Steven P. Tinling

Role of tumor necrosis factor-α in sensorineural hearing loss after bacterial meningitis

Hypothesis: Blockade of tumor necrosis factor-α with tumor necrosis factor-α antibody will reduce the extent of cochlear injury and hearing loss associated with Streptococcus pneumoniae meningitis. Background: Inflammatory mediators play a significant role in the morbidity associated with bacterial meningitis, including hearing loss and labyrinthitis ossificans. Previous studies have shown the attenuation of hearing loss by the nonspecific blockade of such pathways. Methods: Fifty Mongolian gerbils were divided into four groups. Auditory brainstem response testing was conducted to measure hearing thresholds. Streptococcus pneumoniae meningitis was induced in Groups 1 and 2. Group 2 was then given a single intraperitoneal injection of tumor necrosis factor-α antibody, whereas Group 1 received phosphate-buffered saline. Uninfected animals in Groups 3 and 4 were implanted with osmotic pumps that delivered a continuous 8-day intrathecal flow of either tumor necrosis factor-α (Group 4) or phosphate-buffered saline (Group 3). After 6 weeks, auditory brainstem response testing was repeated. The cochleas were harvested and analyzed histomorphometrically. Results: Group 2 animals with Streptococcus pneumoniae meningitis that also received tumor necrosis factor-α antibody developed significantly less hearing loss than Group 1 animals with meningitis alone. The decrease in the average threshold at 4, 8, 16, and 32 kHz was 31, 30, 25, and 28 dB sound pressure level, respectively (p < 0.0092 for each). Furthermore, histomorphometric analysis showed significantly less damage to the organ of Corti, spiral ganglion, spiral ligament, and stria vascularis in Group 2. Conversely, tumor necrosis factor-α induced meningitis animals (Group 3) showed increased hearing loss compared with phosphate-buffered saline controls (Group 4), with p < 0.0001 at all frequencies. Conclusion: Tumor necrosis factor-α plays an important role in cochlear injury after bacterial meningitis. Blockade of tumor necrosis factor-α reduces postmeningitic hearing loss and cochlear injury. Induction of meningitis with intrathecal tumor necrosis factor-α also resulted in hearing loss and cochlear injury similar to bacterial meningitis.

Location and timing of initial osteoid deposition in postmeningitic labyrinthitis ossificans determined by multiple fluorescent labels

Objectives/Hypothesis Variable amounts of fibrosis and neo‐ossification fill the cochlea following bacterial meningitis. The purpose of the study was to delineate the timing and location of initial ossification following pneumococcal meningitis, as well as subsequent remodeling and resorption, over the 3‐month period after infection.

Chronology of labyrinthitis ossificans induced by Streptococcus pneumoniae meningitis.

Objective: Labyrinthitis ossificans consists of novel osteogenesis that fills the normally patent cochlear and vestibular lumen as an end‐stage sequelae to various pathologies. This study was designed to establish the sequence of events and chronology of the osteoneogenesis and calcification. Study Design: A prospective randomized double‐blind study. Methods: By using serial application of different colored fluorochromes, which deposit in newly forming bone, the timing of bone deposition and bone remodeling can be established. Labyrinthitis ossificans was induced in she groups (n = 5) of gerbils by an intrathecal injection of live Streptococcus pneumoniae. Group 1 received no fluorochrome labels, group 2 received one label, group 3 received three labels, and groups 4, 5, and 6 received four labels. The temporal bones were harvested after 2 weeks (group 1), 1 month (group 2), 3 months (group 3), 4 months (group 4), 6 months (group 5), and 12 months (group 6). Results: Sixteen of the 25 animals that received labels developed ossification, demonstrated with fluorescent microscopy. In the animals that developed labyrinthitis ossificans, newly formed disorganized bone began calcifying as early as 3 weeks (label 1) after S pneumoniae injection. Osteoneogenesis continued as evidenced by the presence of the other labels when first applied at 6 weeks (label 2), and 10 weeks (label 3). Ossification, calcification, and remodeling proceeded through a 12‐month course, wherein a reduction of labels was present at 6 months and total disappearance by 12 months. Conclusions: The use of fluorescent stains in this animal model provides a means to establish a timeline of the ossification seen in labyrinthitis ossificans. Key Words: Ossified cochlea, labyrinthitis, deafness, cochlear implant, meningitis.

