Keratin particle-induced osteolysis: a mouse model of inflammatory bone remodeling related to cholesteatoma.

We implanted keratin and poly(methyl methacrylate) (PMMA) particles to the surface of mouse calvariae to produce a quantitative, localized, inflammatory bone remodeling similar to that seen in cholesteatoma. Both types of particles resulted in increased osteoclast density compared with controls. Osteoclasts infiltrated from marrow and vascular spaces and were active at the periphery of these spaces leading to significant bone remodeling, as demonstrated by the incorporation of bone-labelling fluorophores. Osteoclasts were rarely found on the surface of the calvariae, and mineral apposition rate at the ventral surface was not altered in keratin-implanted animals compared with nonoperated controls. While not useful for the study of the root cause of cholesteatoma, this model will allow the study ofpathologic bone remodeling related to cholesteatoma in a genetically defined animal.

Chronic bacterial rhinosinusitis: description of a mouse model.

OBJECTIVES: To survey normal murine sinonasal anatomy and to create a mouse model for chronic bacterial rhinosinusitis. DESIGN: Anatomic, histologic, and pathophysiologic study displaying normal murine sinonasal anatomy and surgically created unilateral sinonasal inflammation. SUBJECTS: Twenty-one 6-week-old, male C57BL/6 mice. INTERVENTIONS: Animals that underwent unilateral maxillary sinus ostial obstruction using Merocel nasal packing, animals with unilateral Bacteroides fragilis inoculation alone, and animals with both ostial obstruction and bacterial inoculation were examined at 4 weeks for histologic evidence of chronic sinonasal inflammation. Experimental interventions were compared with contralateral control sinuses within each animal and with normal and sham-operated controls. RESULTS: Normal mouse paranasal sinuses include maxillary sinuses, ethmoid air cells, and respiratory-type epithelium. In experimental animals, the lateral maxillary sinus wall, nasal septum, and superior turbinelle of the maxillary sinus were examined histologically. Epithelial thickening and disarray, goblet cell hyperplasia, inflammatory infiltrates, and sinonasal fibrosis were present in the experimental sinuses of animals packed with Merocel alone or Merocel with bacterial inoculation. Changes seen with Merocel and bacteria were more dramatic than those with Merocel alone. Sham-operated controls and sinuses inoculated with bacteria alone did not differ significantly from the sinuses of normal animals. CONCLUSION: Unilateral maxillary sinus ostial obstruction using Merocel nasal packing along with B fragilis inoculation results in a persistent, localized bacterial rhinosinusitis in mice.

Invasiveness of fibroblasts from experimental cholesteatomas.

HYPOTHESIS: Cultured fibroblasts derived from experimental gerbil cholesteatoma tissue exhibit an invasive phenotype in comparison with normal fibroblasts. BACKGROUND: Aural cholesteatomas are enlarging accumulations of keratin debris caused by keratinizing squamous epithelium in the middle ear. They characteristically result in the destruction of adjacent tissues, specifically bone erosion. The mechanisms by which cholesteatomas relentlessly invade the structures of the temporal bone are varied, but it has been suggested that one factor contributing to the aggressive nature of cholesteatomas is the transformation of resident fibroblasts into an invasive phenotype. METHODS: The ability of cultured normal and cholesteatoma fibroblasts to invade a basement membrane matrix in a Boyden chamber assay was examined. RESULTS: Less than 1% of gerbil fibroblasts invaded the matrix, compared with almost 10% of the invasive HT-1080 fibrosarcoma cells. Normal and cholesteatoma fibroblasts did not differ from each other in their invasive potential. CONCLUSION: Normal fibroblasts and fibroblasts from induced cholesteatomas do not exhibit the invasive phenotype characteristic of true neoplastic cells.

Neuronal degeneration in the gerbil brainstem is associated with spongiform lesions.

