Acta Oto-Laryngologica
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Vestibular-oculomotor, Opto-oculomotor and Visual Function in the Rat after Long-term Inhalation Exposure to Toluene Per Nylen, Birgitta Larsby, Ann-Christin Johnson, Birgitta Eriksson, Gunnar Höglund & Richard Tham To cite this article: Per Nylen, Birgitta Larsby, Ann-Christin Johnson, Birgitta Eriksson, Gunnar Höglund & Richard Tham (1991) Vestibular-oculomotor, Opto-oculomotor and Visual Function in the Rat after Long-term Inhalation Exposure to Toluene, Acta Oto-Laryngologica, 111:1, 36-43 To link to this article: http://dx.doi.org/10.3109/00016489109137352
Published online: 08 Jul 2009.
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Acta Otolaryngol (Stockh) 1991; 1 1 1: 36-43
Vestibular-oculomotor, Opto-oculomotor and Visual Function in the Rat after Long-term Inhalation Exposure to Toluene PER NYLEN.'.' BIRGITTA LARSBY.3 ANN-CHRISTIN JOHNSON.'.' BIRGITTA ERIKSSON.' GUNNAR HOGLUND'.' and RICHARD THAM'
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Friim the 'Drpcirlmen~of Ncrrroniedicint-. Ntttiond Instirrtrc~qf Oc~rr~pcrtiontrl Hcwltk. S-17\84 Solnet 'Depcirinierrt of Physiology. Knrolimkn Iti.stitiitc. S-10401 Sto(~kIw1ni.crnd 'Depctrrnienr of O t o l o r ~ n g o l o gUniversity ~. Hospircrl. S-581 85 Li17kiipiti.e. Swedc'n
Nplin P, Larsby B. Johnson A-C. Eriksson B. Hoglund G. Tham R. Vestibular-oculomotor. opto-oculomotor and visual function in the rat after long-term inhalation exposure to toluene. Acta Otolaryngol (Stockh) 1991: 111: 36-43. Pigmented rats were exposed to toluene ( I OOO ppm. 21 h/day) for 6 or 11 weeks. The function of the vestibulo- and opto-oculomotor systems was tested one month after the end of the exposure by recording of nystagmus. induced by vestibular or optokinetic stimuli. The eye movements were recorded by a magnetic search coil technique. The optokinetic gain in the exposed animals was reduced compared to :I control group. There was also a slight reduction in gain during sinusoidal oscillatory vestibular stimulation. No effect of the toluene exposure on the gain or duration of nystagmus during acceleratory o r decelerator! rotatory stimulation was demonstrated. nor was there any change in the duration of the optokinetic after-nystagmus. The function of the visual system \%IS tehted 3 to 5 days after exposure by recording the electroretinogram and the visual evoked response. The cunduction velocity in peripheral nerve was also measured. No effect of the toluene exposure on these variables was seen. The results indicate that long-term inhalation of toluene ciiuses ;i long-lasting. possibly permanent. lesion within the ~ehtihulo-cer.ehellum.They gave no evidence that such exposure affects peripheral vestibular o r visual function. Kcr. wr)rr/.c:
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INTRODUCTION Toluene. which is one of the most commonly used industrial solvents. has a low general toxicity. However, acute neurological effects following toluene abuse or accidental exposure have been reported. The symptoms include disorientation. tremor. mood lability. tinnitus. diplopia. hallucinations. dysarthria. ataxia. convulsions. and coma. Chronic symptoms reported after long-term industrial exposure to toluene include changes in visual intelligence. disturbance of sensory function. memory functions and verbal intelligence (for reviews see (1) and (2)). Vestibular hyporeflexia has been reported in workers exposed to toluene (3). and alterations in visual evoked response (VERI have been found among workers exposed to a mixture of industrial solvents. and in rats exposed to toluene for 30 days (4). In the rat. toluene exposure also causes severe auditory impairment. at least in part due to structural alterations in the cochlea ( 5 ) . The pathophysiological mechanisms underlying the clinical symptoms of toluene induced central nervous system (CNS) toxicity are largely unknown. partly due to lack of experimental models. Exceptions are the vestibulo- and opto-oculomotor systems that have proven to be useful for studying acute effects of toluene on the CNS in both humans and rats (6, 7). The present study concerns the long term effects of toluene inhalation on the vestibulooculomotor function and the opto-oculomotor function. The recently developed applica-
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Acta Otolaryngol (Stockh) 111
Vestibular function after exposure to toluene
tion of the magnetic search coil system to small laboratory animals (8). which allows a direct recording of the eye movements, was used. Visual function, which contributes to the opto-oculomotor response, was studied by recording of the electroretinogram and visual evoked potentials.
