Packaging modifications for protecting flavor of extended-shelf-life milk from light.

The effectiveness of titanium dioxide (TiO2)-loaded high-density polyethylene (HDPE) to reduce light-induced oxidation of extended-shelf-life milk (2% total fat) was studied. The objective was to determine differences over time in sensory quality, vitamin retention, and oxidative chemistry as a function of packaging and retail light exposure duration. Effectiveness of packaging for protecting milk quality was assessed by sensory evaluation (triangle tests, untrained panel), changes in volatile compounds, thiobarbituric reactive substances (TBARS), and riboflavin concentration. Milk (2%) was stored in HDPE packages consisting of TiO2 at 3 levels (low: 0.6%; medium: 1.3%; high: 4.3%) at 3 °C for up to 43 d. Light-protected (translucent, foil-wrapped) and light-exposed (translucent) HDPE packages served as controls. The high TiO2-HDPE package provided protection similar to light-protected control package through d 22 of light exposure, with less consistent performance by the medium TiO2 package. The TBARS increased in all treatments during storage. Under the experimental conditions used, a TBARS value of 1.3mg/L could be considered the limiting sensory threshold for differentiating oxidized milk from light-protected milk. Riboflavin concentration decreased 10.5% in the light-protected control and 28.5% in the high TiO2 packaged milk past 29 d of light exposure, but losses were greater than 40% for all other packages. The high TiO2 package protected riboflavin concentration from degradation and controlled aldehyde concentration throughout the test period.

Methods for providing therapeutic agents to treat damaged spiral ganglion neurons.

Sensorineural hearing loss, characterized by damage to sensory hair cells and/or associated nerve fibers is a leading cause of hearing disorders throughout the world. To date, treatment options are limited and there is no cure for damaged inner ear cells. Because the inner ear is a tiny organ housed in bone deep within the skull, access to the inner ear is limited, making delivery of therapeutic agents difficult. In recent years scientists have investigated a number of growth factors that have the potential to regulate survival or recovery of auditory neurons. Coinciding with the focus on molecules that may restore function are efforts to develop novel delivery methods. Researchers have been investigating the use of mini osmotic pumps, viral vectors and stem cells as a means of providing direct application of growth factors to the inner ear. This review summarizes recent findings regarding the molecules that may be useful for restoring damaged spiral ganglion neurons, as well as the advantages and disadvantages of various delivery systems.

Brn3a is a transcriptional regulator of soma size, target field innervation and axon pathfinding of inner ear sensory neurons.

The POU domain transcription factors Brn3a, Brn3b and Brn3c are required for the proper development of sensory ganglia, retinal ganglion cells, and inner ear hair cells, respectively. We have investigated the roles of Brn3a in neuronal differentiation and target innervation in the facial-stato-acoustic ganglion. We show that absence of Brn3a results in a substantial reduction in neuronal size, abnormal neuronal migration and downregulation of gene expression, including that of the neurotrophin receptor TrkC, parvalbumin and Brn3b. Selective loss of TrkC neurons in the spiral ganglion of Brn3a(-/-) cochlea leads to an innervation defect similar to that of TrkC(-/-) mice. Most remarkably, our results uncover a novel role for Brn3a in regulating axon pathfinding and target field innervation by spiral and vestibular ganglion neurons. Loss of Brn3a results in severe retardation in development of the axon projections to the cochlea and the posterior vertical canal as early as E13.5. In addition, efferent axons that use the afferent fibers as a scaffold during pathfinding also show severe misrouting. Interestingly, despite the well-established roles of ephrins and EphB receptors in axon pathfinding, expression of these molecules does not appear to be affected in Brn3a(-/-) mice. Thus, Brn3a must control additional downstream genes that are required for axon pathfinding.

Microwave decalcification of human temporal bones.

