Keywords
riluzole - cisplatin - ototoxicity - auditory brainstem response - transmission electron
microscopy
Introduction
Cisplatin (cis-diamminedichloroplatinum [CDDP]), is a chemotherapeutic agent used
for the treatment of various malignancies, including testicular, ovarian, bladder,
cervical, head and neck, and non-small cell lung cancers. Unfortunately, its use is
associated with dose-limiting side effects, such as nephrotoxicity, neurotoxicity
and ototoxicity.[1] Cisplatin-induced ototoxicity generally manifests as ear pain, tinnitus or sensorineural
hearing loss. The hearing loss is dose-related, cumulative, bilateral and usually
symmetrical, and occurs initially in the higher frequencies. A total of 90.5% of patients
had a significant hearing loss at 8 kHz. By continuation of therapy, the effect extends
to lower frequencies and is often irreversible. It may also progress even once the
cisplatin therapy has been completed or withdrawn.[2]
[3]
Several potential mechanisms of cisplatin-induced ototoxicity have been proposed.
Among these, the most widely described is the coordinate bonding between the atoms
of platinum and DNA that results in the formation of inter- and intra-strand cross-linking
of DNA, induction of p53, and cell cycle arrest. Recent studies have also highlighted
the relationship between reactive oxygen species (ROS) generated by cisplatin administration
and apoptosis. Through the action of ROS, various pathways are believed to be involved
in the induction of the apoptosis of cochlear cells, including the caspase-dependent
pathway (up-regulation of Bcl-2, caspase-3 and caspase-9) or caspase-independent pathway
(activation of pro-apoptotic c-Jun N-terminal kinase [JNK]), lipid peroxidation of
the cell membrane, and induction of Ca2+ influx. In addition, inflammatory pathways have been shown to play a role in cisplatin-induced
apoptosis (including activation of nuclear factor-κb [NF-κb] and activator protein-1
[AP-1]).[4]
[5]
Following cisplatin administration, the most prominent change is seen in the organ
of Corti, in animal studies, in which there is degeneration and loss of sensory cells.
These changes initially start at the first row of outer hair cells (OHCs) in the basal
turn of the cochlea, followed by the other OHC rows and, eventually, the inner hair
cells (IHCs) in the context of prolonged administration or high cisplatin doses.[6]
[7]
[8] In addition to its destructive effect upon the organ of Corti, cisplatin also affects
the stria vascularis,[7] and several studies have demonstrated cisplatin-induced degeneration of spiral ganglion
cells (SGCs)[7]
[8]
[9] and supporting cells.[10]
The present study investigates the potential role of a neuroprotective, antiapoptotic
agent, riluzole (2- amino-6-trifluoromethoxy benzothiazole), in preventing the degeneration
of cochlear cells and thus avoiding the hearing loss resulting from cisplatin-mediated
apoptosis. Riluzole has been approved by the Food and Drug Administration (FDA) for
the treatment of amyotrophic lateral sclerosis.[11] Since it can block voltage-gated Ca2+ and Na+ ion channels as well as activate K+ ion channels and G-protein dependent signal transduction pathways, the major mechanism
of the clinical action of riluzole is thought to be its inhibition of glutamate release
and noncompetitive antagonist effects on glutamate receptors.[12] The potential beneficial effects of riluzole in neuronal cell death have been demonstrated
in animal models of retinal ischemia,[13] spinal cord injury,[14] and noise-induced hearing loss,[15]
[16] in which the cytotoxic mechanisms are believed to base on the glutamate excitotoxic
pathway. The excitotoxic pathway has not been invoked before as a mechanism of cisplatin-induced
ototoxicity; however, recent studies have shown that riluzole has properties independent
of its effect on glutamate release, which may be of relevance in the prevention of
cisplatin ototoxicity. These include the suppression of the generation of ROS and
the inhibition of downstream apoptotic signals,[17]
[18]
[19] as well as the triggering of endogenous protective molecules, such as heat shock
proteins (HSPs) and heme oxygenase-1 (HO-1).[20]
[21]
[22] In this study, we analyzed the hearing thresholds by auditory brainstem responses
(ABRs) and documented cell degeneration in hair cells, the stria vascularis and auditory
nerve fibers, by transmission electron microscopy, to evaluate the effect of riluzole
on cisplatin-induced ototoxicity
Materials and Methods
Animals
This study was approved and monitored by the Experiments Local Ethics Committee (G.
