Effects of nitric oxide on normal and ischemic cochlea of the guinea pig.

To determine whether nitric oxide (NO)/peroxynitrite plays any role in neurodestruction observed in ischemic cochlea of the guinea pig, the effects of NO donors like S-nitrosocysteine (S-NC) and nitroglycerin (NTG), peroxynitrite generators like 3-morpholinosydnonimine (SIN-1), peroxynitrite inhibitors like superoxide dismutase plus catalase (SOD/Cat), as well as NOS inhibitors like N(G)-nitro-l-arginine methyl ether (L-NAME), were tested on normal and ischemic cochleae. Various concentrations of S-NC and SIN-1 were introduced into the perilymphatic space of normal guinea pig cochlea. Quantitative scanning electron microscopy of inner and outer hair cells was carried out 2 days later. To determine the level of NO in the cochlea after 20 to 120 min of ischemia, nitrites/nitrates in the perilymph were measured. The effects of NO on the ischemic cochlea were tested by infusion of SOD/Cat, L-NAME, S-NC, and NTG into the perilymphatic space just before decapitation. Introduction of fixative into the cochlea was delayed for 15 min to investigate the effects of the chemicals on nerve endings at the base of inner hair cells. The results showed that the level of nitrites/nitrates tended to decline with increasing time of ischemia. There was no significant hair cell loss in the cochleae treated with SIN-1 or S-NC. At 15 min after ischemia, most of the nerve endings at the base of the inner hair cells were protected from damage when 1 mM S-NC or NTG was infused into the perilymph. Taken together, the results indicate that NO/peroxynitrite is unlikely to be involved in the neurodestruction in the ischemic cochlea. In fact, exogenous NO may have a neural protective effect.

Ototoxicity of sodium nitroprusside is not due to nitric oxide.

Sodium nitroprusside (SNP) has been used as a donor for nitric oxide (NO) to study the effects of NO on the mammalian cochlea. In the present study, we set out to determine whether NO was the chemical responsible for the ototoxic effects seen after the application of SNP at the round window membrane of the adult guinea pig cochlea. In the first instance, NO released from S-nitrosocysteine, a compound not related to cyanide, has no toxic effect on the hair cells of the cochlea. Light-exposed SNP that could no longer produce NO, light-exposed SNP to which acetylcysteine (ATC) or hydroxycobalamin (HCL) was added to eliminate cyanide, and freshly prepared SNP to which ATC or HCL was added were also tested. Six groups of animals consisting of three animals in each group were used. The single chemical or combination of chemicals stated above was soaked in a piece of gelfoam that was then applied to the round window membrane of the animal under ketamine-xylasine anesthesia. The animals were reanesthetized 3 days later and perfused for scanning electron microscopy and hair cell quantitative analysis. The results showed that, in animals given S-nitrosocysteine, no hair cell loss was noted, while light-exposed SNP led to severe hair cell damage similar to that seen after the administration of fresh SNP. In animals treated with the mixture of light-exposed SNP and ATC or HCL, or fresh SNP with ATC or HCL, ototoxicity was significantly attenuated. These results have convincingly demonstrated that NO at a certain level is not destructive to auditory hair cells and the hair cell loss observed after SNP application is most likely due to the cyanide released from the SNP instead of NO.

Effects of BDNF and NT-3 on hair cell survival in guinea pig cochlea damaged by kanamycin treatment.

The aim of this study was to determine whether neurotrophic factors such as brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) would protect auditory hair cells from ototoxicity by aminoglycoside antibiotic. Twenty-seven Wistar guinea pigs were divided into three groups of nine animals each. BDNF and NT-3 (100 microg/ml) were delivered into the right scala tympani of guinea pig cochlea through a cannula-osmotic pump device. Artificial perilymph (AP) was used as control. Immediately after implantation of the device, each animal was given five successive doses of kanamycin (400 mg/kg). At 15, 30 and 60 days after infusion, surviving inner and outer hair cells were counted at each turn of every cochlea with a Philips 515 scanning electron microscope. Multiple comparison tests were carried out among the groups, using ANOVA and Dunnett T3/Tukey HSD. Protective effects of NT-3 on hair cells were observed at 30 and 60 days after kanamycin injection. BDNF had no protective effect on hair cells at 15 and 60 days, but some at 30 days. This study suggests that NT-3 and BDNF may protect against cochlear hair cell damage caused by kanamycin treatment. Possible mechanisms for the otoprotective effects were discussed. No single mechanism postulated can explain fully the results seen in this study. It is possible that the mechanisms act in concert to produce the observed effects, or there are as yet undiscovered mechanisms or secondary messengers responsible for the otoprotective effects.

Localization of nitric oxide synthase and NADPH-diaphorase in guinea pig and human cochleae.

The distributions of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and nitric oxide synthase (NOS) in mammalian cochlea were studied at light and electron microscope levels by NADPH-d histochemistry and brain NOS (bNOS) immunohistochemistry. The cochleae from 15 albino guinea pigs were perilymphatically fixed with 2% periodate-lysine-paraformaldehyde, decalcified in 10% EDTA and processed for light and electron microscopy after NADPH-d or NOS staining in frozen and vibratome sections respectively. One human cochlea was available for light microscope examination of NADPH-d or bNOS stained sections. Light microscope results revealed that type I neurons and nerve fibers of the spiral ganglion cells were labeled by bNOS immunohistochemistry as well as NADPH-d histochemistry in both guinea pig and human cochleae. At subcellular level, NADPH-d reaction product was localized in the mitochondria of the neuronal cytoplasm and axoplasm and in the cytoplasm of the vascular endothelium. The immunoreaction products of bNOS were evenly distributed in the neuronal cytoplasm and axoplasm. Myelinated and unmyelinated fibers in the intraganglionic spiral bundle and the inner spiral and inner radial fibers below the inner hair cells were labeled for bNOS. The nerve endings below the outer hair cells were not stained. NOS immunoreaction product was also found in the outer hair cells, Schwann cells of myelinated nerve fibers, Deiter's cells, pillar cells and the tympanic lamina cells. No difference was found in the staining pattern of both NADPH-d and NOS reaction products between human and guinea pig cochleae at the light microscope level. The results suggest that NO plays an important role in the maintenance of auditory function in the mammal.

Ototoxicity of sodium nitroprusside.

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 N(G)-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 microM), 0.1 ml of L-NAME (9.3 microM or 18.5 microM) 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-NAME 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.

The role of nitric oxide in facial motoneuronal death.

The present study aimed to determine whether nitric oxide synthase (NOS)/nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity would be induced in facial motoneurons after facial nerve avulsion and if so, whether such activity was related to neuronal death commonly observed after such injury. The left facial nerve in each of 28 Wistar albino rats was avulsed from the facial canal. Ten of them received either daily injections of N omega-nitro-L-arginine methyl ether (L-NAME) or the vehicle. After survival times ranging from 2-50 days, serial brainstem sections were processed for NOS immunocytochemistry and NADPH-d histochemistry respectively. The number of surviving, NOS and NADPH-d positive and NOS negative neurons were compared statistically. Two days after facial nerve avulsion, increased NADPH-d activity was noticed in the facial motoneurons and in the endothelial lining of many dilated blood vessels in the facial motor nucleus (FMN). NOS-positive neurons were not detectable until five days after operation. Both the number and staining intensity of NADPH-d and NOS-positive neurons increased steadily with increasing survival time while the number of surviving neurons decreased after nerve avulsion. Daily administration of L-NAME protected 17% the neurons from death in the affected FMN when examined at 30 days after nerve avulsion, suggesting a neurodestructive property of NO. It was also noticed that some of the surviving neurons were first NOS positive but became NOS negative later.

Compression of the facial nerve caused increased nitric oxide synthase activity in the facial motor nucleus.

Nitric oxide synthase activities in the facial motor nucleus were studied in rats after unilateral compression of the facial nerve. Using a radiometric assay which measured the total soluble nitric oxide synthase activities in the facial motor nucleus and the surrounding tissues, it was found that nitric oxide synthase activities were markedly increased during facial paralysis that resulted from compression of the facial nerve. The subsequent decrease in nitric oxide synthase activities between postoperative days 20 and 40 coincided with the recovery of facial functions. In contrast, staining with NADPH-diaphorase histochemistry revealed that the diaphorase activities in the facial motor neurons were markedly increased between days 20-40 when the total activities as measured biochemically were in decline. However, staining of the vascular endothelium was increased on postoperative day 7 when the total activity was high. It is suggested that the increase in total nitric oxide synthase activities immediately after facial nerve compression may be predominantly endothelial. Since the increase in neuronal NADPH-diaphorase reactivity coincided with the recovery of facial functions, increased neuronal nitric oxide synthase may be a contributing factor to the restoration of facial innervation. The results of this study show that biochemical measurements of soluble nitric oxide synthase activities in tissue homogenates and NADPH-diaphorase histochemical staining in tissue sections may represent two distinct populations of nitric oxide synthase.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of NADPH-diaphorase activity in the facial motoneurons after compression of the facial nerve in the albino rat.

Compression of the rat facial nerve in its bony canal led to increased nitric oxide synthase activity in the facial motoneurons as revealed by NADPH-d histochemistry. The increase occurred steadily from 3 days to 6 weeks, peaking at 4 weeks after compression. Parallel to this was also a gradual return of facial function which reached its maximum at 4-5 weeks after compression.

Glial reaction after facial nerve compression in the facial canal of the albino rat.

The facial nerve of the albino rat was compressed by inserting a thin nylon thread in the facial canal. After a survival period of 1-60 days, the animal was perfused with Ringer's solution followed by a 2% periodate-lysine-paraformaldehyde fixative. Frozen sections from the brain stem containing the facial motor nucleus (FMN) were obtained and stained for OX-42 (against CR3 antigen), OX-18 (against Class I MHC antigen), OX-6 (against Class II MHC antigen) and GFAP (anti-glial fibrillary acidic protein). The first glial reaction in the FMN occurred in the microglia which showed a significant increase in the CR3 immunoreactivity within 24 h after compression. Upregulation of the GFAP was not noticed until 2 days after compression. In each case, the staining reaction was initially light, but increased with time and appeared to peak at 3-4 days for OX-42 and 4-5 days for GFAP. The activated microglia first assumed a perineuronal position but were later displaced by the activated astrocytes. The number of stained microglia was noticeably increased and was most likely the result of proliferation of the resident microglia rather than invasion from the blood stream. The increase in the number of GFAP positive cells was most likely the result of more resident astrocytes being activated, as previous studies have shown the absence of mitotic activity of astrocytes after lesion of the facial nerve. In addition to increased CR3 and GFAP activity there was also an upregulation of Class I MHC antigen in the microglia, as revealed by increased immunostaining against OX-18.(ABSTRACT TRUNCATED AT 250 WORDS)