one of the most common disabilities in the world.1
Its incidence in Cuba is 2.1 per 1,000
primary cause of human auditory loss is the
alteration or death of sensorial receptor cells
of the inner ear, the cochlear hair cells,
located in the organ of Corti (OC).3
Mammalian hair cells cannot regenerate after
loss lead to permanent sensorineural deafness,
which is characterized by loss of hair cells and
retrograde degeneration of the spiral ganglion (SG)
implants are crucial to recover the audition for
patients with sensorineural deafness.5
Their function is based on the stimulation of
the axons of the SG type I neurons.6
Effective cochlear implants which may work with
only 10% of the original population of SG
neurons have been developed.7
Moreover, the preservation degree of SG neurons
is considered decisive for language recognition
after a cochlear implant. SG neuronal
degeneration could be a restrictive factor
against the optimal function of more advanced
implant performance is also influenced by the
loss of the peripheral processes innervating the
OC because the action potentials generation site
needs to be transferred either towards the soma
of the SG neurons or toward the central axon.8
application of therapies based on drugs or on
stem cells to protect9
or to regenerate SG neurons10
as an alternative to overcome deafness, has
motivated recent interest in studying the
ultrastructure of these neurons.
In adult rats,
a decrease of the SG neuronal density starting
from the 8th week of deafness, as well as the
emptying of the osseous spiral lamina due to the
loss of the peripheral processes innervating the
OC since the 4th week, have been shown by light
microscopy in a previous paper.11
The aim of the present work was to study the
ultrastructure of the SG neurons and their
peripheral processes in order to determine the
onset of the ototoxicity-induced degenerative
Adult male Wistar rats weighting from 250 to 300
g were provided by the National Center for
Laboratory Animal Production. At the beginning
of the experiment, the normal function of the
auditory pathway was confirmed in all the
animals by means of brainstem auditory evoked
potentials (BSAEP) and steady state auditory
evoked potentials (SSAEP).
divided into five groups with two rats on each:
control (non-treated rats) and four groups of
rats treated with the ototoxic agents. The
latter were sacrificed after 2, 4, 8 and 16
weeks of deafness.
induction: At the beginning of the
experiment, the four treated groups
simultaneously received intraperitoneal
kanamycine (400 mg/kg) and furosemide (150 mg/kg).12
One week later, hearing loss was confirmed by
means of BSAEP and SSAEP. Those animals that
didn’t answer to 105 dB pspl intensity for BSAEP
or to 105 dB spl for SSAEP, were considered deaf.
processing: The animals were fixed by
vascular perfusion in 10% formalin. A cochlea
was removed from each rat and immediately fixed
by perilymphatic perfusion in 2%
paraformaldehyde and 2% glutaraldehyde (in 0.1
mol/L, pH 7.4 sodium phosphate buffer). The
cochleae were decalcified in 8,3% EDTA, post-fixed
in 1% osmium tetroxide (in the same buffer),
dehydrated in acetone and embedded in Spurr
resin. The samples were cut through the cochlear
horizontal plane with an Ultrotome III (LKB)
ultramicrotome. Semithin cochlear sections dyed
with Stevenel Blue and mounted on glass slides10
were obtained to locate the medial cochlear turn
by light microscopy. ultrathin sections of the
cochlear medial turn were placed on 400 mesh
grids, stained with uranyl acetate and lead
citrate and examined under a Transmission
Electron Microscope Jeol JEM 100S.
cochleae SG neurons showed intact myelin sheaths,
round nuclei, several narrow channels of rough
endoplasmic reticulum (Nissl bodies) and
mitochondria with normal cristae (Fig. 1A).
fourth week of deafness, prominent
irregularities in both the myelin sheath and the
nuclear envelope were observed (Fig. 1B)
together with cytoplasmic matrix clearing:
scarce Nissl bodies (Fig. 1C).
weeks of deafness, the remaining Type I SG
neurons showed cytoplasmic shrinkage related to
their myelin sheaths and conspicuous cytoplasmic
inclusions (Fig. 1D). Nuclear envelope
invaginations, dilated Nissl bodies and
cytoplasmic vacuolization were also evident
after deafness, most of the remaining Type I SG
neurons exhibited complete demyelination, which
resulted in the pathological Type III SG neurons
with dense mitochondria and scarce Nissl bodies
cochleae, the myelinated peripheral processes
showed intact myelin sheaths and extensive
Schwann cell cytoplasm surrounding them (Fig.
