Arch Otorhinolaryngol (1989) 246:235-237 ,ro.i ~ O- Laryngology �9 Springer-Verlag 1989

Melanin in the inner ear*

An experimental study with control and kanamycin-intoxicated colored guinea-pigs

B. Gratacap, A. Attard, A. Laurent, P. Stoebner, D. Smirou, and R. Charachon

Clinique Universitaire d' ORL du CHU de Grenoble, BP 217 X, F-38043 Grenoble, France

Summary. Following several studies on the effects of kanamycin toxicity on the inner ears of guinea pigs, we have studied the importance of melanin in this phe- nomenon. Transmission electron microscopy showed that, under the influence of kanamycin, the inter- mediate strial cells developed a secretory aspect simi- lar to that seen in skin melanocytes. This aspect as yet has never been described for the inner ear cells. A planimetric, morphometric method was also used to determine the strial cell melanin status in control ani- mals. Additional findings in the study confirmed an increase in the number of melanosomes during kana- mycin poisoning. Statistical data are discussed.

Key words: Inner ear - Toxicity - Kanamycin - Mela- nin

Introduction

During the past 10 years, we have carried out several experimental studies to assess the toxic effects of an aminoglycoside (kanamycin) on the inner ear struc- tures of the guinea pig. Our initial aim was to find pos- sible differences between the lesions occurring in the organ of Corti and those in the stria vascularis. We thought that any lesions might concern these two ele- ments, since the path followed by the toxic molecule begins in the general circulation and then goes via the strial vessels to the endolymph and the hair cells [5]. Our study was based on transmission electron micro- scopy.

* Presented at the First European Congress of Oto-Rhino- Laryngology and Cervico-Facial Surgery, Paris, 26-29 Sep- tember 1988 Offprint requests to: B. Gratacap

We found that the classic hair cell lesions seen had certain characteristic changes: injury followed by de- struction, beginning at the outer hair cells and then involving the inner hair cells from the base to the apex of the cochlea. By analogy with kidney lesions, we showed that the toxic drug entered the hair cell and was then accumulated by the lysosomes present. These organelles then increase in number. When their mass becomes too high, their content is dumped into the cell. This destroys the cell by the release of lytic en- zymes. Lysosomal injury of the proximal renal tubular cells and that of the inner ear's hair cells are absolutely identical when studied under electron microscopy [6, 7].

The lesions observed in the stria vascularis are very different, but they appear at the same time as those seen in the organ of Corti. Several protocols, despite using lower doses of aminoglycosides and shorter du- rations of treatment, did not show a sequential evolu- tion of the injury between these different structures.

However, four main changes were observed in the stria vascularis [8]: interstitial swelling; disappearance of the microvilli of the endolymphatic border of the marginal cells; mitochondrial lesions ranging from simple loss of the internal crests to complete destruc- tion with the formation of the classical "myeloid body" (and wrongly taken to be similar to the lesions in the hair cells); changes in melanin metabolism. In this report we want to emphasize these latter changes.

The presence of melanine inside the intermediate cells of the stria has been known for a long time. These cells have the same morphological relationships as with melanocytes found elsewhere, as in the eyes, the meninges and (mainly) the skin. They have the same embryological origin and are derived from the neural crest. They belong to the amine precursor uptake and

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decarboxylation system, giving rise to the melano- somes which are insoluble pigments resulting from a reaction involving dihydroxyphenylalanine. (This is the main principle of the Fontana stain.) The melano- somes or melanin granules are characterized in trans- mission electron microscopy as oval vesicles that are very dense to electrons. They sometimes contain small-sized vesiculo-globular elements which are lighter to electrons. One of the main characteristics of the skin melanocyte is to transfer their melanosomes to the keratinocyte. Those melanocytes not present in the skin are not physiologically able to secrete their melanin and are therefore called "continent".

Our first observations led us to uncover several changes in melanin metabolism during kanamycin poisoning. We then decided to study these findings in the present study.

Materials and methods

Four series of adult male pigmented guinea pigs were used for a total of 28 animals. These included 12 controls and 16 poisoned with kanamycin injected subcutaneously (6 animals with 200 rag/ kg per day for 9-11 days and 10 animals with 400 mg/kg per day for 5-13 days). This antibiotic schedule produced animals with different levels of kanamycin toxicity.

A cortical electrode was always placed in each animal under general anesthesia before giving the kanamycin. Using the method described by Aran and Erre [1], cortical evoked re- sponse audiometry was used to assess the global hearing status of both control and experimental animals, as well as the absence of deafness before intoxication.

Histological fixation was obtained by window perfusion with glutaraldehyde in vivo under general anesthesia. After sacrificing the animal, fixation was completed with osmic acid staining. Cochleas were then dissected under an optical micro- scope to obtain sections of the membranous labyrinth that con- tained each of the three rows of hair cells, These sections were then processed, orientated and embedded in Spurr.

Radial sections perpendicular to the cochlear canal were then obtained. Semithin sections were stained with toluidine blue and by Fontana's method. Ultrathin sections were used for transmission electron microscopy. Seventy-three sections were obtained in all, 58 of which were studied completely: 41 in con- trol animals and 17 in antibiotic-toxic ones. The 58 sections studied were taken in different rows, so as to assess the amount of interindividual differences. Three sections were also taken 100 gm apart within the third row to analyze intraindividual variations as well.