OBJECTIVES/HYPOTHESIS: Morphological and immunohistochemical studies of human temporal bones are challenging as a result of difficulties in obtaining reliably fixed specimens and the lengthy time required for decalcification, typically 4 to 7 months. A novel method of processing human temporal bones using a microwave oven to accelerate decalcification is described. This procedure provides a rapid means of decalcifying temporal bones with good preservation of tissue morphology and antigenicity. METHODS: Human temporal bone specimens obtained at autopsy (n = 12, from specimens aged 43-91 y) were fixed within 6.5 hours of death by transtympanic perilymphatic perfusion of the inner ear. Decalcification was carried out using ethylenediaminetetra-acetic acid (EDTA) in a microwave oven and required only 3 to 6 weeks. Specimens were then dehydrated, embedded in paraffin, sectioned, and mounted on slides for morphological and immunohistochemical evaluation. RESULTS: Microscopic examination revealed no obvious artifacts attributable to the microwave decalcification process. The quality of morphological preservation was largely dependent on the postmortem fixation interval and adequacy of perilymphatic perfusion. Immunohistochemical analysis demonstrated strong positive staining for the enzyme Na,K-ATPase, an integral membrane protein. CONCLUSIONS: This study demonstrates that microwave decalcification provides an efficient and reliable means of processing human temporal bones for histological and histochemical examination. Decalcification time is significantly reduced with no apparent adverse effects on structural preservation or antigenicity.

EphB2 guides axons at the midline and is necessary for normal vestibular function.

Mice lacking the EphB2 receptor tyrosine kinase display a cell-autonomous, strain-specific circling behavior that is associated with vestibular phenotypes. In mutant embryos, the contralateral inner ear efferent growth cones exhibit inappropriate pathway selection at the midline, while in mutant adults, the endolymph-filled lumen of the semicircular canals is severely reduced. EphB2 is expressed in the endolymph-producing dark cells in the inner ear epithelium, and these cells show ultrastructural defects in the mutants. A molecular link to fluid regulation is provided by demonstrating that PDZ domain-containing proteins that bind the C termini of EphB2 and B-ephrins can also recognize the cytoplasmic tails of anion exchangers and aquaporins. This suggests EphB2 may regulate ionic homeostasis and endolymph fluid production through macromolecular associations with membrane channels that transport chloride, bicarbonate, and water.

Analysis of BDNF production in the aging gerbil cochlea.

Degeneration of cochlear neurons is the most commonly observed cellular change in the aging human and gerbil cochlea. Although it is unclear what leads to this neuronal loss, changes in the production of target-derived trophic factors may be the ultimate cause of cochlear neuron degeneration. The present study used an enzyme-linked immunosorbent assay to investigate whether BDNF is produced by the organ of Corti or cochlear ganglia of young, middle aged, or aged gerbils. The results revealed an age-related increase in BDNF in the organ of Corti, but not in the cochlear ganglia.

Selective and transient expression of a native chondroitin sulfate epitope in Deiters' cells, pillar cells, and the developing tectorial membrane.

The tectorial membrane (TM) is an acellular connective tissue overlying the sensory hair cells of the organ of Corti. Association of the tectorial membrane with the stereocilia of the sensory hair cells is necessary for proper auditory function. During development, the mature tectorial membrane is thought to arise by fusion of a "major" and "minor" tectorial membrane (Lim, Hear Res 1986;22:117-146). Several proteins and glycoconjugates have been detected in the developing TM; however, the specific molecules which mediate fusion of the two components of the TM have not been identified. In the present study, a novel monoclonal antibody (TC2) that recognizes a native epitope on glycosaminoglycans enriched in chondroitin-4-sulfate revealed a transient and restricted expression in the developing gerbil TM. The localization patterns suggest that Deiters' and pillar cells secrete a TC2-positive matrix prior to birth that later becomes incorporated into the marginal band and superior layer (cover net) of the TM. The developmental timecourse and patterns of TC2 reactivity suggest that this molecule may play a critical role in the fusion of the minor TM with the major TM.

Comparison of ephrin-A ligand and EphA receptor distribution in the developing inner ear.

Members of the recently discovered Eph family appear to play important roles in a variety of developmental processes including tissue segmentation, cell migration and axonal guidance. To begin to understand the functions of the EphA subclass of receptors and their corresponding GPI-linked (ephrin-A) ligands in the inner ear, a developmental immunohistochemical analysis was completed. The results indicated that the ligands ephrin-A1 and ephrin-A2 were localized mainly at cellular boundaries in the inner ear. Ephrin-A1 was detected mainly in the epithelial cells lining the fluid filled ducts of the inner ear, whereas ephrin-A2 was prominently expressed in connective tissue regions. The receptor EphA4 was detected in vestibular hair cells. EphA5 and EphA7 were detected mainly in cochlear and vestibular supporting cells. These results suggest that these Eph molecules play a role in establishing the formation and cellular organization of the complex inner ear labyrinth. Additionally, all of the ligands and receptors evaluated were expressed in vestibular and cochlear neurons at various developmental stages, suggesting they may play a role in establishing or maintaining innervation to the inner ear.

