Review article: reflux and its consequences--the laryngeal, pulmonary and oesophageal manifestations. Conference held in conjunction with the 9th International Symposium on Human Pepsin (ISHP) Kingston-upon-Hull, UK, 21-23 April 2010.

BACKGROUND: Gastro-oesophageal reflux disease (GERD) is one of the commonest diseases of Western populations, affecting 20 to 30% of adults. GERD is multifaceted and the classical oesophageal symptoms such as heartburn and regurgitation often overlap with atypical symptoms that impact upon the respiratory system and airways. This is referred to as extra-oesophageal reflux disease (EERD), or laryngopharyngeal reflux (LPR), which manifests as chronic cough, laryngitis, hoarseness, voice disorders and asthma. AIM: The 'Reflux and its consequences' conference was held in Hull in 2010 and brought together a multidisciplinary group of experts all with a common interest in the many manifestations of reflux disease to present recent research and clinical progress in GERD and EERD. In particular new techniques for diagnosing reflux were showcased at the conference. METHODS: Both clinical and non-clinical key opinion leaders were invited to write a review on key areas presented at the `Reflux and its consequences' conference for inclusion in this supplement. RESULTS AND CONCLUSION: Eleven chapters contained in this supplement reflected the sessions of the conference and included discussion of the nature of the refluxate (acid, pepsin, bile acids and non-acid reflux); mechanisms of tissue damage and protection in the oesophagus, laryngopharynx and airways. Clinical conditions with a reflux aetiology including asthma, chronic cough, airway disease, LPR, and paediatric EERD were reviewed. In addition methods for diagnosis of reflux disease and treatment strategies, especially with reference to non-acid reflux, were considered.

Inhibition of post-meningitic cochlear injury with cerebrospinal fluid irrigation.

OBJECTIVE: Labyrinthitis ossificans, the pathologic ossification of the otic capsule associated with profound deafness and loss of vestibular function occurs frequently as a sequella of bacterial meningitis and subsequent purulent labyrinthitis. Experimentally, in Streptococcus pneumoniae meningitis, it has been shown that a vigorous inflammatory response to teichoic acids in the bacterial cell wall contributes to cochlear damage and subsequent fibrosis and ossification. The hypothesis of this study is that a dilution of concentration of inflammatory mediators through cerebrospinal fluid (CSF) irrigation will lead to a reduction in both inner ear pathology and permanent hearing loss. STUDY DESIGN AND SETTING: Auditory brainstem response testing was used to determine baseline hearing thresholds in 20 Mongolian gerbils (12 irrigated, 8 sham irrigated animals) at 32 kHz, 16 kHz, 8 kHz, and 4 kHz frequencies. Their thresholds at 14 days and 120 days post-procedure were also obtained. Streptococcus pneumoniae meningitis was induced in both groups of animals by intrathecal (i.t.) injection of bacteria. Both groups received penicillin treatment. Forty-eight hours after inoculation, both groups were implanted with i.t. inflow and outflow catheters. The irrigated group was infused continuously with artificial CSF over 36 hr at a rate of 70 muL/hr and the outflow sampled. The tubing in the sham irrigated group was clamped (without sampling). They were sacrificed at 120 days post-procedure and histomorphometric analysis carried out. The concentration of interleukin 1beta (IL-1beta) for the CSF samples from the irrigated group were compared to samples collected from an additional control group of 8 non-irrigated meningitic gerbils. IL-1beta was chosen to study because it is a potent pro-inflammatory cytokines in bacterial meningitis that is unaffected by the neurosurgical trauma of the experimental protocol. RESULTS: Twenty animals survived the meningitis (6 irrigation, 6 sham irrigation, 8 non-irrigation meningitic controls). At Days 14 and 120 post-infection, the irrigated animals manifested significantly less hearing loss with a mean loss of 28.82 dB compared to the sham irrigation group mean loss of 40.76 dB (P < 0.03). The degree of hearing loss in both groups was frequency-dependent with greater loss at higher frequencies (mean loss = 22.4 dB at 32 kHz, 23.0 dB at 16 kHz, 18.6 dB at 8 kHz, and 12.5 dB at 4 kHz). Histomorphometric analysis demonstrated a marked reduction in degeneration of the spiral ligament, spiral ganglion cells, and stria vascularis in experimental animals as compared to controls. Immunohistochemistry showed a significant reduction in IL-beta1 concentrations in the irrigated animals compared to the non-irrigated, infected controls (P < 0.03). CONCLUSIONS: Irrigation of CSF resulted in a significant reduction in post-meningitic cochlear injury when compared to controls. This model for continuous cerebrospinal fluid irrigation provides a means to evaluate the effects of a dilution of inflammatory mediators on hearing loss and labyrinthitis ossificans after bacterial meningitis. SIGNIFICANCE: Despite advances in the prevention of meningitis and improved antibiotic treatment, bacterial meningitis continues to have significant associated morbidity. This study provides insight into some of the mechanisms responsible for post-meningitic hearing loss and labyrinthitis ossificans and presents a novel approach to reduce these complications.

