- Email: [email protected]
Ototoxicity of sodium nitroprusside
ELSEVIER
Hearing Research 114 (1997) 169-178
Ototoxicity of sodium nitroprusside Run Sheng Ruan ~,*, Seng Kee Leong b, Kian Hian Yeoh ~ a Department of Otolaryngology, National University Hospital, Lower Kent Ridge Road, 119074, Singapore b Department of Anatomy, National UniversiO, of Singapore, 10 Kent Ridge Crescent, 119260, Singapore Received 2 November 1996; revised 15 August 1997; accepted 31 August 1997
Abstract Nitric oxide (NO) not only has normal physiological roles like vasodilation and neurotransmission in the living organism, it could also have possible neurodestructive effects under certain pathological conditions. The present study aimed to determine whether direct exposure of guinea pig cochlea to a NO donor like sodium nitroprusside (SNP), or a nitric oxide synthase (NOS) inhibitor like Ne-nitro-L-arginine methyl ester (L-NAME), would cause damage to the auditory hair cells. A piece of gelfoam was placed on the round window of the right ear of adult albino guinea pigs. It was then soaked with 0.1 ml of SNP (3.4 gM), 0.1 ml of L - N A M E (9.3 gM or 18.5 gM) or 0.1 ml of injection water, the vehicle used to dissolve the above chemicals. Twelve animals receiving SNP were perfused 1 day, 2, 3 and 7 days later, with three animals being used for each survival period. Six animals receiving L-NAME were allowed to survive up to 7 days before perfusion. Eight animals receiving injection water or 0.45% saline were used as controls. With the scanning electron microscope, the inner and outer hair cells were counted over a 1 mm length of the basilar membrane in each turn of every cochlea. The results showed that, in animals treated with L - N A M E at both concentrations stated, no significant loss of either inner or outer hair cells was noted in any part of the cochlea studied. However, as early as 1 day after SNP treatment, a striking loss of inner and outer hair cells was observed in the three lower turns of the cochlea. Damage to the outer hair cells was extended to the apical turn with increasing survival period, but no significant loss of inner hair cells was evident in the apical turn at any of the survival periods studied. To rule out the possibility that the effects were due to the presence of cyanide, a metabolite of SNP, hydroxycobalamin was introduced into the scala tympani of three animals through a cannula-osmotic pump device during SNP treatment. There was no significant difference in the results between the groups with and without hydroxycobalamin infusion 7 days after SNP treatment. The present study suggests that an excessive production of NO in the inner ear could lead to extensive loss of hair cells. © 1997 Elsevier Science B.V.
Key words." Hair cells; Inner hair cells; Outer hair cells; Guinea pig; Nitric oxide; Sodium nitroprusside; L-NAME
1. Introduction Since the d i s c o v e r y o f nitric oxide ( N O ) as an e n d o t h e l i u m - d e r i v e d relaxing f a c t o r a n d as a n e u r o t r a n s m i t ter in b o t h the central a n d p e r i p h e r a l n e r v o u s systems, there has been a t r e m e n d o u s surge in interest in this very simple n o n - c a r b o n c o n t a i n i n g m o l e c u l e in the biological system. N O is p r o d u c e d on d e m a n d f r o m L-arginine b y nitric oxide synthase (NOS), with c o n c o m i t a n t p r o d u c t i o n o f L-citrulline, in the presence o f several * Corresponding author. Tel.: +65 772 5371; Fax: +65 775 3820; E-maih [email protected]
0378-5955 / 97 / $17.00 © 1997 Elsevier Science B,V. All rights reserved
PIIS0378-5955(97)00159-7
co-factors, i n c l u d i n g n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e ( N A D P H ) , a n d o f c a l m o d u l i n to which C a 2+ b i n d s ( K n o w l e s et al., 1989). T h e a b o v e discoveries in the c h e m i s t r y o f N O have led i n v e s t i g a t o r s to use N O S a n d citrulline i m m u n o h i s t o c h e m i s t r y a n d N A D P H diap h o r a s e ( N A D P H - d ) h i s t o c h e m i s t r y to investigate at the light m i c r o s c o p i c level the presence o f N O in the m a m m a l i a n cochlea a n d to correlate it with its functions in the m o d u l a t i o n o f c o c h l e a r b l o o d flow ( C B F ) a n d a u d i t o r y n e u r o t r a n s m i s s i o n ( Z d a n s k i et al., 1994; F e s s e n d e n et al., 1994). O u r c u r r e n t electron m i c r o scopic e x a m i n a t i o n o f N O S i m m u n o s t a i n e d guinea pig a n d h u m a n c o c h l e a r tissue has further c o n f i r m e d the
170
R.S. Ruan et al./Hearing Research 114 (1997) 169-178
existence of NOS in spiral ganglion cells and their synapses with inner hair cells and in the endothelial cells of all the cochlear blood vessels. Previous studies also showed a profound increase in CBF when sodium nitroprusside (SNP), a NO donor, was applied to the round window membrane of the mammalian cochlea (Ohls6n et al., 1992), whereas a competitive NOS inhibitor, NC-nitro-L-arginine methyl ester (L-NAME), resulted in a reduction in CBF (Brechtelsbauer et al., 1993, 1995). Notwithstanding the convincing evidence of the important physiological roles of NO, there is strong evidence that N O could be responsible for neurodestruction in the central nervous system (Moncada et al., 1991; Kader et al., 1993; Ruan et al., 1995). In the auditory system, it has been reported that SNP has an acute ototoxic effect in a concentration- and time-dependent manner (Ohls6n et al., 1993; Chen et al., 1995; Dais et al., 1996; Kong et al., 1996), and the deleterious action of pneumolysin on guinea pig cochlea could be blocked by the administration of L - N A M E (Amaee et al., 1995). To further elucidate the role of NO in the cochlear tissue, the present investigation sought to study whether exposure of the cochlea to excessive NO produced by a NO donor like SNP or reduced NO induced by the administration of a NOS inhibitor like L - N A M E would lead to destruction of the hair cells.
may be the salt content in the SNP solution that resulted in hair cell damage, three animals were treated with 0.45% saline (pH 6) which contained sodium equivalent to that of SNP and allowed to survive for up to 7 days. The left cochleae of all the above animals were compared morphologically with the right cochleae. To rule out the possibility that the hair cell damage observed in animals treated with SNP was due to a SNP metabolite other than NO, for example, the free cyanide, hydroxycobalamin (HCL) was administered into the scala tympani via a cannula-osmotic pump device to detoxify cyanide potentially formed during diffusion of SNP through the round window. The cannula-osmotic pump device was prepared as described by Brown et al. (1993). The pump used was an Alzet 2002 osmotic pump (Alza Corp., Palo Alto, CA) having a flow rate of 0.5 gl/h for a continuous infusion period of 14 days. The pump was loaded with a solution (1 mg/ml) of H C L (Yung Shin, Pharmaceutical Industrial Co. Ltd., Taiwan) and incubated in sterile normal saline at 37°C overnight for activation before implantation. Three guinea pigs were subjected to a cochleostomy to the scala tympani at the level of the basal turn of the cochlea. The tip of a cannula connected to a HCL-loaded osmotic pump was placed in the scala tympani to allow H C L delivery therein, followed by SNP application in the tympanic cavity. The pumps were subcutaneously implanted between the forelegs.