Multimodal in vivo imaging of oral cancer using fluorescence lifetime, photoacoustic and ultrasound techniques

This work reports a multimodal system for label-free tissue diagnosis combining fluorescence lifetime imaging (FLIm), ultrasound backscatter microscopy (UBM), and photoacoustic imaging (PAI). This system provides complementary biochemical, structural and functional features allowing for enhanced in vivo detection of oral carcinoma. Results from a hamster oral carcinoma model (normal, precancer and carcinoma) are presented demonstrating the ability of FLIm to delineate biochemical composition at the tissue surface, UBM and related radiofrequency parameters to identify disruptions in the tissue microarchitecture and PAI to map optical absorption associated with specific tissue morphology and physiology.

Increased proliferation and migration of epithelium in advancing experimental cholesteatomas.

Hypothesis Hyperproliferative and migratory process of keratinocytes are part of the pathogenesis of cholesteatoma. Background Cytokeratin (CK) changes were prominent in the most rapidly expanding regions of cholesteatoma formation. Methods The three types of animal model—canal ligation (CL), retraction pocket (RP), and propylene glycol (PG)—were induced in Mongolian gerbils. The monoclonal antibodies to CK1/10, CK5/6, and CK13/16 were used for immunohistochemistry. The intensity of immunostaining in the pars tensa of the tympanic membrane was measured using the densitometry and compared with respect to the stage of cholesteatoma and the type of animal model. Results With cholesteatoma formation, CK expressions were significantly increased at the peripheral part of the pars tensa, the expanding part of cholesteatoma. Among the CKs tested, the prominent changes were observed in expression of CK13/16, a marker for hyperproliferation. Among the animal models, CK changes of CK5/6 and CK1/10 were most prominent in the CL type, whereas those of CK13/16 were more persistent in the RP type. Conclusion These results suggested that complex alterations of epidermal keratinocytes occur during cholesteatoma formation and that hyperproliferative and migratory processes play important roles in the pathogenesis of cholesteatoma.

Expression Patterns of Cytokeratins in Retraction Pocket Cholesteatomas

Objectives To investigate the patterns of cytokeratin (CK) expression in retraction pocket cholesteatoma.

Inhibition of nitric oxide synthase blocks osteoclastic bone resorption in adaptive bone modeling

In this study, the auditory bulla of the gerbil was pressurized, leading to active modeling of the bone of the bulla wall with a significant increase in osteoclast surface and mineral apposition rate. Systemic infusion of L-N(G)-nitro-arginine-methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), inhibited this modeling process. The percentage osteoclast surface (Oc.S/BS) on the inner surface bulla wall was significantly reduced in the L-NAME-treated animals when compared with pressurized saline-treated bullae. Fluorescent bone surface (BSf) mineral apposition rates (MAR) and bone formation rate (BFR) were not significantly different in the pressurized bullae when the L-NAME group was compared with the control (vehicle only) group. However, L-NAME significantly suppressed BSf in the unpressurized bullae. Therefore, it is likely that nitric oxide is a mediator of osteoclastic resorption due to adaptive bone modeling through one or more of the isoforms of NOS.

The Effects of Superoxide Dismutase in Gerbils with Bacterial Meningitis

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 fibrosis occurred in the IM group (IT vs. IM: P = 0.010; IT vs. control group: P = 0.001). The amount of surviving spiral ganglion cells correlated inversely with the extent of fibrosis (r = −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.

Bone lining cells of the mammalian cochlea

It is generally believed that all bone surfaces are covered by a nearly continuous layer of cells known as bone lining cells (BLC) which separate the general extracellular fluid (GECF) from bone and its fluid compartment. Within the cochlea of some mammals regions of bone matrix are exposed to extracellular fluid. Within the scalae of the cochlea, perilymph is in contact with bone matrix; there is no evidence of a lining endothelium. Within the modiolus and subjacent to the spiral ligament, bone matrix is in contact with GECF. These findings may have importance in understanding calcium homeostasis within the scalae and may relate to the pathophysiology of labyrinthitis ossificans. Additionally, since BLCs probably represent a specific phenotype, the presence of a pure population of BLCs within the scalae may provide a source for the development of a pure culture of this cell.

Human recombinant interleukin-1 receptor antagonist blocks bone resorption induced by interleukin-1β but not interleukin-1α

Both interleukin-1α (IL-1α) and interleukin-1β (IL-1β) are powerful stimulators of bone resorption in vivo and in vitro. Interleukin-1 receptor antagonist (IL-1ra) binds to many interleukin-1 receptors. It does not activate the receptor and effectively blocks the action of IL-1α and IL-1β. In this study, human recombinant IL-1ra, at 100-fold excess, was found to block bone resorption in cultured mouse calvaria due to IL-1β but not IL-1α. These observations may be explained by differential affinities of receptors for IL-1α, IL-1β and rhIL-1ra on target bone cells.