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Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

A large number of perivascular cells expressing both macrophage and melanocyte characteristics (named perivascular-resident macrophage-like melanocytes, PVM/Ms), previously found in the intra-strial fluid-blood barrier, are also found in the blood-labyrinth barrier area of the vestibular system in normal adult cochlea, including in the three ampullae of the semicircular canals (posterior, superior, and horizontal), utricle, and saccule. The cells were identified as PVM/Ms, positive for the macrophage and melanocyte marker proteins F4/80 and GSTα4. Similar to PVM/Ms present in the stria vascularis, the PVM/Ms in the vestibular system are closely associated with microvessels and structurally intertwined with endothelial cells and pericytes, with a density in normal (unstimulated) utricle of 225 ± 43/mm(2); saccule 191 ± 25/mm(2); horizontal ampullae 212 ± 36/mm(2); anterior ampullae 238 ± 36/mm(2); and posterior ampullae 223 ± 64/mm(2). Injection of bacterial lipopolysaccharide into the middle ear through the tympanic membrane causes the PVM/Ms to activate and arrange in an irregular pattern along capillary walls in all regions within a 48-h period. The inflammatory response significantly increases vascular permeability and leakage. The results underscore the morphological complexity of the blood barrier in the vestibular system, with its surrounding basal lamina, pericytes, as well as second line of defense in PVM/Ms. PVM/Ms may be important to maintain blood barrier integrity and initiating local inflammatory response in the vestibular system. 

Characterization and inflammatory response of perivascular-resident macrophage-like melanocytes in the vestibular system.

Normal microvessel structure and function in the cochlea is essential for maintaining the ionic and metabolic homeostasis required for hearing function. Abnormal cochlear microcirculation has long been considered an etiologic factor in hearing disorders. A better understanding of cochlear blood flow (CoBF) will enable more effective amelioration of hearing disorders that result from aberrant blood flow. However, establishing the direct relationship between CoBF and other cellular events in the lateral wall and response to physio-pathological stress remains a challenge due to the lack of feasible interrogation methods and difficulty in accessing the inner ear. Here we report on new methods for studying the CoBF in a mouse model using a thin or open vessel-window in combination with fluorescence intra-vital microscopy (IVM). An open vessel-window enables investigation of vascular cell biology and blood flow permeability, including pericyte (PC) contractility, bone marrow cell migration, and endothelial barrier leakage, in wild type and fluorescent protein-labeled transgenic mouse models with high spatial and temporal resolution. Alternatively, the thin vessel-window method minimizes disruption of the homeostatic balance in the lateral wall and enables study CoBF under relatively intact physiological conditions. A thin vessel-window method can also be used for time-based studies of physiological and pathological processes. Although the small size of the mouse cochlea makes surgery difficult, the methods are sufficiently developed for studying the structural and functional changes in CoBF under normal and pathological conditions. 

Thin and open vessel windows for intra-vital fluorescence imaging of murine cochlear blood flow.

Tight control over cochlear blood flow (CoBF) and the blood-labyrinth barrier (BLB) in the striavascularis is critical for maintaining the ionic, fluid and energy balance necessary for hearing function. Inefficient CoBF and disruption of BLB integrity have long been considered major etiologic factors in a variety of hearing disorders. In this study, we investigate structural changes in the BLB of the striavascularis in age-graded C57BL/6 mice (1 to 21 months) with a focus on changes in two blood barrier accessory cells, namely pericytes (PCs) and perivascular-resident macrophage-like melanocytes (PVM/Ms). Decreased capillary density was detectable at 6 months, with significant capillary degeneration seen in 9- to 21-month-old mice. Reduced capillary density was highly correlated with lower numbers of PCs and PVM/Ms. "Drop-out" of PCs and "activation" of PVM/Ms were seen at 6 months, with drastic changes being observed by 21 months. With newly established in vitro three-dimensional cell-based co-culture models, we demonstrate that PCs and PVM/Ms are essential for maintaining cochlear vascular architecture and stability. 

Structural changes in thestrial blood-labyrinth barrier of aged C57BL/6 mice.