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MATERIAL AND METHODS Exposure. Pigmented rats (DA-HAN, male) aged 4 to 5 months were divided into 2 groups. Group A (for test of the vestibular system): Fifteen rats were exposed to toluene ( I OOO ppm, 21 h/day, 7 daydweek). Five of the rats were exposed for 6 weeks and the remaining 10 rats for 1 1 weeks. Control groups ( 5 and 10 rats, respectively) inhaled air without added toluene. Group B (for test of the visual system and peripheral nerve): Sixteen rats were exposed to toluene ( 1 000 ppm, 21 h/day. 5 dayslweek). Ten of the rats were exposed for 8 weeks and the remaining 6 rats for 12 weeks. Control groups (10 and 6 rats, respectively) inhaled air without added toluene. The exposure technique has been described earlier (9) and is briefly summarized here. The exposure chambers were made of stainless steel and contained 9 cages. Fluid solvent (Toluene GR, Merck) was vapourized on a filter (stainless steel thread). mixed with air and released into the animal compartment. A constant air flow. corresponding to 10 to 15 complete air changes per hour, was obtained by maintaining a slight subatmospheric pressure (-30 Pa) in the chamber. The exposures lasted from I2 a.m. to 9 a.m.. and light was on between 6 a.m. and 6 p.m. Solvent concentration in the chambers was monitored continuously using a gas spectrometer (Miran I A. Wilks, Foxboro). The mean concentration was 1002 (SD=62) ppm. Temperature and humidity in the chambers (recorded daily at the start and end of the exposure) were 21 (SD=0.3) "C and 65 (SD=3.3) 95. respectively. Test of the uestibulo- and opto-ocrrloinotor systerns. Three weeks after the end of the exposure, a nut was fixed to the skull of each rat under ether anaesthesia. The nut was used to restrain the head during the subsequent experiment. After this procedure the rats were given a recovery period of at least one week before recordings were made. Recordings of eye movements were made by a magnetic search coil system (8).Details concerning the stirnulation equipment have been given by Larsby et al. ( 7 ) . The following tests were made: ( a ) Rotatory acceleration/deceleration. The acceleration was 13.3"ls' during 9 s followed by rotation at constant speed (12Oo/s) for at least one minute after the nystagmus had ceased. The turn-table was then decelerated with 13.3"/s2.The stimulation was carried out in ambient light every 5 minutes, using alternating clockwise and counter-clockwise acceleratioddeceleration. The slow phase velocity (SPV) of the rotatory nystagmus was measured and used to calculate velocity gain. expressed as slow-phase eye velocity divided by head (i.e. turn-table) velocity. The duration of the nystagmus from the beginning of stimulation was also calculated. The average of the results of 3 clockwise and 3 counter-clockwise accelerations/decelerationswas calculated. ( b ) Sinusoidal oscillation. The frequency and maximum velocity of the sine wave stimulus was either 0.8 Hz, 90°/s or 0.2 Hz. 90"ls. The stimulation was carried out in darkness and lasted 40 s with an interval of 5 min between each test period. Gain and phase between SPV and head velocity were calculated. The average of 3 stimulations at each frequency was calculated. ( c )Randomized oscillation. The turn-table was moved using an unpredictable. pseudorandomized protocol, which covered the frequency range from 0.1 to 1 .O Hz. The stimulation lasted for 60 s and was made in ambient light every 5 minutes. Fast Fourier transform
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Fig. I . Schematic drawing of visual evoked
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response from normal rat indicating recorded latencies (from stimulus onset to PI, N I and P2) and amplitudes (PI-NI and Nl-P2). ms
and power spectrum analysis techniques were used to calculate gain (SPVhead velocity) and phase. The average of 5 stimulations was calculated. (6) Optokinetic stimulation. The velocities of the visual pattern were 5, 10, 15, 20, 30, 40, 50 and 60%, clockwise and counter-clockwise. The average gain of one clockwise and one counter-clockwise stimulation was calculated at each velocity. The duration of the optokinetic after-nystagmus (OKAN) and the after-after nystagmus (OKAAN) was measured in darkness after the optokinetic stimulation at 20"s. The average duration of OKAN and OKAAN was calculated from 3 registrations in both directions. Test ofthe uisual system and peripheral nerve. The electroretinogram (ERG),the visual evoked response (VER), and the nerve conduction velocity (NCV) in the tail were recorded 2 to 5 days after the end of the toluene exposure. ( a )ERG. The rat was kept in complete darkness for at least 12 h before registration. The preparation for recording was made under dim red light. The left eye was used for registration. Cornea and conjunctiva were anaesthetized (Tetrakain 456, Alcon) and the pupil dilated (Mydriacyl, Alcon and Isopto-Plain, Alcon). The eye was illuminated by a xenon lamp (Zeiss 900 W)and fibre optics. The light was filtered through water and a neutral density filter (2 log units). The intensity of the light beam at the cornea was 9x lo" photons/cm'/s. The stimulus duration was 0.25 s and the interval between each stimulus
Fig. 2. Eye position (upper frucings) and eye velocity (kowrr.frctcings) in normal rat during and after rotatory acceleration (acc; 13%'; left), during continuous sinusoidal oscillation (frequency 0.2 Hz; middle) and during optokinetic stimulation (OKs; 20%; right).
