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Delayed-onset temporary auditory threshold shift following head blow in guinea pigs
Hearing Research 199 (2005) 111–116 www.elsevier.com/locate/heares
Delayed-onset temporary auditory threshold shift following head blow in guinea pigs Naokimi Tokui *, Hideaki Suzuki, Tsuyoshi Udaka, Nobuaki Hiraki, Takeyuki Fujimura, Kazunobu Fujimura, Kazumi Makishima Department of Otorhinolaryngology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan Received 20 April 2004; accepted 12 August 2004 Available online 12 October 2004
Abstract This study attempts to investigate the development of sensorineural hearing loss following a head blow without skull fracture in association with physiological and histopathologic changes in an experimental animal model. With the head in a freely movable position, albino guinea pigs were given a single blow to the occipital region by a head blow device. At 1, 7, and 14 days after the blow, the animalsÕ auditory brainstem response (ABR) and cochlear microphonics (CM) were examined, and both the temporal bone and brain stem were observed by light and electron microscopy. The ABR threshold was unchanged at day 1, was significantly increased at day 7, and was fully recovered at day 14. The I–V and I–II interpeak latencies were significantly prolonged at days 1 and 7, and wave I latency was significantly prolonged at day 7 only. These latencies were recovered to normal limits at day 14. On the other hand, no significant change in CM versus the control group was observed at any point in the measurements. Histopathologically, no abnormal finding was seen at the light microscopic level. However, at the electron microscopic level, there were some injuries to the eighth nerve. At day 1, the lamellar structure of the myelin sheath was irregular, and the periaxonal space was expanded; at day 7, the myelin sheath was disintegrated. At day 14, however, these changes were partially reversed. These results suggest that sensorineural hearing loss following a head blow in this model is attributed to dysfunction of the eighth nerve rather than to cochlear impairment. 2004 Elsevier B.V. All rights reserved. Keywords: Sensorineural hearing loss; Head injury; Head blow device; Disintegration of myelin sheath
1. Introduction Head injury is often accompanied by neurotological damage – such as laceration of the tympanic membrane, hemotympanum, dislocation of the ossicles, temporal bone fracture, and brain stem hemorrhage – that lead to conductive and/or sensorineural hearing losses. Without such obvious macroscopic damage, some patients who have received a head blow still manifest hearing *
Corresponding author. Tel.: +81 93 691 7448; fax: +81 93 601 7554. E-mail address: [email protected] (N. Tokui). 0378-5955/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2004.08.009
loss and tinnitus. In such cases, it is always difficult to clinically clarify the etiopathology of these manifestations even if thorough otological, neurological, and radiological examinations are performed. There are a number of studies on animal models of hearing disorder induced by a head blow. In most of them, the heads of animals were immobilized during the blow. Under this condition, cochlear damage and/ or labyrinthine fractures frequently occur, resulting in sensorineural hearing losses (Wittmaack, 1932; Schuknecht, 1969). In reality, however, the human head is not immobilized during a collision, fall, strike, or traffic accident, and human autopsies after head injury have shown
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that inner ear damage and temporal bone fractures are often absent (Makishima and Snow, 1975a). Makishima and Snow (1975a,b) established an experimental animal model of a head blow against a freely movable head by using a specially designed head blow device. In that experiment, a temporary auditory threshold shift occurs without macroscopic damage to the temporal bone or brain stem, implying that this animal model more accurately mimics real human head injury. Their results suggested that the lesion responsible for auditory disorder following this type of head blow is unlikely to be the cochlea but rather the retrocochlear region. However, the precise pathogenesis of this phenomenon is scarcely understood. The present study was performed to elucidate the pathogenesis of auditory disorder in the same animal model by morphological and electrophysiological methods.
2. Materials and methods 2.1. Head blow device and loading method Adult albino guinea pigs of both sexes, weighing 250– 450 g, were anesthetized with 10 mg/kg pentobarbital sodium administered intraperitoneally in order to reduce body movement during subsequent procedures. The animals were suspended by a string tied to the upper incisor teeth. The other end of the string was loosely tied to a supporting rod. Mechanical loading to the head was made by a head blow device manufactured by Kayaba Industry Co. (Tokyo, Japan) according to previous reports (Makishima and Snow, 1975b; Makishima et al., 1976). The device had a pendulum: a 3-kg iron weight attached to the end of a 1-m-long stainless steel arm. The device allowed the weight to swing down in an arc, striking the occipital portion of the skull at the lowest point of its path. The peak impact force was measured by a force sensor (Crystal Piezoelectric Force Link; Kistler Japan Co., Tokyo, Japan) equipped on the weight, and was expressed in Newtons (N). At the instant the weight impacted the head, the string was released from the supporting rod, so that the force of the blow threw the animal, which landed on a soft sponge pad. The animals were then subjected to electrophysiological measurements and microscopic observations at 1 day (early group), 7 days (intermediate group), or 14 days (late group) after the head blow. In control animals, which were treated as above but without receiving a head blow, no significant differences were seen between days 1, 7, and 14, so all of them were presented as a single group. The study was performed in accordance with the Institutional Animal Care and Use Committee of the University of Occupational and Environmental Health.