Distribution of Eph-related molecules in the developing and mature cochlea.

Receptors and ligands of the Eph family have recently been shown to influence the development of a variety of tissues. In the present study, the temporal and spatial distribution of Eph receptors and ligands were investigated in the embryonic and postnatal cochlea using Northern blot and immunohistochemical analysis. The results of Northern blot experiments revealed that a large number of Eph family members were present in embryonic cochlear and vestibular ganglia. Immunohistochemical studies revealed that ligands and receptors of the GPI subclass were distributed in complementary patterns within the differentiating spiral limbus, inner sulcus and outer sulcus. The distribution of these molecules became more restricted beginning in the first postnatal week. In contrast, members of the transmembrane subclass of Eph ligands were largely associated with cochlear neurons and their target hair cells. Expression of these ligands appeared to increase during the second postnatal week, corresponding to the period of peripheral nerve fiber reorganization in the cochlea. Together, these studies suggest that multiple Eph family members play unique roles in formation of the cochlea.

Developmental changes in growth factors released by the embryonic inner ear.

Recent studies have demonstrated a role for neurotrophins in regulating the survival of developing auditory and vestibular neurons. However, the developmental time-course for neurotrophin production and release by inner ear tissues has not been defined. In the present study, neurotrophin-like activity was evaluated from culture medium conditioned by early- or midembryonic stage inner ears. Examination of the proteinaceous properties of conditioned medium revealed a developmental change in growth factor release by the inner ear. Neurotrophin-like molecules were not detected in medium conditioned by early stage otocysts. In contrast, neurotrophin-like bioactivity was detected in medium conditioned by middevelopmental stage inner ears. Western blot analysis revealed that NT-3 was released by the rat inner ear at midstages of inner ear development. ELISA measurements revealed that both NT-3 and BDNF are produced by the middevelopmental stage inner ear, and that NT-3 protein levels are higher than BDNF levels. These results suggest that there are developmental changes in the release of growth factors by the inner ear.

The role of neurotrophic factors in regulating the development of inner ear innervation.

Several neurotrophins and their receptors regulate the survival of vestibular and cochlear neurons and probably also the efferent and autonomic neurons that innervate the inner ear. Mice lacking either brain-derived neurotrophic factor (BDNF) or its associated receptor, TrkB, lose all innervation to the semicircular canals and have reduced innervation of the outer hair cells in the apical and middle turns of the cochlea. Mice lacking neurotrophin-3 (NT-3) or its receptor, TrkC, lose many spiral ganglion cells predominantly in the basal turn of the cochlea. Nerve fibers from spiral ganglion cells in the middle turn extended to inner hair cells of the base. In mice lacking both BDNF and NT-3, or both TrkB and TrkC, there is a complete loss of innervation to the inner ear. Thus, these two neurotrophins and their associated receptors have been shown to be absolutely necessary for the normal development of afferent innervation of the inner ear. Current research efforts are testing the therapeutic potential for neurotrophins to treat hearing loss.

Effects of neurotrophin and neurotrophin receptor disruption on the afferent inner ear innervation.

Two neurotrophins and their two receptors appear to regulate the survival of vestibular and cochlear neurons in the developing ear. Mice lacking either brain derived neurotrophic factor (BDNF) or its associated receptor, Trk B, show a severe reduction in the number of vestibular neurons and a loss of all innervation to the semicircular canals. Mice lacking NT-3 or its receptor, Trk C, show a severe reduction of spiral neurons in the basal turn of the cochlea. Mice lacking both BDNF and NT-3 or Trk B and Trk C, reportedly lose all innervation to the inner ear. These two neurotrophins and their associated receptors are necessary for the normal afferent innervation of the inner ear.

Degeneration of vestibular neurons in late embryogenesis of both heterozygous and homozygous BDNF null mutant mice.

