A new spontaneous mutation in the mouse protocadherin 15 gene.

We have characterized a new allele of the protocadherin 15 gene (designatedPcdh15(av-6J)) that arose as a spontaneous, recessive mutation in the C57BL/6J inbred strain at Jackson Laboratory. Analysis revealed an inframe deletion in Pcdh15, which is predicted to result in partial deletion of cadherin domain (domain 9) in Pcdh15. Morphologic study revealed normal to moderately defective cochlear hair cell stereocilia in Pcdh15(av-6J) mutants at postnatal day 2 (P2). Stereocilia abnormalities were consistently present at P5 and P10. Degenerative changes including loss of inner and outer hair cells were seen at P20, with severe sensory cell loss in all cochlear turns occurring by P40. The hair cell phenotype observed in the 6J allele between P0 and P20 is the least severe phenotype yet observed in Pcdh15 alleles. However, young Pcdh15(av-6J) mice are unresponsive to auditory stimulation and show circling behavior indicative of vestibular dysfunction. Since these animals show severe functional deficits but have relatively mild stereocilia defects at a young age they may provide an appropriate model to test for a direct role of Pcdh15 in mechanotransduction.

Particulate dermal matrix as an injectable soft tissue replacement material.

Products currently used as injectable soft tissue replacement materials in the dermatologic, plastic and reconstructive, and urological fields exhibit several shortfalls including reactivity, migration, rapid degradation, and necessity of a donor site. This study examines the feasibility of providing a particulate acellular human dermal matrix for injection as a soft tissue replacement material that addresses many of these issues. Animal feasibility studies tested differences in implant performance related to processing techniques, matrix concentration, and volume of the collagen matrix to be injected. Results demonstrated that processing techniques that involve shearing and tearing of the dermal collagen matrix resulted in frayed and damaged collagen bundles and led to rapid resorption or loss of the implant, when injected subcutaneously in a rat model. Processing the collagen matrix in liquid nitrogen resulted in less damage to the collagen matrix and exhibited longer persistence, when compared to the damaged collagen matrix. This particulate matrix also exhibits rapid repopulation by host cells that should enhance revascularization and remodeling. The particulate nature of this processed dermal matrix allows for easy delivery of concentrations up to 330 mg/mL, which exceeds that of other currently used products. This increased concentration should allow for decreased need of "overcorrection" and repeated injections.

The mouse Ames waltzer hearing-loss mutant is caused by mutation of Pcdh15, a novel protocadherin gene.

The neuroepithelia of the inner ear contain hair cells that function as mechanoreceptors to transduce sound and motion signals. Mutations affecting these neuroepithelia cause deafness and vestibular dysfuction in humans. Ames waltzer (av) is a recessive mutation found in mice that causes deafness and a balance disorder associated with the degeneration of inner ear neuroepithelia. Here we report that the gene that harbours the av mutation encodes a novel protocadherin. Cochlear hair cells in the av mutants show abnormal stereocilia by 10 days after birth (P10). This is the first evidence for the requirement of a protocadherin for normal function of the mammalian inner ear.

Neuroepithelial defects of the inner ear in a new allele of the mouse mutation Ames waltzer.

This report presents new findings regarding a recessive insertional mutation in the transgenic line TgN2742Rpw that causes deafness and circling behavior in mice homozygous for the mutation. The mutant locus was mapped to a region on mouse chromosome 10 close to three spontaneous recessive mutations causing deafness: Ames waltzer (av), Waltzer (v), and Jackson circler (jc). Complementation testing revealed that the TgN2742Rpw mutation is allelic with av. Histological and auditory brainstem response (ABR) evaluation of animals that have the new allele balanced with the av(J) allele (called compound heterozygotes, TgN2742Rpw/av(J)) supports our genetic analysis. ABR evaluation shows complete absence of auditory response throughout the life span of TgN2742Rpw/av(J) compound heterozygotes. Scanning electron microscopy revealed abnormalities of inner and outer hair cell stereocilia in the cochleae of TgN2742Rpw mutants at 10 days after birth (DAB). The organ of Corti subsequently undergoes degeneration, leading to nearly complete loss of the cochlear neuroepithelium in older mutants by about 50 DAB. The vestibular neuroepithelia remain morphologically normal until at least 30 DAB. However, by 50 days, degenerative changes are evident in the saccular macula, which progresses to total loss of the saccular neuroepithelium in older animals. The new allele of av reported here will be designated av(TgN2742Rpw).

An animal model for external ear canal inflammation.

