the production of nitric oxide, superoxide, and peroxynitrite by activated inflammatory cells; 3) an increase in excitatory amino acids; and 4) the vascular and hypoxic changes associated with the meningogenic inflammatory process.26 The amount of resultant inflammatory response in the subarachnoid space to S. pneumoniae via these mechanisms is directly proportional to morbidity and mortality.30 REACTIVE OXYGEN SPECIES Vascular and hypoxic changes promote the production of reactive oxygen species. Reactive oxygen species include oxygen-containing molecules with unpaired electrons and metabolites arising from aerobic metabolism. Several critical reactive oxygen species include the superoxide anion O-2; the hydroxyl radical, OH-; and hydrogen peroxide, H2O2. Superoxide can react with nitric oxide, forming peroxynitrite, ONOO-. These species have been shown to indirectly mediate tissue damage by generating complement, lipid, and arachidonic acid-derived chemotactic factors for neutrophils. These active species are generated during postischemic reperfusion of organs, in hyperoxic tissue during acute and chronic inflammation, and during exposure to ionizing radiation. ANTI-INFLAMMATORY AGENTS Host inflammation and the oxygen radicals produced by activated inflammatory cells are directly associated with neuronal injury. Numerous anti-inflammatory agents have the potential to curtail the host inflammatory response to bacterial meningitis. Immunosuppression has resulted in a reduction in hearing loss and other neurologic sequelae associated with bacterial meningitis in several human and animal studies.31-36 Dexamethasone exerts its effects through multiple mechanisms. Dexamethasone decreases prostaglandin E2, tumor necrosis factor, and platelet-activating factor and reduces capillary endothelial cell permeability by inhibiting IL-1.32,36,37 Efficacy of corticosteroid treatment for bacterial meningitis remains controversial, although there is mounting evidence demonstrating a reduction in morbidity with inclusion of corticosteroids in the treatment regimen. Lebel et al32 in a double-blind study compared inclusion of dexamethasone with placebo in the adjunctive management of bacterial meningitis. The investigators found a statistically significant reduction in subsequent sensorineural hearing loss in the steroid-treated group. This finding applied only to
Otolaryngology– Head and Neck Surgery Volume 131 Number 5
meningitis caused by H. influenzae. Prior authors were unable to show the efficacy of steroids as therapy for bacterial meningitis.38,39 A second question that has been attempted to be answered is the role of corticosteroids in preventing labyrinthitis ossificans. Even if steroids were not able to prevent the hearing loss associated with bacterial meningitis, if they could reduce the incidence of associated labyrinthitis ossificans they would play a significant role in allowing for subsequent cochlear implantation in postmeningitis deaf children. Hartnick et al have demonstrated recently some efficacy of systemic steroids in preventing the development of labyrinthitis ossificans in children with pneumococcal meningitis.40 The inflammatory burst which results from the bacteriolytic effects of ampicillin on S. pneumoniae may potentiate the damage due to meningitis and can be prevented with inhibition of the cyclooxygenase pathway.41 DeSautel et al42 demonstrated that the amount of cochlear fibrosis in gerbils following bacterial meningitis was significantly reduced by depleting complement with a component of cobra venom. Amaee et al9 demonstrated that nitric oxide (NO) may play a role in the ototoxicity of pneumolysin, a toxin elaborated from S. pneumoniae. Animals were exposed to intracochlear NO and concurrently treated with SOD or normal saline and SOD conferred marked protection upon the cochlea from the lesions caused by NO donors. SOD has demonstrated therapeutic potential for treating a variety of conditions including inflammation, oxygen toxicity, reperfusion injury, and radiation injury. However, the native enzyme’s short halflife in plasma (6 minutes in mice, 25 minutes in human) limits the enzyme’s effectiveness in many applications.43 Covalent conjugation of SOD with polyethylene glycol (PEG) increases cellular enzyme activities and the circulatory half-lives of these enzymes from less than 10 minutes to 40 hours, thus providing prolonged protection from partially reduced oxygen species.43 SOD has been shown to be effective in prevention of reperfusion injuries in coronary artery occlusion44 and acute bowel infarction.45 The concentrations of SOD used in these studies were between 1000 U/kg and 20,000 U/kg. In summary, S. pneumoniae infection in the central nervous system induces a severe host inflammatory response, which is believed to significantly contribute to labyrinthine end-organ damage and auditory pathway destruction. Oxygen radicals play an important role in the host inflammatory process. The purpose of this study is to investigate the effects of administering SOD, an oxygen radical scavenger, in gerbils with experimentally induced bacterial meningitis, on hearing loss and labyrinthine ossification.
GE et al 565
METHODS The study was conducted with 8-week-old male Mongolian gerbils (Meriones unguiculatus) obtained from Tumblebrook Farm (West Brookfield, MA). The use of the laboratory animals adhered to National Institutes of Health guidelines, with approval by the Animal Use and Care Administrative Advisory Committee of the University of California, Davis. Induction of Meningitis In order to mimic the human experience of exposure of the immune system to Streptococcus pneumoniae prior to hematologic seeding of the central nervous system, the animals were immunized with 0.05 cc of Pneumovax 23 (Merck & Co., Inc., Whitehouse Station, NJ) mixed with an equal amount of Freund’s Complete Adjuvant (Sigma, St. Louis, MO). Three weeks later, auditory thresholds were determined using auditory brainstem responses (ABRs) in three groups of 10 gerbils. Three of the gerbils died during the surgical intervention and were replaced. The animals were anesthetized with intraperitoneal injection of ketamine (Vedco Inc., St. Joseph, MO) (80 mg/kg) and Xylazine (Butler Co., Columbus, OH) (20 mg/kg). All three groups were infected by IT injection of approximately 5 L of 105/mL S. pneumoniae type 3 into the cisterna magna. Following the final injection for each syringe, the remaining solution was cultured to verify bacterial viability and the absence of contamination. Twenty-four hours after the induction of infection, all of the gerbils received daily subcutaneous injections of penicillin G (400 U/g) for 7 days. IT and Intramuscular Administration of SOD PEG-SOD (Sigma) (one milligram protein contains 4000 units PEG-SOD) was dissolved in 20 mL of artificial cerebrospinal fluid (CSF; NaCl 138.70 mM, KCl 3.35 mM, MgCl2 1.16 mM, NaHCO3 20.95 mM, NaH2PO4 . H2O 59.0 mM, glucose 3.39 mM, CaCl2 1.26 mM). On post-op days 1 and 3, group 1 was treated with IT SOD, group 2 was treated with intramuscular (IM) SOD, and group 3 was given IM normal saline. In group 1 a head mount was designed to facilitate the injection (Fig 1). A stainless steel anchoring screw (0-80 x 1/8 in, Small Parts Inc, Miami, FL) was modified by drilling a hole in the center to accommodate 0.025 in Silastic tubing (Dow Corning Corp, Midland, MI). A burr hole was made through the gerbils’ parietal bone. The dura was then entered with a pick and the soft silastic tubing was slid between the dura and the cerebral cortex. The screw was rotated 360 degrees, fixing it to the skull. Dental cement was used to secure
566 GE et al
Fig 1. Head mount implanted in a gerbil’s skull. The insert shows a head mount prior to the implantation.
