221 Hewing Research, 26 (1987) 221-224 Elsevier

HRR 00873

Short Communication

Modified staining technique for surface preparation of the organ of Corti

LibuSe 6lehlovA and LuboS Voldfich Institute of Experimental Medicine, Czechoslovak Academy of Sciences. 12000 Prague 2, Czechoslovakia

(Received 6 June 1986; accepted 30 September 1986)

A simple technique for the staining of the inner ear tissues using toluidine blue and Ehrlich haematoxylin is described. The

method is very convenient for histological evaluation of hair cell counts, for estimation of the number of scars in the reticular lamina,

for observing the arrangement of stereociliar pattern and for the detection of traumatic changes after noise exposure. The method

described proves to be very useful for histological examination of human temporal bones as well and makes possible an immediate

evaluation of the hair cells of the human cochlea and direct correlation of the inner ear pathology with audiometric data.

Staining technique; Human cochlea; Inner ear pathology

Introduction

Ever since the publication of the monograph ‘Structural pattern of the organ of Corti’ by En- gstrGm, Ades and Andersson (1966) twenty years ago, the most preferred method of surface speci- men preparation has been the fixation and stain- ing with osmium tetroxide, and the study of the cytoarchitecture of the organ of Corti using the phase contrast microscopy.

While the osmium tetroxide is necessary for electron microscopic studies, for the routine light microscopic examination the fixation with 10% solution of neutral formaldehyde and staining with toluidine blue and Ehrlich haematoxylin proved to be very useful and gave very good results.

Preparation of temporal bones

Experimental animals After decapitation of the experimental animal,

the temporal bones were removed, bullaea opened and the inner ears were fixed by slow perfusion with 10% neutral solution of formaldehyde through the round and oval windows, and submerged for

Correspondence to: LibuSe ClehlovL, Institute of Experimental

Medicine, Czechoslovak Academy of Sciences, Tfida Lidov$ch

milici 61, 12000 Prage 2, Czechoslovakia.

few days in the fixative. The bony cochlear wall is easily accessible in such experimental animals like guinea-pig, rat or bat and there is no need for any special approach to the cochlear shell before stain- ing.

Humans The preparation of the human cochlea for fixa-

tion and for staining presents more difficulties. Within a few hours after the patients death the

posterior half of the tympanic membrane was reflected forward through the outer ear canal ex- posing the middle ear cavity. The incudo-stapedial joint was cut, the incus pushed aside and the stapes was extracted. After the perforation of the round window membrane with a small hook, 10% neutral solution of formaldehyde was injected through the oval window. The injection was made carefully under low pressure to avoid damage to the structures of the cochlear duct. After 4 to 5 perfusions with 0.5 to 1 ml of fixative each time through the Scala tympani and back through the Scala vestibuli, the middle ear cavity was filled

with the fixative and sealed with a tight plug to prevent the leakage of the fixative. The fixative was left in the labyrinth until an autopsy could be performed. The petrous part of the temporal bone was removed from the scull with a bone saw and submerged in the fixative for several days. After a

0378-5955/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

Fig. 1. Surface specimen of the middle part of the organ of Corti of jaguar (Panfhero omzz). 1.2.3. three rows of outer hair

inner hair cells; g, giant cilia; Bar = 20 pm.

cells:

Fig. 2. Surface specimen of the second turn of the organ of Corti of guinea-pig. 1.2.3, three rows of outer hair cells; i, stereocilia of

inner hair cells are out of focus; N. nuclei of inner hair cells; S, scar in the reticular lamina; Bar = 20 pm.

223

thorough rinse with water the bony capsula around the cochlea was carefully thinned with a dental drill to avoid damage to the membranous labyrinth.

Staining procedure

After a thorough rinse with water and before removing the bony cochlear wall, a small opening was made with a fine hook at the apex in the place of helicotrema. Using a fine Pasteur pippette with few drops of stain, the cochleae, both experimen- tal and human, were perfused through the opening made at the apex, and using capillary forces the stain was allowed to run down the scalaea. A very short, about 5 s, pre-staining with ca. 1% water solution of saturated ethanol solution of toluidine blue was then rinsed with water and followed by, about half minute, staining with Ehrlich haema-

toxylin, this gave the best results. While haema- toxylin stained above all the superficial structures (stereocilia, cuticular plates, Fig. l), toluidine blue penetrated into the tissue and stained cellular bodies (hair cells, pillar cells and supporting cells) and cellular components, e.g. nuclei (Fig. 2). Thus, the focusing on different levels allows the thor- ough examination of the tissue under study.

When the staining was finished and the exces-

sive stain was washed away with water, the dissec- tion was started under the dissecting microscope.

The thinned bone was chipped away with shar- pened steel pick and individual turns of the organ of Corti were removed with a fine iris knife. Individual turns were mounted between an object glass and a cover glass in glycerol.

The advantages of the method

For the routine work at the light microscopic level the method is very quick and easy.

The great advantage is especially evident when the noise exposed material is dissected. The vitally injured tissues become conspicuously stained (Fig. 3) for the stain penetrates deeply into the injured tissues which makes it possible to detect vital

injuries already during dissection under the dis- secting microscope, at the same time to differenti- ate them from preparation artifacts and to com- pare and correlate the injuries in the organ of Corti and in the adjacent tissues like in the stria vascularis, the Reissner’s and the basilar membranes.

Usually it is not possible to fix human temporal

Fig. 3. The rupture in the basilar membrane due to noise overstimulation (one-third octtive band of noise, CF 1 kHz, 142 dB, 10 min;

survival time, 7 days). Guinea-pig, second turn. The basilar membrane is covered with flat epithelial cells. The vitally injured part of the basilar membrane is conspicuously stained.

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Fig. 4. Surface specimen of the second turn of human organ of Corti, Male, age 56. For further explanation see legend to Fig. I.

bones quickly enough for detailed examination in electron microscopy, therefore this method is very convenient for the evaluation of human cochlear pathology (Fig. 4) also for economic reason.

The method described has been routinely used in our Institute for more than fifteen years and applied to several hundreds of animals including

monkey. Altogether 72 human temporal bones were examined using this method.

Reference

EngstrGm, H., Ades, H.W. and Andersson, A. (1966) Structural

pattern of the organ of Corti. A Systematic Mapping of

Sensory Cells and Neural Elements. Almqvist and Wiksell,

guinea-pig, rat, bat, mouse, dog, cat, rabbit and Stockholm.