Assessing fractional hair cell survival in archival human temporal bones

Abstract

Objectives/hypothesis: Histopathological analysis of hair cell survival in human temporal bone sections has historically been binarized such that each hair cell row is rated as either present or absent, thereby greatly underestimating the amount of hair cell loss. Here, we describe and validate a technique to reliably assess fractional hair cell survival in archival sections stained with hematoxylin and eosin (H&E) using high-resolution light microscopy and optical sectioning.

Study design: Technique validation.

Methods: Hair cell counts in archival temporal bone slide sets were performed by several observers using either differential interference contrast (DIC) or confocal microscopy of the endogenous eosin fluorescence in hair cells. As a further cross-check, additional decelloidinized sections were immunostained with hair cell markers myosin VI and VIIa.

Results: Cuticular plates and stereocilia bundles are routinely resolvable in DIC imaging of archival H&E-stained human material using standard research-grade microscopes, allowing highly accurate counts of fractional hair cell survival that are reproducible across observer and can be verified by confocal microscopy.

Conclusions: Reanalysis of cases from the classic temporal bone literature on presbycusis suggests that, contrary to prior reports, differences in audiometric patterns may be well explained by the patterns of hair cell loss.

Level of evidence: NA Laryngoscope, 130:487-495, 2020.

Keywords: Presbycusis; audiometric pattern; hair cell loss.

Figure 1:

The binarization of hair cell counts in temporal bone studies oversimplifies the patterns of hair cell loss. A: The classic approach rates hair cells in each of the 4 rows as present or absent in each section . B: A schematic shows why the number of hair cells per section increases dramatically as the section plane moves from mid-modiolar to tangential. C: In counts of hair cells per section in a serial-section set from a normal neonate, the five numbered peaks correspond to the five tangential cuts through the 2½ cochlear turns in human, as schematized in D. Minimum values (always ≥ 2) are seen in the mid-modiolar sections, where the section plane is perpendicular to the spiraling sensory epithelium.

Figure 2:

High-power DIC (A) and confocal (B) imaging can be used to see stereocilia and cuticular plates in archival human material. A: DIC image of the organ of Corti from the 0.5 kHz region of a normal 2 yr old male. Locations of the in-focus cuticular plates, OHC nuclei and Deiters’ cell (DC) nuclei are indicated by the arrowheads. B_xy: Maximum projection of a confocal z-stack of the same section, using the endogenous fluorescence of eosin to form the image. B_yz: To simulate a “surface view” of the hair cells in this 3D confocal stack, we crop an area just above and below the reticular lamina (yellow dashed lines in B_xy) and re-project it into the yz plane. Positions of the IHCs and OHCs are numbered. Scale bar in A applies to all panels. See Supplementary Figure 1 to step through this section by both DIC and confocal microscopy at multiple focal planes.

Figure 3:

High-power DIC (A) and confocal (B) imaging can reveal fractional OHC loss in a noise-damaged ear. A: DIC image of the organ of Corti from the 2.8 kHz region of a 58-yr old male with occupational noise damage, showing two in-focus cuticular plates and stereocilia bundles (white arrowheads): one IHC and one 1^st-row OHC. In-focus nuclei of OHCs and Deiters’ cells are also indicated with arrowheads, grey and black respectively). Blue-fill arrowheads point to dense eosinophilic portions of the phalangeal scars that fill in the reticular lamina after hair cell loss. B_xy: Maximum projection of a confocal z-stack of the same section, using the endogenous fluorescence of eosin to form the image. B_yz: To simulate a “surface view” of the hair cells, we crop an area just above and below the reticular lamina (yellow dashed lines in B_xy) and re-project it into the yz plane. Positions of the IHCs (3 in-line) and OHCs (one from the 1^st row and one from the 2^nd row 2) are numbered. Scale bar in A applies to all panels. See Supplementary Figure 2 to step through this section by both DIC and confocal at multiple focal planes.

Figure 4:

To assess the robustness and reproducibility of our fractional analysis of hair cell survival, we compared results from two independent observers (B,C). Both observers noted significant fractional hair cell loss (40–60%), especially for OHCs, in the apical half of the cochlea that was missed, by definition, in the binary analysis (D). This case was from a 95 yr-old male with a history of experimental noise exposure, with an audiogram (A) obtained 10 months prior to death .

Figure 5:

Two observers, each blinded to all case-specific information, produce fractional hair-cell survival scores that are highly correlated. Data shown here are averaged over 5% length bins from two complete cases, both different from the one shown in Figure 4: one was a 70 yr-old normal-aging male, the other was a 96 yr-old female with progressive bilateral sensorineural hearing loss. The former case was chosen because it was relatively well preserved; the latter because the preservation was not ideal. Dashed lines indicated best-fit straight lines to the OHC and IHC counts from the two observers, only one of which (Observer 2) participated in the analysis from Figure 4.

Figure 6:

As further confirmation of the accuracy of our assessment of fractional hair cell survival, we compared cytocochleograms in the two ears of one normal-aging 70 yr-old female: one processed as celloidin sections (A,B), the other as a microdissected wholemount of the organ of Corti (C,D). B: A DIC image from 3.9 kHz region of left ear, showing in-focus cuticular plates and stereocilia bundles in the 2^nd and 3^rd row OHCs (blue-fill arrowheads) and a missing OHC in the 1^st row (white-filled arrowhead). D: Confocal image of the myosin-immunostained cuticular plates of hair cells from 3.0 kHz region of the right ear. Arrowheads note the locations of missing IHCs and 1^st-row OHCs. Note two supernumerary IHCs (red-solid filled arrowheads.

Figure 7:

Assessment of fractional hair-cell survival provides a better correlation between histopathology and hearing loss. Case 1 was an 83 yr-old male with a down-sloping audiogram (top panel), classified as “cochlear conductive presbycusis” , because the binary analysis of hair cell survival (middle panels) did not explain the hearing loss. Case 2 was an 89 yr-old male, classified as ‘indeterminate presbycusis’ , because the binary analysis of hair cell loss (middle panels) also did not correlate with the pattern of threshold shift (top panel). In both cases, the DIC-based re-analysis of fractional hair cell survival (bottom panels) provides a more compelling explanation of the audiogram. Case 2 also includes examples of false negatives, where the classic analysis suggested a total loss of hair cells – see Figure 8.

Figure 8:

Imaging of stereocilia and cuticular plates identifies hair cells where prior analyses rated them as missing. Low-power brightfield (A) vs. high-power DIC (B) images of the 3-kHz (12 mm) region of the Case 2 from Figure 7, as shown by the inset in A. Stereocilia bundles (arrowheads in B) clearly document the OHC survival in this region.