2. Materials and methods
2.1. Tissue preparation for scanning electron microscopy (SEM)
A total of 29 albino guinea pigs of both sexes weighing 250-300 g were used in this study. All subjects had a normal Preyer's reflex and were free of middle ear infection as assessed directly under an operating microscope. The animals were anesthetized with an intramuscular injection of ketamine (40 mg/kg) and xylazine (4 mg/kg). Through a post-auricular incision, the right temporal bulla was opened to expose the round window. Under the operating microscope, a small piece of gelfoam was placed on the round window membrane, and 0.1 ml of SNP (Roche) at a concentration of 3.4 IuM (pH 6), or L - N A M E (Sigma) at a concentration of 9.3 gM (pH 4) or 18.5 g M (pH 3.5) was deposited on the gelfoam. Both chemicals were dissolved in injection water (pH 8) just before use. Twelve animals which received SNP were killed 1 day, 2, 3 and 7 days after operation, with three animals being used for each survival period. Six animals, three of which were given L - N A M E at a concentration of 9.3 gM and the other three at 18.5 gM, were killed on the 7th postoperative day. In addition to the above, five animals having gelfoam soaked with injection water were used as control and they were given a survival period of 7 days. To rule out the possibility that it
To quantitate the hair cells in each turn of every cochlea, a modified technique for tissue preparation for SEM assessment was developed. Animals were decapitated under deep anesthesia with ketamine and xylazine. The bullae were opened after removal from the skull. The oval and round windows were punctured and a cold solution of 2.5% phosphate-buffered glutaraldehyde was introduced slowly via the round window to the perilymphatic space. The perfused cochleae were kept in the same fixative for 2 h, then thoroughly rinsed with phosphate buffer. They were afterwards post-fixed in 1% osmium tetroxide for 1 h and transferred to phosphate buffer for dissection. The basilar membrane of the apical turn of the cochlea was exposed by removal of the bony cochlear capsule, the spiral ligament and Reissner's membrane with a pair of fine forceps. On the tympanic side of the cochlea, an opening on the cochlear duct was created at each turn by gently removing part of its lateral wall and the spiral ligament to facilitate the subsequent introduction of chemicals to the organ of Corti. The thiocarbohydrazide (TCH) technique as described previously (Hunter-Duvar, 1978) was used to
R.S. Ruan et al./Hearing Research 114 (1997) 169 178
171
m e m b r a n e was exposed was first examined. The next turn of the basilar membrane was viewed after removing the apical turn of the cochlea. In this way, each cochlea was studied from the apical turn to the basal turn. Photographs of five fields of basilar membrane were taken randomly in each turn for morphological observation and quantitative assessment. The inner and outer hair cells with stereocilia bundles were counted over a 1 m m length of basilar membrane in the photographs. Hair cells without stereocilia attachment or with bleb formation were considered to be damaged and not included in the count. The mean values and standard deviation of the mean were calculated and statistical analysis was performed with Student's t-test between experimental and control animals. Confidence levels of 95% were used to interpret significance. The A N O V A technique was also used to evaluate whether there was any difference in means a m o n g the groups of guinea pigs perfused 7 days after treatment with water, L - N A M E (18.5 BM) and SNP.
3. Results
Fig. 1. SEM photomicrographs of the basilar membrane at the (A) and 200 gm away from the apex (B) of the left cochlea control guinea pig. Note that there are a few scattered outer cells (OHC) in A, and only one row of OHC in B. The arrows cate OHC. The arrowheads indicate the inner hair cells. Scale 10 Bm.
apex in a hair indibar:
provide a conductive coat for the cochlea. Briefly, the cochlea with an opening on every turn of the cochlear duct was incubated for 20 min in a freshly prepared and filtered, saturated solution of T C H . After six washes in distilled water, the cochlea was incubated for 1 h in 1% aqueous osmium tetroxide, followed by six washes in distilled water. The whole procedure was then repeated. All the cochleae were dehydrated carefully through a graded series of ethanol. After two 10 min changes of 100% ethanol, the cochleae were dried in a critical point drier according to standard procedures, then observed in a Philips 515 SEM.
2.2. Numerical analysis The apical turn of the cochlea where the basilar
After recovery from anesthesia, guinea pigs given SNP unexpectedly displayed spectacular postural changes. Their heads were tilted towards the operated side and this was accompanied by a spontaneous nystagmus with the rapid component of eye movement directed toward the unoperated side. These changes were not observed in animals given L - N A M E , injection water or saline. In control animals treated with either water or saline, no morphological difference in the hair cells was observed between the right and left ears (Fig. 3A D, Figs. 4A D and 5A-D). It was evident that only inner hair cells (IHC) and a few scattered outer hair cells (OHC) were present at the apex of the cochleae (Fig. I A). The O H C population increased gradually as the cochlea was traced from the upper to the lower part of its apical turn (Fig. 1B). All three rows of O H C appeared only at a distance of at least 1 m m away from the apex. In animals receiving L - N A M E at both concentrations of 9.3 BM and 18.5 BM, there was no significant loss of either I H C or O H C in any part of the cochlea up to 7 days after treatment (Fig. 6A-D). On the other hand, as early as 1 day after SNP treatment, marked I H C and O H C loss was observed in the lower three turns of the cochlea, but not in the apical turn (Fig. 2A D). It was obvious that, of the three turns of the cochlea showing hair cell damage, the loss of hair cells was more severe in the lower than in the upper turn. In fact, there was complete loss of I H C and O H C in the
172
R.S. Ruan et al./Hearing Research 114 (1997) 169 178
!!~ilm
Fig. 2. SEM photomicrographs o f the basilar m e m b r a n e in the apical (A), upper middle (B), lower middle (C) and basal (D) turns of the cochlea 1 day after exposure to sodium nitroprusside. Note that the inner and outer hairs in the three lower turns were severely damaged. Scale bar: 50 Bm.
Table 1 Survival of hair cells at each turn of the guinea pig cochlea 1-7 days after exposure to L - N A M E , SNP and various control agents Agent
Water Saline L-NAME L-NAME SNP SNP SNP SNP SNP+HCL
N u m b e r of animals
5 3 3 3 3 3 3 3 3
Concentration
0.45% 9.3 BM 18.5BM 3.4 BM 3.4gM 3.4 g M 3.4 BM 1 mg/ml
Survival period (days)
7 7 7 7 1 2 3 7 7
Hair cell counts (mean _+S.D.) Apical turn
Upper middle turn
Lower middle turn Basal turn
IHC
OHC
IHC
OHC
IHC
OHC
IHC
OHC
94+12 101_+9 93_+14 97+11 102_+6 85_+7 98_+4 104_+6 102-+3'
308+27 310_+35 307+31 312-+33 290_+12 306_+7 183_+9" 183_+10" 179-+ 11'
113_+10 98_+8 111_+11 116_+10 65_+ 13" 28_+7* 95_+7* 95_+5* 88_+9*
404_+27 400_+33 411_+29 409_+26 46_+7* 135_+12" 15_+8" 94_+ 16" 91 _+ 13'
ll8ill 100_+9 116_+11 129_+12 37+4* 4_+7* 3_+4* 0 4_+5
448_+31 451+29 443_+38 448_+30 27_+5* 1_+2" 1 _+ 1" 0 0
131_+10 131-+12 127+10 132_+10 17_+5" 0 0 0 0
466_+22 458-+25 463+24 464_+24 24_+5 * 0 0 0 0
*P < 0.05, compared with water-treated cochlea. *P > 0.05, compared with animals at 7 days after SNP treatment.