Middle ear infection (or inflammation) is the most common pathological condition that causes fluid to accumulate in the middle ear, disrupting cochlear homeostasis. Lipopolysaccharide, a product of bacteriolysis, activates macrophages and causes release of inflammatory cytokines. Many studies have shown that lipopolysaccharides cause functional and structural changes in the inner ear similar to that of inflammation. However, it is specifically not known how lipopolysaccharides affect the blood-labyrinth barrier in the stria vascularis (intra-strial fluid-blood barrier), nor what the underlying mechanisms are. In this study, we used a cell culture-based in vitro model and animal-based in vivo model, combined with immunohistochemistry and a vascular leakage assay, to investigate lipopolysaccharide effects on the integrity of the mouse intra-strial fluid-blood barrier. Our results show lipopolysaccharide-induced local infection significantly affects intra-strial fluid-blood barrier component cells. Pericytes and perivascular-resident macrophage-like melanocytes are particularly affected, and the morphological and functional changes in these cells are accompanied by substantial changes in barrier integrity. Significant vascular leakage is found in the lipopolysaccharide treated-animals. Consistent with the findings from the in vivo animal model, the permeability of the endothelial cell monolayer to FITC-albumin was significantly higher in the lipopolysaccharide-treated monolayer than in an untreated endothelial cell monolayer. Further study has shown the lipopolysaccharide-induced inflammation to have a major effect on the expression of tight junctions in the blood barrier. Lipopolysaccharide was also shown to cause high frequency hearing loss, corroborated by previous reports from other laboratories. Our findings show lipopolysaccharide-evoked middle ear infection disrupts inner ear fluid balance, and its particular effects on the intra-strial fluid-blood barrier, essential for cochlear homeostasis. The barrier is degraded as the expression of tight junction-associated proteins such as zona occludens 1, occludin, and vascular endothelial cadherin are down-regulated. 

Lipopolysaccharide-induced middle ear inflammation disrupts the cochlear intra-strial fluid-blood barrier through down-regulation of tight junction proteins.

The vestibular blood-labyrinth barrier (BLB) is comprised of perivascular-resident macrophage-like melanocytes (PVM/Ms) and pericytes (PCs), in addition to endothelial cells (ECs) and basement membrane (BM), and bears strong resemblance to the cochlear BLB in the stria vascularis. Over the past few decades, in vitro cell-based models have been widely used in blood-brain barrier (BBB) and blood-retina barrier (BRB) research, and have proved to be powerful tools for studying cell-cell interactions in their respective organs. Study of both the vestibular and strial BLB has been limited by the unavailability of primary culture cells from these barriers. To better understand how barrier component cells interact in the vestibular system to control BLB function, we developed a novel culture medium-based method for obtaining EC, PC, and PVM/M primary cells from tiny explants of the semicircular canal, sacculus, utriculus, and ampullae tissue of young mouse ears at post-natal age 8-12 d. Each phenotype is grown in a specific culture medium which selectively supports the phenotype in a mixed population of vestibular cell types. The unwanted phenotypes do not survive passaging. The protocol does not require additional equipment or special enzyme treatment. The harvesting process takes less than 2 h. Primary cell types are generated within 7-10 d. The primary culture ECs, PCs, and PVM/M shave consistent phenotypes more than 90% pure after two passages (∼ 3 weeks). The highly purified primary cell lines can be used for studying cell-cell interactions, barrier permeability, and angiogenesis.

Culture media-based selection of endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes from the young mouse vestibular system.

Normal blood supply to the cochlea is critical for hearing. Noise damages auditory sensory cells and has a marked effect on the microvasculature in the cochlear lateral wall. Pericytes in the stria vascularis (strial pericytes) are particularly vulnerable and sensitive to acoustic trauma. Exposure of NG2DsRedBAC transgenic mice (6-8 weeks old) to wide-band noise at a level of 120 dB for 3 h per day for 2 consecutive days produced a significant hearing threshold shift and caused pericytes to protrude and migrate from their normal endothelial attachment sites. The pericyte migration was associated with increased expression of platelet-derived growth factor beta (PDGF-BB). Blockade of PDGF-BB signaling with either imatinib, a potent PDGF-BB receptor (PDGFR) inhibitor, or APB5, a specific PDGFRβ blocker, significantly attenuated the pericyte migration from strial vessel walls. The PDGF-BB-mediated strial pericyte migration was further confirmed in an in vitro cell migration assay, as well as in an in vivo live animal model used in conjunction with confocal fluorescence microscopy. Pericyte migration took one of two different forms, here denoted protrusion and detachment. The protrusion is characterized by pericytes with a prominent triangular shape, or pericytes extending fine strands to neighboring capillaries. The detachment is characterized by pericyte detachment and movement away from vessels. We also found the sites of pericyte migration highly associated with regions of vascular leakage. In particular, under transmission electron microscopy (TEM), multiple vesicles at the sites of endothelial cells with loosely attached pericytes were observed. These data show that cochlear pericytes are markedly affected by acoustic trauma, causing them to display abnormal morphology. The effect of loud sound on pericytes is mediated by upregulation of PDGF-BB. Normal functioning pericytes are required for vascular stability.

