Xiaorui Shi

Upregulated iNOS and oxidative damage to the cochlear stria vascularis due to noise stress.

Our previous work has revealed increased nitric oxide (NO) production in the cochlear perilymph following noise stress. However, it is not clear if the increase of NO is related to iNOS and whether NO-related oxidative stress can cause vascular tissue damage. In this study, iNOS immunoreactivity, NO production, and reactive oxygen species (ROS) in the lateral wall were examined in normal mice and compared with similar animals exposed to 120 dBA broadband noise, 3 h/day, for 2 consecutive days. In the normal animals, iNOS expression was not observed in the vascular endothelium of the stria vascularis and only weak iNOS immunoactivity was detected in the marginal cells. However, expression of iNOS in the wall of the blood vessels of stria vascularis and marginal cells was observed after loud sound stress (LSS). Relatively low levels of NO production and low ROS activity were detected in the stria vascularis in the unstimulated condition. In contrast, NO production was increased and ROS activity was elevated in the stria vascularis after LSS. These changes were attenuated by the iNOS inhibitor, GW 274150. To explore whether noise induces apoptotic processes in the stria vascularis, we examined morphological changes in endothelial- and marginal-cells. In vitro, annexin-V phosphatidylserine (PS) (to label and detect early evidence of apoptosis) was combined with propidium iodide (PI) (to probe plasma membrane integrity). PI alone was used in fixed tissues to detect later stage apoptotic cells by morphology of the nuclei. Following LSS, PS was expressed on cell surfaces of endothelial cells of blood vessels and marginal cells of the stria vascularis. Later stage apoptosis, characterized by irregular nuclei and condensation of nuclei, was also observed in these cells. The data indicate that increased iNOS expression and production of both NO and ROS following noise stress may lead to marginal cell pathology, and the dysfunction of cochlear microcirculation by inducing blood vessel wall damage.

Perivascular-resident macrophage-like melanocytes in the inner ear are essential for the integrity of the intrastrial fluid–blood barrier

The microenvironment of the cochlea is maintained by the barrier between the systemic circulation and the fluids inside the stria vascularis. However, the mechanisms that control the permeability of the intrastrial fluid–blood barrier remain largely unknown. The barrier comprises endothelial cells connected to each other by tight junctions and an underlying basement membrane. In a recent study, we found that the intrastrial fluid–blood barrier also includes a large number of perivascular cells with both macrophage and melanocyte characteristics. The perivascular-resident macrophage-like melanocytes (PVM/Ms) are in close contact with vessels through cytoplasmic processes. Here we demonstrate that PVM/Ms have an important role in maintaining the integrity of the intrastrial fluid–blood barrier and hearing function. Using a cell culture-based in vitro model and a genetically induced PVM/M-depleted animal model, we show that absence of PVM/Ms increases the permeability of the intrastrial fluid–blood barrier to both low- and high-molecular-weight tracers. The increased permeability is caused by decreased expression of pigment epithelial-derived factor, which regulates expression of several tight junction-associated proteins instrumental to barrier integrity. When tested for endocochlear potential and auditory brainstem response, PVM/M-depleted animals show substantial drop in endocochlear potential with accompanying hearing loss. Our results demonstrate a critical role for PVM/Ms in regulating the permeability of the intrastrial fluid–blood barrier for establishing a normal endocochlear potential hearing threshold.

Physiopathology of the Cochlear Microcirculation

Normal blood supply to the cochlea is critically important for establishing the endocochlear potential and sustaining production of endolymph. Abnormal cochlear microcirculation has long been considered an etiologic factor in noise-induced hearing loss, age-related hearing loss (presbycusis), sudden hearing loss or vestibular function, and Menieres disease. Knowledge of the mechanisms underlying the pathophysiology of cochlear microcirculation is of fundamental clinical importance. A better understanding of cochlear blood flow (CoBF) will enable more effective management of hearing disorders resulting from aberrant blood flow. This review focuses on recent discoveries and findings related to the physiopathology of the cochlear microvasculature.

