RESEARCH TECHNIQUES

Barbara A. Bohne, Ph.D.
Gary W. Harding, M.S.E.
Dept. of Otolaryngology, Washington University School of Medicine, St. Louis, MO

The organ of Corti is unique in that it can examined as a whole mount by dividing it into 0.5-1.5 mm long segments, putting the segments on slides in a mounting medium and using a light microscope to "optically section" or focus through the 50-100 um thick organ.

Fig. 1 depicts: A) A radial or cross-section of one turn of the cochlea; and B) A horizontal view of the cochlear duct. In A, the boundaries of scala media (SM) are visible. The basilar membrane (BM) extends from the lip of the osseous spiral lamina (OSL) to the spiral ligament (SPL) and separates scala media from scala tympani (ST). The limbus (L) and tectorial membrane are located on the vestibular lip of the osseous spiral lamina. The organ of Corti (OC) is located on scala-media side of the basilar membrane, just lateral to the osseous spiral lamina. The stria vascularis (StV) forms the lateral boundary of scala media and is anchored to the medial side of the spiral ligament. Reissner's membrane (RM) separates scala media from scala vestibuli (SV). When examined at a radial angle, relatively few sensory cells are visible in the organ of Corti. In B, a short segment of the cochlear duct is illustrated as viewed from Reissner's membrane, looking through scala media and down on the endolymphatic surface of the osseous spiral lamina, organ of Corti and basilar membrane. The spiral ligament and stria vascularis form the lateral edge of the segment. These structures are difficult to examine in this type of preparation because they are oriented perpendicular to the viewing angle. Within the organ of Corti, the heads of the inner and outer pillars (PH) are visible close to the lip of the osseous spiral lamina. The region labeled OHC contains the outer hair cells, Deiters' cells and Hensen cells.

Because multiple regions of damage can occur in the organ of Corti after some experimental treatments, it is important to examine the entire organ from apex to base. Quantitative data are more likely to be accurate, and are easier and less time-consuming to collect when the cochleas are prepared as illustrated in Fig. 1B.

There are two basic techniques for preparing the organ of Corti as a whole-mount, flat preparation: a) Wet-dissection; and b) Plastic-embedding prior to dissection. We use the plastic-embedding technique because it is much less likely to result in dissection artifacts. Our specimens are embedded in Durcupan which is an epoxy resin that does not become brittle with prolonged exposure to heat. Also, this plastic can withstand the electron beam of a transmission electron microscope. By embedding in Durcupan, the entire organ of Corti from apex to base can be prepared for quantitative microscopy. Once lesions are located in the whole mounts, the areas can be semi-thick or thin-sectioned at a radial, tangential or horizontal angle and examined by light or transmission electron microscopy, respectively.

Fig. 2 shows the 3.5-turn chinchilla cochlea with its central modiolus (M). Scala vestibuli (SV), scala media (SM) and scala tympani (ST) are filled with polymerized plastic and are visible because the cochlear bone was dissected away after embedding. Small pieces of double-edged razor blades were used to divide the specimen into 15 segments.

Fig. 3 is a higher power view through the dissection microscope of one dissected segment of the organ of Corti (between horizontal lines). This illustration is similar to that shown schematically in Fig. 1B. BM - basilar membrane; M - modiolus; OC - organ of Corti; OSL - osseous spiral lamina; SPL &: StV - spiral ligament and stria vascularis.

For each segment, the plastic filling scala tympani was trimmed with razor blades to within 0.1-mm of the basilar membrane. The trimmed segments were reembedded, basilar-membrane side down, in 2-mm-thick layers of plastic. To examine the segments, the layers were turned so the basilar membrane faced up and then were mounted in a custom block holder. A small droplet of immersion oil was put over each segment to improve optical clarity before examination. When examining the segments with an oil immersion objective, the objective was immersed in the oil droplet on the surface of the plastic layer. The organ of Corti was then studied with a phase-contrast microscopy by focussing through the tissue with the microscope's fine-focus knob.

Fig. 4 is a low power phase-contrast photomicrograph of one dissected and trimmed segment of the cochlear duct. A fair amount of detail is already visible in the segment at this magnification: BM - basilar membrane lateral to the organ of Corti; MNF - myelinated nerve fibers (i.e., peripheral processes of spiral ganglion cells) in the osseous spiral lamina; OHC - lateral organ of Corti consisting of outer hair cells, Deiters' and Hensen cells; PH - heads of the inner and outer pillar cells; SPL - spiral ligament. The modiolus is just off the screen at the top; Reissner's and the tectorial membranes are present but not visible because they are thin and relatively transparent compared to the organ of Corti.

Fig. 5 shows higher power, phase-contrast microscopic views of the sensory (hair) cells from a young control chinchilla. Panel A is focused on the endolymphatic surface or the reticular lamina of the organ of Corti. Stereocilia can be seen projecting from the apices of the single row of inner hair cells (ihc) and three rows of outer hair cells (ohc 1, 2, 3). Panel B is focused on the bodies of the hair cells. The bodies of the inner hair cells (ihc) are flask-shaped with a centrally located nucleus while the bodies of the outer hair cells (ohc 1, 2, 3) are elongated cylinders with basally located nuclei. The heads of the inner pillars (iph) and outer pillars (oph) separate the row of inner hair cells from the first row of outer hair cells. Note that in control ears from young adults, the cells in the organ of Corti have similar sizes and orientations and lack any age pigment. Disruption of this regular pattern of cell size and orientation is the first sign of damage to the hearing organ.

Last updated 6/15/99.

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