OBJECTIVE: To develop an animal model for testing efficacy of anti-inflammatory drugs designed to treat external ear canal (EAC) disease. METHODS: Histological and morphometric methods were used to characterize EAC inflammation produced by topical application of tetradecanoylphorbol acetate (TPA) in mice. The effects of both single and repeated TPA applications were studied. A treatment trial was performed to evaluate the effects of a ciprofloxacin/hydrocortisone suspension on TPA-induced EAC inflammation. In 10 animals, two bilateral applications of TPA were made, spaced 24 hours apart. Immediately after the second TPA application, otic suspension was applied unilaterally four times over a 48-hour period. The contralateral EACs were left untreated to provide TPA-only controls. RESULTS: Twenty-four hours after a single TPA application, EAC skin showed polymorphonuclear (PMN) leukocyte infiltration, vascular dilation, and thickening of the dermis and epidermis. Dermal and epidermal thickening were more pronounced after two TPA applications and PMN leukocyte infiltration remained high 48 hours after a second TPA placement. After treatment with the otic suspension, PMN leukocyte counts were reduced by an average of 76% relative to EACs that received TPA only. There was also statistically significant reduction of dermal swelling and a trend toward reduced epidermal thickness. Vascular dilation was clearly reduced as well EACs that received four applications of the suspension alone showed no adverse effects compared with those that received saline. CONCLUSION: TPA-induced inflammation of the mouse EAC provides a suitable model for testing the therapeutic efficacy of anti-inflammatory agents being considered for clinical use.

A new mouse insertional mutation that causes sensorineural deafness and vestibular defects.

This article describes a new recessive insertional mutation in the transgenic line TgN2742Rpw that causes deafness and circling behavior in mice. Histologic analysis revealed virtually complete loss of the cochlear neuroepithelium (the organ of Corti) in adult mutant mice. In association with the neuroepithelial changes, there is a dramatic reduction of the cochlear nerve supply. Adult mutants also show morphological defects of the vestibular apparatus, including degeneration of the saccular neuroepithelium and occasional malformation of utricular otoconia. Audiometric evaluations demonstrated that the mice displaying the circling phenotype are completely deaf. Molecular analysis of this mutant line revealed that the transgenic insertion occurred without creating a large deletion of the host DNA sequences. The mutant locus was mapped to a region on mouse chromosome 10, where other spontaneous, recessive mutations causing deafness in mice have been mapped.

Histologic demonstration of glycosaminoglycans in inner ear fluids.

Hyaluronic acid is a glycosaminoglycan (GAG) which has been demonstrated by biochemical and histochemical methods to be present in inner ear fluids and tissues. However, consistent labeling of hyaluronic acid or other GAGs in inner ear fluid compartments in histologic sections has not been previously reported. Staining and characterization of GAGs in the normal inner ear fluids of humans and guinea pigs are described in this report. It was initially observed that alcian blue produces dense staining of the endolymphatic and perilymphatic fluid compartments in celloidin embedded material. Results obtained with alcian blue were subsequently compared to staining obtained with hematoxylin/eosin and periodic acid/Schiff base. Enzyme digestion was also performed with testicular hyaluronidase (bovine and ovine), streptomyces hyaluronidase, chondroitase ABC, and alpha-amylase. Results of the differential staining and enzyme digestion studies suggest that the substances in inner ear fluids that stain with alcian blue are a combination of chondroitin sulfate-4, -5 and/or -6 and hyaluronic acid.

Postnatal persistence of epidermoid rests in the human middle ear.

It is widely believed that congenital cholesteatoma originates from epidermoid cell rests in the temporal bone. Congenital cholesteatomas of the anterior-superior middle ear may arise from such a rest, known as the epidermoid formation (EF), which has been described by Michaels and others. The EF is thought to disappear by 33 weeks' gestation in most cases; however, if it persists beyond fetal life, it may provide a nidus for cholesteatoma formation. The authors studied human temporal bones from individuals ranging from 20 weeks' gestation to 5 years of age to investigate pre- and postnatal occurrence of the EF. A total of 106 temporal bones were surveyed; 76 of these were fetal specimens and 30 were postnatal. EFs were present in 14 (18.4%) of the fetal specimens; they were identified in four (13.3%) of the postnatal temporal bones, with the oldest being 2 years, 7 months of age. Multiple EFs within a single temporal bone were also present in some cases. Although the EFs were composed of cells morphologically identical to those of the external ear canal epidermis, none of the specimens showed keratinization. These findings support the contention that EFs do occasionally persist into postnatal life and may be a source of cholesteatoma. However, a clear transition from epidermoid formation to unequivocal cholesteatoma has not yet been demonstrated.

Electrocochleographic evaluation of the guinea pig model of endolymphatic hydrops.