2A). In deafened cochleae, myelin sheaths and
Schwann cells cytoplasm were altered. Four weeks
after deafness, zones of separation between the
layers of the myelin sheaths and rupture points
of the sheaths were observed (Fig. 2B). Eight
and sixteen weeks after deafness, myelin sheaths
discontinuities with axoplasm efflux were
observed (Fig. 2C). The density of the
peripheral processes suffered a progressive
reduction since the fourth week of deafness
Fig 1. Ultrastructure
of SG cells. A) Control.
B-F) Deafened cochleae. B,
C) 4 weeks, D, E) 8 weeks,
F) 16 weeks. Myelin sheath (arrows),
nucleus (N), mitochondria
(M), Nissl bodies (Nb),
Golgi vesicles (Gv),
cytoplasmic retraction from
the myelin sheath (CR),
cytoplasmic inclusions (small
arrows), vacuoles (V).
Ultrastructure of peripheral
processes innervating the
organ of Corti. A) Control,
B-D) Deafened rats, B) 4
weeks, C) 8 weeks, D) 16
weeks. Myelin sheaths (arrows),
Schwann cells (*).
It has been
proved that the aminoglycoside kanamycin induces
sensorineural deafness, in which the hair cells
loss precedes the degeneration of SG neurons13,14,15,16,17
Furosemide induces clinically relevant
transitory ototoxic damage18
and, as other diuretics, it enhances the
ototoxicity of aminoglycosides.19
cochlear turn was chosen for the electron
microscopical observations because it showed the
greatest neuronal loss in deaf animals related
convey information from the cochlea to the
central auditory system. In normal cochleae, two
types of SG neurons (Type I and Type II) have
been described within the Rosenthal’s canal.20
The present work focused its attention on type I
SG neurons, since they represent approximately
95% of the SG neurons in rat cochleae.21
Besides, they are more susceptible to injury
than type II22 and
are considered of primary relevance in the
application of cochlear implants.23
changes on SG neurons were ultrastructurally
detected in this work 4 weeks before their loss
was revealed by light microscopy.10
Progressive degenerative changes of type I SG
neurons after deafness result in the emergence
of type III neurons, exclusive of pathological
most of the hearing losses are sensorineural24
and it is considered that SG neuronal damage is
due to the failure of the trophic effect exerted
by hair cells over them.25
By light microscopy, the scarcity of peripheral
processes innervating the OC together with the
loss of SG neuronal somata has been demonstrated
in deaf animals.10
In this paper, by electron microscopy, it was
shown that the remaining peripheral processes
undergo progressive degeneration since the
fourth week of deafness. The condition of the OC
supporting cells also influences upon SG
In previous works, degeneration of the OC hair
cells and supporting cells was observed since
the second week of deafness.27
In this work,
degenerative changes on SG neurons were
ultrastructurally detected 4 weeks before the
loss of SG cells and their peripheral processes
were revealed by light microscopy. Provided the
similarities between the inner ear of rodents
and humans,28 the
information from these animal models of deafness
might be taken into account when considering
cochlear implants to patients affected by
cochlear implants are at present the best option
available for deafness treatment, they do not
completely restore the auditive function.
Prevention of hair cell death or their
well as regeneration of the SG neurons to
innervate the new hair cells,9
are among the new therapeutical strategies that
are being studied. The latter approaches
demonstrate the importance of understanding the
morphological changes that occur in the cochlea
submitted to ototoxicity.
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