the screw and tubing to the skull. The scalp was then closed with only a segment of tubing exposed. The tubing was folded and ligated with a suture to prevent CSF leak. During each injection the ligature was removed and 10,000 U/kg of PEG-SOD was injected into the gerbils’ subarachnoid space with a Hamilton syringe. The ligature was then replaced after each injection. In group 2, 10,000 U/kg of PEG-SOD was injected into the gerbils’ lower extremities. In group 3, IM injection of 20 L of normal saline was performed in gerbils’ lower extremities. Auditory Brainstem Evoked Response (ABR) Gerbils were anesthetized intraperitoneally with a mixture of ketamine (80 mg/kg) and Xylazine (20 mg/ kg). Otoscopy was performed in order to screen for otitis externa, myringitis, otitis media with effusion, acute suppurative otitis media, and cholesteatoma. The presence of any of these conditions would have resulted in elimination from the study but was never noted. The temperature of the animals was actively maintained at 37.5oC ⫾ 0.5oC by a digitally controlled heating pad. All recordings were performed in an acoustically insulated and electrically shielded chamber. Auditory thresholds were assessed using auditory brainstem response testing. Acoustic stimuli were digitally produced using the Tucker Davis Technologies (TDT) System II AP2 array processor (Alchua, FL) and S232 device driver and controller. The phase of the stimuli was alternated, canceling cochlear microphonics. Each stimulus had a Gaussian rise and fall time of 3 cycles and a plateau of 9 cycles. The tone bursts were produced with a duty cycle of 101 ms and transduced by a piezoelectric headphone. Sound was delivered to the ear of the gerbil via a 2-mm-diameter
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tube, loosely sealed to the external auditory canal. The acoustic stimulus delivered to the ear canal was assessed with the use of a calibrated 1/4⬙ B&K probe microphone (Norcross, GA). The intensity of the sound was calibrated with a continuous tone at 60 dB sound pressure level (SPL), using the output of the microphone as delivered to both a B&K 2209 SPL Meter and a Hewlett Packard 35670A Dynamic Signal Analyzer (Palo Alto, CA). The averaging capability of the signal analyzer allows the microphone output to be analyzed at 20 to 30 dB below its noise floor. This increases the effective sensitivity of the microphone in the acoustically shielded test chamber to 20 to 25 dB SPL for frequencies from 1 to 70 kHz. The output of stainless steel electrodes placed at the scalp vertex and the soft palate was preamplified with active filtering (300-5000 Hz) by TDT DB4 headstage within the chamber (total gain ⫽ 103). The responses were averaged (10 ms window, 5 s/address) for 500 passes. Auditory brainstem response thresholds were determined in response to 4-kHz, 8-kHz, and 16-kHz tone pips. An ABR was initially obtained in response to 75-dB SPL tone bursts. After replicating the ABR, the stimulus intensity level was then reduced by 10 dB and the process repeated. Subsequent ABRs were measured at successively lower sound levels until a readily identifiable ABR could not be obtained and replicated, at which time the intensity was increased by 5 dB. The threshold was extrapolated as being between the lowest intensity at which it could be clearly observed. All gerbils in this study had a normal pre-op pure tone threshold at 4 kHz, 8 kHz, and 16 kHz as determined by ABR testing. On post-op day four, ABRs were performed on all gerbils and the results were analyzed. After 3 months, the animals were sacrificed and fixed by trans-cardial perfusion with saline followed by 2% paraformaldehyde and 2% glutaraldehyde in 0.5 m sodium cacodylate. The bullae were harvested and decalcified in 0.1 M tetrasodium ethylenediamine tetraacetic acid in the above fixative and then dissected to expose the cochleae. They were then osmicated, dehydrated in graded acetone, and embedded in eponaraldyte. Each cochlea was serially divided into approximately 10 to 15 slices using a 0.04-mm-thick diamond-wafering blade. A mid-modiolar slice was identified and sectioned at 0.5 um and stained with toluidine blue and basic fuchsin. The slides were assessed for the area of cochlear lumen filled by fibrosis and/or neo-ossification in the basal, mid, and apical turns. Video images were captured and calibrated areas measured using SigmaScan Pro® image analysis software (Jandel Corp., San Rafael, CA). The number of surviving spiral ganglion cells was recorded for the basal, mid, and apical turns. The status of the organ of
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Fig 2. Histologic section through the basal turn of the cochlea from a postmeningitic animal that received IT SOD injection, demonstrating no labyrinthitis ossificans, normal organ of Corti, stria vascularis, spiral ligament, and preservation of spiral ganglion cells. SG, spiral ganglion cells; OC, organ of Corti; SV, stria vascularis; SL, spiral ligament. (Magnification 180X; stained with toluidine blue and basic fuchsin.)
Fig 3. Histologic section through the basal turn of the cochlea from a postmeningitic animal that received IT SOD injection, demonstrating no labyrinthitis ossificans. The organ of Corti is structurally intact with some cellular damage (Grade 2), stria vascularis has decreased cellularity and mild apoptotic changes (Grade 2), spiral ligament has decreased matrix and cellularity (Grade 2), and spiral ganglion cells were preserved. SG, spiral ganglion cells; OC, organ of Corti; SV, stria vascularis; SL, spiral ligament. (Magnification 180X; stained with toluidine blue and basic fuchsin.)
Corti, stria vascularis, and the spiral ligament were assessed. The criteria for grading these structures are as follows: Organ of Corti 1) Hair cell status (healthy, incomplete loss, complete loss).
GE et al 567
Fig 4. Histologic section through the basal turn of the cochlea from a postmeningitic animal that received IM SOD injection, demonstrating mild labyrinthitis ossificans. The organ of Corti is structurally damaged with few residual cells (Grade 3), stria vascularis shows significantly decreased cellularity with moderate to severe apoptotic changes (Grade 3), spiral ligament shows paucity of cells (Grade 3), and spiral ganglion cellularity is significantly decreased. SG, spiral ganglion cells; OC, organ of Corti; SV, stria vascularis; SL, spiral ligament. (Magnification 180X; stained with toluidine blue and basic fuchsin.)
Fig 5. Histologic section through the basal turn of the cochlea from a postmeningitic animal that received IM saline, demonstrating severe labyrinthitis ossificans. Severe structural damage to organ of Corti, stria vascularis, and spiral ligament. Spiral ganglion cells were not preserved. (Magnification 180X; stained with toluidine blue and basic fuchsin.)
2) Structure integrity (inner tunnel of Corti, supporting cells). 3) Surrounding structures (basilar membrane, tectorial membrane). 4) Grade 1-4 defined as: Grade 1, normal (Fig 2). Grade 2, structurally intact with some cellular damage (Fig 3). Grade 3, few residual cells, some structural
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Table 1. Percentage of total fibrosis in each group
1 2 3 4 5 6 7 8 9 10 Average
IT Group
IM Group
Control Group
0 * 0 0 10 0 0 0 0 2.5 1.39
0 64.8 69.9 39.9 32.4 73.3 0 0 52.2 0 33.25
100 100 100 89.1 46.2 34.1 69.6 13.7 40.1 100 69.28
IT, intrathecal; IM, intramuscular. *This gerbil died prior to sacrifice.
damage (Fig 4). Grade 4, no residual structure (Fig 5). Stria vascularis 1) Cell density. 2) Cell morphology. 3) Grade 1-4 defined as: Grade 1, normal (Fig 2); Grade 2, decreased cellularity with mild apoptotic changes (Fig 3); Grade 3, significant decrease in cellularity with moderate to severe apoptotic changes (Fig 4); Grade 4, structure destroyed (Fig 5). Spiral Ligament 1) Cell density and morphology. 2) Grade 1-4 defined as: Grade 1, normal (Fig 2); Grade 2, decreased matrix and cellularity (Fig 3); Grade 3, paucity of cells (Fig 4); Grade 4, structure destroyed (Fig 5). RESULTS Histopathology Histologic analysis of the cochlea in a mid-modiolar section in group 1 (IT SOD group, n ⫽ 10) demonstrated 20% of animals had some degree of fibrosis which was limited to the scala tympani of the basal turn. There was no neo-ossification present. The mean area of luminal space containing fibrosis was 1.4%. Sixty percent of the animals in group 2 (IM SOD group, n ⫽ 10) and 100% of the control group (n ⫽ 10) developed fibrosis or neoossification within the cochlea. The mean area of luminal space containing fibrosis or neo-ossification in the IM SOD group was 33.2% and 69.3% in the control group. The degree of labyrinthitis ossificans was significantly less in the IT SOD group compared with the IM SOD and control (P ⫽ 0.010, IT vs IM group; P ⫽ 0.001, IT vs control group) (Table 1). There was also significantly less fibrosis in the IM group vs control group (P ⫽ 0.02, IM vs control group) (Table 1). Fibrosis and neo-ossification filled the lumen of all 3 scala in the severe cases and was more limited to the scala tympani in mild cases.
In the IT SOD group, the histology of the organ of Corti, stria vascularis, and spiral ligament all appeared to be normal or near normal (Figs 2 and 3). There was also a large number of healthy-appearing spiral ganglion cells in all turns of the cochlea despite the severe hearing loss in this group. The mean density of spiral ganglion cells per mm2 was 2815.76 (Table 2). In the IM SOD group, the histology of organ of Corti, stria vascularis, and spiral ligament varied across the entire spectrum from normal-appearing to complete destruction (Fig 4). The mean density of spiral ganglion cells was 1666.14 per mm2 (Table 3). In the control group, there were no normal-appearing structures and nearly all of the gerbils developed significant fibrosis in all turns of their cochleae (Fig 5). Very few animals avoided complete destruction of the organ of Corti, stria vascularis, and spiral ligament. The mean density of residual spiral ganglion cells was 138.70 per mm2 (Table 4). In all groups, the number of surviving spiral ganglion cells was strongly inversely correlated with the amount of fibrosis ( r⫽ -0.75) (Tables 2– 4). There was also a correlation between the amount of fibrosis and the degree of damage of stria vascularis (r ⫽ 0.78), organ of Corti (r⫽ 0.80), and spiral ligament (r ⫽ 0.81) (Tables 2-4). The hearing loss in each frequency correlated with the status of the organ of Corti at the corresponding turn of the cochlea ( r⫽ 0.57) (Tables 2– 4). Consistent with human temporal bone histopathology in postmeningitic patients, there was more fibrosis in the basal turn than in the apical turn. Auditory Function In the IT SOD group, the average deterioration in pure tone thresholds between the preoperative baseline and 4-day postinduction of meningitis at 4 kHz, 8 kHz, and 16 kHz was 41, 44.5, and 38.5 dB, respectively. In the IM SOD the average deterioration in thresholds at the 3 frequencies was 39, 44, and 38.5 dB. In the control group, the average deterioration in thresholds at the 3 frequencies was 53.5, 51.5, and 47 dB, respectively. The control group developed 20% greater deterioration of their pure tone thresholds than the animals treated with IM or IT SOD, although this difference was not statistically significant (P ⫽ 0.23, IT vs control; P ⫽ 0.11, IM vs control) (Table 2). DISCUSSION There are multiple sequelae from bacterial meningitis that affect an individual’s hearing and potential for aural rehabilitation. These sequelae include central auditory pathway injuries, spiral ganglion cell loss, cochlear hair cell and supporting cell damage, stria vascularis and spiral ligament injury, and
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Table 2. IT injection group ABR threshold shift on POD#4 (dB) and histologic analysis Threshold Shift (dB)
Basal Turn
No.