Microwave exposure increases bone demineralization rate independent of temperature.

There is a long-standing controversy regarding an effect of microwaves, independent of increasing temperature, on the rate of bone demineralization. In this study, we exposed standardized samples of gerbil femur to constant microwave exposure while maintaining the demineralizing solution (ethylenediamine tetraacetic acid, EDTA) at 20 degrees C. Random samples were selected at 3 h intervals, embedded in plastic and sectioned for histological evaluation to determine the extent of demineralization. The time to complete demineralization was significantly faster with microwave exposure (33 h) compared to non-exposure on a tissue rotator (45 h) in a limited amount (5 mL/24 h) of EDTA. The presence of bone marrow was a significant barrier to the rate of demineralization and resulted in an asymmetrical pattern of mineral extraction. Samples without bone marrow were completely demineralized after 21 h of exposure to microwaves and EDTA. Additional comparisons were made between samples exposed to an effectively infinite supply of demineralizing agent (bone marrow intact). There was a significant increase in rate with unlimited demineralizing agent with (24 h) or without (30 h) microwaves when compared to tissue demineralized on the rotator. Our results establish a positive effect of microwaves on the rate of bone demineralization which is independent of temperature.

Expression patterns of cytokeratins in retraction pocket cholesteatomas.

OBJECTIVES: To investigate the patterns of cytokeratin (CK) expression in retraction pocket cholesteatoma. STUDY DESIGN: An animal model study. METHODS: Retraction pocket cholesteatomas were induced by electrocautery of the eustachian tube orifice in 24 mongolian gerbils. They were divided into normal and cholesteatoma groups of clinical stages I to IV. The antibodies to pan-cytokeratin CK 1/10, CK 5/6, CK 4, and CK 13/16 were used for immunohistochemical staining. The intensity of staining in each group as measured with densitometry was compared regarding anatomical sites and clinical stages. RESULTS: In retraction pocket cholesteatoma, CK expression was altered only at focal sites such as the pars tensa of the tympanic membrane. The change of CK expression was observed only at certain stages of cholesteatoma formation. In keratinocytes from cholesteatomas, CK 13/16 was overexpressed compared with control specimens, indicating hyperproliferation. The site with the most prominent change in retraction pocket cholesteatoma was somewhat different from that in canal ligation cholesteatoma in a previous study. CONCLUSIONS: The results suggested that aural cholesteatoma is a disease with a spectrum of pathological conditions and that the transmigration and hyperproliferation process of squamous epithelium occurs in areas adjacent to the cholesteatoma.

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 six 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.

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.

Adaptive bone modeling in the middle ear is substance-P dependent.

HYPOTHESIS: Localized bone modeling in the middle ear is substance-P dependent. BACKGROUND: Processes of local bone modeling and remodeling in the middle and inner ear lead to destructive processes such as otosclerosis and chronic otitis media. The cellular events associated with these processes are known, but the mechanisms of the control and activation of the involved cells are not. The authors hypothesized that one of the control mechanisms of local bone modeling is related to the action of a neuropeptide, substance-P and that capsaicin, which depletes substance-P, would block modeling in the gerbil model of adaptive bone modeling. METHODS: One middle ear of each of 24 Mongolian gerbils was pressurized to 10 mmHg to induce adaptive bone modeling. Half of the animals were pretreated with capsaicin and half received vehicle alone. At the end of the 5-day experimental period, the bulla was studied histomorphometrically for osteoclastic and osteoblastic activity. RESULTS: Capsaicin pretreatment inhibited the percent of bone occupied by osteoclasts on the inner surface of the bulla and the rate of mineralization of bone on the outer surface of the bulla. CONCLUSIONS: It is likely that substance-P is a mediator of localized adaptive bone modeling in vivo. Processes of bone modeling and remodeling in the middle and inner ear may be under neural control.

Safety of silver oxide-impregnated silastic tympanostomy tubes.

Otorrhea occurs after the insertion of tympanostomy tubes in as many as 50% of ears. Although topical antibiotic solutions minimize otorrhea in the immediate postoperative period, recurrent otorrhea is sometimes a clinical problem. The antimicrobial effects of silver oxide when impregnated into a tympanostomy tube may decrease the incidence of recurrent otorrhea. This study demonstrates that silver oxide-impregnated silicone elastomer is well tolerated within the middle ear of gerbils when implanted for 1 year, and the tissue reaction is no more than silicon elastomer without silver oxide. When applied directly to the round window of guinea pigs, there was no evidence of ototoxicity of silver oxide as measured by electrocochleography (N-1 thresholds) and cytocochleography (hair cell counts). These animal studies indicate that silver oxide-impregnated silicone elastomeric tympanostomy tubes may be used safely in clinical trials to determine efficacy.

In vivo inhibition of localized bone resorption by human recombinant interleukin-1 receptor antagonist.

Interleukin-1 (IL-1) has been implicated as a primary mediator of bone remodeling; it is a powerful activator of bone resorption both in vivo and in vitro. However, there is no direct evidence that IL-1 plays a role in physiological bone modeling or remodeling. Interleukin-1 receptor antagonist (IL-1ra) is a member of the IL-1 family, which bind to IL-1 receptors and blocks the action of IL-1 alpha and IL-1 beta. We have previously shown that IL-1ra blocks IL-1-induced bone resorption in vitro. Evidence is reported here that human recombinant IL-1ra (hrIL-1ra) inhibits strain-induced modeling in the gerbil auditory bulla while having no significant effect on apposition rate. Pressurization of the auditory bulla to 10 mm Hg above atmospheric pressure increased osteoclast surface from 3.62 to 19.14%. Infusion of hrIL-1ra during pressurization resulted in significant inhibition of osteoclast surface on the pressurized side. These findings suggest that IL-1 is a physiological mediator of bone modeling and that hrIL-1ra inhibits resorption but not apposition in the auditory bulla model.

Human recombinant interleukin-1 receptor antagonist blocks bone resorption induced by interleukin-1 beta but not interleukin-1 alpha.

Both interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) 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-1 alpha and IL-1 beta. In this study, human recombinant IL-1ra, at 100-fold excess, was found to block bone resorption in cultured mouse calvaria due to IL-1 beta but not IL-1 alpha. These observations may be explained by differential affinities of receptors for IL-1 alpha, IL-1 beta and rhIL-1ra on target bone cells.

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.

Apical cochlear nerve exposed to perilymph in the gerbil and rat.

