Investigations of the Microvasculature of the Human Macula Utricle in Meniere's Disease.

The integrity and permeability of the blood labyrinthine barrier (BLB) in the inner ear is important to maintain adequate blood supply, and to control the passage of fluids, molecules and ions. Identifying the cellular and structural components of the BLB, the vascular endothelial cells (VECs), pericytes, and the perivascular basement membrane, is critical to understand the pathophysiology of the inner ear microvasculature and to design efficient delivery of therapeutics across the BLB. A recent study of the normal and pathological ultrastructural changes in the human macula utricle microvasculature demonstrated that the VECs are damaged in Meniere's disease (MD), and further studies identified oxidative stress markers (iNOS and nitrotyrosine) in the VECs. Using fluorescence microscopy, the microvasculature was studied in the macula utricle of patients diagnosed with MD that required transmastoid labyrinthectomy for intractable vertigo (n = 5), and patients who required a translabyrinthine approach for vestibular schwannoma (VS) resection (n = 3). Normal utricles (controls) were also included (n = 3). VECs were identified using rabbit polyclonal antibodies against the glucose transporter-1 (GLUT-1) and pericytes were identified using mouse monoclonal antibodies against alpha-smooth muscle actin (α-SMA). Immunofluorescence (IF) staining was made in half of the utricle and flat mounted. The other half was used to study the integrity of the BLB using transmission electron microscopy (TEM). GLUT-1-IF, allowed delineation of the macula utricle microvasculature (located in the stroma underneath the sensory epithelia) in both MD and VS specimens. Three sizes of vessels were present in the utricle vasculature: Small size (<15 µm), medium size (15-25 µm) and large size >25 µm. α-SMA-IF was present in pericytes that surround the VECS in medium and thick size vessels. Thin size vessels showed almost no α-SMA-IF. AngioTool software was used for quantitative analysis. A significant decreased number of junctions, total vessel length, and average vessel length was detected in the microvasculature in MD specimens compared with VS and control specimens. The deeper understanding of the anatomy of the BLB in the human vestibular periphery and its pathological changes in disease will enable the development of non-invasive delivery strategy for the treatment of hearing and balance disorders.

Otopetrin-2 Immunolocalization in the Human Macula Utricle.

BACKGROUND: In the present study, we investigated the localization of otopetrin-2-a member of the otopetrin family that encodes proton-selective ion channels-in the human macula utricle using immunohistochemistry. METHODS: Macula utricle were acquired at surgery from patients who required transmastoid labyrinthectomy for intractable vertigo due to Meniere's disease (MD; n = 3) and/or vestibular drops attacks (VDA; n = 2) and from temporal bones (n = 2) acquired at autopsy from individuals with no balance disorders. Immunofluorescence staining with otopetrin-2 (rabbit affinity purified polyclonal antibody) and GFAP (mouse monoclonal antibody) to identify vestibular supporting cells was made in formalin fixed cryostat sections or whole microdissected utricle (for flat mount preparations). Secondary antibodies against rabbit and mouse were used for the identification of both proteins. Digital fluorescent images were obtained using a high-resolution laser confocal microscope. RESULTS: Using cryostat sections and flat mount preparations otopetrin-2 immunofluorescence was seen as punctated signal throughout the supporting cells cytoplasm. GFAP immunofluorescence was present in the supporting cell cytoplasm. The distribution of otopetrin-2 was similar in the macula utricle obtained from MD, VDA, or autopsy normative patients. CONCLUSIONS: Otopetrin-2 was localized in supporting cells in a similar fashion that otopetrin-1 previously reported in the mouse macula utricle. The differential expression of otopetrin-2 in the supporting cells of the human macula utricle suggest an important role in the vestibular sensory periphery homeostasis and otolith maintenance.

Modulatory Effects of Mild Carbon Monoxide Exposure in the Developing Mouse Cochlea.

Carbon monoxide (CO) is well known as a highly toxic poison at high concentrations, yet in physiologic amounts it is an endogenous biological messenger in organs such as the internal ear and brain. In this study we tested the hypothesis that chronic very mild CO exposure at concentrations 25-ppm increases the expression of oxidative stress protecting enzymes within the cellular milieu of the developing inner ear (cochlea) of the normal CD-1 mouse. In addition we tested also the hypothesis that CO can decrease the pre-existing condition of oxidative stress in the mouse model for the human medical condition systemic lupus erythematosus by increasing two protective enzymes heme-oxygenase-1 (HO-1), and superoxide dismutase-2 (SOD-2). CD-1 and MRL/lpr mice were exposed to mild CO concentrations (25 ppm in air) from prenatal only and prenatal followed by early postnatal day 5 to postnatal day 20. The expression of cell markers specific for oxidative stress, and related neural/endothelial markers were investigated at the level of the gene products by immunohistochemistry, proteomics and mRNA expression (quantitative real time-PCR). We found that in the CD-1 and MRL/lpr mouse cochlea SOD-2 and HO-1 were upregulated. In this mouse model of autoimmune disease defense mechanism are attenuated, thus mild CO exposure is beneficial. Several genes (mRNA) and proteins detected by proteomics involved in cellular protection were upregulated in the CO exposed CD-1 mouse and the MRL/lpr mouse.

