Human vestibular schwannoma reduces density of auditory nerve fibers in the osseous spiral lamina

Abstract

Hearing loss in patients with vestibular schwannoma (VS) is commonly attributed to mechanical compression of the auditory nerve, though recent studies suggest that this retrocochlear pathology may be augmented by cochlear damage. Although VS-associated loss of inner hair cells, outer hair cells, and spiral ganglion cells has been reported, it is unclear to what extent auditory-nerve peripheral axons are damaged in VS patients. Understanding the degree of damage VSs cause to auditory nerve fibers (ANFs) is important for accurately modeling clinical outcomes of cochlear implantation, which is a therapeutic option to rehabilitate hearing in VS-affected ears. A retrospective analysis of human temporal-bone histopathology was performed on archival specimens from the Massachusetts Eye and Ear collection. Seven patients met our inclusion criteria based on the presence of sporadic, unilateral, untreated VS. Tangential sections of five cochlear regions were stained with hematoxylin and eosin, and adjacent sections were stained to visualize myelinated ANFs and efferent fibers. Following confocal microscopy, peripheral axons of ANFs within the osseous spiral lamina were quantified manually, where feasible, and with a "pixel counting" method, applicable to all sections. ANF density was substantially reduced on the VS side compared to the unaffected contralateral side. In the upper basal turn, a significant difference between the VS side and unaffected contralateral side was found using both counting methods, corresponding to the region tuned to 2000 Hz. Even spiral ganglion cells (SGCs) contralateral to VS were affected by the tumor as the majority of contralateral SGC counts were below average for age. This observation provides histological insight into the clinical observation that unilateral vestibular schwannomas pose a long-term risk of progression of hearing loss in the contralateral ear as well. Our pixel counting method for ANF quantification in the osseous spiral lamina is applicable to other pathologies involving sensorineural hearing loss. Future research is needed to classify ANFs into morphological categories, accurately predict their electrical properties, and use this knowledge to inform optimal cochlear implant programming strategies.

Keywords: Auditory nerve fiber density; Cellmask®; Hearing loss; Human temporal bones; Osseous spiral lamina; Vestibular schwannoma.

Figure 1:

Quantification of human auditory nerve fibers in tangential sections through the osseous spiral lamina. 1A. Cochlear spiral illustrating the location of the five tangential sections studied (LB: lower basal turn; UB: upper basal turn; LM: lower middle turn; UM: upper middle turn; A: apex) and their corresponding frequencies, as estimated using standard cochlear reconstruction techniques and a cochlear map for human (Greenwood, 1990; Stakhovskaya et al., 2007) with parameters modified to yield extreme values of 120 Hz (apical tip) and 20,000 Hz (basal tip). 1B. Light micrograph of a contralateral tangential section of the upper middle turn (corresponding to segment 4 in figure 1A) of patient 4, stained with hematoxylin and eosin; rectangular zoomed inset of osseous spiral lamina. Scale bar: 1 mm. 1C. Scatterplot comparing the manual and pixel counting methods. Data from both methods expressed as a ratio of ipsilateral and contralateral counts, log-transformed. Each circle represents a different quantified section (5 patients, 5 slides per patient), line indicates a linear regression fit. R²: coefficient of determination.

Figure 2:

Auditory nerve fiber counts are significantly decreased in the osseous spiral lamina ipsilateral to vestibular schwannoma. Confocal image of an ipsilateral (2A) and contralateral (2B) tangential section of the osseous spiral lamina in the upper basal region (segment 2 in figure 1A), of patient 5, stained with CellMask^® (red) and anti-ChAT (green). Scale bar: 25 μm. 2C. Boxplot showing auditory nerve fiber counts per 500 μm of the osseous spiral lamina, compared across all cochlear segments for ipsilateral versus contralateral ears, obtained by the manual counting method. Center lines show medians; box limits indicate 25^th and 75^th percentiles; whiskers extend 1.5 times the interquartile range from 25^th and 75^th percentiles; individual data points plotted as open circles. n=5 patients, 50 sections. *p<0.05, **p<0.01. 2D. As 2C, obtained by the pixel counting method. n=7 patients, 70 sections.

Figure 3:

Auditory-nerve fibers (ANF) and spiral ganglion cells (SGC) are evidently decreased in ears ipsilateral to vestibular schwannoma (Figure 3A and 3B). ANF and SGC counts are affected differently by region of the cochlea (3C and 3D). Comparison to historical controls (Makary et al., 2011) suggests both ipsilateral and contralateral fibers are affected by the tumor, as the majority of contralateral SGC counts are below the average SGC counts based on age (Figure 3E). Tangent refers to the five tangents used to count ANFs in tangential sections, corresponding to tangential sections described in Figure 1, ascending in frequency along the x-axes. Segment refers to the four segments of the cochlea in which SGCs were counted (as in Merchant and Nadol, 2010), also ascending in frequency along the x-axes. Each patient is represented by a different color (consistent across panels; solid circles depict ipsilateral data and open circles depict contralateral data) and number (corresponding to patient numbers in table 1).