Subcellular distribution and relative expression of fibrocyte markers in the CD/1 mouse cochlea assessed by semiquantitative immunogold electron microscopy

Abstract

Spiral ligament fibrocytes function in cochlear homeostasis, maintaining the endocochlear potential by participating in potassium recycling, and fibrocyte degeneration contributes to hearing loss. Their superficial location makes them amenable to replacement by cellular transplantation. Fibrocyte cultures offer one source of transplantable cells, but determining what fibrocyte types they contain and what phenotype transplanted cells may adopt is problematic. Here, we use immunogold electron microscopy to assess the relative expression of markers in native fibrocytes of the CD/1 mouse spiral ligament. Caldesmon and aquaporin 1 are expressed more in type III fibrocytes than any other type. S-100 is strongly expressed in types I, II, and V fibrocytes, and α1Na,K-ATPase is expressed strongly only in types II and V. By combining caldesmon or aquaporin 1 with S-100 and α1Na,K-ATPase, a ratiometric analysis of immunogold density distinguishes all except type II and type V fibrocytes. Other putative markers (creatine kinase BB and connective tissue growth factor) did not provide additional useful analytical attributes. By labeling serial sections or by double or triple labeling with combinations of three antibodies, this technique could be used to distinguish all except type II and type V fibrocytes in culture or after cellular transplantation into the lateral wall.

Figure 1.

Transmitted light (A) and immunofluorescence (B–G) microscopy of wax sections of spiral ligament in a midmodiolar plane, labeled with each of the antibodies and viewed by confocal microscopy. (A) The locations of the different fibrocyte types (I–V), the Reissner membrane (RM), and stria vascularis (sv) are shown. This section is the same as the one shown in B. (B) Caldesmon is localized along the side of the ligament adjacent to the bony wall, the location of the type III cells. (C) S-100 is localized throughout the ligament but appears to be absent from a thin line, probably representing the type III cell region (*). (D) AQP1 is localized along the margin of the ligament, where type III cells occur. The difference in appearance of the labeling compared with caldesmon, which localizes to the same region, is probably due to the fact that the AQP1 is a membrane protein while caldesmon is cytoplasmic. The former probably therefore outlines the cells rather than filling them. (E) Na,K-ATPase is localized to type II and type V regions (arrows) and is also strongly expressed in the stria vascularis (sv) but more weakly elsewhere. (F) CK-BB is localized to type II, III, IV, and V regions. (G) CTGF is localized to type II, III, and IV regions. Scale bar = 50 µm.

Figure 2.

Ultrastructural characteristics used to identify the different fibrocyte types. (A) Type I cells are elongated with thin, organelle-poor cytoplasm (cy) and a few fine processes (arrow). (B) Type II cells have dense cytoplasm (cy) containing many organelles and numerous fine processes (arrow). (C) Type III cells are variably shaped, often flattened, with dense, narrow cytoplasm and string-like processes. (D) Type IV cells are also variably rounded or elongated, with a number of fine processes, and thin cytoplasm. Type V cells strongly resemble type II cells, with the cytoplasm (cy) containing many organelles and multiple fine processes (arrow). Scale bars: A and C = 5 µm; B, D, and E = 2 µm.

Figure 3.

Immunogold labeling of type I cells. (A) Labeling for caldesmon (CALD) is sparse and primarily cytoplasmic (arrows). (B) Labeling for AQP1 is practically absent. (C) Labeling for S-100 is strong in the cytoplasm and stronger in the nucleus (n), while mitochondria and chromatin tend to have relatively few gold particles over them in comparison. (D) Labeling for α1Na,K-ATPase is weak but present on the plasma membrane and less commonly in the cytoplasm. It is enriched over the few fine processes that extend from these cells (inset). Scale bars: A, D, and D inset = 1 µm; B and C = 2 µm.

Figure 4.

Immunogold labeling of type II cells. (A) Labeling for caldesmon (CALD) is sparse and both cytoplasmic (arrows) and nuclear (arrowheads). (B) Labeling for AQP1 is also sparse but present on the plasma membrane of the fine processes (arrows) and occasionally in the cytoplasm (arrowhead). (C) Labeling for S-100 is strong in the cytoplasm and stronger in the nucleus (n), while mitochondria (arrows) and chromatin have relatively few gold particles over them. Labeling is also present in the fine processes. (D) Labeling for α1Na,K-ATPase is moderate and specific mostly to the plasma membrane, especially the fine processes, and less commonly in the cytoplasm. Scale bars: A–C = 2 µm; D = 1 µm.

