Histopathologic Changes of the Inner ear in Rhesus Monkeys After Intratympanic Gentamicin Injection and Vestibular Prosthesis Electrode Array Implantation

Abstract

Bilateral vestibular deficiency (BVD) due to gentamicin ototoxicity can significantly impact quality of life and result in large socioeconomic burdens. Restoring sensation of head rotation using an implantable multichannel vestibular prosthesis (MVP) is a promising treatment approach that has been tested in animals and humans. However, uncertainty remains regarding the histopathologic effects of gentamicin ototoxicity alone or in combination with electrode implantation. Understanding these histological changes is important because selective MVP-driven stimulation of semicircular canals (SCCs) depends on persistence of primary afferent innervation in each SCC crista despite both the primary cause of BVD (e.g., ototoxic injury) and surgical trauma associated with MVP implantation. Retraction of primary afferents out of the cristae and back toward Scarpa's ganglion would render spatially selective stimulation difficult to achieve and could limit utility of an MVP that relies on electrodes implanted in the lumen of each ampulla. We investigated histopathologic changes of the inner ear associated with intratympanic gentamicin (ITG) injection and/or MVP electrode array implantation in 11 temporal bones from six rhesus macaque monkeys. Hematoxylin and eosin-stained 10-μm temporal bone sections were examined under light microscopy for four treatment groups: normal (three ears), ITG-only (two ears), MVP-only (two ears), and ITG + MVP (four ears). We estimated vestibular hair cell (HC) surface densities for each sensory neuroepithelium and compared findings across end organs and treatment groups. In ITG-only, MVP-only, and ITG + MVP ears, we observed decreased but persistent ampullary nerve fibers of SCC cristae despite ITG treatment and/or MVP electrode implantation. ITG-only and ITG + MVP ears exhibited neuroepithelial thinning and loss of type I HCs in the cristae but little effect on the maculae. MVP-only and ITG + MVP ears exhibited no signs of trauma to the cochlea or otolith end organs except in a single case of saccular injury due to over-insertion of the posterior SCC electrode. While implanted electrodes reached to within 50-760 μm of the target cristae and were usually ensheathed in a thin fibrotic capsule, dense fibrotic reaction and osteoneogenesis were each observed in only one of six electrode tracts examined. Consistent with physiologic studies that have demonstrated directionally appropriate vestibulo-ocular reflex responses to MVP electrical stimulation years after implantation in these animals, histologic findings in the present study indicate that although intralabyrinthine MVP implantation causes some inner ear trauma, it can be accomplished without destroying the distal afferent fibers an MVP is designed to excite.

Figures

Fig. 1

Representative H&E-stained, light microscopy images of vestibular neuroepithelia from a normal control ear. SCC crista (A) neuroepithelium shows linear array of hair cells (HCs) and support cells arranged between the basement membrane and cuticular plate. Stereocilia (arrows) and cupula are visible. High magnification view (box) of the central zone of the crista (B) shows characteristic appearance of type I and II HCs. Type I HCs are flask shaped with spherical nuclei and surrounded by a single calyx that appears as a clear “halo,” whereas type II HCs are cylindrical without a nerve chalice. Both type I and II HCs reside above a layer of supporting cell nuclei (arrowheads). The abundance of type I compared to type II HCs in the central zone of the crista is evident. The thickness of the neuroepithelium is measured from the basement membrane to the cuticular plate (H). A similarly cellular architecture is observed in the saccule macula (C). “Blebs” (arrows) occurring over the cuticular plate are a result of cellular autolysis during histologic processing. High magnification view (box) of the macula (D) again shows the distribution of type I and II HCs and support cells (arrowheads). The ratio of type I to II HCs is lower than that in the central zone of the crista shown in B.

Fig. 2

Representative ×10 and ×40 (inset) light microscopy images of Rhesus monkey semicircular canal cristae. Normal ears (A–C) show a high ratio of type I to II hair cells (HCs) in the central zone of each crista, with intact stereocilia and cupula, and presence of ampullary nerve fibers. In ITG-only ears (D–F), there is almost complete loss of central zone HCs and stereocilia, with displacement of the cupula (arrow). The neuroepithelium is thinned and cuticular plate absent. There appears to be persistent innervation (asterisk) to each crista, albeit decreased in density, even up to 3 years (D and E) after final ITG injection and abolishment of angular vestibular-ocular reflex. In MVP-only ears (G–I), there is variable presence of type I and II HCs in the central zone. In ITG + MVP ears (J–L), there is consistent thinning of the central zone neuroepithelium with complete loss of type I and II HCs. Again, nerve fibers (asterisk) of each crista are evident. MVP multichannel vestibular prosthesis. Scale bars are 100 and 50 μm (inset). Specimens shown: MRH37x left ear for normal, F247RhE right ear for ITG-only, M0603163RhO left ear for MVP-only, and M067RhF left ear (J), F32RhD left ear (K), and F32RhD right ear (L) for ITG + MVP.