R.S. Ruan et al./Hearing Research 114 (1997) 169 178
173
Fig. 3. SEM photomicrographs of the basilar membrane in the apical (A), upper middle (B), lower middle (C) and basal (D) turns of the cochlea from the unoperated ear. Scale bar: 20 gin.
basal turn of the cochlea at 2 days after SNP administration. Significant O H C loss was noticed in the apical turn at 3 days after exposure to SNP but no significant I H C loss was evident in any of the survival periods studied. In animals with infusion of H C L into the scala tympani, there was also severe loss of hair cells in the lower three turns of the cochlea at 7 days after SNP treatment (Fig. 7A-D). There was, however, no statistical difference in hair cell counts between the groups with and without H C L infusion. The results of O H C and I H C counts in each turn of the cochlea exposed to L - N A M E , SNP and various control agents are shown in Table 1 where the results of Student's t-test are indicated. The results of A N O V A analysis showed, after adjusting for multiple comparison using the Bonferroni correction, that only comparisons between the groups at 7 days after treatment with water and SNP were
found to be statistically significant at the 5% level, which is in agreement with the results of Student's t-test.
4. Discussion
The present study provides evidence that direct exposure of the cochlea to SNP, a N O donor, results in severe damage to the auditory hair cells. It suggests that the presence of excessive N O in the cochlea may lead to extensive loss of hair cells. However, from a pharmacokinetic point of view, free cyanide might be produced during the breakdown of SNP. To determine whether SNP ototoxicity could involve free cyanide, we applied H C L into the scala tympani during SNP diffusion through the round window. In this situation, any free
174
R.S. Ruan et al./Hearing Research 114 (1997) 169 178
Fig. 4. SEM photomicrographs of the basilar membrane in the apical (A), upper middle (B), lower middle (C) and basal (D) turns of the cochlea 7 days after water treatment. Scale bar: 20 pm.
cyanide present will be converted by H C L to the nontoxic cyanocobalamin. The results showed that there was no significant difference in the counts of remaining hair cells between the groups with and without H C L infusion. These results suggest that the hair cell loss observed in this study was not due to free cyanide possibly released from SNP. Dawson et al. (1993) in their in vitro studies noted no neurodestructive effects of K4[Fe(CN)6] which had similar effects to Fe(CN5)z, another metabolite of SNP. This notwithstanding, whether the SNP itself or some product of SNP breakdown is responsible for the ototoxicity observed in this study remains to be clarified. Although the p H of L - N A M E and SNP solutions used in this study was shifted when the chemicals were dissolved in water, it should be noted that the p H shift was greater in the former than in the latter.
In spite of this, L - N A M E caused no significant damage to hair cells. Logically, it is unlikely that the loss of hair cells after SNP treatment could be attributed to the minor shift in the p H of the SNP solution. Recent studies have shown that damage to the cochlea induced by pneumolysin, a cytotoxic agent released from Streptococcus pneumoniae during bacterial meningitis, could be blocked by L - N A M E (Amaee et al., 1995) and the cochlear potentials could be suppressed by SNP applied either to the round window (Ohls6n et al., 1993; K o n g et al., 1996) or by intracochlear perfusion (Chen et al., 1995; Dais et al., 1996). Also, SNP significantly reduced in vivo both O H C voltage-induced length changes and whole-cell outward currents (Chen et al., 1995). These results point to N O as the possible ototoxic agent to the cochlea under certain pathological conditions. On the other hand, overstimulation of ex-
R.S. Ruan et al./Hearing Research 114 (1997) 169 178
175
Fig. 5. SEM photomicrographs of the basilar membrane in the apical (A), upper middle (B), lower middle (C) and basal (D) turns of the cochlea 7 days after treatment with 0.45% saline. Scale bar: 20 gm.
citatory amino acid (EAA) receptors may also play an important role in the pathophysiological changes in the ischemic cochlea. This conclusion is based on multiple studies in ischemic cochleae which showed that acute degeneration of afferent synapses with the IHC occurred with the introduction of glutamate or its analogs into the inner ear, leading to subsequent loss of spiral ganglion neurons (Pujol et al., 1985; Juiz et al., 1989; Puel et al., 1991a). Moreover, ischemia-induced degeneration of afferent synapses could be prevented by prior application of glutamate antagonists (Puel et al., 1991b, 1994), suggesting overstimulation of EAA receptors in ischemic conditions of the cochlea. The relationship between NO formation and EAA receptor activation has been established in the brain (Dawson et al., 1992). Previous studies in vitro have also demonstrated that EAA neurotoxicity in cultures of the cere-
bral cortex, hippocampus and striatum could be mediated by N O formation (Dawson et al., 1991, 1993). Therefore, it is reasonable to postulate that the neurodestructive effects of EAA receptor overstimulation in the cochlea is also mediated by excessive NO formation under certain conditions. The close similarity between the pattern of hair cell loss observed in the present study and that found in noise- and aminoglycoside-induced pathological changes in the cochlea suggests that NO formation may play a role under these conditions also. Although the exact mechanism of NO-induced cytotoxicity remains unclear, it has been suggested that NO could cause ADP-ribosylation of proteins (Brune and Lapetina, 1989), inhibit enzymes involved in D N A synthesis (Hibbs et al., 1989) and react with superoxide ion to form toxic hydroxyl free radicals (Beckman et al.,
176
R.S. Ruan et al./Hearing Research 114 (1997) 16~178
Fig. 6. SEM photomicrographs of the basilar membrane in the apical (A), upper middle (B), lower middle (C) and basal (D) turns of the cochlea 7 days after exposure to L-NAME (18.5 gm). No hair cell loss was noted. Scale bar: 20 gm.