The generation of mice lacking specific neurotrophins permits evaluation of the trophic requirements of particular neuronal populations throughout development. In the present study, we examined the developing vestibulocochlear system to determine the time course of neurotrophin dependence and to determine whether competition occurred among developing cochlear or vestibular neurons for available amounts of either brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5 (NT-4/5). Both cochlear and vestibular neurons were present in mice lacking NT-4/5. In contrast, vestibular neurons decreased in number beginning at mid-stages of inner ear development, in mice lacking BDNF. Early in development (E12.5-13), the size of the vestibular ganglion was normal in bdnf -/- mice. Decreased innervation to vestibular sensory epithelia was detected at E13.5-15, when progressive loss of all afferent innervation to the semicircular canals and reduced innervation to the utricle and saccule were observed. At E16.5-17, there was a reduction in the number of vestibular neurons in bdnf -/- mice. A further decrease in vestibular neurons was observed at P1 and P15. Compared to bdnf -/- mice, mice heterozygous for the BDNF null mutation (bdnf +/-) showed an intermediate decrease in the number of vestibular neurons from E16.5-P15. These data indicate a late developmental requirement of vestibular neurons for BDNF and suggest competition among these neurons for limited supplies of this factor.

Neuronal deficits, not involving motor neurons, in mice lacking BDNF and/or NT4.

Nerve growth factor and other neurotrophins signal to neurons through the Trk family of receptor tyrosine kinases. TrkB is relatively promiscuous in vitro, acting as a receptor for brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT4) and, to a lesser extent, NT3 (refs 3-5). Mice lacking TrkB show a more severe phenotype than mice lacking BDNF, suggesting that TrkB may act as a receptor for additional ligands in vivo. To explore this possibility, we generated mice lacking NT4 or BDNF as well as mice lacking both neurotrophins. Unlike mice lacking other Trks or neurotrophins, NT4-deficient mice are long-lived and show no obvious neurological defects. Analysis of mutant phenotypes revealed distinct neuronal populations with different neurotrophin requirements. Thus vestibular and trigeminal sensory neurons require BDNF but not NT4, whereas nodose-petrosal sensory neurons require both BDNF and NT4. Motor neurons, whose numbers are drastically reduced in mice lacking TrkB, are not affected even in mice lacking both BDNF and NT4. These results suggest that another ligand, perhaps NT3, does indeed act on TrkB in vivo.

Effects of the neurotrophins and CNTF on developing statoacoustic neurons: comparison with an otocyst-derived factor.

During the early stages of auditory development, the inner ear (otocyst) releases an unidentified, diffusible factor that promotes neurite outgrowth from the associated statoacoustic ganglia (SAG). Using a variety of criteria, the present study compared the neurite- and survival-promoting properties of this otocyst-derived factor (ODF) to the neurotrophins NGF, BDNF, NT-3, and NT-4 and ciliary neurotrophic factor (CNTF). Ganglia known to respond to specific growth factors were cultured in the presence of ODF. ODF failed to promote neurite outgrowth from trigeminal, ciliary, sympathetic, or dorsal root ganglia, suggesting that ODF may have properties different from other identified growth factors. In complementary experiments, SAG explants were cultured in ODF, the neurotrophins, and CNTF. The extent of outgrowth was greatest in the presence of ODF and CNTF, with the neurotrophins having little effect. In neuron-enriched, dissociated cell cultures, ODF promoted both survival and outgrowth of SAG neurons. However, neither the neurotrophins nor CNTF, alone or in combination, promoted the survival or outgrowth of dissociated SAG neurons. Thus, the outgrowth seen in the explant cultures appears to be due to indirect effects via the ganglionic nonneuronal cells. The addition of anti-NGF antisera failed to block the activity of chick, rat, or mouse ODF, further indicating that NGF is not the primary component of ODF. Together, the results of this study indicated that the properties of the ODF are not mimicked by the neurotrophins or CNTF.

Developmental regulation of a neurite-promoting factor influencing statoacoustic neurons.

The present study investigated a target-derived, neurite-promoting factor (NPF) released by the developing chick otocyst and its effects on statoacoustic ganglia (SAG). SAG explants cultured in the absence of otocysts produced little neurite outgrowth at all stages of development examined (E4-E13). However, extensive neurite outgrowth was seen when E4-E6 SAG were cultured in the presence of otocysts of the same age. The amount of neurite outgrowth observed in cocultures steadily decreased at later developmental stages. E7-E9 cocultures produced less outgrowth and E10-E13 cocultures produced the least outgrowth compared to E4-E6 cocultures. Additionally, otocysts from older stages were unable to promote outgrowth of E4 SAG. Thus, the level of the factor released by the otocysts declined during development. In contrast, neurite outgrowth was promoted when E10-E15 SAG were cocultured in the presence of younger stage otocysts. Our data indicate that the release of NPF from chick otocysts decreased from E6 to E13, although the ability of SAG neurons to respond to the NPF was maintained throughout development.