Acta Otolaryngol (Stockh) 11 1
Vestibular function after exposure to toluene
Gain 1.0 _I
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Fig. 3. Mean velocity gain elicited by rotatory acceleration/deceleration. Measurements made at least 4 weeks after inhalation exposure to toluene (1000 ppm. 21 Wday, 7 dayslweek for I I weeks). Horizontal bar indicates stimulus (acceleration or deceleration, 13.3%'). Vertical bars indicate one SD. U, rats (n=10) exposed to toluene. acceleration; A-A , same exposed rats, deceleration: 0 -4 control . rats (n= 10). acceleration: &-A. same control rats. deceleration.
30s. Recordings were made using a corneal contact lens made of a transparent silicone elastomere (Sylgard 184). The lens and the reference area (vertex with electrode paste applied) were connected to calomel electrodes by plastic tubes filled with saline. A metal surface electrode was placed on the tail and connected to ground. The response was fed into a DC-amplifier and stored in a computer (Metric 8). The average of 5 ERG recordings were used for calculation of latency and amplitude of a- and b-waves, trough. and c-wave. (b) VER. An active electrode was placed above the visual cortex. and a reference electrode in the midline, 10 mm rostra1 to the vertex. The stimulus was an electronic flash generated by a photo stimulator (Grass PS 22C). Sixty-four stimuli were presented at a rate of 2 stimulils. Three latencies and 2 amplitudes of 3 prominent peaks (see Fig. 1) were calculated from an average of these 64 recordings (amplifier signal averager: Medelec MS 92). (c) NCV. Two surface electrodes, applied on the proximal part of the tail. were used for electric stimulation (intensity 100 V, duration 0.2 ms). The compound action potential in response to one stimulus was recorded by two pairs of needle electrodes (placed 3 cm and 7 cm distal to the negative stimulation electrode) and an amplifier signal averager (Medelec MS 92). One recording was made on each animal. NCV was calculated as the distance between the proximal electrode in each electrode pair divided by the difference in mean latency of the main peak in the short and the long distance recording. Statistical ana/ysis. Differences between groups were analysed using two-factor repeated ANOVA or Student's r-test.
RESULTS A rotatory acceleration of 13"/s2,performed in ambient light, elicited nystagmus both in exposed rats and in controls (Fig. 2. left tracings). Deceleration elicited nystagmus with a lower gain and shorter duration than that recorded during acceleration (Fig. 3). Even after 11 weeks of exposure no difference in gain was established between exposed rats and controls during acceleration or deceleration (ANOVA. p>0.05). nor was there any difference in duration of nystagmus between the two groups during acceleratory or deceleratory stimulation (acceleration: 53+ 14 s vs. 48+8 s. I-test p 0 . 0 5 : deceleration: 7 3 s vs. S F 2 s t-test p0.05). During sinusoidal oscillatory stimulation in the dark at 0.2 Hz (Fig. 2. middle tracings) the gain was lower in the exposed rats than in the controls (Fig. 4. upper right graph: r-test. pO.OS). During randomized
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Fig. 4 . Effect of toluene inhalation on mean gain (upper graph) and phase (lower graph) as a function of frequency of randomized (left) and sinusoidal (right) oscillatory stimulation. Measurements made at least 4 weeks after inhalation exposure to toluene ( I OOO ppm. 21 hiday, 7 daysiweek). Vertical bars indicate one SD. A--A, rats ( n = 3 exposed to toluene for 6 weeks; 0-4. rats (n=10)exposed to toluene for 11 weeks; 0-4,control rats ( 1 1 weeks. n= 10).
oscillatory stimulation in ambient light (Fig. 4, upper left graph) the exposed rats had a lower gain than that of the controls at all frequencies (ANOVA, 6 weeks exposure, p>0.05; 11 weeks exposure, ~(0.05). N o difference in phase (Fig. 4, lower graphs) was established (ANOVA, p>0.05), neither during sinusoidal nor during randomized oscillation. Optokinetic stimulation with a pattern, moving with constant velocity in either direction, elicited nystagmus in exposed as well as in non-exposed rats (Fig. 2 . right tracings). The gain was lower in rats exposed for 6 or 11 weeks compared to controls (ANOVA. Gain 1.0
0.5
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Fig. 5 . Effect of toluene inhalation ( I 000 ppm. 21 hiday, 7 daysiweek) on mean gain of optokinetic nystagmus relative to stimulus velocity. Vertical bars indicate one SD. A-A. rats ( n = 5 ) exposed to toluene f o r 6 weeks: 0-4.rats ( r z = 1 0 1 exposed to toluene for 1 1 weeks: c-4. control rats ( 1 I weeks, n= 10).
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Acta Otolarvnnol (Stockh) 11 1
pO.OS), or in the duration of OKAAN (3t-1 s vs. 3+3 s, p 0 . 0 5 ) . The results of the VER. ERG, and NCV recordings are summarized in Table I. No difference between exposed rats and controls was demonstrated (t-test, p>O.O5) in any of these recordings.