Spongiform lesions arise in dendrites and glia in the brainstem of domestic Mongolian gerbils. Most pronounced within the cochlear nucleus (CN), this disorder is dynamic and progressive; the lesions increase in number, size, and extent with age. It has not been clear whether these spongioid lesions either cause or are associated with significant neural degeneration. In contrast, feral Mongolian gerbils (wild-trapped in Tuva) and their offspring show few spongiform lesions. The Tuvan gerbils provide an appropriate within-species control. We compared degeneration in the brainstem of domestic and Tuvan gerbils using the amino-cupric-silver (ACS) stain of de Olmos et al. [(1994) Neurotoxicol. Teratol., 16:545-561]. Positive histologic controls were provided by cerebellar stab wounds in domestic gerbils and by unilateral kainic acid injections into the CN of Tuvan gerbils. The ACS stain revealed extensive degeneration of axons, terminals, dendrites, and neurons in the brainstem of domestic gerbils. Neurodegeneration was most pronounced in the CN and was coextensive with spongiform lesions. Neurodegeneration was also seen in the trapezoid body, lateral lemniscus, and inferior colliculus, but was less pronounced than in the CN. The cerebellar stab wounds resulted in silver-stained Purkinje cells restricted to the stab wound local region. Kainic acid produced extensive neuronal and spongiform degeneration of the injected CN that was very similar to that spontaneously occurring in domestic gerbils. In contrast, the non-injected CN of Tuvan gerbils showed no neuronal or spongiform degeneration with the ACS stain. We conclude that, in domestic gerbils, the naturally occurring spongiform lesions of the CN and the accompanying neurodegeneration are both results of a common mechanism, most probably excitotoxic.

Quantitative measures reflect degeneration, but not regeneration, in the deafness mouse organ of Corti.

The deafness mouse (dn/dn) is a well known model of hereditary deafness uncomplicated by behavioral and motor disturbances. The organ of Corti in this mouse develops a normal complement of sensory and supporting cell structures, yet animals homozygous for this gene never demonstrate any hearing capacity. They are profoundly deaf from birth. Soon after development, the organ of Corti rapidly degenerates, most sensory cells having vanished by 50 days of age. Published observations have suggested that apical regions of the organ of Corti may regenerate some supporting cell structures by 90 days of age. We have quantified changes in organ of Corti structure from 15 to 130 days of age using several different measures. Measures of peak height and total cross-sectional area. as well as a subjective rating scale, all demonstrate consistent degenerative changes during this time period. No evidence for regeneration of supporting or sensory cell structures is noted, although a surprising degree of variability is present in all regions of the organ of Corti which may account for previous claims.

Distinct roles for sodium, chloride, and calcium in excitotoxic dendritic injury and recovery.

The postsynaptic neuronal dendrite is selectively vulnerable to hypoxic-ischemic brain injury and glutamate receptor overactivation. We explored the glutamate receptor pharmacology and ionic basis of rapid, reversible alterations in dendritic shape which occur in cultured neurons exposed to glutamate. Dendrite morphology was assessed with the fluorescent membrane tracer, DiI, or immunofluorescence labeling of the somatodendritic protein, MAP2. Cortical cultures derived from 15-day-old mouse embryos underwent segmental dendritic beading when exposed to NMDA, AMPA, or kainate, but not to metabotropic glutamate receptor agonists. Varicosity formation in response to NMDA or kainate application was substantially attenuated in reduced sodium buffer (substituted with N-methyl-D-glucamine). Furthermore, veratridine-induced sodium entry mimicked excitotoxic alterations in dendrites and additionally caused varicosity formation in axons. Solutions deficient in chloride (substituted with Na methylsulfate) and antagonists of chloride-permeable GABA/glycine receptors reduced NMDA- or kainate-induced varicosity formation. An increase in dendrite volume was observed as varicosities formed, and varicosity formation was attenuated in sucrose-supplemented hypertonic media. Despite marked structural changes affecting virtually all neurons, dendrite shape returned to normal within 2 h of terminating glutamate receptor agonist application. Neurons exposed to kainate recovered more rapidly than those exposed to NMDA, and neurons exposed to NMDA in calcium-free buffer recovered more rapidly than cells treated with NMDA in normal buffer. While sodium, chloride, and water entry contribute to excitotoxic dendritic injury acutely, calcium entry through NMDA receptors results in lasting structural changes in damaged dendrites.