B. Gratacap et al.: Inner ear melanin

A second lineness with the skin was the appear- ance of a secretory character. At first, melanosomes were found in piles inside intermediate cells. They were then found in the marginal cells. Later on, they were seen to migrate into the cochlear canal and final- ly were found inside cells of Reissner's membrane.

The number of melanosomes was found to increase in the antibiotic-toxic animals. We then aimed to study this last phenomenon with more quantitative data, but we first had to determine the control ani- mals' status.

Three sections 100 gm apart in the third row of sev- eral control animals were studied. For these sections, a planimetric morphometric method with a graphic table and a computer were used. As for all other sec- tions, each whole stria was rebuilt photographically at x 3,000 magnification, so that a global surface area calculation could be carried out. In order to reduce the importance of the incidence of the cut of the section, the vessel and nuclear surface area were substracted from the strial surface area. A second reconstruction was then done with a higher magnification ( x 25,000), so as to be able to count the pigment granules present and to eliminate artifacts such as silver deposits from the photographic products. This procedure gave us the number of melanosomes per unit of surface area of the stria and was the basis four our statistical analyses (Table 1).

In the control animals, the range of the number of melanosomes was very large. However, this disper- sion was not haphazard, as the intraindividual varia- tions (effect of the level of the section) and the interin- dividual variations (effect of the animal) were both found to be statistically significant by A N O V A (F = 16.92 and F = 9.81) for P = 0.05, respectively). In other words, there was wide variability in the number of melanosomes found in the controls: between one animal and another, as well as within the same animal between two turns of the cochlea or even for close sec- tions of the same turn. With this prerequisite in mind, when comparing two homogeneous series of animals, control (A) and antibiotic-toxic (B), without taking in account either the dose or the duration of the intoxica- tion, Student's t-test showed a significant increase in

Results

In antibiotic-toxic animals, there was a qualitative in- crease in the length of the extensions of the inter- mediate cells, as well as a greater intrication with the interdigitations of the marginal cells. These changes were similar to those seen in the skin under the influ- ence of the ultraviolet light.

Table 1. Quantitative results showing the number of melano- somes per 100 gm 2 of a relative surface area of the stria vas- cularis on three sections ($1, $2, $3) 100 gm apart on the third row of a homogeneous series of six control animals (AI-A6)

A1 A2 A3 A4 A5 A6

$1 12.78 3.70 4.78 6.83 12.29 2.17 $2 9.09 6.73 4.30 5.46 9.76 3.64 $3 7.62 4.93 3.57 3.87 8.90 4.38

B. Gratacap et al.: Inner ear melanin 237

Table 2. "Intoxication effect" analysis: number of melanosomes per 100 gm 2 of a relative surface area of the stria vascularis with five control animals (A) and ten toxic animals without taking into count either the dose or the duration of poisoning (B)

1 2 3 4 5 6 7 8 9 10

A 5.21 4.56 2.40 4.44 6.33 B 11.88 6.15 6.30 4.80 7.62 14.52 4.61 7.89 8.18 20.19

Student's t-test: t = 2.01 (ddl = 13), P = 0.05

Table 3. "Dose effect" analysis: number of melanosomes per 100 ~tm 2 of a relative surface area of the stria vascularis with five control animals (A), five toxic animals with 200 mg kanamycin/kg per day (B) and five toxic animals with 400 mg kanamycin/kg per day

1 2 3 4 5

A Control 5.21 4.56 2.40 4.44 6.33 B 200mg/kg/day 11.88 6.15 6.30 4.80 7.62 C 400 mg/kg/day 14.52 4.61 7.89 8.18 22.19

Linear regression coefficient: r = 0.52, P = 0.05

melanin. We have also known for a long t ime the dif- ferences in reactions be tween albino and p igmented animals [2]. Finally, it would appear reasonable to advance the hypothesis that, as with the skin and o ther tissues, melanin might play a significant protect ive role because of its powerful free radical scavenging characteristics [10] or by altering aminoglycosides themselves [4-9].

Acknowledgement. This work was supported by INSERM grant no. 856005.

the n u m b e r of me lanosomes in the toxic animals (Ta- ble 2). Moreove r , when compar ing three homogene - ous series of animals [control (A) , weakly toxic (B) and more strongly toxic (C)], the l inear regression coefficients showed that the n u m b e r of me lanosomes increased significantly with the degree of the poison- ing incurred (Table 3).

Discuss ion

Our findings show that toxic exposure to kanamycin results in the change of in termedia te cells in the inner ear f rom a state of rest to an "act ive" one. This is ac- compan ied by cytological p h e n o m e n a , the mos t strik- ing of which is the appearance of changes having a secretory character . There is also an increase in the n u m b e r of melanosomes . This last finding must be carefully in terpre ted because of the great var ia t ion found in the control animals.

The present s tudy emphasizes the fact that results concerning aminoglycoside intoxication, and even those concerning cochlear metabol ism, mus t refer to

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