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DISCUSSION In the present study, functional variables of the vestibulo- and opto-oculomotor systems were tested one month after the end of a long-term inhalation exposure (6 or 11 weeks) to toluene. The gain during constant angular acceleration or deceleration of the exposed rats was not lower than that of the controls. This indicates that peripheral vestibular function was not affected, since a peripheral impairment would have resulted in a lower gain in the exposed rats. A long-lasting impairment of central vestibular function is indicated by a reduction in gain during sinusoidal oscillation at 0.2 Hz and during unpredictable. randomized oscillatory stimulation (after toluene exposure for I 1 weeks). Since the acceleration and velocity of head rotation were continuously changing during these tests, they are regarded to reveal disturbances in the “direct pathways”, which are responsible for fast changes in the central vestibular system (10). The cerebellar flocculus is regarded to be an essential link within these direct pathways. and reduction in gain is seen in pigmented rats after surgical Table I. Latencies and riinplititdes oj ele~trorerinoRr~ii?i. visual evoked response, and peripheral nerve conduction velocit? in rats &er inhulation exposirre to toirrene 11000 ppm, 21 hlday, 5 dayslweek, I 2 weeks: n=6) No differences between exposed groups and controls were demonstrated (t-test. p>O.O5) Controls Mean E/ecrrnretinogrrirn Latency (ms) a- wave --._ 77 5 b-wave 70 trough 405 c-wave 1210 Amplitude CpV) a-wave 11.0 b-wave 24 I trough 3.0 c-wave 54.0 Visiicil euoked response Latency (ms) PI 36.6 N1 24.1 P2 59.2 Amplitude (pV) PI-N1 19.5 N I-P2 19.6
Exposed
SD
Mean
3.5 4. I 44.7 254
25 70 400 1211
SD
3.2 11.2
28.9 I26
5.7 50.5 11.5 17.7
7.8 256 4.7 70.8
3.9 29.9 20.0 30.2
I .o 1.1 2.4
37.5 24.8 54.3
3.3 ?.3 2.9
6.1 5.4
19.7 18.4
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Acta Otolaryngol (Stockh) 1 11
flocculectomy (11). Hence the reduction in gain after toluene exposure may be due to a cerebellar disturbance. The most prominent finding after the long-term exposure to toluene was a reduced gain during optokinetic stimulation. The pathways of the accessory optic system that convey optokinetic impulses also convey impulses elicited by flash stimulation, at least in rabbits (12). No effect of the toluene exposure on the ERG and VER recordings (using single flash stimuli) was seen, indicating that no gross functional impairment of the peripheral visual system had occurred. The rats used for visual testing were. however, exposed to a somewhat tower total dose than the animals used for optokinetic testing ( 5 dayslweek for 12 weeks instead of 7 days/week for 11 weeks). In addition, Dyer et al. 14), using paired flash stimuli to test the VER, found a larger variation in the latencies of some peaks of the VER after the second flash in rats exposed to toluene. Such possible small changes in VER latencies (up to about 5 ms) can, however, not explain the reduction in gain in the vestibulo- or opto-oculomotor response. This response is built up during several seconds of continuous optokinetic stimulation. As long as the eyes are not following the optic stimulus exactly, there is a retinal slip which causes adjustment of the eye movements. A small change in conduction velocity is unlikely to affect the ability to detect a retinal slip and hence to cause a reduction in gain of optokinetic nystagmus. The observed reduction in gain is more probably explained by a disturbance of the cerebellar function, similar to that indicated during vestibular stimulation. This assumption is supported by several reports suggesting that the flocculus contributes to the regulation of the gain of optokinetic nystagmus (for review, see (13)), and the observation that gain is reduced in monkeys, rabbits and cats after flocculectomy or cerebellectomy (14). The present results. obtained after long-term exposure to toluene, partly differ from those of corresponding measurements during acute exposure. A prolonged duration of the post-acceleratory nystagmus, and of the optokinetic after-nystagmus, has been demonstrated during acute exposure to toluene (7). This finding suggests a disturbance of the velocity storage mechanism that is part of the central processing of optokinetic as well as vestibular signals. No such impairment of the velocity storage mechanism was seen 4 weeks after the end of the long-term exposure made in the present study. In other respects, the observations on long-term exposure are similar to those made on acutely exposed rats. Thus, both types of exposure cause a reduction in the gain during optokinetic stimulation. The results reported here indicate that long-term exposure to toluene causes long-lasting central, possibly cerebellar, dysfunction. These findings are consistent with a report on workers who showed reduced visual suppression after long-term exposure to another benzene derivate, styrene (15). This symptom is found also in patients suffering from chronic toxic encephalopathy, believed to be caused by exposure to organic solvents ( I 6). Cerebellar lesions due to exposure to toluene are suggested also by clinical symptoms and by computer tomography of toluene abusers (17, 18, 19, 20). The results in the present study demonstrate that the opto- and vestibulo-oculomotor systems are useful as experimental models for the understanding of mechanisms underlying chronic functional disturbances caused by organic solvents. The present experimental observations on rats agree with clinical findings on humans, supporting the interpretation that solvent exposure can cause long-lasting impairment of CNS functions.
ACKNOWLEDGEMENTS We thank Maud Hagman and Gunilla Linder for skillful technical assistance. The investigation was supported by the Swedish Work Environment Fund (grants 84-1291 and 85-0479).