4K
8K
16K
OC
1 2 3 4 5 6 7 8 9 10 AV
0 40 5 55 60 60 60 10 60 60 41
10 45 25 50 65 60 55 10 65 60 44.5
0 30 40 50 55 50 45 0 60 55 38.5
1 * 2 1 2 † 2 1 3 2 1.75
SV
Mid Turn
SL
SG
FB
OC
1
1
3041
0
1
1 2 4 1 1 1 2 2 1.67
2 2 3 2 2 2 2 2 2
3418 1292 790 † 2243 2920 2880 2350 2366
0 0 0 7.4 0 0 0 0 0.82
1 1 2 2 2 1 2 2 1.56
SV
Apical Turn
SL
SG
FB
SG
FB
1
1
4054
0
3862
0
1 1 1 1 1 1 2 3 1.33
2 2 2 2 2 2 3 2 2
2833 4311 3524 3067 3181 3006 2620 2561 3239
0 0 0 0 0 0 0 0 0
2566 2323 2935 3317 2881 † 2967 1876 2840
0 0 0 0 0 0 0 0 0
OC, organ of Corti; SV, stria vascularis; SL, spiral ligament; SG, spiral ganglion; #/mm2 FB, % of fibrosis; AV, average. *This gerbil died prior to sacrifice. †Not able to assess due to the oblique cut of the section.
Table 3. IM injection group ABR threshold shift on POD#4 (dB) and histologic analysis Threshold Shift (dB)
Basal Turn
Mid Turn
No.
4K
8K
16K
OC
SV
SL
SG
1 2 3 4 5 6 7 8 9 10 AV
5 55 55 55 25 60 10 5 55 65 39
10 55 55 50 60 65 15 10 55 65 44
5 50 50 40 55 60 15 5 55 50 38.5
1 4 4 3 3 4 2 1 4 2 2.8
1 3 4 4 4 4 1 1 4 1 2.7
2 3 4 3 3 4 1 2 4 2 2.8
3500 659 0 0 0 0 4160 * 167 1773 1139
Apical Turn
FB
OC
SV
SL
SG
0 86 54 38 40 91 0 0 100 0 40.9
1 4 4 2 3 3 2 1 4 2 2.6
1 3 4 2 3 4 1 1 4 1 2.4
2 3 3 2 2 3 1 1 4 2 2.3
3516 243 0 1862 1909 0 3427 3450 237 3875 1851
FB 0 29 87 0 0 27 0 0 100 0 24.3
SG
FB
3059 793 0 2192 2885 823 5752 * 549 * 2006
0 0 100 0 0 0 0 0 100 0 20
OC, organ of Corti; SV, stria vascularis; SL, spiral ligament; SG, spiral ganglion; FB, fibrosis; AV, average. *Not able to assess due to the oblique cut of the section.
Table 4. Control group ABR threshold shift on POD#4 (dB) and histologic analysis Threshold Shift (dB)
Basal Turn
Mid Turn
No.
4K
8K
16K
OC
SV
SL
SG
1 2 3 4 5 6 7 8 9 10 AV
55 60 50 50 55 55 50 55 50 55 53.5
55 50 45 45 55 50 50 55 55 55 51.5
45 45 35 40 55 50 45 50 50 55 47
4 4 4 4 4 4 4 4 4 4 4
4 4 4 4 4 4 4 3 4 4 3.9
4 4 4 4 3 4 3 3 4 2 3.5
0 0 0 0 0 0 0 0 0 0 0
Apical Turn
FB
OC
SV
SL
SG
FB
100 100 5 43 33 100 100 37 100 44 66.2
4 4 4 4 4 4 4 4 4 3 3.9
4 4 4 4 4 4 2 3 4 3 3.6
4 4 4 3 3 4 2 3 4 2 3.3
0 0 0 0 0 0 0 525 0 1107 163.2
100 100 100 100 63 100 41 25 100 0 72.9
SG 0 0 0 0 0 0 0 0 2276 252.8
FB 100 100 34 100 42 100 16 29 100 0 62.1
OC, organ of Corti; SV, stria vascularis; SL, spiral ligament; SG, spiral ganglion; FB, fibrosis; AV, average.
the development of labyrinthitis ossificans. Various mechanisms have been identified that, although necessary in the response to pathogens, contribute to the injury to these structures. Many of the mechanisms result in bystander injury by induction of the local
immune response in the brain and cochlea.22,25-30 Streptococcus pneumoniae cell wall lipoteichoic acids are potent activators of the alternative complement pathway.28,29 An excessive degree of inflammation can result from the explosive release of these
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sub-components of the cell wall and subsequent activation of the complement cascade.41 The blood-brain barrier effectively sequesters the CSF space from serum-associated host defenses and CSF is normally deficient in complement factors C3 and C5, immunoglobulin, and leukocytes, although some resident macrophages are present.29,46-48 The majority of plasma complement components are made in hepatic parenchymal cells.49 However, after cytokine stimulation, reactive astrocytes, microglia, macrophages, and neutrophils develop receptors to the complement anaphlotoxies, C3a and C5a, and participate in the active production of their precursors, C3 and C5.50 Disruption of the blood-brain barrier also contributes to low levels of complement in the CSF.51,52 Activation of the alternative pathway results in the cleavage and activation of C3, producing C3a and C3b. The C3a fragment binds to receptors on mast cells and basophils, resulting in the degranulation of histamine and other cytokines. This results in increased vascular permeability and capillary leakage. C5, when in complex with C3b, can be cleaved by C5 convertase, leading to the formation of C5a and C5b. C5a increases vascular permeability and is a potent chemotaxin in CSF, accelerating leukocytosis.29,53 Complement C5a also induces the release of hydrolytic enzymes from neutrophils with resultant secondary innocent bystander damage. Another mechanism of host-cell injury involves the incorporation of the pneumococcal Forssman antigen into eukaryotic cell membranes, resulting in complement-induced lysis of host cells.54 These mechanisms may explain some of the organ of Corti and spiral ganglion cell injury. In addition to activation of the alternative pathway of the complement cascade, data from in vivo experiments indicate that the S. pneumoniae produce cell-wall components that activate monocytes, leukocytes, cerebrovascular endothelial cells, and astrocytes.20,55 These cells, in turn, produce various pro-inflammatory cytokines such as IL-1, IL-6, IL-8, TNF, and PAF,26,56-59 or express specific receptors on their surface.50 Teichoic and lipoteichoic acids bind the acute phase reactant C-reactive protein, activate procoagulant activity on the surface of endothelial cells, induce cytokines, and initiate the influx of leukocytes.40,54,60 These cytokines and receptors initiate an accelerating cascade of events, resulting in alteration of the bloodbrain barrier, PMN and serum protein infiltration, meningeal inflammation, increased intracranial pressure, and decreased cerebral vascular perfusion.61 Transient and permanent neuronal injury appears to be related to the production of nitric oxide (NO).26 NO is produced by NO synthase (NOS), which con-
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verts arginine and oxygen into citrulline and NO. There are two different types of NOS, constitutive and inducible. Inducible NOS (iNOS) is generated by macrophages in response to specific cytokines. iNOS has been demonstrated to be present in guinea pig cochlea following long-term exposure to bacterial toxins.62 NO has been implicated in causing cochlear damage.63 Microperfusion of the scala tympani with NO donors resulted in reduced cochlear compound action potentials and cochlear microphonics together with hair-cell and organ of Corti damage in adult guinea pigs. Preperfusion of the cochlea with L-methyl arginine, which inhibits the release of NO, or with SOD provides protection from the NO donors.9,63 In the current study, acute hearing loss was not significantly reduced by the administration of SOD. There was, however, a significant decrease in the development of postmeningitic intracochlear fibrosis and neo-ossification. IT SOD also protected the animals from damage to the organ of Corti, stria vascularis, and spiral ligament. Most importantly, IT SOD prevented the loss of spiral ganglion cells. Given the gross preservation of cochlear structures with the IT SOD, it is possible that had longitudinal ABRs been performed, there may have been some recovery of function. Subsequent studies will investigate this possibility. One of the possible explanations for the acute hearing loss in the SOD-treated animals is that the 24-hour delay in administration of the drug may have allowed for inflammatory damage to occur in the organ of Corti. Inhibition of the reactive oxygen species, although too late to interfere with the initial organ of Corti injury, appears to prevent subsequent structural damage as well as blocking the initiation of labyrinthitis ossificans. Maintaining a patent cochlear lumen, despite failure to protect from deafness, may make aural rehabilitation possible with cochlear implant. The inverse correlation between the degree of luminal fibroses and the preservation of spiral ganglion cells also may contribute to the potential success of cochlear implantation. CONCLUSION IT injection of SOD significantly inhibited cochlea fibrosis and neo-ossification following bacterial meningitis by inhibiting oxygen free radicals which form in the course of the inflammatory process. Maintenance of the cochlear lumen and spiral ganglion cell populations improves the potential for aural rehabilitation in postmeningitic deafness. Despite profound deafness, the animals that did not go on to develop labyrinthitis ossificans had greater preservation of spiral ganglion cells as well as the organ of Corti, stria vascularis, and spiral ligament.