Ü. ET - B.30.2.GÜN.0.05.06.00/80-6489). The experimental animals were 24 young adult,
male, albino guinea pigs, weighing between 350 and 400 g. After the attainment of
the ethics committee approval for the study, the guinea pigs were kept for 2 weeks
in the Experimental Animals in Research and Application Center. All animals were housed
in plastic cages with water and food available ad libitum and maintained on a 12-hour light/dark cycle.
Experimental Design and Drug Administration
The animals were divided into three groups, and all were assessed for ABR before drug
administration. A dose of 5 mg/kg/day of cisplatin (Cisplatin DBL; Faulding Pharmaceuticals,
Warwickshire, UK) was administered for 3 days intraperitoneally (i.p.) to induce ototoxicity
in all 3 groups. Group 1 (n = 8) received 2 ml of saline i.p. for 3 days, half an hour before cisplatin. Groups
2 and 3 received, respectively, 6 mg/kg of riluzole hydrochloride (Riluzole hydrochloride
25 mg [Sigma R11]; Sigma Aldrich, Darmstadt, Germany) i.p., and 8 mg/kg of riluzole
hydrochloride i.p., dissolved in saline to give a 2 ml volume on each treatment day,
half an hour before cisplatin, for 3 days.
Wang et al have previously studied guinea pigs exposed to acoustic trauma and determined
that that 6 mg/kg of riluzole i.p. was the minimal effective dose to prevent hearing
loss and cell death in the inner ear. In addition, they noticed side effects, such
as hypotonia and weight loss, at doses of 8 mg/kg and higher.[15] The dose selection in our study was, therefore, based on these data.
Guinea pigs were coded as S1–S8, in group 1; R6.1–R6.8 in group 2; and R8.1–R8.8 in
group 3.
Anesthesia
The animals were anesthetized with 30 mg/kg of ketamine hydrochloride (Ketalar, Eczacıbasi
Ilac Sanayi ve Ticaret A.S, Luleburgaz, Turkey) and 4 mg/kg of xylazine (Rompun, Bayer
Vital, Leverkusen, Germany) given as an intramuscular infusion before the electrophysiological
tests.
Auditory Brainstem Response Evaluation
The ABR evaluations of both ears (n:44) of the guinea pigs were performed with the
Bio-logic Navigator Pro device version 2.2.0 (Bio-Logic Systems Corp., Mundelein,
IL, USA) twice before drug administration, and 24 hours after the last drug administrations.
In the ABR evaluations, we used a 13.00 Hz rate click stimulus, 10 ms analysis time,
1,000 sweeps in averaging, 100–1,500 Hz filtration, rarefaction polarity and then
tone burst stimulus at 8 kHz, 50–1,500 Hz band-pass filtration, and alternating polarity.
Changes in the intensity were made in accordance with the responses achieved in the
70 dB nHL intensity level recordings.
Transmission Electron Microscopic Evaluation
The animals were sacrificed under deep anesthesia after the ABR measurements, and
the right temporal bone of each animal was removed. The cochleae were harvested from
the temporal bone. Tissue samples were decalcified by placing into ethylenediaminetetraacetic
acid (EDTA) solution prepared with 2.5% glutaraldehyde. Tissue samples were then placed
in 1% osmium tetroxide for 1 hour, followed by fixation and staining. After this,
samples were dehydrated with alcohol series and tissues were placed in propylene oxide
for 30 minutes, prior to a 30-minute waiting period in embedding material (AGR1030
epoxy-resin (Araldite CYC212) Kit [Agar Scientific, Stansted, Essex, U.K.]). After
this step, tissues taken into embedding material were placed into a rotator at room
temperature for 2 hours and then transferred into an oven at 40°C for another 2 hours.