Platelet-Derived Growth Factor Subunit B Signaling Promotes Pericyte Migration in Response to Loud Sound in the Cochlear Stria Vascularis.

Can damaged or degenerated vessels be regenerated in the ear? The question is clinically important, as disruption of cochlear blood flow is seen in a wide variety of hearing disorders, including in loud sound-induced hearing loss (endothelial injury), ageing-related hearing loss (lost vascular density), and genetic hearing loss (e.g., Norrie disease: strial avascularization). Progression in cochlear blood flow (CBF) pathology can parallel progression in hair cell and hearing loss. However, neither new vessel growth in the ear, nor the role of angiogenesis in hearing, have been investigated. In this study, we used an established ex vivo tissue explant model in conjunction with a matrigel matrix model to demonstrate for the first time that new vessels can be generated by activating a vascular endothelial growth factor (VEGF-A) signal. Most intriguingly, we found that the pattern of the newly formed vessels resembles the natural 'mesh pattern' of in situ strial vessels, with both lumen and expression of tight junctions. Sphigosine-1-phosphate (S1P) in synergy with VEGF-A control new vessel size and growth. Using transgenic neural/glial antigen 2 (NG2) fluorescent reporter mice, we have furthermore discovered that the progenitors of "de novo" strial vessels are NG2-derived cells. Taken together, our data demonstrates that damaged strial microvessels can be regenerated by reprogramming NG2-derived angiogenic cells. Restoration of the functional vasculature may be critical for recovery of vascular dysfunction related hearing loss.

Vascular regeneration in adult mouse cochlea stimulated by VEGF-A and driven by NG2-derived cells ex vivo.

Acoustic trauma disrupts cochlear blood flow and damages sensory hair cells. Damage and regression of capillaries after acoustic trauma have long been observed, but the underlying mechanism of pathology has not been understood. We show herein that loud sound causes change of phenotype from neural/glial antigen 2 positive/α-smooth muscle actin negative to neural/glial antigen 2 positive/α-smooth muscle actin positive in some pericytes (PCs) on strial capillaries that is strongly associated with up-regulation of transforming growth factor-β1. The acoustic trauma also reduced capillary density and increased deposition of matrix proteins, particularly in the vicinity of transformed PCs. In a newly established in vitro three-dimensional endothelial cell (EC) and PC co-culture model, transformed PCs induced thicker capillary-like branches in ECs and increased collagen IV and laminin expression. Transplantation of exogenous PCs derived from neonatal day 10 mouse cochleae to acoustic traumatized cochleae, however, significantly attenuated the decreased vascular density in the stria. Transplantation of PCs pretransfected with adeno-associated virus 1-vascular endothelial growth factor-A165 under control of a hypoxia-response element markedly promotes vascular volume and blood flow, increased proliferation of PCs and ECs, and attenuated loud sound-caused loss in endocochlear potential and hearing. Our results indicate that loud sound-triggered PC transformation contributes to capillary wall thickening and regression, and young PC transplantation effectively rehabilitates the vascular regression and improves hearing.

Acoustic Trauma Causes Cochlear Pericyte-to-Myofibroblast-Like Cell Transformation and Vascular Degeneration, and Transplantation of New Pericytes Prevents Vascular Atrophy.