Electrocochleography (ECochG) was used to evaluate cochlear function in guinea pigs with experimentally induced endolymphatic hydrops (ELH) before and after osmotic dehydration with either glycerol or urea. We surgically induced ELH in the right ears of 9 guinea pigs, while the right ears of 6 guinea pigs received a sham operation. The left ears of the 15 animals constituted the normal group. Eight weeks after surgery, summating potential (SP) and action potential (AP) amplitudes were measured prior to and following the administration of glycerol or urea. The SPs and SP/AP ratios were reduced in all groups, with no significant differences among groups or between dehydrating agents. Some of the hydropic ears, however, did show an increased AP threshold and a recruitment effect. In measurements from 6 additional animals, serum osmolarity increased more with urea than with glycerol. The guinea pig model remains valuable for investigation of ELH, even though it differs in significant respects from ELH in humans.

Effect of basic fibroblast growth factor on perforated chinchilla tympanic membranes.

Basic fibroblast growth factor (bFGF) is a polypeptide mitogen which stimulates proliferation of epidermal and connective tissue cells. When applied to tympanic membrane perforations it has been reported to enhance healing and produce connective tissue hyperplasia. Previous work with animal models has shown that hyperplastic alterations of the tympanic membrane play an essential role in cholesteatoma development. This study was designed to further investigate the hyperplastic effects of bFGF and to determine if it might induce cholesteatoma formation during the healing process. Ten chinchillas received bilateral tympanic membrane perforations. In each animal, three doses of bFGF (400 nanograms per dose) were applied to the perforated tympanic membrane on one side; the opposite (control) ear received saline alone. The animals were terminated at either two or four weeks and studied histologically. Although the dosage and administration schedule used were consistent with previous studies utilizing other rodent species, there was little evidence that bFGF affected tympanic membrane healing in chinchillas. In both control and bFGF-treated ears, dense connective tissue occupied the lamina propria of the tympanic membrane, providing an effective barrier against ingrowth of skin toward the middle ear. No cholesteatomas developed in any animals included in the study. The results of this work indicate that the risk of cholesteatoma formation following administration of bFGF is minimal when it is applied short-term to acute perforations.

Development of the human external ear.

External ear development is a lengthy and complex process that extends from early embryonic life until well into the postnatal period. Initial development of the auricle and external auditory canal during the fourth and fifth weeks of gestation is closely associated with anatomical changes involving the pharyngeal arch apparatus of the human embryo. The auricle and external canal are well formed by the time of birth but do not attain their full size and adult configuration until about 9 years of age. Sebaceous and modified apocrine glands, which are responsible for cerumen production, begin their development at about 5 months gestation in association with hair follicles in the outer portion of the external canal. Although they appear anatomically mature before birth, these glands do not reach full functional capacity until puberty.

External and middle ear pathology in TGF-alpha-deficient animals.

Transforming growth factor-alpha (TGF-alpha) is a growth-regulatory peptide found in a wide range of embryonic and adult tissues. TGF-alpha is produced by keratinocytes and has been reported to be overexpressed in several epidermal diseases, including middle ear cholesteatoma. This report describes ear pathology in the waved-1 mutant mouse, which is severely deficient in TGF-alpha. Morphologic changes of the external and middle ear were studied histologically in waved-1 mutants 2 weeks to 6.5 months of age. Abnormalities found in the mutants included epidermal hyperplasia of the external ear canal (EAC) and tympanic membrane (TM) and enlargement of specialized sebaceous glands adjacent to the cartilaginous EAC. Sebum and desquamated keratin progressively accumulated within the EAC, displacing the TM into the middle ear. These changes appear similar to those occurring in Mongolian gerbils, which are known to develop cholesteatoma. The alterations found in waved-1 mutants are discussed in relation to the possible involvement of TGF-alpha in cholesteatoma pathogenesis.

Distribution of chlorpyrifos in air and on surfaces following crack and crevice application to rooms.

Measureable levels of chlorpyrifos were seen in air and on horizontal and vertical surfaces over an 84-day sampling period following application by two different methods. Pressurized aerosol applications had the highest airborne levels over the 84-day sampling period, and movement into adjacent, nontreated rooms was seen 7 days after application. Highest surface residues found were located at floor/wall interfaces and were due probably as a result of splash or overspray around treated areas. Residue levels from desk sides were very low and all surface residues were highly variable. One could not predict what surface levels would be based upon airborne concentrations.

Effect of topical hyaluronic acid on experimental cholesteatoma.