2.2. Auditory brain stem response The animals were anesthetized with 35 mg/kg pentobarbital sodium administered intraperitoneally and placed in a prone position with their head fixed in a head-holder. Body temperature was kept at approximately 38 C by a heating pad. After a tracheostomy was performed, the animals were placed on a ventilator and were immobilized with an intramuscular injection of 3 mg/kg tubocurarine chloride. A stainless steel screw electrode was placed on the vertex as a recording electrode. A reference electrode was placed on the right temporal portion, and a ground electrode was placed at the midpoint between the two external auditory canals in the occipital portion. A click or a tone-burst stimulus was delivered to the right ear through an ear bar with a closed sound system. Using a digital synthesizer (1930A; NF Corporation, Yokohama, Japan), a series of 100-ls clicks was generated with a digital synthesizer at a rate of 10 pps. Tone bursts (10 ms with rise–fall times of 1 ms) of 2, 4, 8, 12, or 18 kHz were generated with an external generator (made in our laboratory) at a rate of 5 pps. The responses were amplified 1000· and bandpassfiltered (8–3000 Hz). Each run consisted of 128 responses, which were summated and averaged with an FFT analyzer (SA-74; Rion, Tokyo, Japan) in the time analysis mode. Acoustic stimuli were initially presented at an intensity of 80 dB, and decreased by 10-dB steps well above the threshold and then in 5-dB steps near the threshold until auditory brain stem response (ABR) waves disappeared. The threshold was defined as the minimum sound intensity at which a visible ABR wave was seen in two runs. The latency of each wavelet was measured by the cursor mode of the FFT analyzer. Calibrations of the sound intensity were made with a B & K 4128 probe microphone (Danish Pro Audio, Alleroed, Denmark) in a dummy ear, and were confirmed in the ear canal near the tympanic membrane of each guinea pig at the end of measurement. 2.3. Cochlear microphonics After the auditory brain stem response measurement, the amplitude of cochlear microphonics (CM) was measured to monitor the cochlear function. The right tympanic bulla was opened via a ventral approach and the bony cochlea was exposed. A tiny fenestration (about 150 lm in diameter) was made in the bony wall over the scala tympani near the round window. An enamelinsulated platinum electrode with an exposed tip 100 lm in diameter was inserted into the scala tympani through the surgical fenestration. An identical reference electrode was placed over the surface of the bulla. A ground Ag/AgCl disc electrode was placed on the cervical muscle. A pure tone sound of 4 kHz, ranging from 20
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to 90 dB SPL, was used to measure the amplitude of CM. All responses were double-traced to confirm reproducibility. 2.4. Light microscopy Immediately following the electrophysiological measurements, the animals were perfused transcardially with 10% formaldehyde. Bilateral temporal bones and the brain stem were removed and immersed in the same fixative overnight. After rinsing with 10 mM phosphate buffered saline at pH 7.2, the tissue was decalcified with 0.2 M ethylenediaminetetraacetic acid in 0.1 M phosphate buffer at pH 7.4 (PB) for 7 days and was then dehydrated and embedded in paraffin. Serial 7-lm-thick sections were stained with hematoxylin–eosin or luxol fast blue and viewed at 20–400·. 2.5. Transmission electron microscopy
Fig. 1. The ABR thresholds for tone-burst stimuli. The animals were subjected to ABR measurement at 1 (early group), 7 (intermediate group), and 14 days (late group) after the head blow as described in Section 2. **P < 0.01.
Immediately following the electrophysiological measurements, the animals were perfused transcardially with a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in PB. Bilateral temporal bones and the brain stem were removed and immersed in the same fixative for 2 h, postfixed with 1% osmium tetroxide in PB, dehydrated in a graded series of ethanol, and then embedded in epoxy resin. Ultrathin sections were made on an ultramicrotome, stained with uranyl acetate, and viewed with a JEM 1200EX electron microscope (JEOL, Tokyo, Japan).
Table 1 details the peak and interpeak latencies for the click stimuli at 80 dB peSPL. The I–V and I–II interpeak latencies were significantly prolonged in the early group. They were also significantly prolonged in the intermediate group, along with the wave I latency. In the late group, all three latencies had returned to the normal range. Fig. 2 illustrates representative ABR waveforms of the control and intermediate groups.
2.6. Statistics
3.3. Cochlear microphonics
Data were expressed as means ± SEM, and the statistical significance of differences was tested using Mann– WhitneyÕs U test. Differences were considered significant when the P value was <0.05.
No significant change of the input–output curves of CM was observed in the early, intermediate, or late groups in comparison with the control group (data not shown). 3.4. Macroscopic and light microscopic findings
3. Results 3.1. Impact force The impact force was 119.2 ± 8.1 N (n = 12), 110.8 ± 6.4 N (n = 12), and 100.9 ± 5.7 N (n = 11) in the early, intermediate, and late groups, respectively. There were no significant differences among the three groups.