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References 1. Vernon M. Meningitis and deafness: the problem, its physical, audiological, psychological, and educational manifestations in deaf children. Laryngoscope 1967;77:1856-74. 2. Stutman HR, Marks MI. Bacterial meningitis in children: diagnosis and therapy. Clin Pediatr (Phila) 1987;26:431-8. 3. Nadol JB Jr. Hearing loss as a sequela of meningitis. Laryngoscope 1978;68:739-55. 4. Rosenhall U, Nylen O, Lindberg J, et al. Auditory function after haemophilus influenzae meningitis. Acta Otolaryngol (Stockh) 1978;85:243-7. 5. Keane WM, Potsic WP, Rowe LD, et al. Meningitis and hearing loss in children. Arch Otolaryngol Head Neck Surg 1979;105:9-44. 6. Berlow SJ, Caldarelli DD, Matz GJ, et al. Bacterial meningitis and sensorineural hearing loss: a prospective investigation. Laryngoscope 1980;90:1445-52. 7. Finitzo-Hieber T, Simhadri R, Hieber JP. Abnormalities of the auditory brainstem response in post-meningitic infants and children. Intern J Pediatr Otorhinolaryngol 1981;3:275-86. 8. Eisenberg LS, Luxford WM, Becher TS, et al. Electrical stimulation of the auditory system in children deafened by meningitis. Otolaryngol Head Neck Surg 1984;92:700-5. 9. Amaee FR, Comis SD, Osborne MP, et al. Possible involvement of nitric oxide in the sensorineural hearing loss of bacterial meningitis. Acta Otolaryngol (Stockh) 1997(117):329-36. 10. Dodge PR, Davis H, Feigin RD, et al. Prospective evaluation of hearing impairment as a sequela of acute bacterial meningitis. N Engl J Med 1984;311:869-74. 11. Friedmann I, Arnold W. Pathology of the Ear. New York: Churchill Livingston; 1993. P. 752. 12. Novak MA, Fifer RC, Barkmeir JC. Labyrinthine ossification after meningitis: its implications for cochlear implantation. Otolaryngol Head Neck Surg 1990;103:351-6. 13. Wenger JD, Hightower AW, Facklam FF, et al. Bacterial meningitis in the United States, 1986: A report of a multistate surveillance study. J Infect Dis 1986;162:1316-23. 14. Schlech WF, III JI, Ward JD, et al. Bacterial meningitis in the United States, 1978-1981. The national bacterial meningitis surveillance study. JAMA 1985;253:1749-54. 15. Sugiura S, Paparella MM. The pathology of labyrinthine ossification. Laryngoscope 1967;77:1974-89. 16. Kimura R, Perlman JB. Arterial obstruction of the labyrinth Part I. Cochlear changes. Part II. Vestibular changes. Ann Otol Rhinol Laryngol 1958;67:5-24;25-40 17. Fredrickson JM, Griffith AW, Lindsay JR. Transverse fracture of the temporal bone: a clinical and histopathologic study. Arch Otolaryngol 1963;78:770-84. 18. Ward PH. The histopathology of auditory and vestibular disorders in head trauma. Ann Otol Rhinol Laryngol 1969;78:227-38. 19. Benitez JT, Bouchard KR, Lane-Szopo D. Pathology of deafness and disequilibrium in head injury: A human temporal bone study. Am J Otol 1980;1:163-7. 20. Paparella MM, Sugiura S. The pathology of suppurative labyrinthitis. Ann Otol Rhinol Laryngol 1967;76:554-86. 21. Brodie HA, Thompson TC, Vassilian L. Induction of labyrinthitis ossificans after pneumococcal meningitis: An animal model. Otolaryngol Head Neck Surg 1998;118:15-21. 22. Bhatt SM, Laurentano A, Cabellos C. Progression of hearing loss in experimental pneumococcal meningitis: correlation with cerebrospinal fluid cytochemistry. J Infect Dis 1993;167:675-83. 23. Tuomanen E. Molecular and cellular mechanisms of pneumococcal meningitis. Ann N Y Acad Sci 1997;797:42-52. 24. Blank AL, Davis GL, Van de Water TR, et al. Acute Streptococcus pneumoniae meningogenic labyrinthitis. Arch Otolaryngol Head Neck Surg 1994;120:1342-6. 25. Kesser BW, Hashisaki GT, Spindel JH, et al. Time course of hearing loss in an animal model of pneumococcal meningitis. Otolaryngol Head Neck Surg 1999;120:628-37. 26. Pfister HW, Fontana A, Tauber MG, et al. Mechanisms of brain injury in bacterial meningitis: workshop summary. Clin Infect Dis 1994;19:63-79.
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27. Tuomanen E. Breaching the blood-brain barrier. Sci Am 1993;168:80-4. 28. Tuomanen E, Tomasz A, Hengstler B. The relative role of bacterial cell wall and capsule in the induction of inflammation in pneumococcal meningitis. J Infect Dis 1985;151:535-40. 29. Tuomanen E, Hengstler B, Zak O, et al. Nonsteroidal antiinflammatory agents in the therapy for experimental pneumococcal meningitis. J Infect Dis 1987;155:985-90. 30. McAlister CK, O’Donoghue JM, Teaty HN. Experimental pneumococcal meningitis. II. Characterization and quantitation of the inflammatory process. J Infect Dis 1975;132:355-60. 31. Bhatt SM, Cabellos C, Nadol JB Jr, et al. The impact of dexamethasone on hearing loss experimental pneumococcal meningitis. Pediatr Infect Dis J 1994;14:93-6. 32. Lebel MH, Freij BJ, Syrogiannopoulos GA, et al. Dexamethasone therapy for bacterial meningitis. N Engl J Med 1988;319:964-71. 33. Girgis NI, Farid Z, Mikhail IA, et al. Dexamethasone treatment for bacterial meningitis in children and adults. Pediatr Infect Dis J 1989;8:848-51. 34. Odio CM, Faingezicht I, Paris M, et al. The beneficial effects of early dexamethasone administration in infants and children with bacterial meningitis. N Engl J Med 1991;324:1525-31. 35. Kennedy WA, Howyt JJ, McCracken GH Jr. The role of corticosteroid therapy in children with pneumococcal meningitis. Am J Dis Child 1991;145:1374-8. 36. Mustafa MM, Lebel MH, Ramilo O, et al. Correlation of interleukin-1( and cachectin concentration in cerebrospinal fluid and outcome from bacterial meningitis. J Pediatr 1989;115:208-13. 37. Mustafa MM, Ramilo O, Saez-Llorens X, et al. Cerebrospinal fluid, prostaglandins, interleukin-1(, and tumor necrosis factor in bacterial meningitis. Clinical and laboratory correlation in placebo-treated and dexamethasone-treated patients. Am J Dis Child 1990;144:883-7. 38. DeLemos RA, Hahherty RJ. Corticosteroids as an adjunct to treatment in bacterial meningitis: a controlled clinical trial. Pediatrics 1969;44:30-4. 39. Belsey MA, Hoffpauir CW, Smith MH. Dexamethasone in the treatment of acute bacterial meningitis: the effect of study design on the interpretation of results. Pediatrics 1969;44:503-13. 40. Hartnick CJ, Kim HY, Chute PM, et al. Preventing labyrinthitis ossificans: the role of steroids. Arch Otolaryngol Head Neck Surg 2001;127:180-3. 41. Tuomanen E, Hengstler B, Rich R, et al. Nonsteroidal antiinflammatory agents in the therapy for experimental pneumococcal meningitis. J Infect Dis 1987;155:985-90. 42. DeSautel MG, Brodie HA. Effects of depletion of complement in the development of labyrinthitis ossificans. Laryngoscope 1999;109:1674-8. 43. Somack R, Saifer MG, William LD. Preparation of long-acting superoxide dismutase using high molecular weight polyethylene glycol (41k-72k daltons). Free Radic Res Commun 1991;1213(Pt 2):553-62. 44. Tamura Y, Saito S, Hatano M, et al. Limitation of myocardial infarct size by polyethylene glycol– conjugated superoxide dismutase in a canine model of 90 min coronary occlusion followed by 4 days of reperfusion. Respiration and Circulation 1989;37:201-8. 45. Dalsing MC, Grosfeld JL, Shiffler MA, et al. Superoxide dismutase: a cellular protective enzyme in bowel ischemia. J Surg Res 1983;34:589-96. 46. Harris JP, Ryan AF. Fundamental immune mechanisms of the brain and inner ear. Otolaryngol Head Neck Surg 1995;112(6):639-53. 47. Quagliarollo VJ, Long WJ, Scheld WM. Morphologic alterations of the blood-brain barrier with experimental meningitis in the rat: temporal sequence and role of encapsulation. J Clin Invest 1986;77:1084-95. 48. Jahnke K. The blood-perilymph barrier. Arch Otorhinolaryngol 1980;228:29-34. 49. Roitt IM, Brostoff J, Male DK. Immunology. St. Louis: C.V. Mosby Company; 1985. P. 266. 50. Gasque P, Singhrao SK, Neal JW, et al. The receptor for complement anaphylatoxin C3a is expressed by myeloid cells and
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51.