Immunohistochemical techniques for the human inner ear.

In this review, we provide a description of the recent methods used for immunohistochemical staining of the human inner ear using formalin-fixed frozen, paraffin and celloidin-embedded sections. We also show the application of these immunohistochemical methods in auditory and vestibular endorgans microdissected from the human temporal bone. We compare the advantages and disadvantages of immunohistochemistry (IHC) in the different types of embedding media. IHC in frozen and paraffin-embedded sections yields a robust immunoreactive signal. Both frozen and paraffin sections would be the best alternative in the case where celloidin-embedding technique is not available. IHC in whole endorgans yields excellent results and can be used when desiring to detect regional variations of protein expression in the sensory epithelia. One advantage of microdissection is that the tissue is processed immediately and IHC can be made within 1 week of temporal bone collection. A second advantage of microdissection is the excellent preservation of both morphology and antigenicity. Using celloidin-embedded inner ear sections, we were able to detect several antigens by IHC and immunofluorescence using antigen retrieval methods. These techniques, previously applied only in animal models, allow for the study of numerous important proteins expressed in the human temporal bone potentially opening up a new field for future human inner ear research.

Neuroglobin expression in the cochlea of rat pups exposed to chronic very mild carbon monoxide (25ppm) in air during and after the prenatal period.

The distribution of neuroglobin (Ngb) was investigated in the normal rat cochlea using immunohistochemistry and non-radioactive insitu hybridization. We also determined whether chronic, very mild CO exposure at 25ppm in air over the gestational and postnatal period alters the expression of Ngb. Pregnant rats were exposed chronically to CO from gestational days 5-20. Four groups were made as follows: prenatal exposure to CO only; prenatal exposure to CO followed by postnatal exposure from postnatal days (5) P5 to P20; rat pups were exposed to CO from P5 to P20; controls (air without CO). In normal adult rats and control group pups, Ngb was found in spiral ganglia neurons, fibrocytes of the spiral ligament, and supporting cells of the organ of Corti. Ngb was not present in the stria vascularis and the inner and outer hair cells. At P20 Ngb immunoreactivity and transcript expression decreased in spiral ganglia neurons and the spiral ligament in the prenatal and pre- and postnatal groups. This decrease was not observed in the postnatal group. Ngb-IR did not decrease in supporting cells in any CO group. Cytochrome-C immunoreactivity followed Ngb distribution in normal controls and CO treated groups. A decrease in Ngb in spiral ganglia neurons and rat spiral ligament, but not in supporting cells, following CO exposure supports the idea that chronic, mild exposure to CO may create a vulnerable cellular environment predisposed to adverse cochlear development.

Neuroglobin, cytoglobin, and transcriptional profiling of hypoxia-related genes in the rat cerebellum after prenatal chronic very mild carbon monoxide exposure (25 ppm).

The expression of neuroglobin (Ngb) and cytoglobin (Cygb), two recently discovered globins with a potential neuroprotective activity against hypoxia and oxidative stress, was investigated in the cerebellum of young rats (postnatal day 20) after being exposed to chronic mild carbon monoxide (CO) at 25 ppm during prenatal (group A), prenatal and postnatal (group B), the postnatal period only (group C), and air (group D). The expression of genes associated with hypoxia signaling pathways was also investigated in the rat cerebella by real-time RT-PCR after CO exposure. Ngb and Cygb mRNAs did not change in any CO-exposed group. Quantitative immunohistochemistry showed no significant change in Ngb protein; however, there was a significant increase of Cygb protein in rats from groups A, B, and C when compared with group D. In group B, genes related to the generation of reactive oxygen species (Nos2) and lipid metabolism (Apat2) were upregulated. In contrast, no changes were found in the expression of 8 genes typically upregulated by hypoxic conditions (Angptl4, Arnt2, Casp1, Crebbp, Hif1a, Hif3a, Mt3, or Vegfa) in any CO-exposed group, suggesting that hypoxia-related gene expression is not altered by this mild CO exposure. Cygb but not Ngb may protect cerebellar cells from the chronic presence of CO exposure during prenatal and postnatal development.