When the animals were sacrificed and dissected, no fractures of the temporal bone or skull base were found in any of them. The brain including the brain stem were macroscopically intact as well. Likewise, at the light
Table 1 The peak and interpeak latencies for click stimuli at 80 dB peSPL Control
3.2. Auditory brain stem response The thresholds of ABR for the tone-burst stimuli are shown in Fig. 1. The threshold was unchanged in the early group, significantly increased at all frequencies in the intermediate group, and fully recovered in the late group.
Wave I latency I–II IPL II–V IPL I–V IPL
Early
Intermediate 0.08*
1.96 ± 0.05
1.99 ± 0.04
2.14 ±
1.01 ± 0.05 4.06 ± 0.06 5.07 ± 0.07
1.16 ± 0.05* 4.35 ± 0.11 5.51 ± 0.11**
1.26 ± 0.32** 4.17 ± 0.68 5.44 ± 0.09**
Late 1.94 ± 0.03 1.13 ± 0.06 4.12 ± 0.08 5.26 ± 0.06
Data values are mean ± SEM (ms). IPL, interpeak latency. * P < 0.05. ** P < 0.01.
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3.5. Transmission electron microscopic finding At the electron microscopic level, some eighth nerves were damaged, as shown in Fig. 3. In the early group, the lamellar structure of the myelin sheath was irregular and the periaxonal space was expanded. In the intermediate group, the myelin sheath was disintegrated and the periaxonal space was still enlarged. In the late group, these changes were partially reversed. In contrast, no abnormal finding was observed in the cochlea or brain stem in any of the groups.
4. Discussion
Fig. 2. Representative ABR waveforms (click stimuli at 80 dB peSPL). (a) Control group: wave I latency = 1.89 ms, I–II interpeak latency = 0.94 ms, I–V interpeak latency = 5.12 ms. (b) Intermediate group (day 7): wave I latency = 2.42 ms, I–II interpeak latency = 1.35 ms, I–V interpeak latency = 5.24 ms.
microscopic level, no hemorrhages, lacerations, or destroyed tissues were observed in the cochlea, eighth nerve, or brain stem of any of the animals.
In the present experiment, no fractures of the temporal bone or skull base were observed, and neither light nor electron microscopic observations showed cochlear damage. The cochlea was not only morphologically but also electrophysiologically intact, as shown by the normal CM. On the other hand, ABR showed temporary auditory threshold shifts and prolongation of wave I latency and I–II interpeak latency (Table 1). Waves I and II of ABR are thought to be derived from the distal intracanalar and intracranial portions of the eighth nerve, respectively (Moller and Jannetta, 1983; DÕHooge et al., 1999). Therefore, the present results of ABR strongly suggest damage to the eighth nerve, which was confirmed by elec-
Fig. 3. Transmission electron micrographs of the eighth nerve (scale bar = 1 lm). (a) Control group. (b) Early group (day 1): irregularity of the lamellar structure of the myelin sheath and expansion of the periaxonal space are seen. (c) Intermediate group (day 7): disintegration of the myelin sheath is observed, and the expansion of the periaxonal space remains. (d) Late group (day 14): both the disintegration of the myelin sheath and the expansion of the periaxonal space are partially reversed.
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tron microscopic observations of the disintegrated myelin sheath and the expanded periaxonal space. It is known that steep acceleration/deceleration of the skull upon a head blow, particularly to the occipital region, causes the brain to rotate and generates shear strain on the cranial nerves (Denny-Brown and Russell, 1941; Holbourn, 1943; Pudentz and Shelden, 1946; Gurdjian, 1970). The eighth nerve is most vulnerable to such mechanical stress, possibly because the nerve enters the petrous portion of the temporal bone immediately after emerging from the brain stem (Ylikoski et al., 1982; Goksu et al., 1992; Feneley and Murthy, 1994). The seventh nerve, which takes a similar route as the eighth nerve between the brain stem and temporal bone, is also vulnerable to injury from head concussion. However, because this nerve has a long path along the facial canal in the temporal bone, seventh nerve palsy following a head blow is ascribed not only to shear strain loaded to the root of the nerve but also to compression and circulatory disorder in the facial canal. Because multiple factors involved in the mechanism of seventh nerve palsy would render its analysis complicated, we did not investigate seventh nerve damage in the present study. Sekiya and Moller (1987, 1988) studied the mechanism underlying the occurrence of hearing loss after neurosurgery of the cerebellopontine angle in animal models. In dogs and monkeys, they directly loaded pressure onto the cerebellum and observed disintegration of the myelin sheath of the eighth nerve, which is consistent with the present results. The integrity of myelin is important for the electrophysiological function of the eighth nerve (Ylikoski and House, 1981; Sekiya and Moller, 1988; Spoendlin and Schrott, 1990; Scaioli et al., 1992; Zhou et al., 1995). Myelin-deficient mutant hamsters manifested prolonged wave I latency and an elevated auditory threshold in ABR measurement (Naito et al., 1999). Those authors suggested that disorders of the myelin sheath induce changes in conduction velocity and, therefore, desynchronization of neuronal firing in the auditory pathway. In addition, it is known that progressive hearing loss in patients with hereditary motor and sensory neuropathy originates from the degeneration of the myelin sheath of the eighth nerve (Scaioli et al., 1992). Our results also showed that the auditory threshold shift was temporary, which is consistent with the observation that the morphological change of the eighth nerve was reversible. Previous reports documented that considerable proportions of sensorineural hearing losses in patients with cerebral concussion without fracture recovered within six months after injury (Podoshin and Fradis, 1975). Recovery of demyelinated nerves has also been reported in other nervous systems. Ludwin (1978) studied a cuprizone-induced demyelination model of mice, and found that the superior cerebellar peduncles are specifically demyelinated and that remyelination occurs within 1–2 weeks after the removal of cuprizone.