Tissues were embedded into horizontal embedding blocks within the same mixture. Sections
were then prepared in 1 µm sizes from these blocks by LKB Leica ultramicrotome (Leica,
Germany, Reichert SuperNova) and stained with toluidine blue (Code: T3260; Sigma Aldrich,
Darmstadt, Germany). The slides were examined with photolight microscope (Leica, Germany,
DM4000B Image Analyze System) with plus camera (Leica, Germany, DFC 280). The relevant
areas were marked to be examined and thin sections in size of 0.2–0.5 µm were prepared
on formyl-coated copper grids. Counter-staining was achieved with uranyl acetate and
lead citrate and the sections were evaluated and captured using an electron microscope
(Carl Zeiss EVO LS10, Carl Zeiss Microscopy Ltd., Cambridge, UK).
The degeneration of the stria vascularis, hair cells and auditory nerve fibers of
18 guinea pigs (n = 6 in each group) was assessed under TEM through established degeneration criteria
and scored as follows: 0, absent; 1, weak; 2, medium; and 3, severe, for each animal
by two histologists. Previously, histologists have used this scoring for two studies.[23]
[24] The histologists were blinded as to the treatment of the guinea pigs they studied.
Statistical Analysis
The SPSS for Windows, Version 20.0. software (IMB Corp., Armonk, NY, USA) was used
for the statistical analysis. The differences among the groups were assessed with
Kruskal-Wallis analysis of variance, and pairwise comparisons of groups were evaluated
by Mann-Whitney U-test to identify the group responsible for differences. Statistical
significance was set at p < 0.05.
Results
Auditory Brainstem Responses
Two guinea pigs from group 2 and group 3, respectively coded as R6.8 and R8.1, died
after the 2nd day of drug administration. Therefore, we analyzed ABR thresholds in all groups before
and after drug administration statistically over both ears of 22 animals for the click
and the 8 kHz-frequency stimuli.
For click stimulus, mean ± standard error of the mean (SEM) values were in group 1,
2, 3 respectively 3.75 ± 1.25, 2.86 ± 1.63, 0 ± 1.04 before drug administiration and
17.5 ± 1.93, 10 ± 2.34, 10 ± 1,81 after drug administiration. For 8,000 hz stimulus,
mean ± SEM values were in group 1,2,3 respectively - 0,62 ± 1.7, - 0,71 ± 1.95, −2.14 ± 1.54
before drug administration and 23.75 ± 2.72, 5 ± 2.72, 10 ± 1.81 after drug administration.
No significant difference was found between baseline ABR thresholds of the groups
at both stimuli (p > 0.05). After drug administration, in group 2, click ABR thresholds were similar
(p > 0.05) and 8 kHz ABR thresholds were significantly lower (p < 0.05) in comparison with the control group. In group 3, click ABR thresholds were
similar (p > 0.05) and 8 kHz ABR thresholds were significantly lower (p < 0.05) in comparison with the control group. There was no significant difference
between group 2 and 3 with regard to posttreatment click and 8 kHz ABR thresholds
(p > 0.05) ([Fig. 1a] and [1b]).
Fig. 1 Mean and SEM ABR values (pretreatment and posttreatment) in dB SPL for click (A)
and 8 kHz tone burst stimuli (B) among the groups. Abbreviations: ABR: auditory brainstem
response; SEM: standard error of the mean; SPL: sound pressure level
Histological Findings
Transmission electron microscopic inspection of hair cells (OHCs, IHCs), the stria
vascularis and auditory nerve fibers were performed for 18 guinea pigs. The degeneration
criteria for each one was determined by two histologists who were blinded to the groups
and scored the structural and cellular deterioration as shown in [Tables 1], [2], and [3]. Each value in the relevant box of the tables signifies the degeneration scores
of the involved specimens of six cochleae for each group. Histologists observed that
in group 1, degenerative findings were evident at both outer ([Fig. 2a]) and inner hair cells ([Fig. 2b]). Marginal, intermediate and basal cells and fibroblasts, which form the stria vascularis,
revealed significant degenerative appearances ([Fig. 2c]). Vascular structures of the epithelium were noted to have decreased. In the auditory
nerve fibers, myelin lamellae separations, myelin figure formations in axons and inter-axonal
areas, giant vacuolar formations in Schwann cells' cytoplasm and accumulations of
electron-dense material in the myelin sheets were observed. Unlike group 1, the hair
cell structures in animals from group 2 were substantially preserved in terms of the
criteria stated in [Table 1]. Moderate loss of the stereocilia of outer and inner hair cells were observed in
this group ([Figs. 3a] and [3b]). In group 2, a better view was determined at the level of the fine structures of
the stria vascularis although giant vacuoles were still present ([Fig. 3c]); the same degenerative findings were found for both groups 1 and 2. Finally, in
group 3, OHCs, IHCs and the stria vascularis showed normal fine structural features
with the exception of some vacuolar formations ([Figs. 4a], [4b], and [4c]). Furthermore, in the auditory nerve fibers, whereas separations in the myelin sheath
were significantly decreased, myelin formations were still observed in some axons
and in the cytoplasm of Schwann cells.
Fig. 2 Transmission electron microscopy micrographs of OHCs, IHCs and the stria vascularis
of group 1 (Uranyl acetate& Lead citrate). (a) OHCs in group 1: 
: the loss of stereocilia, v: vacuoles, →: crystalysis in mitochondria, 
: degeneration of afferent innervation, 
: myelin figures (b) IHCs in group 1: : the loss of stereocilia, v: vacuoles, →: crystalysis in mitochondria, 
: lipid droplets surrounded by electron condensed matter (c) Stria vascularis in group 1: M: marginal cell, I: intermediate cell, B: basal cell,
F: fibroblasts, v: vacuoles. Abbreviations: IHCs: inner hair cells; OHCs: outer hair
cells.
Fig. 3 Transmission electron microscopy micrographs of OHCs, IHCs and the stria vascularis
of group 2 (Uranyl acetate& Lead citrate). (a) OHCs in group 2: : the loss of stereocilia, v: vacuoles →: mitochondria showing regular settlement
under the cell membrane, : normal afferent innervation (b) IHCs in group 2: : stereocilia, →: mitochondrion (c) Stria vascularis in group 2: M: marginal cell, İ: intermediate cell, B: basal cell,
v: vacuoles, 
: irregular formations in the apical portion. Abbreviations: IHCs: inner hair cells;
OHCs: outer hair cells.
Fig. 4 Transmission electron microscopy micrographs of OHCs, IHCs and the stria vascularis
of group 3 (a) OHCs in group 3: : stereocilia, v: vacuoles, →: regular settlement of mitochondria under the cell membrane
(b) IHCs in group 3: : stereocilia, →: mitochondrion (c) Stria vascularis in group 3: M: marginal cell, I: intermediate cell, B: basal cell.
Abbreviations: IHCs: inner hair cells; OHCs: outer hair cells.
Table 1
Degenerative criteria and scores of outer hair cells and inner hair cells
|
Degenerative criteria
|
Outer hair cells (OHCs)
|
Inner hair cells (IHCs)
|
|
Group 1
|
Group 2
|
Group 3
|
Group 1
|
Group 2
|
Group 3
|
|
Loss of stereocilia
|
2, 2, 1, 3, 2, 3
|
2, 2, 1, 2, 3, 1
|
0, 0, 2, 1, 1, 1
|
2, 3, 1, 3, 3, 2
|
2, 1, 3, 2, 2, 2
|
0, 1, 1, 2, 2, 1
|
|
Deterioration in stereocilia
|
3, 2, 1, 3, 2, 2
|
2, 2, 1, 2, 2, 2
|
0, 1, 0, 0, 0, 1
|
3, 2, 2, 2, 2, 2
|
2, 1, 1, 1, 2, 2
|
0, 1, 0, 1, 0, 1
|
|
Vacuolar formations
|
2, 2, 2, 3, 2, 2
|
2, 2, 1, 1, 1, 2
|
1, 0, 0, 0, 1, 1
|
3, 3, 3, 2, 3, 3
|
2, 3, 3, 3, 3, 2
|
2, 1, 1, 1, 1, 1
|
|
Connection sides disorders
|
2, 1, 1, 1, 1, 1
|
1, 1, 1, 0, 1, 1
|
0, 0, 0, 1, 0, 0
|
2, 1, 2, 1, 1, 2
|
1, 1, 1, 2, 1, 1
|
1, 1, 0, 0, 0, 0
|
|
Disruption in mitochondrial settlement
|
3, 3, 3, 2, 3, 3
|
1, 1, 1, 2, 1, 1
|
1, 0, 0, 0, 0, 1
|
3, 3, 3, 3, 3, 3
|
1, 1, 1, 0, 1, 1
|
0, 0, 0, 0, 1, 0
|
|
Crystalysis in mitochondria
|
2, 1, 1, 1, 1, 1
|
1, 1, 1, 1, 1, 1
|
0, 0, 1, 0, 0, 0
|
2, 1, 1, 1, 0, 2
|
1, 1, 1, 1, 0, 1
|
1, 0, 0, 1, 0, 1
|
|
Deterioration in afferent innervation
|
2, 1, 1, 2, 2, 1
|
1, 1, 1, 0, 1, 1
|
0, 1, 0, 0, 0, 0
|
2, 3, 2, 2, 2, 2
|
2, 1, 1, 1, 2, 1
|
1, 0, 1, 1, 1, 1
|
|
Lipid droplets
|
1, 0, 0, 1, 2, 2
|
1, 0, 0, 1, 1, 0
|
0, 0, 1, 0, 0, 0
|
1, 1, 1, 0, 1, 1
|
1, 1, 1, 0, 0, 1
|
1, 0, 0, 0, 0, 0
|
Table 2
Degenerative criteria and scores of the stria vascularis
|
Degenerative criteria
|
Stria vascularis
|
|
Group 1
|
Group 2
|
Group 3
|
|
Giant vacuolar formations
|
3, 3, 3, 3, 3, 3
|
3, 2, 3, 2, 3, 2
|
2, 1, 1, 1, 1, 1
|
|
Crystalysis
|
3, 2, 3, 3, 3, 3
|
3, 2, 2, 2, 2, 2
|
2, 1, 1, 1, 2, 2
|
|
Myelin figure formations
|
2, 3, 2, 2, 2, 2
|
2, 2, 2, 1, 2, 2
|
1, 2, 2, 2, 1, 1
|
|
Accumulation of electron-dense bodies
|
2, 2, 2, 3, 2, 2
|
2, 2, 2, 2, 2, 2
|
2, 1, 1, 1, 2, 2
|
|
Reduction of the vessels
|
3, 3, 3, 3, 3, 3
|
3, 2, 3, 2, 3, 3
|
2, 1, 1, 1, 1, 1
|
|
Connection sides disorders
|
3, 2, 2, 2, 2, 2
|
2, 1, 2, 2, 1, 1
|
1, 0, 1, 0, 0, 0
|
|
Irregular cell membrane formation at the apical region of marginal cells
|
0, 0, 1, 0, 1, 1
|
3, 3, 3, 2, 3, 2
|
0, 0, 1, 1, 0, 1
|
Table 3
Degenerative criteria and scores of auditory nerve fibers
|
Degenerative criteria
|
Auditory nerve fibers
|
|
Group 1
|
Group 2
|
Group 3
|
|
The separation of myelin
|
3, 3, 3, 3, 3, 3
|
3, 2, 2, 3, 3, 3
|
1, 2, 2, 2,1, 1
|
|
Myelin figure formations in inter-axons field
|
3, 3, 3, 2, 3, 3
|
3, 3, 3, 2, 3, 3
|
2, 3, 3, 2, 2, 2
|
|
Myelin figure formation in axons
|
3, 2, 3, 3, 3, 3
|
2, 3, 3, 2, 3, 3
|
2, 2, 2, 2, 2, 1
|
|
Giant vacuolar formations in cytoplasm of Schwann cells
|
3, 2, 2, 2, 2, 2
|
2, 1, 2, 2, 2, 1
|
1, 2, 1, 1, 1, 1
|
|
Accumulation of electro-dense bodies in myelin sheath
|
2, 1, 1, 1, 2, 1
|
1, 0, 1, 1, 1, 1
|
1, 0, 0, 0, 1, 0
|
Discussion
The present study is the first study to investigate the effects of riluzole on cisplatin-
induced ototoxicity. For click stimulus, ABR thresholds were similar among the groups
before and after drug administration. For the 8 kHz-frequency stimulus, in the groups
receiving cisplatin+ riluzole, there were significantly lower ABR thresholds when