Evidence for oxidative stress in the developing cerebellum of the rat after chronic mild carbon monoxide exposure (0.0025% in air).

BACKGROUND: The present study was designed to test the hypothesis that chronic very mild prenatal carbon monoxide (CO) exposure (25 parts per million) subverts the normal development of the rat cerebellar cortex. Studies at this chronic low CO exposure over the earliest periods of mammalian development have not been performed to date. Pregnant rats were exposed chronically to CO from gestational day E5 to E20. In the postnatal period, rat pups were grouped as follows: Group A: prenatal exposure to CO only; group B: prenatal exposure to CO then exposed to CO from postnatal day 5 (P5) to P20; group C: postnatal exposure only, from P5 to P20, and group D, controls (air without CO). At P20, immunocytochemical analyses of oxidative stress markers, and structural and functional proteins were assessed in the cerebellar cortex of the four groups. Quantitative real time PCR assays were performed for inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) nitric oxide synthases. RESULTS: Superoxide dismutase-1 (SOD1), SOD2, and hemeoxygenase-1 (HO-1) immunoreactivity increased in cells of the cerebellar cortex of CO-exposed pups. INOS and nitrotyrosine immunoreactivity also increased in blood vessels and Purkinje cells (PCs) of pups from group-A, B and C. By contrast, nNOS immunoreactivity decreased in PCs from group-B. Endothelial NOS immunoreactivity showed no changes in any CO-exposed group. The mRNA levels for iNOS were significantly up-regulated in the cerebellum of rats from group B; however, mRNA levels for nNOS and eNOS remained relatively unchanged in groups A, B and C. Ferritin-H immunoreactivity increased in group-B. Immunocytochemistry for neurofilaments (structural protein), synapsin-1 (functional protein), and glutamic acid decarboxylase (the enzyme responsible for the synthesis of the inhibitory neurotransmitter GABA), were decreased in groups A and B. Immunoreactivity for two calcium binding proteins, parvalbumin and calbindin, remained unchanged. The immunoreactivity of the astrocytic marker GFAP increased after prenatal exposure. CONCLUSION: We conclude that exogenously supplied CO during the prenatal period promotes oxidative stress as indicated by the up-regulation of SOD-1, SOD-2, HO-1, Ferritin-H, and iNOS with increased nitrotyrosine in the rat cerebella suggesting that deleterious and protective mechanisms were activated. These changes correlate with reductions of proteins important to cerebellar function: pre-synaptic terminals proteins (synapsin-1), proteins for the maintenance of neuronal size, shape and axonal quality (neurofilaments) and protein involved in GABAergic neurotransmission (GAD). Increased GFAP immunoreactivity after prenatal CO-exposure suggests a glial mediated response to the constant presence of CO. There were differential responses to prenatal vs. postnatal CO exposure: Prenatal exposure seems to be more damaging; a feature exemplified by the persistence of markers indicating oxidative stress in pups at P20, following prenatal only CO-exposure. The continuation of this cellular environment up to day 20 after CO exposure suggests the condition is chronic. Postnatal exposure without prenatal exposure shows the least impact, whereas prenatal followed by postnatal exposure exhibits the most pronounced outcome among the groups.

Acid-sensing ionic channels in the rat vestibular endorgans and ganglia.

Acid-sensing ionic channels (ASICs) are members of the epithelial Na+ channel/degenerin (ENaC/DEG) superfamily. ASICs are widely distributed in the central and peripheral nervous system. They have been implicated in synaptic transmission, pain perception, and the mechanoreception in peripheral tissues. Our objective was to characterize proton-gated currents mediated by ASICs and to determine their immunolocation in the rat vestibular periphery. Voltage clamp of cultured afferent neurons from P7 to P10 rats showed a proton-gated current with rapid activation and complete desensitization, which was carried almost exclusively by sodium ions. The current response to protons (H+) has a pH0.5 of 6.2. This current was reversibly decreased by amiloride, gadolinium, lead, acetylsalicylic acid, and enhanced by FMRFamide and zinc, and negatively modulated by raising the extracellular calcium concentration. Functional expression of the current was correlated with smaller-capacitance neurons. Acidification of the extracellular pH generated action potentials in vestibular neurons, suggesting a functional role of ASICs in their excitability. Immunoreactivity to ASIC1a and ASIC2a subunits was found in small vestibular ganglion neurons and afferent fibers that run throughout the macula utricle and crista stroma. ASIC2b, ASIC3, and ASIC4 were expressed to a lesser degree in vestibular ganglion neurons. The ASIC1b subunit was not detected in the vestibular endorgans. No acid-pH-sensitive currents or ASIC immunoreactivity was found in hair cells. Our results indicate that proton-gated current is carried through ASICs and that ionic current activated by H+ contributes to shape the vestibular afferent neurons' response to its synaptic input.