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REFERENCES I . WHO. Environmental Health Criteria 52: Toluene. World Health Organization. Geneva, 1985. 2. Benignus VA. Neurobehavioral effects of toluene: a review. Neurobehav Toxicol Teratol 1981: 3: 407-15. -~ 3. Coscia CG, Tabaro G. Albera C et al. Alterazioni vestibolari nell’esposizione a toluene. Med Lav 1983; 74: 23-9. 4. Dyer RS, Muller KE. Janssen R, Barton CN. Boyes WK, Benignus VA. Neurophysiological effects of 30 days chronic exposure to toluene in rats. Neurobehav Toxicol Teratol 1984: 6: 363-8. 5 . Sullivan MJ, Rarey KE, Conolly RB. Ototoxicity of toluene in rats. Neurotoxicol Teratol 1989; 10: 525-30. 6. HydBn D, Larsby B, Andersson H, Odkvist LM, Liedgren SRC. Tham R. Impairment of visuovestibular interactions in humans exposed to toluene. ORL 1983: 45: 262-9. 7. Larsby B, Tham R, Eriksson B, 6dkvist LM. The effect of toluene on the vestibulo- and optooculomotor system in rats. Acta Otolaryngol (Stockh) 1986: 101: 422-8. 8. Hess BJM, Precht W, Reber A, Cazin L. Horizontal optokinetic ocular nystagmus in the pigmented rat. Neurosci 1985; 15: 97-107. 9. Johnson AC, Juntunen L. Nylkn P. Borg E, Hoglund G. Effect of interaction between noise and toluene on auditory function in the rat. Acta Otolaryngol (Stockh) 1988: 105: 5643. 10. Raphan T, Cohen B. Velocity storage and the ocular response to multidimensional vestibular stimuli. In: Berthoz A, Melvill L, Jones G, eds. Adaptive mechanisms in gaze-control. Facts and theories. Amsterdam: Elsevier Science Publisher. 1985: 12343. 11. Lannou J, Hess BJM, Precht W, Reber A. Effects of bilateral flocculectomy on the horizontal vestibulo-ocular reflex (HVOR) in the rat. Neurosci Lett 1985: Suppl 22: 275. 12. Soodak RE, Simpson JI. The accessory optic system of rabbit. 1. Basic visual response properties. J Neurophys 1988; 60: 2037-54. 13. Van Neerven J, Pompeiano 0, Collewijn H. Depression of the vestibulo-ocular and optokinetic responses by intrafloccular microinjection of GABA-A and GABA-B agonists in the rabbit. Arch Ital Biol 1989: 127: 243-63. 14. Precht W, Blanks RHI, Strata P. Montarolo P. On the role of the subprimate cerebellar flocculus in the optokinetic reflex and visual-vestibular interaction. In: Bloedel JR. Dichgans J. Precht W. eds. Cerebellar functions. Heidelberg: Springer-Verlag. 1985: 86-107. 15. Moller C, Odkvist LM, Larsby B. Tharn R. Ledin T. Bergholtz LM. Otoneurological findings in workers exposed to styrene. Scand J Work Environ Health 1990. In press. 16. Moller C, Odkvist LM, The11 J. et al. Otoneurological findings in psycho-organic syndrome caused by industrial soIvent exposure. Acta Otolaryngol (Stockhl 1989: 107: 5-12. 17. Fornazzari L, Wilkinson DA, Kapur BM. Carlen PL. Cerebellar. cortical and functional impairment in toluene abusers. Acta Neurol Scand 1983: 67: 319-29. 18. Malm G , Lying-Tunell U. Cerebellar dysfunction related to toluene sniffing. Acta Neurol Scand 1980; 62: 188-90. 19. Streicher HZ, Gabow PA, Moss AH. Kono D, Kaehny WD. Syndromes of toluene sniffing in adults. Ann Int Med 1981: 94: 758-62. 20. Lazar RB, Ho SU, Melen 0. Daghestani AN. Multifocal central nervous system damage caused by toluene abuse. Neurology 1983; 33: 1337-40. Manriscript received April 4 , 1990: mcepted Max 10. 1990
Address for correspondence: Per NylCn. National Institute of Occupational Health. S-17184 Solna. Sweden
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ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Vestibular-oculomotor, Opto-oculomotor and Visual Function in the Rat after Long-term Inhalation Exposure to Toluene Per Nylen, Birgitta Larsby, Ann-Christin Johnson, Birgitta Eriksson, Gunnar Höglund & Richard Tham To cite this article: Per Nylen, Birgitta Larsby, Ann-Christin Johnson, Birgitta Eriksson, Gunnar Höglund & Richard Tham (1991) Vestibular-oculomotor, Opto-oculomotor and Visual Function in the Rat after Long-term Inhalation Exposure to Toluene, Acta Oto-Laryngologica, 111:1, 36-43 To link to this article: http://dx.doi.org/10.3109/00016489109137352
Published online: 08 Jul 2009.