The electrochemical and fluorescence detection of nitric oxide in the cochlea and its increase following loud sound

A nitric oxide (NO)-selective sensor (tip diameter 30 microm) was inserted into the perilymph of the basal turn of the guinea pig cochlea. The basal level and stimulation-induced changes of NO were measured. The mean (+/-S.E.M.) basal level of NO was 273+/-42.9 nM. Following perilymphatic perfusion of the artificial perilymph containing NO synthase (NOS) substrate L-arginine (100 microM) combined with cofactor (6R)-5,6,7,8-tetrahydrobiopterin dihydrochloride (100 microM), a rapid and significant increase of NO to a mean concentration of 392+/-32.3 nM (P < 0.01, n = 10) was recorded. In contrast, a significant decrease of mean NO concentration to 180+/-32.7 nM (P < 0.01, n = 10) was observed following the perfusion of the NOS-inhibiting agent N(G)-nitro-L-arginine methyl ester (100 microM). No change in the NO concentration was found following the perfusion of either artificial perilymph or N(G)-monomethyl-D-arginine (100 microM) solution employed as controls. Broadband noise exposure (3 h/day at 120 dBA SPL) for three consecutive days produced an increase in NO concentration to 618+/-60.7 nM (P < 0.05, n = 10) in the perilymph. In addition, by using specific dyes for NO, 4,5-diaminofluoresceine diacetate and for the reactive oxygen species (ROS), dihydrorhodamine 1,2,3, the distribution of NO in the whole mounts of the organ of Corti and the production of ROS in vivo in the organ of Corti were investigated in both control (n = 5) and noise-exposed (n = 5) animals. The more intense NO and ROS fluorescence was observed in both the inner and outer hair cells in the noise-exposed groups. It is proposed that both the basal level and the increase in NO concentration following the addition of substrate (L-arginine) are produced by the constitutive NOS while the elevated NO and ROS following noise exposure indicate that NO may be involved in noise-induced hearing loss.

Altered expression of inducible nitric oxide synthase (iNOS) in the cochlea.

Using immunohistochemistry and Western blot, the expression of inducible nitric oxide synthase (iNOS) in the lateral wall and organ of Corti was examined in normal (unstimulated) and stimulated mice and guinea pigs. The stimuli were: (1). injection of bacterial lipopolysaccharide (LPS, 5 mg/ml) into the middle ear through the tympanic membrane and (2). exposure to a 110 dB SPL (A-weighted) broadband noise, 3 h/day, for three consecutive days. For the unstimulated condition, weak iNOS expression was found in the vascular endothelium, marginal cells, nerve fibers, stereocilia of hair cells and Hensens cells of the organ of Corti. More intense iNOS fluorescence signals were observed in cochlear tissues (particularly in hair cells and stria vascularis marginal cells) in animals exposed to loud sound or treated with LPS. Although the precise roles of iNOS expression in normal cochlear function have yet to be determined, enhanced iNOS expression following noise exposure and LPS suggests its participation in cochlear pathophysiology, including noise- and inflammatory factor-induced hearing loss.

Resident macrophages in the cochlear blood-labyrinth barrier and their renewal via migration of bone-marrow-derived cells

A large population of perivascular cells was found to be present in the area of the blood-labyrinth barrier in the stria vascularis of normal adult cochlea. The cells were identified as perivascular resident macrophages (PVMs), as they were positive for several macrophage surface molecules including F4/80, CD68, and CD11b. The macrophages, which were closely associated with microvessels and structurally intertwined with endothelial cells and pericytes, constitutively expressed scavenger receptor classes A1 and B1 and accumulated blood-borne proteins such as horseradish peroxidase and acetylated low-density lipoprotein. The PVMs were demonstrated to proliferate slowly, as evidenced by the absence of 5-bromo-2-deoxyuridine (BrdU)-positive PVMs at 3–14 days in normal mice injected with BrdU. However, in irradiated mice, the majority of the PVMs turned over via bone-marrow-cell migration within a 10-month time-frame. The existence of PVMs in the vascular wall of the blood-labyrinth barrier might therefore serve as a source for progenitor cells for postnatal vasculogenesis and might contribute to the repair of damaged vessels in the context of a local inflammatory response.

AIF and endoG translocation in noise exposure induced hair cell death.