Time course of auditory impairment in mice lacking the electroneutral sodium bicarbonate cotransporter NBC3 (slc4a7).

Mice with a targeted disruption of the gene encoding the stilbene-insensitive electroneutral sodium bicarbonate cotransporter (NBC3; slc4a7) exhibit cochlear and retinal degeneration. To establish the progressive nature of sensory cells loss in slc4a7-/- deficient mice, we studied the morphology of cochleas of slc4a7-/- and slc4a7+/+ mice from postnatal day two (P2) to ninety (P90). Cell death was evaluated in slc4a7-/- cochleas using the TUNEL technique and caspase-3 immunoreactivity. The time course of NBC3 expression in the cochlea was assessed by immunohistochemistry using an antibody against NBC3. Between P2 and P8, slc4a7-/- mice cochlea exhibit normal morphology. There was a normal complement of inner and outer hair cells from the hook to the apical region. At P15, slc4a7-/- mice cochlea inner and outer hair cells were still present at the hook region, and vacuoles were seen underneath Hensen's cells. At P21, inner and outer hair cells were degenerated in this region. Between P30 and P90, there was a pronounced loss of hair cells and spiral ganglia neurons. Morphological analysis of the spiral ligament showed a progressive loss of type II and IV fibrocytes beginning at day 21. Transmission electron microscopy observations at P30 and P90 revealed that type II and IV fibrocytes showed shrinkage and vacuolization. In addition, hair cells were deteriorated with evidence of shrinkage and picnotic nuclei. TUNEL staining showed apoptotic cells at P8 in the organ of Corti at the basal region of the cochlea. At P15, caspase-3 immunoreactivity was present in supporting cells of the organ of Corti. NBC3 mild immunoreactivity was detected in the organ of Corti at P11. There was an increase in the expression of NBC3 in the spiral ligament between P17 and P19. From P21 to P90, NBC3 expression was confined to the spiral ligament and inner and outer sulcus cells. The vestibular sensory epithelia from slc4a7-/- mice were normal from P2 to P90. Damage of the sensory epithelia at the high frequency zone of the cochlea suggests that NBC3 may play an important physiological role in this region.

Limiting iron availability confers neuroprotection from chronic mild carbon monoxide exposure in the developing auditory system of the rat.

Iron deficiency and chronic mild carbon monoxide (CO) exposure are nutritional and environmental problems that can be experienced simultaneously. We examined the effects of chronic mild CO exposure and iron availability on auditory development in the rat. We propose that chronic mild CO exposure creates an oxidative stress condition that impairs the spiral ganglion neurons. The CO-exposed rat pups had decreased neurofilament proteins and increased copper, zinc-superoxide dismutase (SOD1) in the spiral ganglion neurons. We conclude that the increased amount of SOD1 causes an increase in hydrogen peroxide production that allows the Fenton reaction to occur. This reaction uses both iron and hydrogen peroxide to generate hydroxyl radicals and leads to the development of oxidative stress that impairs neuronal integrity. However, rat pups with decreased iron and CO exposure (ARIDCO) exhibited in their cochlea an up-regulation of transferrin, whereas their expression of neurofilament proteins and SOD1 were similar to control. Consequently, reduced iron availability and the normal expression of SOD1 do not promote oxidative stress in the cochlea. By using basal c-Fos expression as a marker for cellular activation we found a significant reduction in c-Fos expression in the central nucleus of the inferior colliculus in iron-adequate rat pups exposed to CO. By contrast, rather than being reduced, c-Fos expression in the ARIDCO group is the same as for controls. We conclude that the cochlea of rat pups with normal iron availability is selectively affected by mild CO exposure, causing a chronic oxidative stress, whereas limiting iron availability ameliorates the effect caused by mild CO exposure by averting conditions that facilitate oxidative stress.

Mutations in the gene encoding filamin B disrupt vertebral segmentation, joint formation and skeletogenesis.