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Article views: 2
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ioto20 Download by: [University of Cincinnati Libraries]
Date: 23 March 2016, At: 19:10
Acta Otolaryngol (Stockh) 1991; 1 1 1: 36-43
Vestibular-oculomotor, Opto-oculomotor and Visual Function in the Rat after Long-term Inhalation Exposure to Toluene PER NYLEN.'.' BIRGITTA LARSBY.3 ANN-CHRISTIN JOHNSON.'.' BIRGITTA ERIKSSON.' GUNNAR HOGLUND'.' and RICHARD THAM'
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Friim the 'Drpcirlmen~of Ncrrroniedicint-. Ntttiond Instirrtrc~qf Oc~rr~pcrtiontrl Hcwltk. S-17\84 Solnet 'Depcirinierrt of Physiology. Knrolimkn Iti.stitiitc. S-10401 Sto(~kIw1ni.crnd 'Depctrrnienr of O t o l o r ~ n g o l o gUniversity ~. Hospircrl. S-581 85 Li17kiipiti.e. Swedc'n
Nplin P, Larsby B. Johnson A-C. Eriksson B. Hoglund G. Tham R. Vestibular-oculomotor. opto-oculomotor and visual function in the rat after long-term inhalation exposure to toluene. Acta Otolaryngol (Stockh) 1991: 111: 36-43. Pigmented rats were exposed to toluene ( I OOO ppm. 21 h/day) for 6 or 11 weeks. The function of the vestibulo- and opto-oculomotor systems was tested one month after the end of the exposure by recording of nystagmus. induced by vestibular or optokinetic stimuli. The eye movements were recorded by a magnetic search coil technique. The optokinetic gain in the exposed animals was reduced compared to :I control group. There was also a slight reduction in gain during sinusoidal oscillatory vestibular stimulation. No effect of the toluene exposure on the gain or duration of nystagmus during acceleratory o r decelerator! rotatory stimulation was demonstrated. nor was there any change in the duration of the optokinetic after-nystagmus. The function of the visual system \%IS tehted 3 to 5 days after exposure by recording the electroretinogram and the visual evoked response. The cunduction velocity in peripheral nerve was also measured. No effect of the toluene exposure on these variables was seen. The results indicate that long-term inhalation of toluene ciiuses ;i long-lasting. possibly permanent. lesion within the ~ehtihulo-cer.ehellum.They gave no evidence that such exposure affects peripheral vestibular o r visual function. Kcr. wr)rr/.c:
cerehrlltti~i.vrs~ihrrlw.s.vsfrrii, oiptokiiie~ic.sy.sti'nr. ci.siitil e w i k i d piitcviticrl. i~l~,i.triir~.tiriiigrmn. tierve condirctioii uelociry.
INTRODUCTION Toluene. which is one of the most commonly used industrial solvents. has a low general toxicity. However, acute neurological effects following toluene abuse or accidental exposure have been reported. The symptoms include disorientation. tremor. mood lability. tinnitus. diplopia. hallucinations. dysarthria. ataxia. convulsions. and coma. Chronic symptoms reported after long-term industrial exposure to toluene include changes in visual intelligence. disturbance of sensory function. memory functions and verbal intelligence (for reviews see (1) and (2)). Vestibular hyporeflexia has been reported in workers exposed to toluene (3). and alterations in visual evoked response (VERI have been found among workers exposed to a mixture of industrial solvents. and in rats exposed to toluene for 30 days (4). In the rat. toluene exposure also causes severe auditory impairment. at least in part due to structural alterations in the cochlea ( 5 ) . The pathophysiological mechanisms underlying the clinical symptoms of toluene induced central nervous system (CNS) toxicity are largely unknown. partly due to lack of experimental models. Exceptions are the vestibulo- and opto-oculomotor systems that have proven to be useful for studying acute effects of toluene on the CNS in both humans and rats (6, 7). The present study concerns the long term effects of toluene inhalation on the vestibulooculomotor function and the opto-oculomotor function. The recently developed applica-
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Vestibular function after exposure to toluene
tion of the magnetic search coil system to small laboratory animals (8). which allows a direct recording of the eye movements, was used. Visual function, which contributes to the opto-oculomotor response, was studied by recording of the electroretinogram and visual evoked potentials.