: Connexin 43 (Cx43) is a protein constituent of gap junctions (GJs) in various barrier cells, especially astrocytes and microglia of the blood-brain-barrier (BBB), where it plays an important role in intercellular communication and regulation of the barrier. Despite the importance of Cx43 in other blood barriers, not much attention has been paid to expression and function of Cx43 in the blood-labyrinth-barrier (BLB) of the stria vascularis in the cochlea. : We used multiple research approaches, including immunocytochemical staining, patch-clamp dye loading technique, real-time quantitative reverse transcription (RT)-PCR, western blot, measurement of endocochlear potential (EP) with an electrode through the scala media, and auditory brainstem response to test hearing function. : We found Cx43 expressed in vascular endothelial cells (ECs) and perivascular resident macrophages (PVMs) in the stria vascularis of adult C57BL/6 mouse cochleae. In particular, we found Cx43 expressed in foot processes of PVMs at points of contact with the endothelium. Consistent with Cx43 expression , we also found Cx43 expressed in EC-EC and EC-PVM interfaces in a co-cultured cell line model. Using a patch-clamp dye loading technique, we demonstrated that Alexa Fluor® 568 dye injected into PVMs diffuses to connected neighboring ECs. The functional coupling between the ECs and PVMs is blocked by 18α-Glycyrrhetinic acid (18α-GA), a GJ blocker. Suppression of Cx43 with small interfering RNA (siRNA)  significantly elevated hearing threshold and caused the EP to drop and the blood barrier to become more permeable. In further study, using  primary EC cell line models, we demonstrated that suppression of Cx43 disrupts intercellular tight junctions (TJs) in the EC monolayer and increases endothelial monolayer permeability. : Taken together, these findings underscore the importance of Cx43 expression in the normal ear for maintaining BLB integrity, normal EP, and hearing function.

Suppression of Connexin 43 Leads to Strial Vascular Hyper-Permeability, Decrease in Endocochlear Potential, and Mild Hearing Loss.

Millions of people are affected by hearing loss. Hearing loss is frequently caused by noise or aging and often associated with loss of pericytes. Pericytes populate the small vessels in the adult cochlea. However, their role in different types of hearing loss is largely unknown. Using an inducible and conditional pericyte depletion mouse model and noise-exposed mouse model, we show that loss of pericytes leads to marked changes in vascular structure, in turn leading to vascular degeneration and hearing loss. In vitro, using advanced tissue explants from pericyte fluorescence reporter models combined with exogenous donor pericytes, we show that pericytes, signaled by VEGF isoform A165 (VEGFA165), vigorously drive new vessel growth in both adult and neonatal mouse inner ear tissue. In vivo, the delivery of an adeno-associated virus serotype 1-mediated (AAV1-mediated) VEGFA165 viral vector to pericyte-depleted or noise-exposed animals prevented and regenerated lost pericytes, improved blood supply, and attenuated hearing loss. These studies provide the first clear-cut evidence that pericytes are critical for vascular regeneration, vascular stability, and hearing in adults. The restoration of vascular function in the damaged cochlea, including in noise-exposed animals, suggests that VEGFA165 gene therapy could be a new strategy for ameliorating vascular associated hearing disorders.

VEGFA165 gene therapy ameliorates blood-labyrinth barrier breakdown and hearing loss.

Perivascular-resident macrophage-like melanocytes (PVM/Ms) can upregulate the expression of tight junction-related proteins in endothelial cells (ECs) by secreting pigment epithelial-derived factor (PEDF), and thereby regulate the permeability of the intrastrial fluid-blood barrier critical for maintaining inner ear homeostasis. This study aimed to investigate the effects of long non-coding RNA (lncRNA) Rian on cell growth of PVM/Ms and PVM/Ms regulation of intrastrial fluid-blood barrier integrity mediated by PEDF. Rian was downregulated in the aged cochlea from 12-month-old C57BL/6 mice. Rian overexpression inhibited cell apoptosis and promoted cell viability of hypoxia-injured PVM/Ms as well as increased the concentration and expression of PEDF secreted by PVM/Ms. In contrast, Rian silencing exerted the opposite effects. Furthermore, in a cell co-culture model of ECs and PVM/Ms, Rian overexpression in PVM/Ms increased the expression of the junction-associated proteins in co-cultured ECs, and this effect was abrogated by blockade of PEDF by anti-PEDF in PVM/Ms. Further mechanistical investigation revealed that Rian promoted STAT3 nuclear translocation and activation by binding to FUS, and thereby promoted the secretion of PEDF. Collectively, Rian attenuates PVM/Ms injury and strengthens the ability of PVM/Ms to maintain the integrity of the endothelial barrier by promoting PEDF expression.

Long non-coding RNA Rian promotes the expression of tight junction proteins in endothelial cells by regulating perivascular-resident macrophage-like melanocytes and PEDF secretion.

Jinhui Zhang

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy