NERVE ENDINGS OF THE ORGAN OF CORTI AUTHORS: DOMINGGUS MANGAPE DEPARTMENT OF OTOLARYNGOLOGY HIROSHIMA UNIVERSITY

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1 Ear Res Jpn 13 NERVE ENDINGS OF THE ORGAN OF CORTI AUTHORS: DOMINGGUS MANGAPE YASUO HARADA NOBUHARU TAGASHIRA DEPARTMENT OF OTOLARYNGOLOGY HIROSHIMA UNIVERSITY SCHOOL OF MEDICINE. Nerve endings of the organ of Corti D. Mangape* Y. Harada* and N. Tagashira* *Department of Otolaryngology, Hiroshima University School of Medicine Microdissection have been used in attempt to observe the inner fluid spaces that the organ of corti has. By using this method, structures exposed into this fluid spaces could be examined and recorded under scanning electron microscope. A group of guinea pigs were treated with intraperitoneal inlection of streptomycin 250 mg/kg/day and another group were given kanamycin 400 mg/kg/day. Both groups were treated for 10 days, and than decapitated following the day of the last inlection. Microdissection using a small double-sided adhesive tape was used. This method is simple, easy to perform and provides a good possibility to observe the lateral wall of the space of Nuel. Our previous findings in the studies of normal nerve endings of outer hair cells were reviewed as the control. Although there were clearly evidence of ultra - structural changes in the cilia and their hair cells, nerve endings on the lower pole of outer hair cells seemed to be resistance to the effect of streptomycin or kanamycin. Further studies are in progress at our department to solve more this problem.

2 Ear Res Jpn 13 NERVE ENDINGS OF THE ORGAN OF CORTI There have been many reports about the cochlear pathology in experimental animals after intoxication with ototoxic antibiotics. Cochlear pathologies following ototoxicity have been investigated for many years, and it has been established that degeneration cells and nerves are the main histologic findings. The purpose of this study is to observed the effect of ototoxic drugs on the inner side of organ of Corti. Emphasis is placed on the nerve endings of the outer hair cells. Success in observing the normal structure of these nerve endings in our previous study, had led us to propose this study. Materials and methods Guinea pigs with normal Preyer reflex, gr body weight were used. One group was treated with intraperitoneal injection of streptomycin (SM) 250 mg/kg/day, and another group was given kanamycin (KM) 400 mg/kg/day. Both groups were treated for 10 days. The animals were decapitated following the day of the last injection. Normal findings from the previous study were reviewed as the control. After intraperitoneal anesthesia by pentobarbital sodium, the animals were decapitated and the temporal bones were removed from the skull. After removal the stapes, the round window membrane was perforated and the Perilymphatic space was perfused with 2% phosphate buffered glutaraldehyde. This procedure was done as quickly as possible. Specimens were left in the

3 Ear Res Jpn 13 fixative for 24 hours at 4 Ž. Under the microscopic view, the bony cochlea capsule were carefully removed together with the tectorial membrane and the Reissner's membrane. Cochlea was postfixed with 2% alcohol, followed by iso-amyl acetate. The cochlea was further critical point dried using CO2. In the dry state, the cochlea was mounted on the stub and gold sputter coated. Before dissection, scanning electron microscope was employed to observe the loss of hair cell cilia. Dissection was done using a small double-sided adhesive tape. The tape was put on the lateral part of the organ of Corti and after a few seconds the tape was pulled out along with the part of the organ of Corti. The weak point at which the organ of Corti is frequently separated are the space of Nuel and the tunnel of Corti. Separation of these structures enabled detailed observation of the nerve endings of outer hair cells. Dissected specimens were sputter coated for scanning electron microscopy. Results Before microdissection, the cochlea was observed under scanning electron microscope. The loss of hair cell cilia was observed in all guinea pigs, although great variations were noticed among individual animals. In the streptomycin group, the middle turns showed the loss of cilia in the outer-most row of outer hair cells (Fig. 1), while the upper turns in some guinea pigs showed the loss of cilia in all rows of outer hair cells (Fig. 2). In the kanamycin group, the

