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The source of the Endocochlear Potential was originally demonstrated by Tasaki & Spiropolisin 1959. They opened scala media at the apex and touched an electrode to different tissues that bounded it. They found that an electrode showed the highest positive voltage when contacting striavascularis, the highly vascular tissue in the lateral wall of the cochlea.
The stria comprises two epithelial layers: • A layer of marginal cells • A layer of intermediate/basal cells. • Between the two layers lies an extracellular space termed the intrastrial space (IS) • Which is thus surrounded by the • apical membranes of intermediate cells and the basolateral membranes of marginal cells.
The intrastrial fluid–blood barrier separates the striavascularis(SV) from peripheral circulation. • The integrity of the barrier is critical for maintaining inner ear homeostasis, especially for sustaining the endocochlear potential (EP), an essential driving force for hearing function.
Classically, the intrastrial fluid–blood barrier comprises basement membrane and endothelial cells that connect to each other with tight junctions to form a diffusion barrier that prevents most blood-borne substances from entering the ear. • Recently, found that the intrastrial fluid–blood barrier also includes a large number of pericytesand perivascular- resident macrophage-like melanocytes (PVM/Ms)
What are Endothelial Cells? • The endothelium is the thin layer of cells that lines the interior surface of blood vessels and lymphatic vessels, forming an interface between circulating blood or lymph in the lumen and the rest of the vessel wall.
What are Pericytes? • Pericytes are contractile cells that wrap around endothelial cells • Pericytes communicate with endothelial cells • Pericytes regulate capillary blood flow, the clearance and phagocytosis of cellular debris
What are perivascular-resident macrophage like melanocytes (PVM/Ms)? • Perivascular of/relating to tissues surrounding small blood vessels • Macrophage a phagocytic tissue cell of the immune system that functions in the destruction of foreign antigens • Melanocytes specialized skin cell that produces the protective skin-darkening pigment melanin
PVM/Ms are a hybrid cell type • PVM/Ms express several melanocyte markers • PVM/Ms are resident macrophages, because the cells are positive for several macrophage surface molecules, including: • F4/80, CD68, and CD11b.
Astrocytes, Glial Cells, PVM/Ms • Astrocytes in the blood-brain barrier and Glial Cells in the blood-retina barrier play a functional role in maintaining the regulation of barrier integrity. • If these cells are compromised, the barrier loses tight-junction proteins, become leaky, and tissue edema results. • IDEA: PVM/Ms have a similar functional role as Astrocytes and Glial cells
Hypothesis • Test whether communication between Endothelial Cells (ECs) and PVM/Ms are essentialfor: • Barrier function • Microvessel permeability • Tested in both in vitro and in vivo models
Model • In Vitro: Primary endothelial and PVM/M cell lines from mouse cochlea • In Vivo: Ablating PVM/Ms. • Utilized a transgenic mouse model with a diphtheria toxin (DT) receptor under control of a human integrin αM promoter (CD11b) enabled transient depletion of the PVM/Ms
3D Confocal images show a large population of PVM/Ms sandwiched between marginal and basal cell layers of the SV. • (White) PVM/Msare labeled with an antibody for F4/80 • (Red) Cytoskeleton is labeled with phalloidin
A 3D reconstructed confocal image of the StriaVascularis Left Image: PVM/Ms situated in/under subepithelial marginal cells Right Image: PVM/Ms no direct contact with basal cells
FigC: Overview of PVM/Msare highly invested on the abluminal surface of capillaries FigD: Close Up of PVM/Ms, structurally intertwined with endothelial tube that interface with the capillary wall • Capillaries are labeled with antibody for collagen IV
Express melanocyte marker proteins: cytosolic GSTα4 GST Kir4.1
Consistent with the findings from whole-mounted tissue, GST, GSTα4, and Kir 4.1 also are expressed in primary cultured PVM/Ms. Hence, PVM/Ms have characteristics of melanocytes!!
Primary cultured ECs and PVM/Msunder differential interference contrast (DIC) microscopy are identified for marker proteinsvMf (ECs) and F4/80 (PVM/Ms) under confocal fluorescent microscopy • The EC monolayer expresses tight-junction proteins ZO-1 and occludin
Permeability was assessed by measuring the flux of 70-kDa fluorescent dextran across the endothelial monolayer for both the co-culture model and two control models (null endothelial monolayer and confluent EC monolayer)
EC monolayer leakage was determined by measuring the intensity of FITC-dextran fluorescence in the basolateral chamber • Permeability of the EC monolayer increases when PVM/Ms are absent • PVM/Msstrengthen the integrity of the endothelial barrier
In Vivo Time… • A transgene encoding a DT receptor was used for transient depletion of PVM/Ms. • The mice were assigned randomly to receive DT or control (saline) injections • 5 Day Regimen…of Intravenously Injections…
Ablation of PVM/Ms results in marked vascular leakage Immediately following the 5-d DT treatment, permeability assays using a range of low- and high-molecular-mass fluorescent tracers, including Evans blue, cadaverine Alexa Fluor-555, BSAAlexa Fluor-555, and IgG-Alexa 568, were found to accumulate in the cochlear lateral wall tissue of PVM/M-ablated animals but not in control animals
The in vivoresults corroborate in vitro results showing that absence of PVM/Ms weakens the endothelial barrier • Confirms previous findings that disruption of the intrastrial fluid–blood barrier results in significant hearing loss
mRNA levels for Tight Junction Proteins (ZO-1, occludin, and ve-cadherin), assessed with PCR, were decreased dramatically in the absence of PVM/Ms (EC only)
Protein levels for Tight Junctions (ZO-1, occludin, and ve-cadherin) analyzed by in-cell Western blotting, also showed a marked decrease
Immunohistochemical examination by confocal microscopy showed a less dense distribution of tight- and adherens-junction proteins between ECs in the absence of PVM/Ms This indicates that PVM/Mshave broad effects on expression of junction proteins, which link directly to endothelial barrier permeability.
Pigment epithelium-derived factor • Model of the PEDF/PEDFR signaling pathway • Regulates the expression of junction proteins • PVM/Mscontrol vascular permeability through this PEDF/PEDFR signaling pathway.
Take Away Message • PVM/Msare a hybrid phenotype with macrophage and melanocyte characteristics • PVM/Ms are important for hearing thresholds and EP • PVM/Mscontrol the integrity of the intrastrialfluid–blood barrier in the SV by affecting the expression of tight- and adherens-junction proteins • PVM/Msstabilize the intrastrialfluid–blood barrier • PEDF (Pigment epithelium-derived factor) is as an essential signaling molecule