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MATERIAL AND METHODS Exposure. Pigmented rats (DA-HAN, male) aged 4 to 5 months were divided into 2 groups. Group A (for test of the vestibular system): Fifteen rats were exposed to toluene ( I OOO ppm, 21 h/day, 7 daydweek). Five of the rats were exposed for 6 weeks and the remaining 10 rats for 1 1 weeks. Control groups ( 5 and 10 rats, respectively) inhaled air without added toluene. Group B (for test of the visual system and peripheral nerve): Sixteen rats were exposed to toluene ( 1 000 ppm, 21 h/day. 5 dayslweek). Ten of the rats were exposed for 8 weeks and the remaining 6 rats for 12 weeks. Control groups (10 and 6 rats, respectively) inhaled air without added toluene. The exposure technique has been described earlier (9) and is briefly summarized here. The exposure chambers were made of stainless steel and contained 9 cages. Fluid solvent (Toluene GR, Merck) was vapourized on a filter (stainless steel thread). mixed with air and released into the animal compartment. A constant air flow. corresponding to 10 to 15 complete air changes per hour, was obtained by maintaining a slight subatmospheric pressure (-30 Pa) in the chamber. The exposures lasted from I2 a.m. to 9 a.m.. and light was on between 6 a.m. and 6 p.m. Solvent concentration in the chambers was monitored continuously using a gas spectrometer (Miran I A. Wilks, Foxboro). The mean concentration was 1002 (SD=62) ppm. Temperature and humidity in the chambers (recorded daily at the start and end of the exposure) were 21 (SD=0.3) "C and 65 (SD=3.3) 95. respectively. Test of the uestibulo- and opto-ocrrloinotor systerns. Three weeks after the end of the exposure, a nut was fixed to the skull of each rat under ether anaesthesia. The nut was used to restrain the head during the subsequent experiment. After this procedure the rats were given a recovery period of at least one week before recordings were made. Recordings of eye movements were made by a magnetic search coil system (8).Details concerning the stirnulation equipment have been given by Larsby et al. ( 7 ) . The following tests were made: ( a ) Rotatory acceleration/deceleration. The acceleration was 13.3"ls' during 9 s followed by rotation at constant speed (12Oo/s) for at least one minute after the nystagmus had ceased. The turn-table was then decelerated with 13.3"/s2.The stimulation was carried out in ambient light every 5 minutes, using alternating clockwise and counter-clockwise acceleratioddeceleration. The slow phase velocity (SPV) of the rotatory nystagmus was measured and used to calculate velocity gain. expressed as slow-phase eye velocity divided by head (i.e. turn-table) velocity. The duration of the nystagmus from the beginning of stimulation was also calculated. The average of the results of 3 clockwise and 3 counter-clockwise accelerations/decelerationswas calculated. ( b ) Sinusoidal oscillation. The frequency and maximum velocity of the sine wave stimulus was either 0.8 Hz, 90°/s or 0.2 Hz. 90"ls. The stimulation was carried out in darkness and lasted 40 s with an interval of 5 min between each test period. Gain and phase between SPV and head velocity were calculated. The average of 3 stimulations at each frequency was calculated. ( c )Randomized oscillation. The turn-table was moved using an unpredictable. pseudorandomized protocol, which covered the frequency range from 0.1 to 1 .O Hz. The stimulation lasted for 60 s and was made in ambient light every 5 minutes. Fast Fourier transform
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Fig. I . Schematic drawing of visual evoked
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response from normal rat indicating recorded latencies (from stimulus onset to PI, N I and P2) and amplitudes (PI-NI and Nl-P2). ms
and power spectrum analysis techniques were used to calculate gain (SPVhead velocity) and phase. The average of 5 stimulations was calculated. (6) Optokinetic stimulation. The velocities of the visual pattern were 5, 10, 15, 20, 30, 40, 50 and 60%, clockwise and counter-clockwise. The average gain of one clockwise and one counter-clockwise stimulation was calculated at each velocity. The duration of the optokinetic after-nystagmus (OKAN) and the after-after nystagmus (OKAAN) was measured in darkness after the optokinetic stimulation at 20"s. The average duration of OKAN and OKAAN was calculated from 3 registrations in both directions. Test ofthe uisual system and peripheral nerve. The electroretinogram (ERG),the visual evoked response (VER), and the nerve conduction velocity (NCV) in the tail were recorded 2 to 5 days after the end of the toluene exposure. ( a )ERG. The rat was kept in complete darkness for at least 12 h before registration. The preparation for recording was made under dim red light. The left eye was used for registration. Cornea and conjunctiva were anaesthetized (Tetrakain 456, Alcon) and the pupil dilated (Mydriacyl, Alcon and Isopto-Plain, Alcon). The eye was illuminated by a xenon lamp (Zeiss 900 W)and fibre optics. The light was filtered through water and a neutral density filter (2 log units). The intensity of the light beam at the cornea was 9x lo" photons/cm'/s. The stimulus duration was 0.25 s and the interval between each stimulus
Fig. 2. Eye position (upper frucings) and eye velocity (kowrr.frctcings) in normal rat during and after rotatory acceleration (acc; 13%'; left), during continuous sinusoidal oscillation (frequency 0.2 Hz; middle) and during optokinetic stimulation (OKs; 20%; right).
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Vestibular function after exposure to toluene
Gain 1.0 _I
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Fig. 3. Mean velocity gain elicited by rotatory acceleration/deceleration. Measurements made at least 4 weeks after inhalation exposure to toluene (1000 ppm. 21 Wday, 7 dayslweek for I I weeks). Horizontal bar indicates stimulus (acceleration or deceleration, 13.3%'). Vertical bars indicate one SD. U, rats (n=10) exposed to toluene. acceleration; A-A , same exposed rats, deceleration: 0 -4 control . rats (n= 10). acceleration: &-A. same control rats. deceleration.