The filamins are cytoplasmic proteins that regulate the structure and activity of the cytoskeleton by cross-linking actin into three-dimensional networks, linking the cell membrane to the cytoskeleton and serving as scaffolds on which intracellular signaling and protein trafficking pathways are organized (reviewed in refs. 1,2). We identified mutations in the gene encoding filamin B in four human skeletal disorders. We found homozygosity or compound heterozygosity with respect to stop-codon mutations in autosomal recessive spondylocarpotarsal syndrome (SCT, OMIM 272460) and missense mutations in individuals with autosomal dominant Larsen syndrome (OMIM 150250) and the perinatal lethal atelosteogenesis I and III phenotypes (AOI, OMIM 108720; AOIII, OMIM 108721). We found that filamin B is expressed in human growth plate chondrocytes and in the developing vertebral bodies in the mouse. These data indicate an unexpected role in vertebral segmentation, joint formation and endochondral ossification for this ubiquitously expressed cytoskeletal protein.

Mild carbon monoxide exposure diminishes selectively the integrity of the cochlea of the developing rat.

Rat pups were chronically exposed to carbon monoxide (CO) concentrations (12 or 25 ppm) in air starting at day 8, through 22 days of age, to examine the changes in the peripheral auditory system. Gastrostomy-reared rat pups, with or without CO exposure, were used and compared with mother-reared pups. The organ of Corti and the neurons of the spiral ganglion were analyzed for their morphology by using immunochemical and histological techniques. The inner and outer hair cells in the organ of Corti of animals exposed to 12 and 25 ppm CO were not different from the controls. However, at 25 ppm CO exposure, the nerve terminals innervating the inner hair cells were swollen. The somata of neurons in the spiral ganglion showed mild changes in the cytoplasm, and signs of mild vacuolization were observed in myelin covering their central processes. Synaptophysin, a marker for synaptic vesicles, and choline acetyltransferase, a marker for cholinergic terminals, showed no difference in immunoreactivity in CO exposed animals at 12 and at 25 ppm when compared with their age-matched controls. Also, Na(+)K(+) ATPase immunoreactivity patterns were normal compared with controls. Three enzymes were significantly reduced at the 25 ppm CO exposure: Cytochrome oxidase, NADH-TR, and calcium ATPase were decreased in both the organ of Corti and the neurons of the spiral ganglion, and decreased immunostaining for the neurofilament and myelin basic proteins was found. We conclude that components of the cochlea are selectively affected by mild chronic CO exposure during development.

Immunolocalization of voltage-gated calcium channel alpha1 subunits in the chinchilla cochlea.

The immunohistochemical localization of alpha1A, alpha1B, alpha1C, alpha1D, and alpha1E voltage-gated calcium channel subunits was investigated in the chinchilla organ of Corti and spiral ganglia with the use of specific antipeptide antibodies. The inner and outer hair cells were immunoreactive for alpha1A and alpha1D subunit antibodies. Alpha1C immunoreactivity localized to the nerve terminals innervating inner hair cells and the basal pole of the outer hair cell. There was only non-specific staining to alpha1B and alpha1E. Supporting cells were non-immunoreactive. Spiral ganglia neurons were alpha1B, alpha1C, and alpha1D immunoreactive. A few spiral ganglia neurons were alpha1E immunoreactive. The importance of alpha1D, the pore-forming subunit of the L-type channel, in outer and inner hair cell function has been clearly demonstrated in electrophysiological, molecular biological, and knockout models. The presence of alpha1A, the pore-forming subunit of the P/Q type channels, has not previously been demonstrated in inner and outer hair cells, and its function in the cochlear hair cell is unknown.

Subcellular immunolocalization of NMDA receptor subunit NR1, 2A, 2B in the rat vestibular periphery.

The immunohistochemical localization of the NMDA glutamate receptor subunits NR1, NR2A, and NR2B was investigated in the rat vestibular periphery at the light and electron microscopy level using specific antipeptide antibodies. The afferent calyceal terminals and nerve fibers innervating type I vestibular hair cells were strongly NR1, NR2A, and NR2B immunoreactive. Under electron microscopy, the basolateral type I hair cell membrane was NR1 immunoreactive. The type II hair cell and its afferent boutons were NR1, NR2A, and NR2B non-immunoreactive. Nearly all of Scarpa's ganglion neurons were NR1 immunoreactive, but there was a subset of NR2A non-immunoreactive neurons. Additionally, the larger sized Scarpa's ganglia neurons were NR2B immunoreactive, while the smaller neurons were non-immunoreactive. These findings are strong evidence for functional NMDA receptor mediation or modulation of afferent excitatory neurotransmission from type I but not type II vestibular hair cells to the primary afferent nerve. The receptor subtype(s) may be a combination of NR1/NR2A, NR1/NR2B, and/or NR1/NR2A/NR2B.