Figure 5.

Immunogold labeling of type III cells. (A) Labeling for caldesmon (CALD) is moderate and present in both the cytoplasm and nucleus. (B) Labeling for AQP1 is also moderate but present primarily on membranes of the cell body and processes (inset) and occasionally in the cytoplasm (arrowhead). (C) Labeling for S-100 is weak in the cytoplasm and slightly stronger in the nucleus. (D) Labeling for α1Na,K-ATPase is weak to moderate and mostly in the cytoplasm, although with some plasma membrane labeling. Scale bars: A–C = 2 µm; B inset and D = 1 µm.

Figure 6.

Immunogold labeling of type IV cells. (A) Labeling for caldesmon (CALD) is weak and present in both the cytoplasm (arrows) and nucleus (arrowheads). (B) Labeling for AQP1 is virtually absent. (C) Labeling for S-100 is moderate to strong in the cytoplasm and slightly stronger in the nucleus. (D) Labeling for α1Na,K-ATPase is weak to moderate and in the cytoplasm (white arrows), nucleus (white arrowheads), and on the plasma membrane (black arrows). Scale bars = 2 µm.

Figure 7.

Immunogold labeling of type V cells. (A) Labeling for caldesmon (CALD) is weak and present mainly in the cytoplasm (arrows). (B) Labeling for AQP1 is weak to moderate and primarily on the membranes of fine processes near the cell body (arrowheads) and wider processes that extend into the scala vestibuli (sv) (inset). (C) Labeling for S-100 is moderate to strong in the cytoplasm of the cell body and fine processes, sparing the mitochondria (inset). (D) Labeling for α1Na,K-ATPase is moderate and primarily on the plasma membrane in both fine processes and the wider processes extending into scala vestibuli (sv) (inset). Scale bars: A–C = 2 µm; B inset, D, and D inset = 1 µm; C inset = 0.25 µm.

Figure 8.

Labeling for CK-BB in type V fibrocytes. The labeling is weaker in the cell body (cb) than the processes (p), which extend out into the scala vestibuli (sv). Scale bar = 1 µm.

Figure 9.

Histograms showing the relative labeling density in fibrocytes from each sample (left) and the mean across all four samples (right) for caldesmon (above line) (samples 1B, 2B, 3B, and 5A), AQP1, S-100, and α1Na,K-ATPase (below line) (samples Hide, 4109, 4178, and 4179). Caldesmon and AQP1 are most highly expressed in type III fibrocytes, with the latter to a greater extent. S-100 is expressed to a similar extent in types I, II, and V and is only weakly present in types III and IV. α1Na,K-ATPase is expressed most strongly and to a similar degree in type II and type V fibrocytes and more weakly in types I, III, and IV. Bars = SEM.

Figure 10.

Histograms showing the ratio of labeling density for α1Na,K-ATPase on the cell body to labeling density on fine processes for all types of fibrocytes in all samples (upper) and the mean across the four samples (lower). In nearly all samples and fibrocytes, the ratio is less than one, showing a tendency for α1Na,K-ATPase to be enriched on the plasma membrane of the fine processes rather than the cell body. From the mean values, this ratio is lowest for type II cells and highest for type I cells. Bars = SEM.

Figure 11.

Triple labeling for AQP1, Na,K-ATPase, and S-100. (A) In type II fibrocytes, labeling with all three markers is visible, as indicated by the large, intermediate, and small arrows indicating 15 nm (S-100), 10 nm (AQP1), and 5 nm (Na,KATPase) gold particles, respectively. (B) In type III fibrocytes, only the 10 nm and 5 nm particles are visible in this example because of the lower expression of S-100. Scale bars = 200 nm. (C) Histogram showing the relative amount of labeling for each antibody in the triple-labeled sample. Counts were conducted in 10 images of each fibrocyte type in one section and the proportion of each particle size displayed for each type. The patterns are unique for each type, but note that the most similar pattern is found between type II and type V fibrocytes, which are hard to distinguish.