52. 53.
54. 55. 56.
nonmyeloid cells in inflamed human central nervous system: analysis in multiple sclerosis and bacterial meningitis. J Immunol 1998;160:3543-54. Zwahlen A, Nydegger UE, Vaudaux P, et al. Complementmediated opsonic activity in normal and infected human cerebrospinal fluid: early response during bacterial meningitis. J Infect Dis 1982;145(5):635-46. Morgan BP, Gasque P. Expression of complement in the grain: role in health and disease. Immunol Today 1996;17(10):461-6. Ernst JD, Hartiala KT, Goldstein IM, et al. Complement (C5)derived chemotactic activity accounts for accumulation of polymorphonuclear leukocytes in cerebrospinal fluid of rabbits with pneumococcal meningitis. Infect Immun 1984;46(1):81-6. Hummell DS, Swirt AJ, Tomasz A, et al. Activation of the alternative complement pathway by pneumococcal lipoteichoic acid. Infect Immun 1985;47:384-7. Tuomanen E, Liu H, Hengstler B, et al. The induction of meningeal inflammation by components of the pneumococcal cell wall. J Infect Dis 1985;151(5):859-68. Waage A, Halstensen A, Shalaby R, et al. Local production of tumor necrosis factor (, interleukin 1 and interleukin 6 in meningococcal meningitis. J Exp Med 1989;170:1859-67.
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57. Beutler B, Krochin N, Milsark IW, et al. Control cachectin (tumor necrosis factor) synthesis: mechanisms of endotoxin resistance. Science 1986;232:977-80. 58. Movat HZ. Tumor necrosis factor and interleukin-1: role in acute inflammation and microvascular injury. J Lab Clin Med 1987;110:668-81. 59. Tracey KJ, Lowry SF, Cerami A. Cachectin: a hormone that triggers acute shock and chronic cachexia. J Infect Dis 1988;157:413-20. 60. Fischer J, Tomasz A. Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae. J Bacteriol 1985; 163:46-54. 61. Schaad UB, Kaplan SL, Mccracken GH Jr. Steroid therapy for bacterial meningitis. Clin Infect Dis 1995;20:685-90. 62. Takumida M, Anniko M, Popa R. Possible involvement of free radicals in lipopolysaccharide-induced labyrinthitis in the guinea pig: a morphological and functional investigation. ORL J Otorhinolaryngol Relat Spec 1998;Sep-Oct;60(5):246-53. 63. Farzin RA, Comis SD, Osborn MP, et al. Possible involvement of nitric oxide in the sensorineural hearing loss of bacterial meningitis. Acta Otolaryngol (Stockh) 1997;117:329-36.
VOLUME 131
NUMBER 5
AWARD WINNERS The effects of superoxide dismutase in gerbils with bacterial meningitis NORMAN N. GE,
MD,
SHAUNA A. BRODIE, STEVEN P. TINLING,
BACKGROUND: Inflammatory products, such as oxygen radicals generated during the course of bacterial meningitis, can damage nerve endings, hair cells, and/or supporting cells in the cochlea. Superoxide dismutase (SOD), an O2-scavenger, has been shown to play an important role in the protection against radical toxicity in various animal experiments. OBJECTIVE: To study the antioxidant effects of SOD on the inflammatory response of gerbils with bacterial meningitis. STUDY DESIGN: Meningitis was induced in three groups of 10 gerbils by intrathecal (IT) injection of Streptococcus pneumoniae into the cisterna magna. Group 1 received IT SOD, group 2 received intramuscular (IM) SOD, and group 3, the control group, received IM normal saline. Histologic data and auditory brainstem responses (ABR) were obtained from each gerbil. RESULTS: Fibrosis and/or neo-ossification were near absent in the IT SOD group and significantly less
From the Department of Otolaryngology–Head and Neck Surgery, University of California, Davis Medical Center, Davis, CA. Presented at the Annual Meeting of the American Academy of Otolaryngology–Head and Neck Surgery, San Diego, CA, September 22-25, 2002. Supported by the Resident Research Grant from the American Academy of Otolaryngology, Head and Neck Surgery Foundation. Winner of 1st place, 2002 Resident Research Award, Clinical Science. Reprint requests: Hilary A. Brodie, MD, PhD, 2521 Stockton Blvd, Suite 7200, Department of Otolaryngology–Head and Neck Surgery, UC Davis, Sacramento, CA 95817; e-mail, [email protected]. 0194-5998/$30.00 Copyright © 2004 by the American Academy of Otolaryngology–Head and Neck Surgery Foundation, Inc. doi:10.1016/j.otohns.2004.03.046
MA,
and HILARY A. BRODIE,
MD, PHD,
Davis, California
fibrosis occurred in the IM group (IT vs. IM: P ⴝ 0.010; IT vs. control group: P ⴝ 0.001). The amount of surviving spiral ganglion cells correlated inversely with the extent of fibrosis (r ⴝ -0.753, P < 0.00001). CONCLUSIONS: IT injection of SOD significantly reduced cochlear fibrosis and neo-ossification, reduced the spiral ganglion cell loss, and decreased damage of the cochlear components following bacterial meningitis. (Otolaryngol Head Neck Surg 2004;131:563-72.)
B
acterial meningitis is the most common cause of both acquired profound bilateral sensorineural hearing loss in childhood and labyrinthitis ossificans.1-3 The reported incidence of hearing loss following meningitis ranges from 6% to 37%, with an estimated 5% suffering from profound deafness.4-8 Inflammatory products, such as oxygen radicals generated during the course of bacterial meningitis, can damage nerve endings, hair cells, and/or supporting cells in the cochlea.9 However, the mechanism responsible for the development of hearing loss in hosts with meningitis has not been fully elucidated. The effects of antiinflammatory agents in humans and animals with bacterial meningitis are under investigation. Among many anti-inflammatory agents, superoxide dismutase (SOD) has been used in numerous animal studies for its scavenger effect on free oxygen radicals and anti-inflammatory properties. Intrathecal (IT) administration has been shown to decrease brain damage during acute ischemic events, but its protective effects in hosts with bacterial meningitis have not been reported. 563
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HEARING LOSS IN BACTERIAL MENINGITIS Historically the three most common organisms responsible for bacterial meningitis are Hemophilus influenza (64%), Streptococcus pneumoniae (16%), and Nisseria meningitides (10%).10-12 The relative frequencies of these bacterial infections are changing with the advent of newer vaccines. The incidence of hearing loss following meningitis is greatest with S. pneumoniae (31%) and lowest with H. influenza (6%). The mortality rate of S. pneumoniae meningitis (19% in children; 20%-30% in adults) is also the highest of the three infecting organisms.13-14 As high as 80% of patients with profound postmeningitic deafness have been noted to have some degree of labyrinthine ossification.8 Bacterial meningitis and the associated inflammatory process may damage various components of the auditory pathway, (i.e., from the cochlea to the auditory cortex). Temporal bone histopathologic studies15-19 have indicated that the likely cause of such sensorineural hearing loss (SNHL) is serous or suppurative labyrinthitis. PATHOLOGY OF LABYRINTHITIS OSSIFICANS Following the onset of labyrinthitis, the inflammatory process proceeds through three characteristic stages: acute, fibrous, and ossification.20-21 In the acute stage, the perilymphatic spaces are infiltrated by bacteria, polymorphonuclear leukocytes (PMN), and a serofibrinous exudate. However, some experimental evidence suggests that the acute inflammatory process may begin before bacteria are present within the inner ear.22 The fibrous stage, which follows the formation of granulation tissue with associated angiogenesis, is composed of hypertrophic fibroblasts and collagen deposition. The ossification stage is characterized by the formation of osteoid and subsequent mineralization. This last stage progresses with bone formation and remodeling, which obliterates the perilymphatic and endolymphatic spaces. The temporal progression of bone formation in labyrinthitis ossificans has not been fully delineated. Ossification in humans has been noted to occur within a year after meningitis even though the hearing loss resulting from meningitis may occur as early as 48 hours after infection.12,23 A cellular infiltrate via the cochlear aqueduct has been noted to develop between several hours and 3 days following the onset of infection in animal models.24,25 INFLAMMATORY PROCESS In bacterial meningitis, a vigorous inflammatory response occurs as a result of the triggering of local host defenses by components of the bacterial cell
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wall.23,26,27 One of the factors that results in higher morbidity and mortality of S. pneumoniae as compared with other organisms is that lipoteichoic acids of the S. pneumoniae cell wall are potent activators of the alternative complement pathway.28,29 Additional pathogenic mechanisms include: 1) cytotoxic bacterial cell wall components; 2) the production of nitric oxide, superoxide, and peroxynitrite by activated inflammatory cells; 3) an increase in excitatory amino acids; and 4) the vascular and hypoxic changes associated with the meningogenic inflammatory process.26 The amount of resultant inflammatory response in the subarachnoid space to S. pneumoniae via these mechanisms is directly proportional to morbidity and mortality.30 REACTIVE OXYGEN SPECIES Vascular and hypoxic changes promote the production of reactive oxygen species. Reactive oxygen species include oxygen-containing molecules with unpaired electrons and metabolites arising from aerobic metabolism. Several critical reactive oxygen species include the superoxide anion O-2; the hydroxyl radical, OH-; and hydrogen peroxide, H2O2. Superoxide can react with nitric oxide, forming peroxynitrite, ONOO-. These species have been shown to indirectly mediate tissue damage by generating complement, lipid, and arachidonic acid-derived chemotactic factors for neutrophils. These active species are generated during postischemic reperfusion of organs, in hyperoxic tissue during acute and chronic inflammation, and during exposure to ionizing radiation. ANTI-INFLAMMATORY AGENTS Host inflammation and the oxygen radicals produced by activated inflammatory cells are directly associated with neuronal injury. Numerous anti-inflammatory agents have the potential to curtail the host inflammatory response to bacterial meningitis. Immunosuppression has resulted in a reduction in hearing loss and other neurologic sequelae associated with bacterial meningitis in several human and animal studies.31-36 Dexamethasone exerts its effects through multiple mechanisms. Dexamethasone decreases prostaglandin E2, tumor necrosis factor, and platelet-activating factor and reduces capillary endothelial cell permeability by inhibiting IL-1.32,36,37 Efficacy of corticosteroid treatment for bacterial meningitis remains controversial, although there is mounting evidence demonstrating a reduction in morbidity with inclusion of corticosteroids in the treatment regimen. Lebel et al32 in a double-blind study compared inclusion of dexamethasone with placebo in the adjunctive management of bacterial meningitis. The investigators found a statistically significant reduction in subsequent sensorineural hearing loss in the steroid-treated group. This finding applied only to