1990). Thus, it is possible that a cascade of events may take place involving overactivation of E A A receptors resulting in an excessive formation of N O which ultimately leads to neurodestruction. The present study revealed that the loss of hair cells was always greater in the lower turn when the cochlea was exposed to SNP. It may be argued that in our experiment, N O may not have diffused uniformly throughout the perilymph. The area proximal to the round window could have received a higher concentration of N O as the gelfoam soaked with the N O donor was placed on the round window membrane. However, all the animals which received SNP presented the typical clinical picture of a unilateral peripheral destruction of the vestibular organ immediately after the animals recovered from anesthesia, indicating a direct action of N O on the vestibular receptors, the ampullae and cris-
tae. The route taken by N O to reach these receptors is most likely through diffusion from the scala tympani into the cochlear duct via the basilar membrane, but entry across the lateral wall of the cochlear duct could also be possible. If N O could still cause damage to the vestibular receptors after traversing all the turns of the cochlea, it is unlikely that, by the time it reached the upper turns, its concentration would be so diminished as to be unable to cause damage to the hair cells there. It is more likely that for some reason yet to be elucidated, the hair cells in the upper turn are more resistant to the destructive action of NO. Previous studies have shown that the application of L - N A M E on the round window of the cochlea did not affect the c o m p o u n d action potential thresholds of the cochlea (Brechtelsbauer et al., 1995). Our observations also showed no morphological changes in the cochlea
R.S. Ruan et al./Hearing Research 114 (1997) 169 178
177
Fig. 7. SEM photomicrographs of the basilar membrane in the apical (A), upper middle (B), lower middle (C) and basal (D) turns of the cochlea 7 days after exposure to sodium nitroprusside with hydroxycobalamin infusion into the scala tympani, Severe damage to the inner and outer hair cells was also noted in the three lower turns. Scale bar: 10 gin.
of animals treated with L-NAME, suggesting that, though excessive production of N O could destroy the hair cells of the cochlea, some deprivation of NO had no apparent effects on them. Lastly, it should be mentioned that, in the normal guinea pig cochlea, the three rows of O H C are incomplete at the apical turn for a distance of about 1 mm from the apex. Thus, the number of O H C at the apical thn is less than that at the lower turns of the cochlea in a given length of basilar membrane. This should not be mistaken for hair cell loss in any quantitative study of hair cells in the guinea pig.
Acknowledgments This study was supported by Grant RP 950393/N from the National Medical Research Council of Singapore.
References Amaee, F.R., Comis, S.D., Osborne, M.P., 1995. Ag~-Methyl-L-arginine protects the guinea pig cochlea from the cytotoxic effects of pneumolysin. Acta Otolaryngol. (Stockh.) 115, 386 391. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A., 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87, 1620 1624. Brechtelsbauer, P.B., Nuttall, A.L., Miller, J.M., 1993. Nitric oxide synthase blocker N(;-nitro-t=arginine decrease cochlear blood flow. Assoc. Res. Otolaryngol. 368, P92. Brechtelsbauer, P.B., Nuttall, A.L., Miller, J.M., 1995. Basal nitric oxide production in the cochlear vasculature. Otolaryngol. Head Neck Surg. 111, P70. Brown, J.N., Miller, J.M., Altschuler, R.A., Nuttall, A.L., 1993. Osmotic pump implant for chronic infusion of drugs into the inner ear. Hear. Res. 70, 167 172. Brune, B,, Lapetina, E.G., 1989. Activation of a cytosolic ADP-ribosyltransferase by nitric oxide-generating agent. J. Biol. Chem. 264, 8455-8458. Chen, C., Nenov, A., Skellett, R., Fallon, M., Bright, L.. Norris,
178
R.S. Ruan et al./Hearing Research 114 (1997) 16~178
C.H., Bobbin, R.P., 1995. Nitroprusside suppresses cochlear potentials and outer hair cell responses. Hear. Res. 87, 1-8. Dais, C.G.D., Prazma, J., Ball, S.S., Zdanski, C., Carrasco, V., Pillsbury, H.C., 1996. Effect of sodium nitroprusside on compound action potential thresholds in the gerbil cochlea. Hear. Res. 99, 1-6. Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M., Snyder, S.H., 1991. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissue. Proc. Natl. Acad. Sci. USA 88, 7797 7801. Dawson, T.M., Dawson, V.L., Snyder, S.H., 1992. A novel neuronal messenger molecule in brain: The free radical, nitric oxide. Ann. Neurol. 32, 297-311. Dawson, V.L., Dawson, T.M., Bartley, D.A., Uhl, G.R., Snyder, S.H., 1993. Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures. J. Neurosci. 13, 2651-2661. Fessenden, J.D., Coling, D.E., Schacht, J., 1994. Detection and characterization of nitric oxide synthase in the mammalian cochlea. Brain Res. 668, 9-15. Hibbs, J.B., Jr., Taintor, R.R., Vavrin, Z., Rachlin, E.M., 1989. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem. Biophys. Res. Commun. 157, 2811-2814. Hunter-Duvar, I.M., 1978. Electron microscopic assessment of the cochlea. Acta Otolaryngol. Suppl. (Stockh.) 351, 3-24. Juiz, J.M., Rueda, J., Merchan, J.A., Sala, M.L., 1989. The effects of kainic acid on the cochlear ganglion of the rat, Hear. Res. 40, 6574. Kader, A., Frazzini, V.I., Soloman, R.A., Triflatti, R.R., 1993. Nitric oxide production during focal cerebral ischemia in rat. Stroke 24, 1709-1716. Knowles, R.G., Palacios, M., Palmer, R.M.J., Moncada, S., 1989. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc. Natl. Acad. Sci. USA 86, 5159-5162.
Kong, W.J., Ren, T.Y., Nuttall, A.L., 1996. Electrophysiological and morphological evaluation of the acute ototoxicity of sodium nitroprusside. Hear. Res. 99, 22-30. Moncada, S., Palmer, R.M.J., Higgs, E.A., 1991. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev. 43, 109-142. Ohls~n, K.A., Didier, A., Baldwin, D., Miller, J.M., Nuttall, A.L., 1992. Cochlear blood flow in response to dilating agents. Hear. Res. 58, 19-25. Ohls6n, A., Hultcrantz, E., Engstrom, B., 1993. The effect of topical application of vasodilation agents on cochlear electrophysiology. Acta Otolaryngol. 113, 55~61. Puel, J.-L., Ladrech, S., Chabert, R., Pujol, R., Eybalin, M., 1991a. Electrophysiological evidence for the presence of NMDA receptors in the guinea pig cochlea. Hear, Res. 51, 255-264. Puel, J.-L., Pujol, R., Ladrech, S., Eybalin, M., 1991b. o~-Amino-3hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) electrophysiological and neurotoxic effects in the guinea pig cochlea. Neuroscience 45, 63-72. Puel, J.-L., Pujol, R., Tribillac, F., Ladrech, S., Eybalin, M., 1994. Excitatory amine acid antagonists protect cochlear auditory neurons from excitotoxicity. J. Comp. Neurol. 341, 241-256. Pujol, R., Lenoir, M., Robertson, D., Eybalin, M., Johnstone, B.M., 1985. Kainic acid selectivity alters auditory dendrites connected with cochlear inner hair cells. Hear. Res. 18, 145 151. Ruan, R.S., Leong, S.K., Yeoh, K.H., 1995. The role of nitric oxide in facial motoneuronal death. Brain Res. 698, 163-168. Zdanski, C.J., Prazma, J., Petrusz, P., Grossman, G., Raynor, E., Smith, T.L., Pillsbury, H.C., 1994. Nitric oxide synthase is an active enzyme in the spiral ganglion cells of the rat cochlea. Hear. Res. 79, 39-47.