Spongiform degeneration of the gerbil cochlear nucleus: an ultrastructural and immunohistochemical evaluation.

Observations from ultrastructural and immunohistochemical studies suggest that spongiform lesions in the gerbil cochlear nucleus are derived principally from dendrites. Almost one-fifth of the lesion profiles examined ultrastructurally exhibited synaptic contacts with axon terminals. In addition, approximately 80% of lesions are immunopositive for the dendrite-specific microtubule associated protein, MAP2. Ultrastructural studies showed a small percentage (8%) of lesions were derived from myelinated axons, although none were immunohistochemically labelled with antibodies to the tau protein. Staining with the astrocyte-specific markers GFAP, S-100 and vimentin yielded equivocal results, but did not support a major role for astrocytes in lesion formation. The histological profile matches that seen in some other well characterized types of spongiform degeneration.

Progression of cochlear and retinal degeneration in the tubby (rd5) mouse.

Mice homozygous for a defect of the tub (rd5) gene exhibit cochlear and retinal degeneration combined with obesity, and resemble certain human autosomal recessive sensory deficit syndromes. To establish the progressive nature of sensory cell loss associated with the tub gene, and to differentiate tub-related losses from those associated with the C57 background on which tub arose, we evaluated cochleas and retinas from tub/tub, tub/+, and +/+ mice, aged 2 weeks to 1 year by light and electron microscopy. Cochleas from mice of all three genotypes show progressive inner (IHC) and outer hair cell (OHC) loss. Relative to tub/+ and +/+ animals, however, tub homozygotes show accelerated OHC loss, affecting the extreme cochlear base (hook region) by 1 month, and the apex by 6 months. IHC loss in tub/tub animals is accelerated in the basal half of the cochlea, affecting the hook region by 6 months. Spiral ganglion cell losses were observed only in tub/tub mice, and only in the cochlear base. Retinas of tub/tub mice are abnormal at maturity, exhibiting shortened photoreceptor outer segments by 2 weeks, and progressive photoreceptor loss thereafter. Because the tub mutation causes degeneration of sensory cells in the ear and eye but has no other neurological effects, tubby mice hold unique promise for the study of human syndromic sensory loss.

Calpain activation contributes to dendritic remodeling after brief excitotoxic injury in vitro.

The calcium-dependent protease calpain may contribute to neuronal death in acute neurological insults and may be activated very early in the neuronal injury cascade. We assessed the role of calpain in a model of rapid, reversible dendritic injury in murine cortical cultures. Brief sublethal NMDA exposure (10-30 microM for 10 min) resulted in focal swellings, or varicosities, along the length of neuronal dendrites as visualized with the lipophilic membrane tracer Dil or with immunostaining using antibodies to the somatodendritic protein MAP2. These varicosities appeared within minutes of NMDA exposure and recovered spontaneously within 2 hr after NMDA removal. Addition of the calpain inhibitors MDL28,170, calpain inhibitors I and II, and leupeptin (all 1-100 microM) had little effect on the development of NMDA-induced dendrite injury. However, the resolution of varicosities was substantially delayed by addition of calpain inhibitors after sublethal excitotoxic exposure. Using Western blots and immunocytochemistry, we observed reactivity for a calpain-specific spectrin proteolytic fragment during the period of recovery from dendritic swelling, but not during its formation. Spectrin breakdown product immunoreactivity could be blocked by the calpain inhibitor MDL28,170 and appeared in neuronal cell bodies and neurites in a time course that paralleled dendritic recovery. These observations suggest that calcium-dependent proteolysis contributes to recovery of dendritic structure after NMDA exposure. Calpain activation is not necessarily detrimental and may play a role in dendritic remodeling after neuronal injury.