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More recently, Van Reempts et al. (1993) demonstrated that remyelination of the demyelinated sciatic nerve began after approximately 1week, and was completed within 1 month. The onset of damage to the eighth nerve was delayed as shown in Figs. 1 and 3. Feldmann (1987) actually reported human cases with delayed-onset sensorineural hearing loss after head trauma. This phenomenon may be attributed to the delayed reaction of the injured nerve. Blight (1985) proposed two pathogeneses of spinal cord damage by mechanical stress. One is the immediate reaction directly induced by the stress. The other is the delayed reaction derived from biochemical processes that occur several days after injury. For example, the production of cytokines and the release of neurotransmitters, such as glutamate and nitric oxide, may participate in delaying the reaction (Blight and Decrescito, 1986; MacDermott et al., 1986; Panter et al., 1990; Dawson et al., 1991, 1993). 5. Conclusions Electrophysiological and histopathological reactions were studied in a guinea pig model of sensorineural hearing loss following a blow to the head in a freely movable position, mimicking real human head injury. Threshold shifts and prolongations of the I–V and I–II interpeak latencies and of the wave I latency were observed in ABR measurements, whereas no significant changes in CM were detected. Electron microscopic observation of the eighth nerve revealed that the myelin sheath was irregular and the periaxonal space enlarged. Such changes occurred with a delay and were reversible. These results suggest that sensorineural hearing loss in this model is attributable to eighth nerve dysfunction rather than to cochlear impairment. The mechanism underlying eighth nerve damage in the present experimental model needs to be further investigated by biochemical, cell physiological, and molecular biological methods in future studies. Acknowledgements This research was partially supported by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Young Scientists (B), 169791033, 2004. References Blight, A.R., 1985. Delayed demyelination and macrophage invasion: a candidate for secondary cell damage in spinal cord injury. Cent. Nerv. Syst. Trauma 2, 299–315. Blight, A.R., Decrescito, V., 1986. Morphometric analysis of experimental spinal cord injury in the cat: the relation of injury intensity to survival of myelinated axons. Neuroscience 19, 321–341.
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Dawson, V.L., Dawson, T.M., London, E.D., Bredt, D.S., Snyder, S.H., 1991. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc. Natl. Acad. Sci. USA 88, 6368– 6371. 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. Denny-Brown, J.P., Russell, W.R., 1941. Experimental cerebral concussion. Brain 64, 93–164. DÕHooge, R., Coenen, R., Gieselmann, V., Lullmann-Rauch, R., De Deyn, P.P., 1999. Decline in brainstem auditory-evoked potentials coincides with loss of spiral ganglion cells in arylsulfatase Adeficient mice. Brain Res. 847, 352–356. Feldmann, H., 1987. Sudden hearing loss with delayed onset following head trauma. Acta Otolaryngol. 103, 379–383. Feneley, M.R., Murthy, P., 1994. Acute bilateral vestibulo-cochlear dysfunction following occipital fracture. J. Laryngol. Otol. 108, 54– 56. Goksu, N., Haziroglu, R., Kemaloglu, Y., Karademir, N., Bayramoglu, I., Akyildiz, N., 1992. Anatomy of the guinea pig temporal bone. Ann. Oto. Rhinol. Laryngol. 101, 699–704. Gurdjian, E.S., 1970. Movements of the brain and brain stem from impact induced linear and angular acceleration. Trans. Am. Neurol. Assoc. 95, 248–249. Holbourn, A.H.S., 1943. Mechanics of head injuries. Lancet 2, 438– 441. Ludwin, S.K., 1978. Central nervous system demyelination and remyelination in the mouse: an ultrastructural study of cuprizone toxicity. Lab. Invest. 9, 597–612. MacDermott, A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J., Barker, J.L., 1986. NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones. Nature 321, 519–522. Makishima, K., Snow, J.B., 1975a. Pathogenesis of hearing loss in head injury. studies in man and experimental animals. Arch. Otolaryngol. 101, 426–432. Makishima, K., Snow, J.B., 1975b. Electrophysiological responses from the cochlea and inferior colliculus in guinea pigs after head injury. Laryngoscope 85, 1947–1957. Makishima, K., Sobel, S.F., Snow, J.B., 1976. Histopathologic correlates of otoneurologic manifestations following head trauma. Laryngoscope 86, 1303–1314.