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meningitis caused by H. influenzae. Prior authors were unable to show the efficacy of steroids as therapy for bacterial meningitis.38,39 A second question that has been attempted to be answered is the role of corticosteroids in preventing labyrinthitis ossificans. Even if steroids were not able to prevent the hearing loss associated with bacterial meningitis, if they could reduce the incidence of associated labyrinthitis ossificans they would play a significant role in allowing for subsequent cochlear implantation in postmeningitis deaf children. Hartnick et al have demonstrated recently some efficacy of systemic steroids in preventing the development of labyrinthitis ossificans in children with pneumococcal meningitis.40 The inflammatory burst which results from the bacteriolytic effects of ampicillin on S. pneumoniae may potentiate the damage due to meningitis and can be prevented with inhibition of the cyclooxygenase pathway.41 DeSautel et al42 demonstrated that the amount of cochlear fibrosis in gerbils following bacterial meningitis was significantly reduced by depleting complement with a component of cobra venom. Amaee et al9 demonstrated that nitric oxide (NO) may play a role in the ototoxicity of pneumolysin, a toxin elaborated from S. pneumoniae. Animals were exposed to intracochlear NO and concurrently treated with SOD or normal saline and SOD conferred marked protection upon the cochlea from the lesions caused by NO donors. SOD has demonstrated therapeutic potential for treating a variety of conditions including inflammation, oxygen toxicity, reperfusion injury, and radiation injury. However, the native enzyme’s short halflife in plasma (6 minutes in mice, 25 minutes in human) limits the enzyme’s effectiveness in many applications.43 Covalent conjugation of SOD with polyethylene glycol (PEG) increases cellular enzyme activities and the circulatory half-lives of these enzymes from less than 10 minutes to 40 hours, thus providing prolonged protection from partially reduced oxygen species.43 SOD has been shown to be effective in prevention of reperfusion injuries in coronary artery occlusion44 and acute bowel infarction.45 The concentrations of SOD used in these studies were between 1000 U/kg and 20,000 U/kg. In summary, S. pneumoniae infection in the central nervous system induces a severe host inflammatory response, which is believed to significantly contribute to labyrinthine end-organ damage and auditory pathway destruction. Oxygen radicals play an important role in the host inflammatory process. The purpose of this study is to investigate the effects of administering SOD, an oxygen radical scavenger, in gerbils with experimentally induced bacterial meningitis, on hearing loss and labyrinthine ossification.
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METHODS The study was conducted with 8-week-old male Mongolian gerbils (Meriones unguiculatus) obtained from Tumblebrook Farm (West Brookfield, MA). The use of the laboratory animals adhered to National Institutes of Health guidelines, with approval by the Animal Use and Care Administrative Advisory Committee of the University of California, Davis. Induction of Meningitis In order to mimic the human experience of exposure of the immune system to Streptococcus pneumoniae prior to hematologic seeding of the central nervous system, the animals were immunized with 0.05 cc of Pneumovax 23 (Merck & Co., Inc., Whitehouse Station, NJ) mixed with an equal amount of Freund’s Complete Adjuvant (Sigma, St. Louis, MO). Three weeks later, auditory thresholds were determined using auditory brainstem responses (ABRs) in three groups of 10 gerbils. Three of the gerbils died during the surgical intervention and were replaced. The animals were anesthetized with intraperitoneal injection of ketamine (Vedco Inc., St. Joseph, MO) (80 mg/kg) and Xylazine (Butler Co., Columbus, OH) (20 mg/kg). All three groups were infected by IT injection of approximately 5 L of 105/mL S. pneumoniae type 3 into the cisterna magna. Following the final injection for each syringe, the remaining solution was cultured to verify bacterial viability and the absence of contamination. Twenty-four hours after the induction of infection, all of the gerbils received daily subcutaneous injections of penicillin G (400 U/g) for 7 days. IT and Intramuscular Administration of SOD PEG-SOD (Sigma) (one milligram protein contains 4000 units PEG-SOD) was dissolved in 20 mL of artificial cerebrospinal fluid (CSF; NaCl 138.70 mM, KCl 3.35 mM, MgCl2 1.16 mM, NaHCO3 20.95 mM, NaH2PO4 . H2O 59.0 mM, glucose 3.39 mM, CaCl2 1.26 mM). On post-op days 1 and 3, group 1 was treated with IT SOD, group 2 was treated with intramuscular (IM) SOD, and group 3 was given IM normal saline. In group 1 a head mount was designed to facilitate the injection (Fig 1). A stainless steel anchoring screw (0-80 x 1/8 in, Small Parts Inc, Miami, FL) was modified by drilling a hole in the center to accommodate 0.025 in Silastic tubing (Dow Corning Corp, Midland, MI). A burr hole was made through the gerbils’ parietal bone. The dura was then entered with a pick and the soft silastic tubing was slid between the dura and the cerebral cortex. The screw was rotated 360 degrees, fixing it to the skull. Dental cement was used to secure
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Fig 1. Head mount implanted in a gerbil’s skull. The insert shows a head mount prior to the implantation.
the screw and tubing to the skull. The scalp was then closed with only a segment of tubing exposed. The tubing was folded and ligated with a suture to prevent CSF leak. During each injection the ligature was removed and 10,000 U/kg of PEG-SOD was injected into the gerbils’ subarachnoid space with a Hamilton syringe. The ligature was then replaced after each injection. In group 2, 10,000 U/kg of PEG-SOD was injected into the gerbils’ lower extremities. In group 3, IM injection of 20 L of normal saline was performed in gerbils’ lower extremities. Auditory Brainstem Evoked Response (ABR) Gerbils were anesthetized intraperitoneally with a mixture of ketamine (80 mg/kg) and Xylazine (20 mg/ kg). Otoscopy was performed in order to screen for otitis externa, myringitis, otitis media with effusion, acute suppurative otitis media, and cholesteatoma. The presence of any of these conditions would have resulted in elimination from the study but was never noted. The temperature of the animals was actively maintained at 37.5oC ⫾ 0.5oC by a digitally controlled heating pad. All recordings were performed in an acoustically insulated and electrically shielded chamber. Auditory thresholds were assessed using auditory brainstem response testing. Acoustic stimuli were digitally produced using the Tucker Davis Technologies (TDT) System II AP2 array processor (Alchua, FL) and S232 device driver and controller. The phase of the stimuli was alternated, canceling cochlear microphonics. Each stimulus had a Gaussian rise and fall time of 3 cycles and a plateau of 9 cycles. The tone bursts were produced with a duty cycle of 101 ms and transduced by a piezoelectric headphone. Sound was delivered to the ear of the gerbil via a 2-mm-diameter
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tube, loosely sealed to the external auditory canal. The acoustic stimulus delivered to the ear canal was assessed with the use of a calibrated 1/4⬙ B&K probe microphone (Norcross, GA). The intensity of the sound was calibrated with a continuous tone at 60 dB sound pressure level (SPL), using the output of the microphone as delivered to both a B&K 2209 SPL Meter and a Hewlett Packard 35670A Dynamic Signal Analyzer (Palo Alto, CA). The averaging capability of the signal analyzer allows the microphone output to be analyzed at 20 to 30 dB below its noise floor. This increases the effective sensitivity of the microphone in the acoustically shielded test chamber to 20 to 25 dB SPL for frequencies from 1 to 70 kHz. The output of stainless steel electrodes placed at the scalp vertex and the soft palate was preamplified with active filtering (300-5000 Hz) by TDT DB4 headstage within the chamber (total gain ⫽ 103). The responses were averaged (10 ms window, 5 s/address) for 500 passes. Auditory brainstem response thresholds were determined in response to 4-kHz, 8-kHz, and 16-kHz tone pips. An ABR was initially obtained in response to 75-dB SPL tone bursts. After replicating the ABR, the stimulus intensity level was then reduced by 10 dB and the process repeated. Subsequent ABRs were measured at successively lower sound levels until a readily identifiable ABR could not be obtained and replicated, at which time the intensity was increased by 5 dB. The threshold was extrapolated as being between the lowest intensity at which it could be clearly observed. All gerbils in this study had a normal pre-op pure tone threshold at 4 kHz, 8 kHz, and 16 kHz as determined by ABR testing. On post-op day four, ABRs were performed on all gerbils and the results were analyzed. After 3 months, the animals were sacrificed and fixed by trans-cardial perfusion with saline followed by 2% paraformaldehyde and 2% glutaraldehyde in 0.5 m sodium cacodylate. The bullae were harvested and decalcified in 0.1 M tetrasodium ethylenediamine tetraacetic acid in the above fixative and then dissected to expose the cochleae. They were then osmicated, dehydrated in graded acetone, and embedded in eponaraldyte. Each cochlea was serially divided into approximately 10 to 15 slices using a 0.04-mm-thick diamond-wafering blade. A mid-modiolar slice was identified and sectioned at 0.5 um and stained with toluidine blue and basic fuchsin. The slides were assessed for the area of cochlear lumen filled by fibrosis and/or neo-ossification in the basal, mid, and apical turns. Video images were captured and calibrated areas measured using SigmaScan Pro® image analysis software (Jandel Corp., San Rafael, CA). The number of surviving spiral ganglion cells was recorded for the basal, mid, and apical turns. The status of the organ of
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Fig 2. Histologic section through the basal turn of the cochlea from a postmeningitic animal that received IT SOD injection, demonstrating no labyrinthitis ossificans, normal organ of Corti, stria vascularis, spiral ligament, and preservation of spiral ganglion cells. SG, spiral ganglion cells; OC, organ of Corti; SV, stria vascularis; SL, spiral ligament. (Magnification 180X; stained with toluidine blue and basic fuchsin.)
