The mammalian cochlea

The morphology of the hair cells from the inner ear end organs of the domestic pig (Sus scrofa) have been studied using Scanning and Transmission Electron Microscopy, revealing ultrastructural hair cells from the cochlea, saccule and utricle. The pathway of the nerve fibres from the outer hair cells of the organ of Corti to the spiral ganglion of the VIII nerve have also been studied, revealing that each outer hair cell is innervated by peripheral fibres from the spiral ganglion of the auditory nerve; a finding contrary to a number of published studies. This is the first time that the inner ear ultrastructure from S. scrofa has been studied using transmission microscopy, thus providing useful anatomical information in relation to the cellular morphology of the afferent receptors from a “healthy” mammalian ear. Anatomical information in relation to the morphology and innervation of the afferent receptors of the cochlea is of importance both in the diagnosis of trauma to the hearing system and in the development of cochlea implants and other biotechnological systems associated with the enhancement of hearing.

1. Introduction

Current literature shows a paucity of information on consistent and meticulous removal of inner ear parts necessary to identify damage to the ultrastructure symptomatic of hearing and balance loss. Contained within the periotic is the cochlea, a spiral tube that is coiled approximately two and one-half turns around a hollow central pillar (the modiolus) and the vestibular end organs. The saccule, utricle and semi-circular canals make up the vestibular system, whilst the canal of the cochlea is divided into three longitudinal compartments, the scala tympani, scala media (containing the organ of Corti) and scala vestibuli (Corti, 1851; Retzius, 1884). Cytoskeletal layout is stringently specified during the development of the mammalian organ of Corti (Henderson et al., 1995; Tucker et al., 1998 and 1999); however, the long fibrous nature of the pillar cells indicates that they may serve an additional role by containing the innervating nerves to the outer hair cells.

This is the first time that the ultrastructure in the ear of S. scrofa has been studied and this work provides anatomical information, useful in the pathological diagnosis of hair cell loss and fine nerve damage. Damage to ultrastructure can be caused by intense sources of anthropogenic noise, induced chemically by antibiotics such as gentamicin sulphate (Lombarte et al., 1993), or in response to reactive oxygen species (ROS) from a number of environmental pollutants. The production or chemical activation of free radicals may lead to oxidative stress and ultimately to permanent cellular damage (Nicholls & Budd, 2000). Various techniques have been used to reveal internal and external hair cell structures; Spoendlin (1969) used a light microscope to examine the cochlea innervating nerve distributions of the cat, and Wever et al. (1971) used a similar methodology for examining the cochlea from the bottlenose dolphin (Tursiops truncates). Lu & Popper (1998) examined the polarisation of ciliary bundles in the end organs of the sleeper goby (Dormitator latifrons) using immunocytochemicals and a confocal imaging technique. However several authors (e.g. Corwin, 1981; Popper, 1981; Plat & Popper, 1984; Lovell et al., 2005 b,c,d) have used the Scanning Electron Microscope (SEM) to study surface detail of the inner ear ultrastructure, and this was the approach adopted here. Internal cellular structures were studied using Transmission Electron Microscopy (TEM), in accordance with Saidel et al. (1995) and Lovell et al. (2005 c).

2. Materials and methods

SEM preparation methodology

The periotic bone containing the inner ears from 14 mature domestic pigs were removed during processing for the meat industry within 1 hour of the animal’s death, immersed in chilled fixative (2.5% Glutaraldehyde) and refrigerated. The periotic bone was trimmed to a small block and the outer bone layer removed using a fine cutting wheel; the ears and surrounding tissue were subsequently immersed in a watch glass containing a solution of 2.5 % S-Carboxymethyl-L-Cysteine in sodium chloride used to hydrolyse the mucus surrounding the otoconia and hair cells. The saccule, utricle and cochlea were dissected from the labyrinth using a fine scalpel, then dehydrated through a graded ethanol series ranging from 35% through 50%, 70% and 90% to absolute ethanol, prior to desiccation using the critical point drying method used by Lovell et al., (2005b). Fully desiccated end organs were subsequently mounted on a specimen stub using a carbon tab, and coated with c. 8 nm of gold in an Emitech K 550 sputter coater (working at approximately 5 x 10^-6 Torr). The processed specimens were investigated and photographed using a JEOL JSM 5600 scanning electron microscope operated at 15 kv, and a 15 mm working distance.

3. Results

General dissection

Partial removal of the bone covering the inner ear from S. scrofa (Figure 1.a) reveals the canals of cochlea surrounded by the dense auditory periotic bone. The anterior part of the capsule contains the cochlea (Figure 1.a); a spiral tube coiled around the modiolus. Further removal of the remaining bone covering the posterior part of the inner ear reveals the membranous labyrinth of the vestibule (Figure 1.b), which contains the saccule, utricle and the semi-circular canals; each filled with endolymph, a substance possessing viscous and ionic properties that flows around the semicircular canals aiding the sense of balance.