Hearing Research 114 (1997) 169-178
Ototoxicity of sodium nitroprusside Run Sheng Ruan ~,*, Seng Kee Leong b, Kian Hian Yeoh ~ a Department of Otolaryngology, National University Hospital, Lower Kent Ridge Road, 119074, Singapore b Department of Anatomy, National UniversiO, of Singapore, 10 Kent Ridge Crescent, 119260, Singapore Received 2 November 1996; revised 15 August 1997; accepted 31 August 1997
Abstract Nitric oxide (NO) not only has normal physiological roles like vasodilation and neurotransmission in the living organism, it could also have possible neurodestructive effects under certain pathological conditions. The present study aimed to determine whether direct exposure of guinea pig cochlea to a NO donor like sodium nitroprusside (SNP), or a nitric oxide synthase (NOS) inhibitor like Ne-nitro-L-arginine methyl ester (L-NAME), would cause damage to the auditory hair cells. A piece of gelfoam was placed on the round window of the right ear of adult albino guinea pigs. It was then soaked with 0.1 ml of SNP (3.4 gM), 0.1 ml of L - N A M E (9.3 gM or 18.5 gM) or 0.1 ml of injection water, the vehicle used to dissolve the above chemicals. Twelve animals receiving SNP were perfused 1 day, 2, 3 and 7 days later, with three animals being used for each survival period. Six animals receiving L-NAME were allowed to survive up to 7 days before perfusion. Eight animals receiving injection water or 0.45% saline were used as controls. With the scanning electron microscope, the inner and outer hair cells were counted over a 1 mm length of the basilar membrane in each turn of every cochlea. The results showed that, in animals treated with L - N A M E at both concentrations stated, no significant loss of either inner or outer hair cells was noted in any part of the cochlea studied. However, as early as 1 day after SNP treatment, a striking loss of inner and outer hair cells was observed in the three lower turns of the cochlea. Damage to the outer hair cells was extended to the apical turn with increasing survival period, but no significant loss of inner hair cells was evident in the apical turn at any of the survival periods studied. To rule out the possibility that the effects were due to the presence of cyanide, a metabolite of SNP, hydroxycobalamin was introduced into the scala tympani of three animals through a cannula-osmotic pump device during SNP treatment. There was no significant difference in the results between the groups with and without hydroxycobalamin infusion 7 days after SNP treatment. The present study suggests that an excessive production of NO in the inner ear could lead to extensive loss of hair cells. © 1997 Elsevier Science B.V.
Key words." Hair cells; Inner hair cells; Outer hair cells; Guinea pig; Nitric oxide; Sodium nitroprusside; L-NAME
1. Introduction Since the d i s c o v e r y o f nitric oxide ( N O ) as an e n d o t h e l i u m - d e r i v e d relaxing f a c t o r a n d as a n e u r o t r a n s m i t ter in b o t h the central a n d p e r i p h e r a l n e r v o u s systems, there has been a t r e m e n d o u s surge in interest in this very simple n o n - c a r b o n c o n t a i n i n g m o l e c u l e in the biological system. N O is p r o d u c e d on d e m a n d f r o m L-arginine b y nitric oxide synthase (NOS), with c o n c o m i t a n t p r o d u c t i o n o f L-citrulline, in the presence o f several * Corresponding author. Tel.: +65 772 5371; Fax: +65 775 3820; E-maih [email protected]
0378-5955 / 97 / $17.00 © 1997 Elsevier Science B,V. All rights reserved
PIIS0378-5955(97)00159-7
co-factors, i n c l u d i n g n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e ( N A D P H ) , a n d o f c a l m o d u l i n to which C a 2+ b i n d s ( K n o w l e s et al., 1989). T h e a b o v e discoveries in the c h e m i s t r y o f N O have led i n v e s t i g a t o r s to use N O S a n d citrulline i m m u n o h i s t o c h e m i s t r y a n d N A D P H diap h o r a s e ( N A D P H - d ) h i s t o c h e m i s t r y to investigate at the light m i c r o s c o p i c level the presence o f N O in the m a m m a l i a n cochlea a n d to correlate it with its functions in the m o d u l a t i o n o f c o c h l e a r b l o o d flow ( C B F ) a n d a u d i t o r y n e u r o t r a n s m i s s i o n ( Z d a n s k i et al., 1994; F e s s e n d e n et al., 1994). O u r c u r r e n t electron m i c r o scopic e x a m i n a t i o n o f N O S i m m u n o s t a i n e d guinea pig a n d h u m a n c o c h l e a r tissue has further c o n f i r m e d the
170
R.S. Ruan et al./Hearing Research 114 (1997) 169-178
existence of NOS in spiral ganglion cells and their synapses with inner hair cells and in the endothelial cells of all the cochlear blood vessels. Previous studies also showed a profound increase in CBF when sodium nitroprusside (SNP), a NO donor, was applied to the round window membrane of the mammalian cochlea (Ohls6n et al., 1992), whereas a competitive NOS inhibitor, NC-nitro-L-arginine methyl ester (L-NAME), resulted in a reduction in CBF (Brechtelsbauer et al., 1993, 1995). Notwithstanding the convincing evidence of the important physiological roles of NO, there is strong evidence that N O could be responsible for neurodestruction in the central nervous system (Moncada et al., 1991; Kader et al., 1993; Ruan et al., 1995). In the auditory system, it has been reported that SNP has an acute ototoxic effect in a concentration- and time-dependent manner (Ohls6n et al., 1993; Chen et al., 1995; Dais et al., 1996; Kong et al., 1996), and the deleterious action of pneumolysin on guinea pig cochlea could be blocked by the administration of L - N A M E (Amaee et al., 1995). To further elucidate the role of NO in the cochlear tissue, the present investigation sought to study whether exposure of the cochlea to excessive NO produced by a NO donor like SNP or reduced NO induced by the administration of a NOS inhibitor like L - N A M E would lead to destruction of the hair cells.
may be the salt content in the SNP solution that resulted in hair cell damage, three animals were treated with 0.45% saline (pH 6) which contained sodium equivalent to that of SNP and allowed to survive for up to 7 days. The left cochleae of all the above animals were compared morphologically with the right cochleae. To rule out the possibility that the hair cell damage observed in animals treated with SNP was due to a SNP metabolite other than NO, for example, the free cyanide, hydroxycobalamin (HCL) was administered into the scala tympani via a cannula-osmotic pump device to detoxify cyanide potentially formed during diffusion of SNP through the round window. The cannula-osmotic pump device was prepared as described by Brown et al. (1993). The pump used was an Alzet 2002 osmotic pump (Alza Corp., Palo Alto, CA) having a flow rate of 0.5 gl/h for a continuous infusion period of 14 days. The pump was loaded with a solution (1 mg/ml) of H C L (Yung Shin, Pharmaceutical Industrial Co. Ltd., Taiwan) and incubated in sterile normal saline at 37°C overnight for activation before implantation. Three guinea pigs were subjected to a cochleostomy to the scala tympani at the level of the basal turn of the cochlea. The tip of a cannula connected to a HCL-loaded osmotic pump was placed in the scala tympani to allow H C L delivery therein, followed by SNP application in the tympanic cavity. The pumps were subcutaneously implanted between the forelegs.