Activation of caspases is a key element in the apoptotic process. However, mitochondria also play an important role via the release of proapoptotic proteins. This study investigated the roles of mitochondria-related apoptosis inducing factor (AIF) and endonuclease G (endoG), mitochondrion-specific nucleases, as well as caspase-3, an important mediator of apoptosis, in noise exposure induced hair cell death. Guinea pigs were exposed for 4h/day to broadband noise at 122 dB SPL for 2 days. After the noise exposure, the cochleae were examined for the activity of caspase-3 with carboxyfluorescein-labeled fluoromethyl ketone (FMK)-peptide inhibitors. The cochleae were further examined for AIF and endoG translocation from the mitochondria by immunohistochemistry. Noise exposure triggered activation of caspase-3 in apoptotic hair cells. In the normal organ of Corti, AIF and endoG were co-localized to the mitochondria. After noise exposure, AIF translocated into the nuclei of apoptotic and necrotic hair cells. The translocation of endoG from mitochondria into the nucleus was also found in apoptotic OHCs. These findings indicate that mitochondria-released proapoptotic proteins, AIF and endoG, are important factors in a noise-induced hair cell death pathway.

Nitric oxide distribution and production in the guinea pig cochlea.

Production sites and distribution of nitric oxide (NO) were detected in cochlear lateral wall tissue, the organ of Corti and in isolated outer hair cells (OHCs) from the guinea pig using the fluorescent dye, 4,5-diaminofluorescein diacetate. Fluorescent signal, indicating the presence of NO, was found in the afferent nerves and their putative endings near inner hair cells (IHCs) and putative efferent nerve endings near OHCs, the IHCs and OHCs, the endothelial cells of blood vessels of the spiral ligament, the stria vascularis, and the spiral blood vessels of the basilar membrane. An increased NO signal was observed following exposure to the substrate for NO, L-arginine, while exposure to NO synthase inhibitors resulted in a decrease in NO signal. Observation of OHCs at the subcellular level revealed differentially strong fluorescent signals at the locations of cuticular plate, the subcuticular plate region, the infranuclear region, and the region adjacent to the lateral wall. The findings indicate the presence of NO in the cochlea and suggest that NO may play an important role in both regulating vascular tone and mediating neurotransmission in guinea pig cochlea.

Endothelial cell, pericyte, and perivascular resident macrophage-type melanocyte interactions regulate cochlear intrastrial fluid-blood barrier permeability.

The integrity of the fluid–blood barrier in the stria vascularis is critical for maintaining inner ear homeostasis, especially for sustaining the endocochlear potential, an essential driving force for hearing function. However, the mechanisms that control intrastrial fluid–blood barrier permeability remain largely unknown. At the cellular level, the intrastrial fluid–blood barrier comprises cochlear microvascular endothelial cells connected to each other by tight junctions (TJs), an underlying basement membrane, and a second line of support consisting of cochlear pericytes and perivascular resident macrophage-type melanocytes. In this study, we use a newly established primary cell culture-based in vitro model to show that endothelial cells, pericytes, and perivascular resident macrophage-type melanocytes interact to control intrastrial fluid–blood barrier permeability. When the endothelial cell monolayer was treated with pericyte—or perivascular resident macrophage-type melanocyte—conditioned media, the permeability of the endothelial cell monolayer was significantly reduced relative to an untreated endothelial cell monolayer. Further study has shown the pericytes and perivascular resident macrophage-type melanocytes to regulate TJ expression in the endothelial cell monolayer. The new cell culture-based in vitro model offers a unique opportunity to obtain information on the organ-specific characteristics of the cochlear blood/tissue barrier. Our finding demonstrates the importance of signaling among pericytes, endothelial cells, and perivascular resident macrophage-type melanocytes to the integrity of the intrastrial fluid–blood barrier.

The Cochlear Pericytes

Objectives: Cochlear pericytes are not well characterized. The aim of this study was to further advance the characterization of cochlear pericyte location and distribution, with particular focus on pericyte‐related proteins on the capillaries of the cochlear lateral wall that are functionally integral to structure, contraction, and gap junction transport. Materials and Methods: Cochlear pericytes were identified by the immunofluorescence labeling of pericyte marker proteins, including alpha–smooth muscle actin (α‐SMA), desmin, Thy‐1, tropomyosin, and NG2, and by morphological identification, using fluorescence, electron, and differential interference contrast microscopy. Results: Pericytes were predominately found in the capillary network of the cochlear lateral wall, with considerable morphological heterogeneity across different types of microvessels. For example, pericytes on the vessels of the spiral ligament (V/SL) strongly expressed a gap junction protein, connexin 40, and were positive for α‐SMA, tropomyosin, and desmin. In contrast, pericytes on the vessels of the stria vascularis (V/SV) were positive for desmin, and were negative for α‐SMA and tropomyosin. Conclusions: The capillary networks of the cochlear lateral wall comprise a rich population of pericytes. These pericytes are morphologically heterogeneous, with protein expression potentially indicative of function.