Figure 1.a. The labyrinth of the inner ear from S. scrofa with the covering periotic bone partially removed. 1.b. the saccule and utricle with the covering bone fully removed prior to dissection and preparation for SEM microscopy (Bars = 1 mm) (total basilar membrane length averaged from 12 samples = 32 mm)

Hair cell morphology

The mammalian cochlea has two types of hair cell, inner hair cells (Figure 2.a) and outer hair cells (Figure 2.b). Inner hair cells are arranged in a single row around the inner lamina, and the cilia resemble the ultrastructure from vestibular type hairs. Outer hair cells are arranged in a band of three to four rows orientated toward the outer wall of the cochlea, each bearing over 60 cilia and arranged in a crescent formed from three to four consecutively shorter rows.

Figure 2.a Inner hair cells orientated to allow for cilia length measurements 2.b. outer hair cells from the upper basal region of the cochlea

Figures 3.a through 3.f shows ultrastructural hair cell proliferations from the upper apical tip to the lower basal region of the cochlea from S. scrofa, fixed in glutaraldehyde 1 hour after death from the third batch of ears where the periotic was immersed in fixative without any additional preparation. The Inner Hair Cells (IHC) present in a single row arranged with cilia approximately 10 µm to 15 µm at the upper apical tip of the cochlea, and shorter hairs (approximately 2 µm to 3 µm) in the lower basal segment. The Outer Hair Cells (OHC) present in three ordered rows, separated from the inner hair cells by a ridge approximately 20 µm in width, formed by the fibres of the innervating nerves and blanketed by a layer of gelatinous mucous.

Figure 3.a. Hair cells (aprox. 10 to 15 µm) from the tip of the upper apical, 3.b hair cells (aprox. 8 to 10 µm) from the upper apical, 3.c hair cells (aprox. 6 µm) from the lower apical, 3.d hair cells (aprox. 5 µm) from the upper basal, 3.e hair cells (aprox. 4 µm) from the lower basal region, 3.f hair cells (aprox. 3 µm) close to the end of the lower basal region. IHC. inner hair cells, OHC. outer hair cells

The ridge separating the outer cells from the inner cells is formed from the converging pillar cells from both the tip and base of the afferent. The ratio between the hair cells and the nerve fibre is generally three outer hair cells to one descending pillar fibre.

Figure 4. SEM image of a section through the organ of Corti showing the pillar cells forming the middle (mt.) and inner tunnels (it.) viewed from above. bnf. basal pillar fibre, icc. inner cell cilia, occ. outer cell cilia, tnf. tip fibres

Figure 5. SEM image of a section through the organ of Corti showing the pillar cells forming the middle (mt.) and inner tunnels (it.) viewed from below. bnf. basal pillar fibres, dnf. descending pillar fibres, icc. inner cell cilia, tnf. tip fibre

The descending pillar fibres connect to the spiral ganglion of the VIII nerve through orifices in the outer edge of the hollow bony lamina (Figure 6).

Figure 6. Orifices in the bony lamina through which the descending fibres connect to the spiral ganglion of the VIII nerve

Figure 7 presents a methylene blue section through the cochlea from S. scrofa, showing the organ of Corti and spiral ganglion from the cochlea nerve. The inner part of the basilar membrane (zona arcuata) extends from the tympanic lip of the spiral lamina and supports the organ of Corti, whilst the outer (zona pectinata) connects to the spiral ligament below the spiral prominence of the stria vascularis. The canal below the basilar membrane is the scala tympani, and above the Reissner's membrane is the scala vestibuli. Both canals are filled with perilymph and are continuous with one another by means of a tiny opening, the helicotrema, located at the apex of the cochlea. The duct between the scala tympani and scala vestibuli is the scala media and contains the organ of Corti, defined by the hatched area in Figure 7.

Figure 7. Methylene blue section through the cochlea canal from S. scrofa; the hatched line around the organ of Corti represents the area from where the ultra-thin TEM sections were taken. bma. basilar membrane zona arcuata, bmp. basilar membrane zona pectinata, rm. Reissners membrane, sg. spiral ganglion, sl. spiral lamina, slg. spiral ligament, slm. spiral limbus, sp. spiral prominence, sv. stria vascularis, tm. tectorial membrane

Discussion

The use of the Scanning Electron Microscope (SEM) in the examination of the ultrastructure responsible for the mediation of auditory stimuli has been used to considerable effect on lower vertebrates such as fish (Platt 1977; Lovell et al., 2005b), and invertebrates (Lovell et al., 2005a), though no SEM examinations have, so far, been conducted on the inner ear ultrastructure from S. scrofa. A procedure for the fast removal of the complete cochlea and other end organs of the inner ear undamaged has been demonstrated here. It is essential that the periotic is rapidly immersed in chilled fixative (2.5% glutaraldehyde in 0.1 M cacodylate buffer with 3.5% sodium chloride), then refrigerated to inhibit sample decomposition (the sample must not be frozen, as ice crystals will destroy the ultrastructure).

All mammalian cochleae appear to function according to the same basic principles; however, the effective frequency range differs between species (Fay, 1988). For example, the range of audible frequencies is about 20 Hz to 16 kHz in the human cochlea, about 300 Hz to 45 kHz in S. scrofa (Heffner and Heffner, 1990). Table 1 presents the outlying audible frequencies, along with the cochlea length measurements.

Table 1. Comparison between cochlea length and audible frequency range

Species

Cochlea

Length (mm)

Low (Hz)

High (Hz)

Human

16000

S. scrofa

300

45000

Link to the Study of the Mammalian Vestibular Organs

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