2. Materials and methods
2.1. Tissue preparation for scanning electron microscopy (SEM)
A total of 29 albino guinea pigs of both sexes weighing 250-300 g were used in this study. All subjects had a normal Preyer's reflex and were free of middle ear infection as assessed directly under an operating microscope. The animals were anesthetized with an intramuscular injection of ketamine (40 mg/kg) and xylazine (4 mg/kg). Through a post-auricular incision, the right temporal bulla was opened to expose the round window. Under the operating microscope, a small piece of gelfoam was placed on the round window membrane, and 0.1 ml of SNP (Roche) at a concentration of 3.4 IuM (pH 6), or L - N A M E (Sigma) at a concentration of 9.3 gM (pH 4) or 18.5 g M (pH 3.5) was deposited on the gelfoam. Both chemicals were dissolved in injection water (pH 8) just before use. Twelve animals which received SNP were killed 1 day, 2, 3 and 7 days after operation, with three animals being used for each survival period. Six animals, three of which were given L - N A M E at a concentration of 9.3 gM and the other three at 18.5 gM, were killed on the 7th postoperative day. In addition to the above, five animals having gelfoam soaked with injection water were used as control and they were given a survival period of 7 days. To rule out the possibility that it
To quantitate the hair cells in each turn of every cochlea, a modified technique for tissue preparation for SEM assessment was developed. Animals were decapitated under deep anesthesia with ketamine and xylazine. The bullae were opened after removal from the skull. The oval and round windows were punctured and a cold solution of 2.5% phosphate-buffered glutaraldehyde was introduced slowly via the round window to the perilymphatic space. The perfused cochleae were kept in the same fixative for 2 h, then thoroughly rinsed with phosphate buffer. They were afterwards post-fixed in 1% osmium tetroxide for 1 h and transferred to phosphate buffer for dissection. The basilar membrane of the apical turn of the cochlea was exposed by removal of the bony cochlear capsule, the spiral ligament and Reissner's membrane with a pair of fine forceps. On the tympanic side of the cochlea, an opening on the cochlear duct was created at each turn by gently removing part of its lateral wall and the spiral ligament to facilitate the subsequent introduction of chemicals to the organ of Corti. The thiocarbohydrazide (TCH) technique as described previously (Hunter-Duvar, 1978) was used to
R.S. Ruan et al./Hearing Research 114 (1997) 169 178
171
m e m b r a n e was exposed was first examined. The next turn of the basilar membrane was viewed after removing the apical turn of the cochlea. In this way, each cochlea was studied from the apical turn to the basal turn. Photographs of five fields of basilar membrane were taken randomly in each turn for morphological observation and quantitative assessment. The inner and outer hair cells with stereocilia bundles were counted over a 1 m m length of basilar membrane in the photographs. Hair cells without stereocilia attachment or with bleb formation were considered to be damaged and not included in the count. The mean values and standard deviation of the mean were calculated and statistical analysis was performed with Student's t-test between experimental and control animals. Confidence levels of 95% were used to interpret significance. The A N O V A technique was also used to evaluate whether there was any difference in means a m o n g the groups of guinea pigs perfused 7 days after treatment with water, L - N A M E (18.5 BM) and SNP.
3. Results
Fig. 1. SEM photomicrographs of the basilar membrane at the (A) and 200 gm away from the apex (B) of the left cochlea control guinea pig. Note that there are a few scattered outer cells (OHC) in A, and only one row of OHC in B. The arrows cate OHC. The arrowheads indicate the inner hair cells. Scale 10 Bm.
apex in a hair indibar:
provide a conductive coat for the cochlea. Briefly, the cochlea with an opening on every turn of the cochlear duct was incubated for 20 min in a freshly prepared and filtered, saturated solution of T C H . After six washes in distilled water, the cochlea was incubated for 1 h in 1% aqueous osmium tetroxide, followed by six washes in distilled water. The whole procedure was then repeated. All the cochleae were dehydrated carefully through a graded series of ethanol. After two 10 min changes of 100% ethanol, the cochleae were dried in a critical point drier according to standard procedures, then observed in a Philips 515 SEM.
2.2. Numerical analysis The apical turn of the cochlea where the basilar
After recovery from anesthesia, guinea pigs given SNP unexpectedly displayed spectacular postural changes. Their heads were tilted towards the operated side and this was accompanied by a spontaneous nystagmus with the rapid component of eye movement directed toward the unoperated side. These changes were not observed in animals given L - N A M E , injection water or saline. In control animals treated with either water or saline, no morphological difference in the hair cells was observed between the right and left ears (Fig. 3A D, Figs. 4A D and 5A-D). It was evident that only inner hair cells (IHC) and a few scattered outer hair cells (OHC) were present at the apex of the cochleae (Fig. I A). The O H C population increased gradually as the cochlea was traced from the upper to the lower part of its apical turn (Fig. 1B). All three rows of O H C appeared only at a distance of at least 1 m m away from the apex. In animals receiving L - N A M E at both concentrations of 9.3 BM and 18.5 BM, there was no significant loss of either I H C or O H C in any part of the cochlea up to 7 days after treatment (Fig. 6A-D). On the other hand, as early as 1 day after SNP treatment, marked I H C and O H C loss was observed in the lower three turns of the cochlea, but not in the apical turn (Fig. 2A D). It was obvious that, of the three turns of the cochlea showing hair cell damage, the loss of hair cells was more severe in the lower than in the upper turn. In fact, there was complete loss of I H C and O H C in the
172
R.S. Ruan et al./Hearing Research 114 (1997) 169 178
!!~ilm
Fig. 2. SEM photomicrographs o f the basilar m e m b r a n e in the apical (A), upper middle (B), lower middle (C) and basal (D) turns of the cochlea 1 day after exposure to sodium nitroprusside. Note that the inner and outer hairs in the three lower turns were severely damaged. Scale bar: 50 Bm.
Table 1 Survival of hair cells at each turn of the guinea pig cochlea 1-7 days after exposure to L - N A M E , SNP and various control agents Agent
Water Saline L-NAME L-NAME SNP SNP SNP SNP SNP+HCL
N u m b e r of animals
5 3 3 3 3 3 3 3 3
Concentration
0.45% 9.3 BM 18.5BM 3.4 BM 3.4gM 3.4 g M 3.4 BM 1 mg/ml
Survival period (days)
7 7 7 7 1 2 3 7 7
Hair cell counts (mean _+S.D.) Apical turn
Upper middle turn
Lower middle turn Basal turn
IHC
OHC
IHC
OHC
IHC
OHC
IHC
OHC
94+12 101_+9 93_+14 97+11 102_+6 85_+7 98_+4 104_+6 102-+3'
308+27 310_+35 307+31 312-+33 290_+12 306_+7 183_+9" 183_+10" 179-+ 11'
113_+10 98_+8 111_+11 116_+10 65_+ 13" 28_+7* 95_+7* 95_+5* 88_+9*
404_+27 400_+33 411_+29 409_+26 46_+7* 135_+12" 15_+8" 94_+ 16" 91 _+ 13'
ll8ill 100_+9 116_+11 129_+12 37+4* 4_+7* 3_+4* 0 4_+5
448_+31 451+29 443_+38 448_+30 27_+5* 1_+2" 1 _+ 1" 0 0
131_+10 131-+12 127+10 132_+10 17_+5" 0 0 0 0
466_+22 458-+25 463+24 464_+24 24_+5 * 0 0 0 0
*P < 0.05, compared with water-treated cochlea. *P > 0.05, compared with animals at 7 days after SNP treatment.
