Immunocytochemical traits of type IV fibrocytes and their possible relations to cochlear function and pathology

Abstract

One of the more consistent and least understood changes in the aging human cochlea is the progressive loss of fibrocytes within the spiral ligament. This report presents an animal model for type IV fibrocyte loss, along with immunocytochemical evidence that noise-induced loss of these cells may account for previously unexplained hearing losses. The remarkably low threshold for noise-induced loss of type IV fibrocytes, approximately 24 dB less than the threshold for adjacent hair cell destruction, may account for the prevalence of missing fibrocytes in humans. In mice, changes in the spectrum of traumatizing noise had little effect upon the site of loss of the fibrocytes, suggesting that the primary site of damage that induced the loss was the basal-most cochlear turn, a site expected to be damaged by all three noise bands. Type IV fibrocytes were found to immunostain for connective tissue growth factor (CTGF) and for transforming growth factor beta receptor 3, a receptor that is known to activate CTGF expression. Type IV fibrocytes lack immunostaining for adenosine triphosphatase and connexins that are key players in potassium ion uptake and transmission, which suggests that they play little, if any, role in potassium recycling from perilymphatic space to the endolymphatic space. Consequently, their loss probably does not directly reduce this process. Immunostaining for a receptor for CTGF, low-density-lipoprotein-related protein 1, indicated that CTGF acts as an autocrine and a paracrine agent within the cochlea. The lack of CTGF paracrine effects following noise-induced loss of type IV fibrocytes may account for previously unexplained hearing losses.

FIG. 1

A–D A control ear immunostained for CTGF (antibody concentration 1.1 μg/mL) showing staining of type IV fibrocytes in the lower basal (lower 1) and upper basal (upper 1) half turns. Only a few faint cells are evident in the lower second (lower 2) and upper second (upper 2) half turns. Immunopositive root cells are conspicuous in the basal turn and Deiters cells become increasingly darkly stained in the second turn. Nomarski optics. The dashed rectangle in panel D indicates the field shown in individual panels of Figures 3 and 4. The calibration bar in panel C is 100 μm.

FIG. 2

Cytochemical traits of type IV fibrocytes and related cells. Panel A shows upper basal turn type IV fibrocytes staining for the TGFβ3 receptor (primary antibody concentration 0.2 μg/mL, BT amplification). Panel B shows staining for CTGF (1.1 μg/mL) for comparison to panel A. Panel C shows staining for Na⁺ and K⁺-ATPase (1:19 K dilution). The stria vascularis (SV) and type II fibrocytes (II) are darkly stained but only one cell in the region of type IV fibrocytes (IV) is positive, in contrast to NKCC1. Panel D shows staining for NKCC1 (1:9 K dilution) with a distribution very similar to that of Na⁺, K⁺-ATPase with the exception that the type IV fibrocyte area is darkly stained. Panel E shows type III fibrocytes immunostained for aquaporin 1 (0.06 μg/mL); type III fibrocytes are situated at the peripheral margin of the spiral ligament against the bone. There is staining within the type IV fibrocytes region but this appears to be processes of type III fibrocytes because no aquaporin-1-positive cells were ever found in this region. Panel F shows staining for connexin 26 (0.56 μg/mL). The region occupied by types III and IV fibrocytes are conspicuous by their lack of staining for connexin 26. Panel G shows staining for LRP1 (0.28 μg/mL, BT amplification) in a control ear. Types I and IV fibrocytes are positive, as well as stria vascularis, spiral limbus, and ganglion cells. The inset shows a higher magnification of ganglion cells. The calibration bar in A is 50 μm and applies to panels A–F.

FIG. 3

A–C Extreme sensitivity of CTGF immunostaining to noise exposures (left column) as compared with matched control ears with conductive hearing loss (right column). All images are of upper basal turn. The top pair of images shows a dramatic decrease in staining in the left ear following a 110-dB 4–8-kHz exposure with a 4-day survival. No changes were evident in Azure-stained sections of this case (not shown). The middle pair of images shows that 6 h following a 94-dB 8–16-kHz exposure there was no staining apparent in type IV fibrocytes in the upper basal turn. The bottom pair of images are corresponding Azure-stained material of the same mouse shown in the middle panel. Only the earliest signs of noise-induced change are evident in the Azure-stained material (cells within the ellipse). The calibration bar is 50 μm.

FIG. 4

Azure-stained sections of the type IV fibrocyte region of the upper basal turn. The dashed box in Figure 1D indicates the region covered by the micrographs of the present figure. The left column shows the range of cellular changes with increasing survival times following a 2-h exposure to 96-dB 8–16-kHz noise to the left ears of three mice. The adjacent images in the right column show the corresponding areas in the right ears, which had conductive hearing losses. The changes in cell morphology indicated by the arrowheads range from rounding and slight shrinkage of nuclei (panel A) to small cellular debris (panel B) to missing cells (panel C). BC indicates the basilar crest, the attachment point of the basilar membrane with the spiral ligament. The calibration bar in B is 15 μm.

FIG. 5

Representative plots of type IV fibrocyte loss produced by noise exposures with three different frequency bands. Thin horizontal lines represent sections through the cochlea spaced 100 μm apart. The numbers indicate the degree of cell loss, with 0 being no cell loss and 3 being complete loss. There were no cell losses in the lower basal turn (farthest right) and lower second turn. No appreciable differences were found between mice exposed to 4–8- and 8–12-kHz noise bands. In none of the mice exposed to 20–40-kHz noise were there missing cells in the basal-most upper basal turn (arrows in the lower figure).