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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 ...

Oregon Health & Science University

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.

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

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.

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

This protocol describes a growth medium-based approach for obtaining cochlear endothelial cells (ECs), pericytes (PCs) and perivascular resident macrophage-like melanocytes (PVM/Ms) from the stria vascularis of mice aged between P10 and P15 (P, postnatal day). The procedure does not involve mechanical or enzymatic digestion of the sample tissue. Explants of stria vascularis, 'mini-chips', are selectively cultured in growth medium, and primary cell lines are obtained in 7-10 d. The method is simple and reliable, and it provides high-quality ECs, PVM/Ms and PCs with a purity >90% after two passages. This protocol is suitable for producing primary culture cells from organs and tissues of small volume and high anatomical complexity, such as the inner ear capillaries. The highly purified primary cell lines enable cell culture-based in vitro modeling of cell-cell interactions, barrier control function and drug action.

Isolation and culture of endothelial cells, pericytes and perivascular resident macrophage-like melanocytes from the young mouse ear.

Tissue perivascular resident macrophages (PVM/Ms), a hybrid cell type with characteristics of both macrophages and melanocytes, are critical for establishing and maintaining the endocochlear potential (EP) required for hearing. The PVM/Ms modulate expression of tight- and adherens-junction proteins in the endothelial barrier of the stria vascularis (intrastrial fluid-blood barrier) through secretion of a signaling molecule, pigment epithelium growth factor (PEDF). Here, we identify a significant link between abnormalities in PVM/Ms and endothelial barrier breakdown from acoustic trauma to the mouse ear. We find that acoustic trauma causes activation of PVM/Ms and physical detachment from capillary walls. Concurrent with the detachment, we find loosened tight junctions between endothelial cells and decreased production of tight- and adherens-junction protein, resulting in leakage of serum proteins from the damaged barrier. A key factor in the intrastrial fluid-blood barrier hyperpermeability exhibited in the mice is down-regulation of PVM/M modulated PEDF production. We demonstrate that delivery of PEDF to the damaged ear ameliorates hearing loss by restoring intrastrial fluid-blood barrier integrity. PEDF up-regulates expression of tight junction-associated proteins (ZO-1 and VE-cadherin) and PVM/M stabilizing neural cell adhesion molecule (NCAM-120). These studies point to the critical role PVM/Ms play in regulating intrastrial fluid-blood barrier integrity in healthy and noise-damaged ears.

Perivascular macrophage-like melanocyte responsiveness to acoustic trauma--a salient feature of strial barrier associated hearing loss.

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.

The high prevalence/incidence of hearing loss (HL) in humans makes it the most common sensory defect. The majority of the cases are of genetic origin. Non-syndromic hereditary HL is extremely heterogeneous. Genetic approaches have been instrumental in deciphering genes that are crucial for auditory function. In this study, we first used NADf chip to exclude the implication of known North-African mutations in HL in a large consanguineous Tunisian family (FT13) affected by autosomal recessive non-syndromic HL (ARNSHL). We then performed genome-wide linkage analysis and assigned the deafness gene locus to ch:5q23.2-31.1, corresponding to the DFNB60 ARNSHL locus. Moreover, we performed whole exome sequencing on FT13 patient DNA and uncovered amino acid substitution p.Cys113Tyr in SLC22A4, a transporter of organic cations, cosegregating with HL in FT13 and therefore the cause of ARNSHL DFNB60. We also screened a cohort of small Tunisian HL families and uncovered an additional deaf proband of consanguineous parents that is homozygous for p.Cys113Tyr carried by the same microsatellite marker haplotype as in FT13, indicating that this mutation is ancestral. Using immunofluorescence, we found that Slc22a4 is expressed in stria vascularis (SV) endothelial cells of rodent cochlea and targets their apical plasma membrane. We also found Slc22a4 transcripts in our RNA-seq library from purified primary culture of mouse SV endothelial cells. Interestingly, p.Cys113Tyr mutation affects the trafficking of the transporter and severely alters ergothioneine uptake. We conclude that SLC22A4 is an organic cation transporter of the SV endothelium that is essential for hearing, and its mutation causes DFNB60 form of HL. 

A mutation in SLC22A4 encoding an organic cation transporter expressed in the cochlea strial endothelium causes human recessive non-syndromic hearing loss DFNB60.

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.

Lingling Neng

Oregon Hearing Research Center,
Department of Otolaryngology/Head & Neck Surgery,
Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck Surgery

Oregon Health & Science University

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

Lingling Neng

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Oregon Health & Science University

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

Oregon Hearing Research Center,
Department of Otolaryngology/Head & Neck Surgery,
Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck Surgery