There is considerable controversy regarding the origin and composition of perilymph in the scala vestibuli and scala tympani and the barriers and transport mechanisms between them. To elucidate the anatomical separation between perilymph and the extracellular fluid which surrounds the cochlear nerve in Mongolian gerbils (Meriones unguiculatus) and Sprague Dawley rats (Rattus rattus) we examined the modiolus, osseous spiral lamina and the bone of the perilymphatic scalae using light and electron microscopy. Although the cochlear nerve, spiral ganglion and their extracellular fluid are separated from perilymph by the bone of the modiolus in the middle and basal turns, no bone separates these neural structures from perilymph in the apical turn in the two species examined. Instead, the spiral ganglion and axonal elements of the apical turn were covered by a continuation of the bone lining cell layer of the scala tympani. Gaps between lining cells appeared to provide direct communication between the perilymphatic fluid and the extracellular fluid investing the cochlear nerve. Various authors have described openings in modiolar bone in both the scala vestibuli and the scala tympani. While the bone and cellular covering of the modiolus in the basal middle turns of the cochlea is not presumed to be a complete barrier to fluid exchange between the two scalae and the modiolar canal it can be expected to impose some limitation on the rate of passive diffusion. Therefore our data indicates that in the apical turn of the Mongolian gerbil and Sprague-Dawley rat there may be a more significant communication between perilymph and the extracellular fluid and neural elements of the apical modiolar canal than previously reported.

The intrinsic response of the cricoid cartilage to vertical division.

The explanation for the separation in the cricoid cartilage after the anterior cricoid split procedure has been a matter of speculation. Whether it is caused by extrinsic factors such as stenting, contraction of laryngeal muscles, fibrosis, or whether it is due to the intrinsic nature of the cricoid cartilage itself has yet to be defined. Whole organ in vitro cultures of juvenile gerbil cricoid cartilages were utilized to study the intrinsic response of cricoid cartilage to anterior vertical division. Our results showed the cricoid cartilage to immediately separate, and that separation to markedly increase during whole organ culture. These results suggest that while extrinsic factors may modify the gap after the anterior cricoid split procedure, the intrinsic nature of the cricoid cartilage itself results in the gap formation.

Risedronate activity in the fetal and neonatal mouse.

Otosclerosis, chronic otitis media with and without cholesteatoma, and Paget's disease of bone are just a few of the many diseases of the ear that exhibit abnormalities of bone modeling and remodeling. These diseases result in chronic infection, vestibular dysfunction, and hearing loss. Bisphosphonates are a promising new class of drugs potentially useful in the treatment of these disorders. Currently used in diseases with high rates of bone turnover (Paget's disease of bone, hypercalcemia of malignancy, and osteoporosis), they have been found to be strong inhibitors of bone resorption. A third generation bisphosphonate, 2-(3-pyrindyl)-hydroxyethylidene bisphosphonate (risedronate) is being investigated for toxicity, increased efficacy, and oral administration. In this study the in vitro and in vivo anti-resorptive activity of risedronate was quantified by measuring calcium release in a neonatal mouse calvarial culture system. This model was used to test direct in vitro effects, in vivo exposure in neonatal mice, and the possible effects of in utero and lacteal exposure. Calcium release activated by parathyroid hormone (PTH) was significantly inhibited when risedronate was only present in the pre-incubation media. When risedronate was administered subcutaneously to neonatal mice it resulted in a significant decrease in PTH-activated calcium release in explanted calvaria in vitro. Transplacental and lactational transfer of biologically effective risedronate was not demonstrated in this study; however, a paradoxic increase in PTH-stimulated calcium release in vitro from calvaria theoretically exposed transplacentally and lacteally was noted. This effect was unexplained by the data.

Incomplete coverage of mammalian bone matrix by lining cells.

A functional "membrane" is thought to exist that separates the general extracellular fluid from the bone extracellular fluid. Many investigators have concluded that bone lining cells form this barrier. It is posited that alterations in this barrier may lead to the recruitment of osteoclasts and contribute to the control of extracellular calcium homeostasis. The present ultrastructural studies, however, demonstrated that significant portions of the resting bone are in contact with the general extracellular fluid. In certain regions of the cochlea, up to 88% of the bone surface is in contact with extracellular fluid. These anatomic observations require a critical reevaluation of current theories of the physiologic roles of bone lining cells in osteoclast recruitment and calcium homeostasis.