Fig. 3

Representative ×10 and ×40 (inset) light microscopy images of Rhesus monkey otolith end organ maculae. Normal ears (A and B) show utricular and saccular maculae with type I and II hair cells (HCs), intact stereocilia (inset), and presence of otoconia. In an ITG-only ear treated just 20 days prior to euthanasia, the utricular (C) and saccular (D) maculae both appear remarkably healthy, with minimal HC loss, preserved neuroepithelial height, and intact stereocilia and otoconia. This is in contrast to evidence of cristae injury from the same specimen in Figure 2D–F. In MVP-only ears (E and F), or those that received ITG injection followed by MVP implantation (G and H), preservation of HCs, stereocilia, and otoconia are again demonstrated. However, in an ear with surgical trauma to the saccule as a result of PSCC electrode insertion (F), thinning of the neuroepithelium and overlying otoconia, with decreased HCs and almost complete loss of stereocilia, is observed. MVP multichannel vestibular implant. Scale bars are 100 and 50 μm (inset). Specimens shown: MRH37x left ear for normal, M067RhF right ear for ITG-only, M0603163RhO left ear for MVP-only, and M067RhF left ear (G) and F32RhD right ear (H) for ITG + MVP.

Fig. 4

Representative image of stapes footplate and cochlea in a rhesus monkey that underwent intratympanic gentamicin (ITG) injection and MVP implantation. Image of the oval window (A) shows intact stapes footplate (arrow, folding due to histologic artifact) and adjacent structures, including facial nerve (FN), cochlea (Co), incus (I, fractured during histologic processing), utricle (U), and saccule (S). Cochlea (B) in the same animal also appears grossly intact. Inset (C) shows scala tympani (ST), scala vestibuli (SV), and scale media (SM). Intact tectorial membrane (arrowhead), inner, and outer hair cells (arrows) are visible. Light microscopic images of H&E-stained tissue acquired under ×2.5–×20 magnification. Scale bars are 500 μm (A and B) and 100 μ (C). Specimen shown: F32RhD right ear.

Fig. 5

Neuroepithelial height (top) and vestibular hair cell (HC) surface density (bottom) of each vestibular end organ for each treatment group. Compared to normals, ITG-only ears showed a decrease in neuroepithelial height and HC density in the semicircular canal (SCC) cristae, but not in the otolithic end organ maculae. Similarly, in ITG + MVP ears, the ototoxic effects of gentamicin appeared to be attenuated in otolith maculae compared to SCC cristae. Variability in HC density and neuroepithelial height in MVP-only ears reflects spectrum of surgical trauma.

Fig. 6

Histopathology of electrode array tracts. Typically, a thin fibrotic capsule (arrow) surrounds the electrode tract (asterisk), terminating adjacent to an superior semicircular canal (SSCC) crista (A). The electrode tract is seen traversing through the surgical fenestration into the SSCC ampulla (S). In B, the orientation of an horizontal semicircular (HSCC) electrode tract (asterisk) and its fibrous capsule (arrow) are seen in relation to other adjacent structures, including the HSCC ampulla (H), SSCC ampulla (S), facial nerve (FN), and Scarpa’s ganglion (SG). The close proximity of the HSCC electrode tract to the SSCC ampullary nerve is evident, demonstrating the anatomic basis for current spread between HSCC and SSCC ampullary nerves. In one monkey (C), osteoneogenesis (arrow) was found adjacent to an electrode tract (asterisk) in the HSCC ampulla (H). In another monkey (D), a vigorous fibrosis reaction (arrow) is seen adjacent to a posterior semicircular canal (P) electrode tract (asterisk). Osteoneogenesis and fibrosis may be due to electrode insertion or surgical placement of bone paté and/or fascia around labyrinthotomies during implantation. In all specimens, existent ampullary nerve innervation to crista was evident, even in cases that suffered surgical destruction of the crista during implantation (B). There was no evidence of granulomatous inflammation, hydrops, or extension of fibrosis into the vestibule in any specimens. H&E-stained images captured at ×2.5–×10 magnification under light microscopy. Specimens shown: M0603163RhO left ear (A and C), F32RhD right (B), and left (D) ears.

Fig. 7

Electrode array placement. A Posterolateral view of 3D CT surface reconstruction showing electrode array leads implanted in the left labyrinth of a rhesus monkey via the mastoid cavity. a lead to anterior and horizontal ampullae; b lead to posterior ampulla; c common crus reference electrode; d neck reference electrode; M mandibular ramus; Z zygomatic arch; ANT, POST, SUP, INF anterior, posterior, superior, inferior. B Oblique CT cut through the plane of the basal turn of the cochlea [Co], showing bifurcated electrode array [a] entering the ampullae of the superior [s-scc] and horizontal [h-scc] semicircular canals. Part of the neck reference electrode [d] is also visible, but the posterior SCC electrode array is not included in this section. AM, PL anteromedial, posterolateral. C Image of forked electrode arrays for h-scc and s-scc ampullas (1), posterior SCC (2), and common crus reference (inset) electrodes. A and B reproduced with permission from Dai et al. .