4 Ear Res Jpn and middle turns showed a light damage in their cilia, but the upper turns showed severe damage in all cilia (Fig. 3). Observation on the nerve endings of the outer hair cells, was unfortunately limited to observe the nerve endings in only first row of outer hair cells. This limitation was due to microdissection using in this study which enabled to open only the space of Nuel or the tunnel of Corti. On the other hand the cochlear changes after ototoxic intoxication appear mainly on the outer row of outer hair cells. In both groups, nerve endings on the lower pole of outer hair cells seem to be resistant to streptomycin and kanamycin. In all turns, they appeared to remain in the same size and shape as normal. A few nerve endings showed small holes on their surface (Fig. 4). It was difficult to distinguish wether these defects xere due to ototoxic effect or artifact by preparation. Although nerve endings showed no remarkable change, the hair cells and nerve fibers revealed some changes. Figure 5a shows degenerated hair cell, while figure 5b shows loss of one outer hair cell. The initial stage of collapse in the outer hair cell can be seen in figure 5c. There was evidence that the hair cells, the nerve fibers and the supporting structures were easily damaged after ototoxic intoxication (Fig. 6). Discussion Marked variations of guinea pig in response to streptomycin or kanamycin were noticed in this study. Ylikoski (1) reported the great variations among individual animals regarding the effect

5 Ear Res Jpn 13 ototoxic antibiotics. Therefore, individual variation must be always taken into consideration when damage of the organ of Corti is evaluated. The most frequently affected dy ototoxic drug was the outer row of outer hair cells. Dayal (2) reported that the hair cell loss were consistently seen primarily in the outer-most row of outer hair cells in the apical turn of the cochlea. Toyoda et al (3) found that the outer hair cells in the basal coil of the cochlea were the most severely damaged in kanamycin intoxicated guinea pigs. The first row of outer hair cells showed the loss of cilia only in a few locations. The exposed nerve endings remain almost unaffected. Even after microdissection it was not possible to locate the damaged cilia, therefore relationship between the structures of the nerve endings and the damage of corresponding hair cell still remain unclear. Futhermore, the possibility of artifact during preparation, exists. Nerve endings in all turns appeared to be resistant to the effect of ototoxic drugs since there was no evidence of pathologic changes in their structure. Using TEM, Lim (4) found that sensory cells were disintegrated whereas nerve endings seemed intact in the kanamycin treated animals. Nerve ending degeneration appear to be secondary to the sensory cell degeneration (4). Degeneration of nerve fibers following perilymphatic perfusion with 20% SM was reported by Terayama et al (5). The authors also found that the hair cells, the nerve fibers and the supporting structures were easily damaged, thus resulting in very few intact hair cells in the ototoxic treated guine

6 References 1. Ylikoski, J.: Correlative studies on the cochlear pathology and hearing loss after intoxication with ototoxic antibiotics. Acta Otolaryngol. (Stockh) 78/suppl 326, Dayal, V. S.: Combined effect of noise and kanamycin. Ann. Otol. 80/6: , Toyoda, Y. et al: Quantitative analysis of kanamycin ototoxicosis. Acta Otolaryngol. 84/3-4: , Lim, D. J.: U1trastructural cochlear changes following acoustic hyperstimulation and ototoxicity. Ann. Otol. 85: , Terayama, Y. et al: Ultrastructural changes of the nerve elements following disruption of the organ of Corti. I. Nerve elements in the organ of Corti. Acta Otolaryngol (Stockh) 83: , 1977

7 16 Ear Fig. 1 Hair cell cilia in streptomycin A. Lower treated of the third guinea turn. Res Jpn pig 3 outer hair cells of the third row lost their cilia B. Upper of the second turn. Almost all cells in the third are damaged Fig. 2 Upper of the third turn of SM treated Loss of cilia can be seen in many guinea cells of each turn. pig. row 13

8 Ear Fig. 3 Kanamycin treated guinea Res pig A. Lower of the third turn shows B. Upper of the fourth damage turn shows in only a few cells severe damage in all rows Fig. 4 Nerve disruption and small holes on the surface of nerve endings A. Upper of the basal turn (SM) B. Lower of the basal turn (SM) C. Lower of the basal turn (SM) D. Lower of the second turn (KM) Jpn 13 17

9 18 Ear Res Jpn Fig. 5 Degenerated hair cells with intact nerve endings A. Upper of the third turn (SM). Degenerated hair cells are seen in the left. B. Lower of the third turn (SM). Loss of hair cell in the middle. C. Upper of the third turn (KM). Hair cell in the right is in the initial stage of collapse. Fig. 6 Damaged hair cells, nerve fibers and the supporting structures. 13