Møller, A.R., Jannetta, P.J., 1983. Auditory evoked potentials recorded from the cochlear nucleus and its vicinity in man. J. Neurosurg. 59, 1013–1018. Naito, R., Murofushi, T., Mizutani, M., Kaga, K., 1999. Auditory brainstem responses, electrocochleograms, and cochlear microphonics in the myelin deficient mutant hamster ÔbtÕ. Hear. Res. 136, 44–48. Panter, S.S., Yum, S.W., Faden, A.I., 1990. Alteration in extracellular amino acids after traumatic spinal cord injury. Ann. Neurol. 27, 96–99. Podoshin, L., Fradis, M., 1975. Hearing loss after head injury. Arch. Otolaryngol. 101, 15–18. Pudentz, R.H., Shelden, C.H., 1946. The lucite calvarium-A method for direct observation of the brain. J. Neurosurg. 3, 487–505. Scaioli, V., Pareyson, D., Avanzini, G., Sghirlanzoni, A.F., 1992. F response and somatosensory and brainstem auditory evoked potential studies in HMSN type I and II. J. Neurol. Neurosurg. Psychiatry 55, 1027–1031. Schuknecht, H.F., 1969. Mechanism of inner ear injury from blows to the head. Ann. Otol. Rhinol. Laryngol. 78, 253–262. Sekiya, T., Moller, A.R., 1987. Cochlear nerve injuries caused by cerebellopontine angle manipulations. an electrophysiological and morphological study in dogs. J. Neurosurg. 67, 244–249. Sekiya, T., Moller, A.R., 1988. Effects of cerebellar retractions on the cochlear nerve: an experimental study on rhesus monkeys. Acta Neurochir. (Vienna) 90, 45–52. Spoendlin, H., Schrott, A., 1990. Quantitative evaluation of the human cochlear nerve. Acta Otolaryngol. Suppl. 470, 61–69. Van Reempts, J., Van Deuren, B., Ashton, D., Borgers, M., 1993. A new model of photochemically induced acute and reversible demyelination in the peripheral nervous system. Exp. Neurol. 120, 283–290. Wittmaack, K., 1932. Uber die traumatische Labyrinthdegeneration. Arch. Ohren Nasen Kehlkoopfheil 313, 59–124 (in German). Ylikoski, J., House, J.W., 1981. Demyelinating disease as the assumed cause of hearing loss and vertigo. a case report with light- and electron-microscopic findings. Arch. Otorhinolaryngol. 230, 161–170. Ylikoski, J., Palva, T., Sanna, M., 1982. Dizziness after head trauma: clinical and morphologic findings. Am. J. Otol. 3, 343–352. Zhou, R., Assouline, J.G., Abbas, P.J., Messing, A., Gantz, B.J., 1995. Anatomical and physiological measures of auditory system in mice with peripheral myelin deficiency. Hear. Res. 88, 87–97.
Delayed-onset temporary auditory threshold shift following head blow in guinea pigs Naokimi Tokui *, Hideaki Suzuki, Tsuyoshi Udaka, Nobuaki Hiraki, Takeyuki Fujimura, Kazunobu Fujimura, Kazumi Makishima Department of Otorhinolaryngology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan Received 20 April 2004; accepted 12 August 2004 Available online 12 October 2004
Abstract This study attempts to investigate the development of sensorineural hearing loss following a head blow without skull fracture in association with physiological and histopathologic changes in an experimental animal model. With the head in a freely movable position, albino guinea pigs were given a single blow to the occipital region by a head blow device. At 1, 7, and 14 days after the blow, the animalsÕ auditory brainstem response (ABR) and cochlear microphonics (CM) were examined, and both the temporal bone and brain stem were observed by light and electron microscopy. The ABR threshold was unchanged at day 1, was significantly increased at day 7, and was fully recovered at day 14. The I–V and I–II interpeak latencies were significantly prolonged at days 1 and 7, and wave I latency was significantly prolonged at day 7 only. These latencies were recovered to normal limits at day 14. On the other hand, no significant change in CM versus the control group was observed at any point in the measurements. Histopathologically, no abnormal finding was seen at the light microscopic level. However, at the electron microscopic level, there were some injuries to the eighth nerve. At day 1, the lamellar structure of the myelin sheath was irregular, and the periaxonal space was expanded; at day 7, the myelin sheath was disintegrated. At day 14, however, these changes were partially reversed. These results suggest that sensorineural hearing loss following a head blow in this model is attributed to dysfunction of the eighth nerve rather than to cochlear impairment. 2004 Elsevier B.V. All rights reserved. Keywords: Sensorineural hearing loss; Head injury; Head blow device; Disintegration of myelin sheath
1. Introduction Head injury is often accompanied by neurotological damage – such as laceration of the tympanic membrane, hemotympanum, dislocation of the ossicles, temporal bone fracture, and brain stem hemorrhage – that lead to conductive and/or sensorineural hearing losses. Without such obvious macroscopic damage, some patients who have received a head blow still manifest hearing *
Corresponding author. Tel.: +81 93 691 7448; fax: +81 93 601 7554. E-mail address: [email protected] (N. Tokui). 0378-5955/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2004.08.009
loss and tinnitus. In such cases, it is always difficult to clinically clarify the etiopathology of these manifestations even if thorough otological, neurological, and radiological examinations are performed. There are a number of studies on animal models of hearing disorder induced by a head blow. In most of them, the heads of animals were immobilized during the blow. Under this condition, cochlear damage and/ or labyrinthine fractures frequently occur, resulting in sensorineural hearing losses (Wittmaack, 1932; Schuknecht, 1969). In reality, however, the human head is not immobilized during a collision, fall, strike, or traffic accident, and human autopsies after head injury have shown
112
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that inner ear damage and temporal bone fractures are often absent (Makishima and Snow, 1975a). Makishima and Snow (1975a,b) established an experimental animal model of a head blow against a freely movable head by using a specially designed head blow device. In that experiment, a temporary auditory threshold shift occurs without macroscopic damage to the temporal bone or brain stem, implying that this animal model more accurately mimics real human head injury. Their results suggested that the lesion responsible for auditory disorder following this type of head blow is unlikely to be the cochlea but rather the retrocochlear region. However, the precise pathogenesis of this phenomenon is scarcely understood. The present study was performed to elucidate the pathogenesis of auditory disorder in the same animal model by morphological and electrophysiological methods.