compared with the group receiving cisplatin+ saline after drug administration. For
these outcomes, we believe that riluzole may have positive effect on hearing. On the
other hand, no significant difference ABR thresholds was obtained between the groups
receiving cisplatin + riluzole. After drug administiration, we found relatively better
mean ABR value in the group receiving 6 mg/kg of riluzole. The low counts of guinea
pigs may be the reason of this result.
In addition, the TEM evaluation demonstrated that both doses of riluzole attenuated
the degeneration of hair cells and the stria vascularis, known to be major target
organs of cisplatin-induced ototoxicity in the cochlea. Unlike the ABR results, better
fine structural features were observed in the groups receiving riluzole, especially
in the group receiving 8 mg/kg of riluzole. For this outcome, we believe that riluzole
has a positive effect on preserving cells. Although there was discordance between
the ABR results of groups 2 and 3, we can say that riluzole may have positive effects
on hearing and preserving the cochlea.
The reason for morphological preservation of the stria vascularis and hair cells is
unclear. Previously, the protective effect of riluzole on hair cells and ganglion
neurons exposed to acoustic trauma was observed by Wang et al in an animal model.
Their interpretation focused on an anti-excitotoxic mechanism for the protection of
neuronal cells; this mechanism was not applicable to the preservation of hair cells,
however, with riluzole being thought to act directly on hair cells via another, as
yet unidentified, pathway.[15] We could elucidate the preservation of both hair cells and stria vascularis in our
study with the same viewpoint.
There is no consensus in the current literature regarding the primary target organ
of cisplatin in the cochlea. Van Ruijven et al demonstrated that cisplatin affected
primarily OHC loss; this is followed by myelin sheath detachment of SGCs. Both processes
then run in parallel without any obvious change in the stria vascularis.[6]
[7] In contrast, several studies have shown that damage to the stria vascularis precedes
OHC loss in cisplatin ototoxicity, with Ocho et al proposing that hair cell damage
is secondary to strial degeneration.[25] Thomas et al demonstrated the accumulation of platinum-DNA adducts by immunofluorescence,
particularly in the nuclei of marginal cells in the stria vascularis, and demonstrated
that OHCs had no accumulation of cisplatin-DNA adducts in the period between 4 and
48 hours after infusion of cisplatin in guinea pigs, thus concluding that the main
target of cisplatin-induced ototoxicity is the stria vascularis.[26] Thomas et al noticed that damage of the stria vascularis results in impaired potassium
secretion into the endolymphatic space, and a decreased endocochlear potential that
affects the sensorineural transduction of hair cells. Therefore, if the stria vascularis
is considered as the main and the primary target organ, riluzole may have prevented
the cisplatin-induced reduction of the endocochlear potential and precluded hair cell
degeneration by initially providing protection to the stria vascularis.