Systemic osteoclast activation by localized pressure.

It has been demonstrated that positive air pressure applied to the bulla of the Mongolian gerbil results in significant osteoclastic activation and bone resorption. In this study, continuous positive air pressure of 15 mm Hg was applied to the right bulla for 1 week. A significant increase in osteoclast number and surface were noted in the contralateral, unpressurized bulla as well as in a distant site, the upper extremity (radius). Possible mechanisms are discussed by which a local stimulus (pressure) may cause distant osteoclast activation.

Fine morphology of bony dysplasia of the murine ear: comparisons with otosclerosis.

Dysplastic bony lesions that bear certain similarities to otosclerosis occur spontaneously in LP/J inbred mice. These lesions develop after the fifth week of life and progressively enlarge; they are present in 90% of animals by 90 weeks of age. In the present study, the fine morphology of these lesions was investigated. Dysplastic lesions in the middle ear of LP/J mice appeared to begin as subperiosteal accumulations of amorphous material. These accumulations occurred adjacent to the bone surface below the epithelium of the middle ear. This amorphous material was often associated with macrophages on the adjacent epithelial surface. The material appeared to be replaced with collagen fibers in a disorganized manner, forming a homogeneous base (osteoid) on which calcification occurred. The fine morphology and periodicity of the collagen appeared normal. The lesions then calcified by forming calcospherites that became confluent. During this period, transformed fibroblasts (osteoblasts) appeared in the calcifying matrix, resulting in a lesion made of bone. These lesions could sometimes be seen replacing normal bony contours but most often were exophytic. The lesions at all stages of development may be associated with macrophages and effusions in the middle ear. The lesions in the middle ear of LP/J mice appeared to develop by an active process of bone formation; in contrast, otosclerosis is an active process of bone resorption and formation.

Bone resorption induced by middle-ear implants.

The pressure produced by expanding aural cholesteatomas has been implicated as a causal factor in the induction of osteoclastic resorption of adjoining bone. This concept is supported by observations of osteoclastic bone resorption produced by expansive tympanic implants. We induced osteoclastic bone resorption in gerbils with tympanic implants of autologous and homologous cartilage, silicone rubber, and Teflon, which exerted pressure only by forces of gravity and surface tension. We estimated that the pressure exerted by these implants ranged from 2.1 X 10(-3) to 8.0 X 10(-3) dynes/sq cm (1.6 to 6.0 mm Hg). These pressures are within the range of pressures known to be exerted by cholesteatomas.

Basal lamina breaks in the histogenesis of cholesteatoma.

Aural cholesteatomas may arise in the middle ear by a variety of mechanisms; in some cases it appears that cholesteatomas arise within or behind an intact tympanic membrane. We have observed that microcholesteatomas arise within the tympanic membrane of mongolian gerbils which have keratin accumulations on the lateral surface of the tympanic membrane. An ultrastructural study of the keratinizing epithelium of these animals showed that breaks in the basal lamina allow pseudopods of epithelial cells to extend into the lamina propria and form epithelial cones. The basal lamina later reconstituted itself. Keratinization may occur within these ingrowing cones forming microcholesteatomas. This sequence of events may explain the occurrence of intratympanic cholesteatomas in humans in the absence of invagination or perforation.

Pressure-induced bone resorption in the middle ear.

Osteoclastic activity is seen in areas of bone resorption which are the result of experimental and human cholesteatoma. Many factors may induce osteoclasts, including transmitted pressure. The purpose of this study was to determine if transmitted pressure, in the absence of cholesteatoma, could cause localized bone resorption in the middle ear. Surgical grade silicone was implanted into the middle ear of gerbils without cholesteatoma. Bone resorption was observed only in areas where the implants exerted pressure on bone. It was estimated that pressures of 50 to 120 mm Hg (6.7 X 10(4) to 16 X 10(4) dynes/cm2) resulted in the induction of osteoclastic bone resorption.