2. Materials and methods 2.1. Head blow device and loading method Adult albino guinea pigs of both sexes, weighing 250– 450 g, were anesthetized with 10 mg/kg pentobarbital sodium administered intraperitoneally in order to reduce body movement during subsequent procedures. The animals were suspended by a string tied to the upper incisor teeth. The other end of the string was loosely tied to a supporting rod. Mechanical loading to the head was made by a head blow device manufactured by Kayaba Industry Co. (Tokyo, Japan) according to previous reports (Makishima and Snow, 1975b; Makishima et al., 1976). The device had a pendulum: a 3-kg iron weight attached to the end of a 1-m-long stainless steel arm. The device allowed the weight to swing down in an arc, striking the occipital portion of the skull at the lowest point of its path. The peak impact force was measured by a force sensor (Crystal Piezoelectric Force Link; Kistler Japan Co., Tokyo, Japan) equipped on the weight, and was expressed in Newtons (N). At the instant the weight impacted the head, the string was released from the supporting rod, so that the force of the blow threw the animal, which landed on a soft sponge pad. The animals were then subjected to electrophysiological measurements and microscopic observations at 1 day (early group), 7 days (intermediate group), or 14 days (late group) after the head blow. In control animals, which were treated as above but without receiving a head blow, no significant differences were seen between days 1, 7, and 14, so all of them were presented as a single group. The study was performed in accordance with the Institutional Animal Care and Use Committee of the University of Occupational and Environmental Health.
2.2. Auditory brain stem response The animals were anesthetized with 35 mg/kg pentobarbital sodium administered intraperitoneally and placed in a prone position with their head fixed in a head-holder. Body temperature was kept at approximately 38 C by a heating pad. After a tracheostomy was performed, the animals were placed on a ventilator and were immobilized with an intramuscular injection of 3 mg/kg tubocurarine chloride. A stainless steel screw electrode was placed on the vertex as a recording electrode. A reference electrode was placed on the right temporal portion, and a ground electrode was placed at the midpoint between the two external auditory canals in the occipital portion. A click or a tone-burst stimulus was delivered to the right ear through an ear bar with a closed sound system. Using a digital synthesizer (1930A; NF Corporation, Yokohama, Japan), a series of 100-ls clicks was generated with a digital synthesizer at a rate of 10 pps. Tone bursts (10 ms with rise–fall times of 1 ms) of 2, 4, 8, 12, or 18 kHz were generated with an external generator (made in our laboratory) at a rate of 5 pps. The responses were amplified 1000· and bandpassfiltered (8–3000 Hz). Each run consisted of 128 responses, which were summated and averaged with an FFT analyzer (SA-74; Rion, Tokyo, Japan) in the time analysis mode. Acoustic stimuli were initially presented at an intensity of 80 dB, and decreased by 10-dB steps well above the threshold and then in 5-dB steps near the threshold until auditory brain stem response (ABR) waves disappeared. The threshold was defined as the minimum sound intensity at which a visible ABR wave was seen in two runs. The latency of each wavelet was measured by the cursor mode of the FFT analyzer. Calibrations of the sound intensity were made with a B & K 4128 probe microphone (Danish Pro Audio, Alleroed, Denmark) in a dummy ear, and were confirmed in the ear canal near the tympanic membrane of each guinea pig at the end of measurement. 2.3. Cochlear microphonics After the auditory brain stem response measurement, the amplitude of cochlear microphonics (CM) was measured to monitor the cochlear function. The right tympanic bulla was opened via a ventral approach and the bony cochlea was exposed. A tiny fenestration (about 150 lm in diameter) was made in the bony wall over the scala tympani near the round window. An enamelinsulated platinum electrode with an exposed tip 100 lm in diameter was inserted into the scala tympani through the surgical fenestration. An identical reference electrode was placed over the surface of the bulla. A ground Ag/AgCl disc electrode was placed on the cervical muscle. A pure tone sound of 4 kHz, ranging from 20
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to 90 dB SPL, was used to measure the amplitude of CM. All responses were double-traced to confirm reproducibility. 2.4. Light microscopy Immediately following the electrophysiological measurements, the animals were perfused transcardially with 10% formaldehyde. Bilateral temporal bones and the brain stem were removed and immersed in the same fixative overnight. After rinsing with 10 mM phosphate buffered saline at pH 7.2, the tissue was decalcified with 0.2 M ethylenediaminetetraacetic acid in 0.1 M phosphate buffer at pH 7.4 (PB) for 7 days and was then dehydrated and embedded in paraffin. Serial 7-lm-thick sections were stained with hematoxylin–eosin or luxol fast blue and viewed at 20–400·. 2.5. Transmission electron microscopy
Fig. 1. The ABR thresholds for tone-burst stimuli. The animals were subjected to ABR measurement at 1 (early group), 7 (intermediate group), and 14 days (late group) after the head blow as described in Section 2. **P < 0.01.