R.S. Ruan et al./Hearing Research 114 (1997) 169 178
173
Fig. 3. SEM photomicrographs of the basilar membrane in the apical (A), upper middle (B), lower middle (C) and basal (D) turns of the cochlea from the unoperated ear. Scale bar: 20 gin.
basal turn of the cochlea at 2 days after SNP administration. Significant O H C loss was noticed in the apical turn at 3 days after exposure to SNP but no significant I H C loss was evident in any of the survival periods studied. In animals with infusion of H C L into the scala tympani, there was also severe loss of hair cells in the lower three turns of the cochlea at 7 days after SNP treatment (Fig. 7A-D). There was, however, no statistical difference in hair cell counts between the groups with and without H C L infusion. The results of O H C and I H C counts in each turn of the cochlea exposed to L - N A M E , SNP and various control agents are shown in Table 1 where the results of Student's t-test are indicated. The results of A N O V A analysis showed, after adjusting for multiple comparison using the Bonferroni correction, that only comparisons between the groups at 7 days after treatment with water and SNP were
found to be statistically significant at the 5% level, which is in agreement with the results of Student's t-test.
4. Discussion
The present study provides evidence that direct exposure of the cochlea to SNP, a N O donor, results in severe damage to the auditory hair cells. It suggests that the presence of excessive N O in the cochlea may lead to extensive loss of hair cells. However, from a pharmacokinetic point of view, free cyanide might be produced during the breakdown of SNP. To determine whether SNP ototoxicity could involve free cyanide, we applied H C L into the scala tympani during SNP diffusion through the round window. In this situation, any free
174
R.S. Ruan et al./Hearing Research 114 (1997) 169 178
Fig. 4. SEM photomicrographs of the basilar membrane in the apical (A), upper middle (B), lower middle (C) and basal (D) turns of the cochlea 7 days after water treatment. Scale bar: 20 pm.
cyanide present will be converted by H C L to the nontoxic cyanocobalamin. The results showed that there was no significant difference in the counts of remaining hair cells between the groups with and without H C L infusion. These results suggest that the hair cell loss observed in this study was not due to free cyanide possibly released from SNP. Dawson et al. (1993) in their in vitro studies noted no neurodestructive effects of K4[Fe(CN)6] which had similar effects to Fe(CN5)z, another metabolite of SNP. This notwithstanding, whether the SNP itself or some product of SNP breakdown is responsible for the ototoxicity observed in this study remains to be clarified. Although the p H of L - N A M E and SNP solutions used in this study was shifted when the chemicals were dissolved in water, it should be noted that the p H shift was greater in the former than in the latter.
In spite of this, L - N A M E caused no significant damage to hair cells. Logically, it is unlikely that the loss of hair cells after SNP treatment could be attributed to the minor shift in the p H of the SNP solution. Recent studies have shown that damage to the cochlea induced by pneumolysin, a cytotoxic agent released from Streptococcus pneumoniae during bacterial meningitis, could be blocked by L - N A M E (Amaee et al., 1995) and the cochlear potentials could be suppressed by SNP applied either to the round window (Ohls6n et al., 1993; K o n g et al., 1996) or by intracochlear perfusion (Chen et al., 1995; Dais et al., 1996). Also, SNP significantly reduced in vivo both O H C voltage-induced length changes and whole-cell outward currents (Chen et al., 1995). These results point to N O as the possible ototoxic agent to the cochlea under certain pathological conditions. On the other hand, overstimulation of ex-
R.S. Ruan et al./Hearing Research 114 (1997) 169 178
175
Fig. 5. SEM photomicrographs of the basilar membrane in the apical (A), upper middle (B), lower middle (C) and basal (D) turns of the cochlea 7 days after treatment with 0.45% saline. Scale bar: 20 gm.
citatory amino acid (EAA) receptors may also play an important role in the pathophysiological changes in the ischemic cochlea. This conclusion is based on multiple studies in ischemic cochleae which showed that acute degeneration of afferent synapses with the IHC occurred with the introduction of glutamate or its analogs into the inner ear, leading to subsequent loss of spiral ganglion neurons (Pujol et al., 1985; Juiz et al., 1989; Puel et al., 1991a). Moreover, ischemia-induced degeneration of afferent synapses could be prevented by prior application of glutamate antagonists (Puel et al., 1991b, 1994), suggesting overstimulation of EAA receptors in ischemic conditions of the cochlea. The relationship between NO formation and EAA receptor activation has been established in the brain (Dawson et al., 1992). Previous studies in vitro have also demonstrated that EAA neurotoxicity in cultures of the cere-
bral cortex, hippocampus and striatum could be mediated by N O formation (Dawson et al., 1991, 1993). Therefore, it is reasonable to postulate that the neurodestructive effects of EAA receptor overstimulation in the cochlea is also mediated by excessive NO formation under certain conditions. The close similarity between the pattern of hair cell loss observed in the present study and that found in noise- and aminoglycoside-induced pathological changes in the cochlea suggests that NO formation may play a role under these conditions also. Although the exact mechanism of NO-induced cytotoxicity remains unclear, it has been suggested that NO could cause ADP-ribosylation of proteins (Brune and Lapetina, 1989), inhibit enzymes involved in D N A synthesis (Hibbs et al., 1989) and react with superoxide ion to form toxic hydroxyl free radicals (Beckman et al.,
176
R.S. Ruan et al./Hearing Research 114 (1997) 16~178
Fig. 6. SEM photomicrographs of the basilar membrane in the apical (A), upper middle (B), lower middle (C) and basal (D) turns of the cochlea 7 days after exposure to L-NAME (18.5 gm). No hair cell loss was noted. Scale bar: 20 gm.