PURPOSE: Inflammation and connective tissue hyperplasia are believed to be important etiological factors in cholesteatoma pathogenesis. Previous work has shown that topically applied hyaluronic acid can reduce connective tissue proliferation in healing wounds and accelerate healing of tympanic membrane perforations. This study was undertaken to determine whether the antiproliferative effect of hyaluronic acid may inhibit propylene glycol-induced cholesteatoma in an animal model. MATERIALS AND METHODS: A 60% propylene glycol solution was injected bilaterally into the middle ear cavities of 20 adult chinchillas. The control group (N = 10) received propylene glycol alone. In addition to propylene glycol injections, the experimental group (N = 10) received repeated bilateral topical applications of 1.5% hyaluronic acid onto the tympanic membranes. Animals were killed at 4 weeks for gross and light microscopic examination. RESULTS: Seven control and 10 experimental animals survived the full 1-month study period. At the end of that time, cholesteatoma was found in 71% (10/14) of control ears and 70% (14/20) of experimental ears. Tympanic membrane structure did not differ significantly between groups by light microscopy and, in all animals, cholesteatomas originated by migration of hyperplastic epidermis through the tympanic membrane, as has been observed in previous studies using this animal model. CONCLUSION: Under the conditions of this study, topical hyaluronic acid had no significant effect on cholesteatoma formation.

Ototoxicity of topical ticarcillin and clavulanic acid in the chinchilla.

BACKGROUND: Currently available topical otic preparations contain a variety of antibiotics and other ingredients that are potentially damaging to the middle and inner ear. There is therefore a need to identify agents that are safe as well as effective for topical otologic use. In pursuit of that goal, we used an animal model to evaluate the ototoxic potential of the broad-spectrum, penicillin derivative ticarcillin--both alone and combined with clavulanic acid (a beta-lactamase inhibitor). METHODS: Twenty chinchillas served as subjects. Ten of the animals were given a single middle ear application of ticarcillin; the remaining 10 animals received ticarcillin disodium plus clavulanate potassium (Timentin). Five animals from each of the two groups were killed after 1 week to assess short-term effects and the other five animals in each group were kept for 4 weeks before their temporal bones were removed for histologic study. RESULTS: Significant toxic effects, involving both the middle and inner ear, were observed in all experimental groups. Alterations of the middle ear at 1 week included inflammation, hemorrhage, and effusions. Middle ear cholesteatomas were observed at 4 weeks. Inner ear changes seen at 1 and 4 weeks included hair cell loss, supporting cell degeneration, and strial damage. CONCLUSION: The study results indicate that ticarcillin should not be considered for further evaluation as a possible antibiotic for use in ototopical preparations.

The effect of cyclophosphamide on development of experimental cholesteatoma.

BACKGROUND: Recent work has shown that middle ear application of propylene glycol in chinchillas produces an inflammatory reaction resulting in cholesteatoma formation in 50% to 70% of animals. This study was done to determine if cyclophosphamide, an immune suppressor and anti-inflammatory agent, is capable of inhibiting cholesteatoma development in the animal model. METHODS: Ten adult chinchillas received systemic cyclophosphamide (20 mg/kg per day) for 14 days. On days 5, 8, and 11 of that period, the animals also received bilateral middle ear applications of 60% propylene glycol (0.2 mL per ear). Four weeks after the first propylene glycol application, the animals were killed for histologic evaluation. RESULTS: Eight of the 10 animals survived the full 4-week study period, providing 16 temporal bones for evaluation. It was found that cyclophosphamide reduced the leukocyte counts; however, middle ear inflammation appeared unaffected. Cholesteatoma occurred in eight (50%) of the 16 ears studied, and histologic findings were essentially identical to those previously seen in animals given propylene glycol alone. CONCLUSION: Under the conditions of this study, cyclophosphamide had no effect on cholesteatoma development in the animal model.

Pathology of otitis media.

Otitis media is characterized by inflammation of the middle ear. The pathologic changes seen in this condition tend to occur on a continuum, progressing from acute and subacute stages to the chronic phase, in which irreversible tissue damage is observed. The earliest morphological changes involve the lamina propria of the middle ear mucosa and include increased capillary permeability, edema, and leukocytic infiltration. During the late acute to subacute stages, the mucosa also tends to show a marked increase in numbers of ciliated and secretory epithelial cells. As the inflammatory process enters the chronic phase, there is a continuing shift in the population of infiltrating leukocytes toward increasing numbers of mononuclear cells that secrete substances that facilitate tissue destruction and fibrosis. There is also development and proliferation of granulation tissue, which is intimately involved in the process of bone erosion. As granulation tissue matures, it becomes denser and less vascular--a process that leads to permanent fibrosis and formation of adhesions that may significantly compromise middle ear function. Other pathologic entities occasionally associated with chronic otitis media include cholesteatoma, cholesterol cysts and granuloma, and tympanosclerosis, all of which may contribute to irreversible alterations of middle ear structure.