Immediately following the electrophysiological measurements, the animals were perfused transcardially with a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in PB. Bilateral temporal bones and the brain stem were removed and immersed in the same fixative for 2 h, postfixed with 1% osmium tetroxide in PB, dehydrated in a graded series of ethanol, and then embedded in epoxy resin. Ultrathin sections were made on an ultramicrotome, stained with uranyl acetate, and viewed with a JEM 1200EX electron microscope (JEOL, Tokyo, Japan).
Table 1 details the peak and interpeak latencies for the click stimuli at 80 dB peSPL. The I–V and I–II interpeak latencies were significantly prolonged in the early group. They were also significantly prolonged in the intermediate group, along with the wave I latency. In the late group, all three latencies had returned to the normal range. Fig. 2 illustrates representative ABR waveforms of the control and intermediate groups.
2.6. Statistics
3.3. Cochlear microphonics
Data were expressed as means ± SEM, and the statistical significance of differences was tested using Mann– WhitneyÕs U test. Differences were considered significant when the P value was <0.05.
No significant change of the input–output curves of CM was observed in the early, intermediate, or late groups in comparison with the control group (data not shown). 3.4. Macroscopic and light microscopic findings
3. Results 3.1. Impact force The impact force was 119.2 ± 8.1 N (n = 12), 110.8 ± 6.4 N (n = 12), and 100.9 ± 5.7 N (n = 11) in the early, intermediate, and late groups, respectively. There were no significant differences among the three groups.
When the animals were sacrificed and dissected, no fractures of the temporal bone or skull base were found in any of them. The brain including the brain stem were macroscopically intact as well. Likewise, at the light
Table 1 The peak and interpeak latencies for click stimuli at 80 dB peSPL Control
3.2. Auditory brain stem response The thresholds of ABR for the tone-burst stimuli are shown in Fig. 1. The threshold was unchanged in the early group, significantly increased at all frequencies in the intermediate group, and fully recovered in the late group.
Wave I latency I–II IPL II–V IPL I–V IPL
Early
Intermediate 0.08*
1.96 ± 0.05
1.99 ± 0.04
2.14 ±
1.01 ± 0.05 4.06 ± 0.06 5.07 ± 0.07
1.16 ± 0.05* 4.35 ± 0.11 5.51 ± 0.11**
1.26 ± 0.32** 4.17 ± 0.68 5.44 ± 0.09**
Late 1.94 ± 0.03 1.13 ± 0.06 4.12 ± 0.08 5.26 ± 0.06
Data values are mean ± SEM (ms). IPL, interpeak latency. * P < 0.05. ** P < 0.01.
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3.5. Transmission electron microscopic finding At the electron microscopic level, some eighth nerves were damaged, as shown in Fig. 3. In the early group, the lamellar structure of the myelin sheath was irregular and the periaxonal space was expanded. In the intermediate group, the myelin sheath was disintegrated and the periaxonal space was still enlarged. In the late group, these changes were partially reversed. In contrast, no abnormal finding was observed in the cochlea or brain stem in any of the groups.
4. Discussion
Fig. 2. Representative ABR waveforms (click stimuli at 80 dB peSPL). (a) Control group: wave I latency = 1.89 ms, I–II interpeak latency = 0.94 ms, I–V interpeak latency = 5.12 ms. (b) Intermediate group (day 7): wave I latency = 2.42 ms, I–II interpeak latency = 1.35 ms, I–V interpeak latency = 5.24 ms.
microscopic level, no hemorrhages, lacerations, or destroyed tissues were observed in the cochlea, eighth nerve, or brain stem of any of the animals.
In the present experiment, no fractures of the temporal bone or skull base were observed, and neither light nor electron microscopic observations showed cochlear damage. The cochlea was not only morphologically but also electrophysiologically intact, as shown by the normal CM. On the other hand, ABR showed temporary auditory threshold shifts and prolongation of wave I latency and I–II interpeak latency (Table 1). Waves I and II of ABR are thought to be derived from the distal intracanalar and intracranial portions of the eighth nerve, respectively (Moller and Jannetta, 1983; DÕHooge et al., 1999). Therefore, the present results of ABR strongly suggest damage to the eighth nerve, which was confirmed by elec-
Fig. 3. Transmission electron micrographs of the eighth nerve (scale bar = 1 lm). (a) Control group. (b) Early group (day 1): irregularity of the lamellar structure of the myelin sheath and expansion of the periaxonal space are seen. (c) Intermediate group (day 7): disintegration of the myelin sheath is observed, and the expansion of the periaxonal space remains. (d) Late group (day 14): both the disintegration of the myelin sheath and the expansion of the periaxonal space are partially reversed.