Kangaroo rats exhibit spongiform degeneration of the central auditory system similar to that found in gerbils.

Kangaroo rats develop spongiform degeneration of the central auditory system similar to that seen in the gerbil. Light microscopic and transmission electron microscopic study of the cochlear nucleus and auditory nerve root (ANR) of Dipodomys deserti and D. merriami show that spongiform lesions develop in dendrites and oligodendrocytes of the cochlear nucleus and in oligodendrocytes of the ANR that are morphologically indistinguishable from those extensively described in the Mongolian gerbil, Meriones unguiculatus. As in Mongolian gerbils, the spongiform degeneration in Dipodomys were much more numerous in animals continually exposed to modest levels of low-frequency noise (< 75 dB SPL). The kangaroo rats with extensive spongiform degeneration also show slightly, but significantly, elevated auditory brainstem evoked response (ABR) thresholds to low-frequency stimuli, a result also found in Mongolian gerbils. These results suggest that the elevated ABR thresholds may be the result of spongiform degeneration. Because low-frequency noise-induced spongiform degeneration has now been shown in the cochlear nucleus of animals from separate families of Rodentia (Heteromyidae and Muridae), the possibility should be investigated that similar noise-induced degenerative changes occur in the central auditory system of other mammals with good low-frequency hearing.

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.

Exposure to low frequency noise during rearing induces spongiform lesions in gerbil cochlear nucleus: high frequency exposure does not.

Spongiform lesions of the gerbil cochlear nucleus are reduced in number and extent by rearing in acoustic isolation compared with rearing while exposed to normal colony low-frequency background noise. This study tested whether rearing under exposure to noise bands of moderate intensity would increase the number and extent of cochlear nucleus spongiform lesions. Gerbils were reared from weaning to young adulthood in acoustic isolation chambers while continually exposed to moderately intense bands of either high frequency or low frequency noise. Exposure to low frequency noise resulted in lesion number and area densities that were more than twice those seen in gerbils exposed to high frequency noise. Lesion extent in the low frequency group was similar to that in colony-reared gerbils; lesion extent in the high frequency group was similar to gerbils reared in acoustic isolation. Comparisons within the posterior ventral cochlear nucleus revealed that the differences in lesion extent were most pronounced in the middle and dorsal-medial portions, the regions that are most responsive to middle and high frequencies. These finding suggest that the regional restriction of spongiform lesions within the cochlear nucleus does not have a tonotopic basis.

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.

Glial populations in the juvenile and adult Mongolian gerbil: relationship to spongiform degeneration of the ventral cochlear nucleus.

The gerbil cochlear nucleus is subject to a spongiform degeneration, the progression of which is dependent on auditory functional activity. The most affected region is the ventrolateral aspect of the caudal posterior ventral cochlear nucleus (PVCN). Lesion density and glial changes were quantified in this region for two age groups. Spongiform lesions increased significantly in area density from 4% in 60-day-old gerbils to 14% in 6-month-old gerbils. In spite of this significant increase in tissue damage, no gliosis was found. A significant age-related decrease in oligodendrocyte density was found in the PVCN.

Acoustic isolation reduces degeneration of the ventral cochlear nuclei in Mongolian gerbils.