Fig 3. Histologic section through the basal turn of the cochlea from a postmeningitic animal that received IT SOD injection, demonstrating no labyrinthitis ossificans. The organ of Corti is structurally intact with some cellular damage (Grade 2), stria vascularis has decreased cellularity and mild apoptotic changes (Grade 2), spiral ligament has decreased matrix and cellularity (Grade 2), and spiral ganglion cells were preserved. SG, spiral ganglion cells; OC, organ of Corti; SV, stria vascularis; SL, spiral ligament. (Magnification 180X; stained with toluidine blue and basic fuchsin.)
Corti, stria vascularis, and the spiral ligament were assessed. The criteria for grading these structures are as follows: Organ of Corti 1) Hair cell status (healthy, incomplete loss, complete loss).
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Fig 4. Histologic section through the basal turn of the cochlea from a postmeningitic animal that received IM SOD injection, demonstrating mild labyrinthitis ossificans. The organ of Corti is structurally damaged with few residual cells (Grade 3), stria vascularis shows significantly decreased cellularity with moderate to severe apoptotic changes (Grade 3), spiral ligament shows paucity of cells (Grade 3), and spiral ganglion cellularity is significantly decreased. SG, spiral ganglion cells; OC, organ of Corti; SV, stria vascularis; SL, spiral ligament. (Magnification 180X; stained with toluidine blue and basic fuchsin.)
Fig 5. Histologic section through the basal turn of the cochlea from a postmeningitic animal that received IM saline, demonstrating severe labyrinthitis ossificans. Severe structural damage to organ of Corti, stria vascularis, and spiral ligament. Spiral ganglion cells were not preserved. (Magnification 180X; stained with toluidine blue and basic fuchsin.)
2) Structure integrity (inner tunnel of Corti, supporting cells). 3) Surrounding structures (basilar membrane, tectorial membrane). 4) Grade 1-4 defined as: Grade 1, normal (Fig 2). Grade 2, structurally intact with some cellular damage (Fig 3). Grade 3, few residual cells, some structural
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Table 1. Percentage of total fibrosis in each group
1 2 3 4 5 6 7 8 9 10 Average
IT Group
IM Group
Control Group
0 * 0 0 10 0 0 0 0 2.5 1.39
0 64.8 69.9 39.9 32.4 73.3 0 0 52.2 0 33.25
100 100 100 89.1 46.2 34.1 69.6 13.7 40.1 100 69.28
IT, intrathecal; IM, intramuscular. *This gerbil died prior to sacrifice.
damage (Fig 4). Grade 4, no residual structure (Fig 5). Stria vascularis 1) Cell density. 2) Cell morphology. 3) Grade 1-4 defined as: Grade 1, normal (Fig 2); Grade 2, decreased cellularity with mild apoptotic changes (Fig 3); Grade 3, significant decrease in cellularity with moderate to severe apoptotic changes (Fig 4); Grade 4, structure destroyed (Fig 5). Spiral Ligament 1) Cell density and morphology. 2) Grade 1-4 defined as: Grade 1, normal (Fig 2); Grade 2, decreased matrix and cellularity (Fig 3); Grade 3, paucity of cells (Fig 4); Grade 4, structure destroyed (Fig 5). RESULTS Histopathology Histologic analysis of the cochlea in a mid-modiolar section in group 1 (IT SOD group, n ⫽ 10) demonstrated 20% of animals had some degree of fibrosis which was limited to the scala tympani of the basal turn. There was no neo-ossification present. The mean area of luminal space containing fibrosis was 1.4%. Sixty percent of the animals in group 2 (IM SOD group, n ⫽ 10) and 100% of the control group (n ⫽ 10) developed fibrosis or neoossification within the cochlea. The mean area of luminal space containing fibrosis or neo-ossification in the IM SOD group was 33.2% and 69.3% in the control group. The degree of labyrinthitis ossificans was significantly less in the IT SOD group compared with the IM SOD and control (P ⫽ 0.010, IT vs IM group; P ⫽ 0.001, IT vs control group) (Table 1). There was also significantly less fibrosis in the IM group vs control group (P ⫽ 0.02, IM vs control group) (Table 1). Fibrosis and neo-ossification filled the lumen of all 3 scala in the severe cases and was more limited to the scala tympani in mild cases.
In the IT SOD group, the histology of the organ of Corti, stria vascularis, and spiral ligament all appeared to be normal or near normal (Figs 2 and 3). There was also a large number of healthy-appearing spiral ganglion cells in all turns of the cochlea despite the severe hearing loss in this group. The mean density of spiral ganglion cells per mm2 was 2815.76 (Table 2). In the IM SOD group, the histology of organ of Corti, stria vascularis, and spiral ligament varied across the entire spectrum from normal-appearing to complete destruction (Fig 4). The mean density of spiral ganglion cells was 1666.14 per mm2 (Table 3). In the control group, there were no normal-appearing structures and nearly all of the gerbils developed significant fibrosis in all turns of their cochleae (Fig 5). Very few animals avoided complete destruction of the organ of Corti, stria vascularis, and spiral ligament. The mean density of residual spiral ganglion cells was 138.70 per mm2 (Table 4). In all groups, the number of surviving spiral ganglion cells was strongly inversely correlated with the amount of fibrosis ( r⫽ -0.75) (Tables 2– 4). There was also a correlation between the amount of fibrosis and the degree of damage of stria vascularis (r ⫽ 0.78), organ of Corti (r⫽ 0.80), and spiral ligament (r ⫽ 0.81) (Tables 2-4). The hearing loss in each frequency correlated with the status of the organ of Corti at the corresponding turn of the cochlea ( r⫽ 0.57) (Tables 2– 4). Consistent with human temporal bone histopathology in postmeningitic patients, there was more fibrosis in the basal turn than in the apical turn. Auditory Function In the IT SOD group, the average deterioration in pure tone thresholds between the preoperative baseline and 4-day postinduction of meningitis at 4 kHz, 8 kHz, and 16 kHz was 41, 44.5, and 38.5 dB, respectively. In the IM SOD the average deterioration in thresholds at the 3 frequencies was 39, 44, and 38.5 dB. In the control group, the average deterioration in thresholds at the 3 frequencies was 53.5, 51.5, and 47 dB, respectively. The control group developed 20% greater deterioration of their pure tone thresholds than the animals treated with IM or IT SOD, although this difference was not statistically significant (P ⫽ 0.23, IT vs control; P ⫽ 0.11, IM vs control) (Table 2). DISCUSSION There are multiple sequelae from bacterial meningitis that affect an individual’s hearing and potential for aural rehabilitation. These sequelae include central auditory pathway injuries, spiral ganglion cell loss, cochlear hair cell and supporting cell damage, stria vascularis and spiral ligament injury, and
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Table 2. IT injection group ABR threshold shift on POD#4 (dB) and histologic analysis Threshold Shift (dB)
Basal Turn
No.