Whereas it is well known that riluzole is a neuroprotective agent, and is generally
considered an anti-glutamatergic drug, its mode of action is not entirely clear. Based
on recent studies, we deduced that riluzole could also manifest protective effects
in the context of cisplatin-induced ototoxicity via its antioxidant and antiapoptotic
properties.[17]
[18] Roth et al demonstrated that, independent of its effect on glutamate release, riluzole
could be protective against manganese (Mn) toxicity by suppressing ROS activation
and could also avert downstream apoptotic signals by suppressing the activation of
caspase 3 and JNK phosphorylation.[17] Additionally, riluzole can inhibit methylmercury (MeHg) induction of oxidative stress
by triggering glutathione (GSH) synthesis; GSH is one of the most abundant and essential
thiol tripeptides present in mammalian cells for scavenging ROS, and reduces oxidative
stress,[27] which in turn may be a potential mechanism for the protective effect of riluzole.
Unfortunately, we were not able to accrue data to investigate this specific pathway
in our study.
One of the cisplatin-induced cytotoxic pathways, following the generation of ROS,
is poly-(ADP-ribose) polymerase-1 (PARP-1) mediated cell death, which requires JNK
to induce apoptosis. Heme oxygenase-1 (HO-1) and heat shock proteins (HSPs) 70 and
90 inhibit JNK phosphorylation and protect against cisplatin-induced ototoxicity.[4]
[5] Piperine, salicylates and the calcium channel blocker flunarizine are known to be
protective agents against cisplatin-induced apoptosis by induction of HO-1.[28]
[29]
[30] Recent studies have shown that the combination of saline with riluzole at optimal
doses stimulates the expression of the endogenous protective genes nuclear factor
erythroid 2-related factor (NRF2) and HO-1.[21]
[30] In addition, riluzole can increase the amount of heat shock factor 1 (HSF-1) present
in the neuronal cell system to boost HSPs.[20]
[21] The riluzole's mechanism of protection of the cochlea against cisplatin ototoxicity
by may also involve inhibition of JNK phosphorylation by increased levels of expression
of endogenous protective genes.
Cisplatin administration induces the generation of ROS by employing the nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase 3 (NOX3), an isoform of NADPH oxidase
that regulates stress-related genes in the cochlea, such as transient receptor potential
vanilloid 1 (TRPV 1) and kidney injury molecule-1 (KIM-1), and initiates apoptosis in the cochlea. The expression of TRPV 1 contributes to cell death by increasing the influx of Ca2+ into the cell and subsequently activating caspases.[4] Accordingly, the action of riluzole on voltage-gated calcium channels in cisplatin-induced
cytotoxicity has also been considered. Long lasting (L)-type calcium channels, gated
by high voltage, are expressed in the lateral wall, the organ of Corti and the SGN.[31]
[32]
[33] In cultured embryonic rat motor neurons, riluzole has been found to block L-type
high voltage-activated (HVA) Ca2+ channels,[34] and administration of a T-type calcium channel antagonist (flunarizine) decreased
apoptosis when compared with control in cisplatin-treated cells.[30] Perhaps the protective effect of riluzole on cisplatin-induced apoptosis is in part
due to its capacity to block calcium channels.
Conclusion
This is a preliminary study for evaluating the potential ability of riluzole to act
as an otoprotective drug in the face of cisplatin ototoxicity. The results suggest
that riluzole may have positive effects on hearing and on the degeneration of the
cochlea in guinea pigs. However, additional studies are needed to confirm these results
and the mechanism of action of riluzole.