1990). Thus, it is possible that a cascade of events may take place involving overactivation of E A A receptors resulting in an excessive formation of N O which ultimately leads to neurodestruction. The present study revealed that the loss of hair cells was always greater in the lower turn when the cochlea was exposed to SNP. It may be argued that in our experiment, N O may not have diffused uniformly throughout the perilymph. The area proximal to the round window could have received a higher concentration of N O as the gelfoam soaked with the N O donor was placed on the round window membrane. However, all the animals which received SNP presented the typical clinical picture of a unilateral peripheral destruction of the vestibular organ immediately after the animals recovered from anesthesia, indicating a direct action of N O on the vestibular receptors, the ampullae and cris-
tae. The route taken by N O to reach these receptors is most likely through diffusion from the scala tympani into the cochlear duct via the basilar membrane, but entry across the lateral wall of the cochlear duct could also be possible. If N O could still cause damage to the vestibular receptors after traversing all the turns of the cochlea, it is unlikely that, by the time it reached the upper turns, its concentration would be so diminished as to be unable to cause damage to the hair cells there. It is more likely that for some reason yet to be elucidated, the hair cells in the upper turn are more resistant to the destructive action of NO. Previous studies have shown that the application of L - N A M E on the round window of the cochlea did not affect the c o m p o u n d action potential thresholds of the cochlea (Brechtelsbauer et al., 1995). Our observations also showed no morphological changes in the cochlea
R.S. Ruan et al./Hearing Research 114 (1997) 169 178
177
Fig. 7. SEM photomicrographs of the basilar membrane in the apical (A), upper middle (B), lower middle (C) and basal (D) turns of the cochlea 7 days after exposure to sodium nitroprusside with hydroxycobalamin infusion into the scala tympani, Severe damage to the inner and outer hair cells was also noted in the three lower turns. Scale bar: 10 gin.
of animals treated with L-NAME, suggesting that, though excessive production of N O could destroy the hair cells of the cochlea, some deprivation of NO had no apparent effects on them. Lastly, it should be mentioned that, in the normal guinea pig cochlea, the three rows of O H C are incomplete at the apical turn for a distance of about 1 mm from the apex. Thus, the number of O H C at the apical thn is less than that at the lower turns of the cochlea in a given length of basilar membrane. This should not be mistaken for hair cell loss in any quantitative study of hair cells in the guinea pig.
Acknowledgments This study was supported by Grant RP 950393/N from the National Medical Research Council of Singapore.
References Amaee, F.R., Comis, S.D., Osborne, M.P., 1995. Ag~-Methyl-L-arginine protects the guinea pig cochlea from the cytotoxic effects of pneumolysin. Acta Otolaryngol. (Stockh.) 115, 386 391. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A., 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87, 1620 1624. Brechtelsbauer, P.B., Nuttall, A.L., Miller, J.M., 1993. Nitric oxide synthase blocker N(;-nitro-t=arginine decrease cochlear blood flow. Assoc. Res. Otolaryngol. 368, P92. Brechtelsbauer, P.B., Nuttall, A.L., Miller, J.M., 1995. Basal nitric oxide production in the cochlear vasculature. Otolaryngol. Head Neck Surg. 111, P70. Brown, J.N., Miller, J.M., Altschuler, R.A., Nuttall, A.L., 1993. Osmotic pump implant for chronic infusion of drugs into the inner ear. Hear. Res. 70, 167 172. Brune, B,, Lapetina, E.G., 1989. Activation of a cytosolic ADP-ribosyltransferase by nitric oxide-generating agent. J. Biol. Chem. 264, 8455-8458. Chen, C., Nenov, A., Skellett, R., Fallon, M., Bright, L.. Norris,
178
R.S. Ruan et al./Hearing Research 114 (1997) 16~178
C.H., Bobbin, R.P., 1995. Nitroprusside suppresses cochlear potentials and outer hair cell responses. Hear. Res. 87, 1-8. Dais, C.G.D., Prazma, J., Ball, S.S., Zdanski, C., Carrasco, V., Pillsbury, H.C., 1996. Effect of sodium nitroprusside on compound action potential thresholds in the gerbil cochlea. Hear. Res. 99, 1-6. Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M., Snyder, S.H., 1991. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissue. Proc. Natl. Acad. Sci. USA 88, 7797 7801. Dawson, T.M., Dawson, V.L., Snyder, S.H., 1992. A novel neuronal messenger molecule in brain: The free radical, nitric oxide. Ann. Neurol. 32, 297-311. Dawson, V.L., Dawson, T.M., Bartley, D.A., Uhl, G.R., Snyder, S.H., 1993. Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures. J. Neurosci. 13, 2651-2661. Fessenden, J.D., Coling, D.E., Schacht, J., 1994. Detection and characterization of nitric oxide synthase in the mammalian cochlea. Brain Res. 668, 9-15. Hibbs, J.B., Jr., Taintor, R.R., Vavrin, Z., Rachlin, E.M., 1989. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem. Biophys. Res. Commun. 157, 2811-2814. Hunter-Duvar, I.M., 1978. Electron microscopic assessment of the cochlea. Acta Otolaryngol. Suppl. (Stockh.) 351, 3-24. Juiz, J.M., Rueda, J., Merchan, J.A., Sala, M.L., 1989. The effects of kainic acid on the cochlear ganglion of the rat, Hear. Res. 40, 6574. Kader, A., Frazzini, V.I., Soloman, R.A., Triflatti, R.R., 1993. Nitric oxide production during focal cerebral ischemia in rat. Stroke 24, 1709-1716. Knowles, R.G., Palacios, M., Palmer, R.M.J., Moncada, S., 1989. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc. Natl. Acad. Sci. USA 86, 5159-5162.
Kong, W.J., Ren, T.Y., Nuttall, A.L., 1996. Electrophysiological and morphological evaluation of the acute ototoxicity of sodium nitroprusside. Hear. Res. 99, 22-30. Moncada, S., Palmer, R.M.J., Higgs, E.A., 1991. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev. 43, 109-142. Ohls~n, K.A., Didier, A., Baldwin, D., Miller, J.M., Nuttall, A.L., 1992. Cochlear blood flow in response to dilating agents. Hear. Res. 58, 19-25. Ohls6n, A., Hultcrantz, E., Engstrom, B., 1993. The effect of topical application of vasodilation agents on cochlear electrophysiology. Acta Otolaryngol. 113, 55~61. Puel, J.-L., Ladrech, S., Chabert, R., Pujol, R., Eybalin, M., 1991a. Electrophysiological evidence for the presence of NMDA receptors in the guinea pig cochlea. Hear, Res. 51, 255-264. Puel, J.-L., Pujol, R., Ladrech, S., Eybalin, M., 1991b. o~-Amino-3hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) electrophysiological and neurotoxic effects in the guinea pig cochlea. Neuroscience 45, 63-72. Puel, J.-L., Pujol, R., Tribillac, F., Ladrech, S., Eybalin, M., 1994. Excitatory amine acid antagonists protect cochlear auditory neurons from excitotoxicity. J. Comp. Neurol. 341, 241-256. Pujol, R., Lenoir, M., Robertson, D., Eybalin, M., Johnstone, B.M., 1985. Kainic acid selectivity alters auditory dendrites connected with cochlear inner hair cells. Hear. Res. 18, 145 151. Ruan, R.S., Leong, S.K., Yeoh, K.H., 1995. The role of nitric oxide in facial motoneuronal death. Brain Res. 698, 163-168. Zdanski, C.J., Prazma, J., Petrusz, P., Grossman, G., Raynor, E., Smith, T.L., Pillsbury, H.C., 1994. Nitric oxide synthase is an active enzyme in the spiral ganglion cells of the rat cochlea. Hear. Res. 79, 39-47.