4K
8K
16K
OC
1 2 3 4 5 6 7 8 9 10 AV
0 40 5 55 60 60 60 10 60 60 41
10 45 25 50 65 60 55 10 65 60 44.5
0 30 40 50 55 50 45 0 60 55 38.5
1 * 2 1 2 † 2 1 3 2 1.75
SV
Mid Turn
SL
SG
FB
OC
1
1
3041
0
1
1 2 4 1 1 1 2 2 1.67
2 2 3 2 2 2 2 2 2
3418 1292 790 † 2243 2920 2880 2350 2366
0 0 0 7.4 0 0 0 0 0.82
1 1 2 2 2 1 2 2 1.56
SV
Apical Turn
SL
SG
FB
SG
FB
1
1
4054
0
3862
0
1 1 1 1 1 1 2 3 1.33
2 2 2 2 2 2 3 2 2
2833 4311 3524 3067 3181 3006 2620 2561 3239
0 0 0 0 0 0 0 0 0
2566 2323 2935 3317 2881 † 2967 1876 2840
0 0 0 0 0 0 0 0 0
OC, organ of Corti; SV, stria vascularis; SL, spiral ligament; SG, spiral ganglion; #/mm2 FB, % of fibrosis; AV, average. *This gerbil died prior to sacrifice. †Not able to assess due to the oblique cut of the section.
Table 3. IM injection group ABR threshold shift on POD#4 (dB) and histologic analysis Threshold Shift (dB)
Basal Turn
Mid Turn
No.
4K
8K
16K
OC
SV
SL
SG
1 2 3 4 5 6 7 8 9 10 AV
5 55 55 55 25 60 10 5 55 65 39
10 55 55 50 60 65 15 10 55 65 44
5 50 50 40 55 60 15 5 55 50 38.5
1 4 4 3 3 4 2 1 4 2 2.8
1 3 4 4 4 4 1 1 4 1 2.7
2 3 4 3 3 4 1 2 4 2 2.8
3500 659 0 0 0 0 4160 * 167 1773 1139
Apical Turn
FB
OC
SV
SL
SG
0 86 54 38 40 91 0 0 100 0 40.9
1 4 4 2 3 3 2 1 4 2 2.6
1 3 4 2 3 4 1 1 4 1 2.4
2 3 3 2 2 3 1 1 4 2 2.3
3516 243 0 1862 1909 0 3427 3450 237 3875 1851
FB 0 29 87 0 0 27 0 0 100 0 24.3
SG
FB
3059 793 0 2192 2885 823 5752 * 549 * 2006
0 0 100 0 0 0 0 0 100 0 20
OC, organ of Corti; SV, stria vascularis; SL, spiral ligament; SG, spiral ganglion; FB, fibrosis; AV, average. *Not able to assess due to the oblique cut of the section.
Table 4. Control group ABR threshold shift on POD#4 (dB) and histologic analysis Threshold Shift (dB)
Basal Turn
Mid Turn
No.
4K
8K
16K
OC
SV
SL
SG
1 2 3 4 5 6 7 8 9 10 AV
55 60 50 50 55 55 50 55 50 55 53.5
55 50 45 45 55 50 50 55 55 55 51.5
45 45 35 40 55 50 45 50 50 55 47
4 4 4 4 4 4 4 4 4 4 4
4 4 4 4 4 4 4 3 4 4 3.9
4 4 4 4 3 4 3 3 4 2 3.5
0 0 0 0 0 0 0 0 0 0 0
Apical Turn
FB
OC
SV
SL
SG
FB
100 100 5 43 33 100 100 37 100 44 66.2
4 4 4 4 4 4 4 4 4 3 3.9
4 4 4 4 4 4 2 3 4 3 3.6
4 4 4 3 3 4 2 3 4 2 3.3
0 0 0 0 0 0 0 525 0 1107 163.2
100 100 100 100 63 100 41 25 100 0 72.9
SG 0 0 0 0 0 0 0 0 2276 252.8
FB 100 100 34 100 42 100 16 29 100 0 62.1
OC, organ of Corti; SV, stria vascularis; SL, spiral ligament; SG, spiral ganglion; FB, fibrosis; AV, average.
the development of labyrinthitis ossificans. Various mechanisms have been identified that, although necessary in the response to pathogens, contribute to the injury to these structures. Many of the mechanisms result in bystander injury by induction of the local
immune response in the brain and cochlea.22,25-30 Streptococcus pneumoniae cell wall lipoteichoic acids are potent activators of the alternative complement pathway.28,29 An excessive degree of inflammation can result from the explosive release of these
570 GE et al
sub-components of the cell wall and subsequent activation of the complement cascade.41 The blood-brain barrier effectively sequesters the CSF space from serum-associated host defenses and CSF is normally deficient in complement factors C3 and C5, immunoglobulin, and leukocytes, although some resident macrophages are present.29,46-48 The majority of plasma complement components are made in hepatic parenchymal cells.49 However, after cytokine stimulation, reactive astrocytes, microglia, macrophages, and neutrophils develop receptors to the complement anaphlotoxies, C3a and C5a, and participate in the active production of their precursors, C3 and C5.50 Disruption of the blood-brain barrier also contributes to low levels of complement in the CSF.51,52 Activation of the alternative pathway results in the cleavage and activation of C3, producing C3a and C3b. The C3a fragment binds to receptors on mast cells and basophils, resulting in the degranulation of histamine and other cytokines. This results in increased vascular permeability and capillary leakage. C5, when in complex with C3b, can be cleaved by C5 convertase, leading to the formation of C5a and C5b. C5a increases vascular permeability and is a potent chemotaxin in CSF, accelerating leukocytosis.29,53 Complement C5a also induces the release of hydrolytic enzymes from neutrophils with resultant secondary innocent bystander damage. Another mechanism of host-cell injury involves the incorporation of the pneumococcal Forssman antigen into eukaryotic cell membranes, resulting in complement-induced lysis of host cells.54 These mechanisms may explain some of the organ of Corti and spiral ganglion cell injury. In addition to activation of the alternative pathway of the complement cascade, data from in vivo experiments indicate that the S. pneumoniae produce cell-wall components that activate monocytes, leukocytes, cerebrovascular endothelial cells, and astrocytes.20,55 These cells, in turn, produce various pro-inflammatory cytokines such as IL-1, IL-6, IL-8, TNF, and PAF,26,56-59 or express specific receptors on their surface.50 Teichoic and lipoteichoic acids bind the acute phase reactant C-reactive protein, activate procoagulant activity on the surface of endothelial cells, induce cytokines, and initiate the influx of leukocytes.40,54,60 These cytokines and receptors initiate an accelerating cascade of events, resulting in alteration of the bloodbrain barrier, PMN and serum protein infiltration, meningeal inflammation, increased intracranial pressure, and decreased cerebral vascular perfusion.61 Transient and permanent neuronal injury appears to be related to the production of nitric oxide (NO).26 NO is produced by NO synthase (NOS), which con-
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verts arginine and oxygen into citrulline and NO. There are two different types of NOS, constitutive and inducible. Inducible NOS (iNOS) is generated by macrophages in response to specific cytokines. iNOS has been demonstrated to be present in guinea pig cochlea following long-term exposure to bacterial toxins.62 NO has been implicated in causing cochlear damage.63 Microperfusion of the scala tympani with NO donors resulted in reduced cochlear compound action potentials and cochlear microphonics together with hair-cell and organ of Corti damage in adult guinea pigs. Preperfusion of the cochlea with L-methyl arginine, which inhibits the release of NO, or with SOD provides protection from the NO donors.9,63 In the current study, acute hearing loss was not significantly reduced by the administration of SOD. There was, however, a significant decrease in the development of postmeningitic intracochlear fibrosis and neo-ossification. IT SOD also protected the animals from damage to the organ of Corti, stria vascularis, and spiral ligament. Most importantly, IT SOD prevented the loss of spiral ganglion cells. Given the gross preservation of cochlear structures with the IT SOD, it is possible that had longitudinal ABRs been performed, there may have been some recovery of function. Subsequent studies will investigate this possibility. One of the possible explanations for the acute hearing loss in the SOD-treated animals is that the 24-hour delay in administration of the drug may have allowed for inflammatory damage to occur in the organ of Corti. Inhibition of the reactive oxygen species, although too late to interfere with the initial organ of Corti injury, appears to prevent subsequent structural damage as well as blocking the initiation of labyrinthitis ossificans. Maintaining a patent cochlear lumen, despite failure to protect from deafness, may make aural rehabilitation possible with cochlear implant. The inverse correlation between the degree of luminal fibroses and the preservation of spiral ganglion cells also may contribute to the potential success of cochlear implantation. CONCLUSION IT injection of SOD significantly inhibited cochlea fibrosis and neo-ossification following bacterial meningitis by inhibiting oxygen free radicals which form in the course of the inflammatory process. Maintenance of the cochlear lumen and spiral ganglion cell populations improves the potential for aural rehabilitation in postmeningitic deafness. Despite profound deafness, the animals that did not go on to develop labyrinthitis ossificans had greater preservation of spiral ganglion cells as well as the organ of Corti, stria vascularis, and spiral ligament.
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