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tron microscopic observations of the disintegrated myelin sheath and the expanded periaxonal space. It is known that steep acceleration/deceleration of the skull upon a head blow, particularly to the occipital region, causes the brain to rotate and generates shear strain on the cranial nerves (Denny-Brown and Russell, 1941; Holbourn, 1943; Pudentz and Shelden, 1946; Gurdjian, 1970). The eighth nerve is most vulnerable to such mechanical stress, possibly because the nerve enters the petrous portion of the temporal bone immediately after emerging from the brain stem (Ylikoski et al., 1982; Goksu et al., 1992; Feneley and Murthy, 1994). The seventh nerve, which takes a similar route as the eighth nerve between the brain stem and temporal bone, is also vulnerable to injury from head concussion. However, because this nerve has a long path along the facial canal in the temporal bone, seventh nerve palsy following a head blow is ascribed not only to shear strain loaded to the root of the nerve but also to compression and circulatory disorder in the facial canal. Because multiple factors involved in the mechanism of seventh nerve palsy would render its analysis complicated, we did not investigate seventh nerve damage in the present study. Sekiya and Moller (1987, 1988) studied the mechanism underlying the occurrence of hearing loss after neurosurgery of the cerebellopontine angle in animal models. In dogs and monkeys, they directly loaded pressure onto the cerebellum and observed disintegration of the myelin sheath of the eighth nerve, which is consistent with the present results. The integrity of myelin is important for the electrophysiological function of the eighth nerve (Ylikoski and House, 1981; Sekiya and Moller, 1988; Spoendlin and Schrott, 1990; Scaioli et al., 1992; Zhou et al., 1995). Myelin-deficient mutant hamsters manifested prolonged wave I latency and an elevated auditory threshold in ABR measurement (Naito et al., 1999). Those authors suggested that disorders of the myelin sheath induce changes in conduction velocity and, therefore, desynchronization of neuronal firing in the auditory pathway. In addition, it is known that progressive hearing loss in patients with hereditary motor and sensory neuropathy originates from the degeneration of the myelin sheath of the eighth nerve (Scaioli et al., 1992). Our results also showed that the auditory threshold shift was temporary, which is consistent with the observation that the morphological change of the eighth nerve was reversible. Previous reports documented that considerable proportions of sensorineural hearing losses in patients with cerebral concussion without fracture recovered within six months after injury (Podoshin and Fradis, 1975). Recovery of demyelinated nerves has also been reported in other nervous systems. Ludwin (1978) studied a cuprizone-induced demyelination model of mice, and found that the superior cerebellar peduncles are specifically demyelinated and that remyelination occurs within 1–2 weeks after the removal of cuprizone.
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More recently, Van Reempts et al. (1993) demonstrated that remyelination of the demyelinated sciatic nerve began after approximately 1week, and was completed within 1 month. The onset of damage to the eighth nerve was delayed as shown in Figs. 1 and 3. Feldmann (1987) actually reported human cases with delayed-onset sensorineural hearing loss after head trauma. This phenomenon may be attributed to the delayed reaction of the injured nerve. Blight (1985) proposed two pathogeneses of spinal cord damage by mechanical stress. One is the immediate reaction directly induced by the stress. The other is the delayed reaction derived from biochemical processes that occur several days after injury. For example, the production of cytokines and the release of neurotransmitters, such as glutamate and nitric oxide, may participate in delaying the reaction (Blight and Decrescito, 1986; MacDermott et al., 1986; Panter et al., 1990; Dawson et al., 1991, 1993). 5. Conclusions Electrophysiological and histopathological reactions were studied in a guinea pig model of sensorineural hearing loss following a blow to the head in a freely movable position, mimicking real human head injury. Threshold shifts and prolongations of the I–V and I–II interpeak latencies and of the wave I latency were observed in ABR measurements, whereas no significant changes in CM were detected. Electron microscopic observation of the eighth nerve revealed that the myelin sheath was irregular and the periaxonal space enlarged. Such changes occurred with a delay and were reversible. These results suggest that sensorineural hearing loss in this model is attributable to eighth nerve dysfunction rather than to cochlear impairment. The mechanism underlying eighth nerve damage in the present experimental model needs to be further investigated by biochemical, cell physiological, and molecular biological methods in future studies. Acknowledgements This research was partially supported by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Young Scientists (B), 169791033, 2004. References Blight, A.R., 1985. Delayed demyelination and macrophage invasion: a candidate for secondary cell damage in spinal cord injury. Cent. Nerv. Syst. Trauma 2, 299–315. Blight, A.R., Decrescito, V., 1986. Morphometric analysis of experimental spinal cord injury in the cat: the relation of injury intensity to survival of myelinated axons. Neuroscience 19, 321–341.
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