The role of auditory experience in the development of spongiform degeneration in the cochlear nuclei of Mongolian gerbils was studied by comparing results of animals exposed to either high or low levels of ambient noise. Gerbils reared in a typical vivarium experienced higher levels of ambient noise than animals reared in acoustic isolation chambers. Animals reared in the colony room showed a much greater number density and area density of spongiform lesions in the CN than did gerbils reared in acoustic isolation. The differences in the number and extent of spongiform lesions between the two groups of gerbils appeared to reflect their differences in exposure to ambient noise. These differences in lesion number and extent were most pronounced in the tonotopic regions of the PVCN which correspond to the greatest differences in the spectral characteristics of the ambient noise to which the animals were exposed. These results were compared with results previously obtained from gerbils with loss of hearing experimentally induced by a conductive block or by sensorineural damage. The lesion numbers and extent reflected the auditory experience of each group; in descending order, colony-reared, isolate, conductive-block, sensorineural loss. These results strongly support the hypothesis that this gerbilline encephalopathy is directly related to auditory functional activity.

Trauma-induced proliferation of astrocytes in the brains of young and aged rats.

The time-course and magnitude of astrocyte proliferation following neural trauma was evaluated in young adult (3 months) and mid-aged (16-19 months) male Fischer 344 rats. One to 4 days after a needle wound was made through the cortex and the hippocampus, rats received three intraperitoneal injections of 3H-thymidine at 8 hour intervals and were sacrificed 1 hour after the last injection. For astrocyte quantification, 3H-thymidine autoradiography was combined with immunohistochemical staining for glial fibrillary acidic protein followed by semithin sectioning. In areas of the cortex and hippocampus adjacent to the wound, astrocytes were categorized as unlabeled or labeled with silver grains over the nuclei. Labeling index and numerical density of astrocytes were determined using stereological methods. The results showed that in both young and older rats, astrocyte proliferation is an early glial response to neural trauma, occurring during the first 4 postlesion days and contributing to an increase in astrocyte population. Regional differences in labeling index and numerical density suggest heterogeneity in the proliferative capacity of astrocyte subpopulations in the rat brain. Compared with young animals, older rats demonstrated greater labeling in the cortex but not in the hippocampus. Thus, aging is associated with region-specific increase in astrocyte reactivity to trauma possibly due to increased availability of mitogens or enhanced sensitivity of astrocytes to mitogenic signals.

Application of glial fibrillary acidic protein immunohistochemistry in the quantification of astrocytes in the rat brain.

Immunohistochemical staining for glial fibrillary acidic protein (GFAP) was employed as a tool for quantification of astrocytes in the rat brain. One-micron-methacrylate sections were prepared from 70-micron slices stained for GFAP by using a preembedding staining procedure. Numbers/unit area of astrocytes and nonastrocytes were determined for cortex, corpus callosum, and hippocampal neuropil. In each, counts from GFAP-stained, toluidine-blue-counterstained sections were compared with counts obtained from sections stained with toluidine blue alone. Numbers of nonastrocytes and total glia in all three regions were comparable in both groups of sections. Astrocyte counts in the cortex and hippocampus also showed no significant differences between the two groups. In contrast, the number of astrocytes in the corpus callosum was significantly lower in GFAP-stained, toluidine-blue-counterstained sections than in sections stained with toluidine blue alone. GFAP immunohistochemistry is a useful tool for the quantification of astrocytes in semi-thin plastic sections of rat brain.

Auditory experience affects degeneration of the ventral cochlear nucleus in Mongolian gerbils.

Gerbils exhibit a unique encephalopathy characterized by spongioform lesions in the neuropil of the cochlear nucleus and that others have recently described. The present results suggest that the course of this degenerative disorder is affected by acoustic experience. In gerbils in which acoustic stimulation was limited postnatally, the number and the extent of these lesions was dramatically reduced. Monaural deprivation reduced lesion number and extent only in the ipsilateral cochlear nucleus; binaural deprivation affected both cochlear nuclei. The lesions were most evident in certain portions of the cochlear nucleus, the caudal anterior ventral cochlear nucleus and the posterior ventral cochlear nucleus, sparing the rostral pole of the anterior ventral cochlear nucleus. In the affected regions the lesions were topologically restricted to the low and middle frequency regions. The apparent tonotopic distribution of lesions was associated with frequent exposure to low and middle frequency ambient noise.