30s. Recordings were made using a corneal contact lens made of a transparent silicone elastomere (Sylgard 184). The lens and the reference area (vertex with electrode paste applied) were connected to calomel electrodes by plastic tubes filled with saline. A metal surface electrode was placed on the tail and connected to ground. The response was fed into a DC-amplifier and stored in a computer (Metric 8). The average of 5 ERG recordings were used for calculation of latency and amplitude of a- and b-waves, trough. and c-wave. (b) VER. An active electrode was placed above the visual cortex. and a reference electrode in the midline, 10 mm rostra1 to the vertex. The stimulus was an electronic flash generated by a photo stimulator (Grass PS 22C). Sixty-four stimuli were presented at a rate of 2 stimulils. Three latencies and 2 amplitudes of 3 prominent peaks (see Fig. 1) were calculated from an average of these 64 recordings (amplifier signal averager: Medelec MS 92). (c) NCV. Two surface electrodes, applied on the proximal part of the tail. were used for electric stimulation (intensity 100 V, duration 0.2 ms). The compound action potential in response to one stimulus was recorded by two pairs of needle electrodes (placed 3 cm and 7 cm distal to the negative stimulation electrode) and an amplifier signal averager (Medelec MS 92). One recording was made on each animal. NCV was calculated as the distance between the proximal electrode in each electrode pair divided by the difference in mean latency of the main peak in the short and the long distance recording. Statistical ana/ysis. Differences between groups were analysed using two-factor repeated ANOVA or Student's r-test.
RESULTS A rotatory acceleration of 13"/s2,performed in ambient light, elicited nystagmus both in exposed rats and in controls (Fig. 2. left tracings). Deceleration elicited nystagmus with a lower gain and shorter duration than that recorded during acceleration (Fig. 3). Even after 11 weeks of exposure no difference in gain was established between exposed rats and controls during acceleration or deceleration (ANOVA. p>0.05). nor was there any difference in duration of nystagmus between the two groups during acceleratory or deceleratory stimulation (acceleration: 53+ 14 s vs. 48+8 s. I-test p 0 . 0 5 : deceleration: 7 3 s vs. S F 2 s t-test p0.05). During sinusoidal oscillatory stimulation in the dark at 0.2 Hz (Fig. 2. middle tracings) the gain was lower in the exposed rats than in the controls (Fig. 4. upper right graph: r-test. pO.OS). During randomized
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Fig. 4 . Effect of toluene inhalation on mean gain (upper graph) and phase (lower graph) as a function of frequency of randomized (left) and sinusoidal (right) oscillatory stimulation. Measurements made at least 4 weeks after inhalation exposure to toluene ( I OOO ppm. 21 hiday, 7 daysiweek). Vertical bars indicate one SD. A--A, rats ( n = 3 exposed to toluene for 6 weeks; 0-4. rats (n=10)exposed to toluene for 11 weeks; 0-4,control rats ( 1 1 weeks. n= 10).
oscillatory stimulation in ambient light (Fig. 4, upper left graph) the exposed rats had a lower gain than that of the controls at all frequencies (ANOVA, 6 weeks exposure, p>0.05; 11 weeks exposure, ~(0.05). N o difference in phase (Fig. 4, lower graphs) was established (ANOVA, p>0.05), neither during sinusoidal nor during randomized oscillation. Optokinetic stimulation with a pattern, moving with constant velocity in either direction, elicited nystagmus in exposed as well as in non-exposed rats (Fig. 2 . right tracings). The gain was lower in rats exposed for 6 or 11 weeks compared to controls (ANOVA. Gain 1.0
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Fig. 5 . Effect of toluene inhalation ( I 000 ppm. 21 hiday, 7 daysiweek) on mean gain of optokinetic nystagmus relative to stimulus velocity. Vertical bars indicate one SD. A-A. rats ( n = 5 ) exposed to toluene f o r 6 weeks: 0-4.rats ( r z = 1 0 1 exposed to toluene for 1 1 weeks: c-4. control rats ( 1 I weeks, n= 10).
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pO.OS), or in the duration of OKAAN (3t-1 s vs. 3+3 s, p 0 . 0 5 ) . The results of the VER. ERG, and NCV recordings are summarized in Table I. No difference between exposed rats and controls was demonstrated (t-test, p>O.O5) in any of these recordings.
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DISCUSSION In the present study, functional variables of the vestibulo- and opto-oculomotor systems were tested one month after the end of a long-term inhalation exposure (6 or 11 weeks) to toluene. The gain during constant angular acceleration or deceleration of the exposed rats was not lower than that of the controls. This indicates that peripheral vestibular function was not affected, since a peripheral impairment would have resulted in a lower gain in the exposed rats. A long-lasting impairment of central vestibular function is indicated by a reduction in gain during sinusoidal oscillation at 0.2 Hz and during unpredictable. randomized oscillatory stimulation (after toluene exposure for I 1 weeks). Since the acceleration and velocity of head rotation were continuously changing during these tests, they are regarded to reveal disturbances in the “direct pathways”, which are responsible for fast changes in the central vestibular system (10). The cerebellar flocculus is regarded to be an essential link within these direct pathways. and reduction in gain is seen in pigmented rats after surgical Table I. Latencies and riinplititdes oj ele~trorerinoRr~ii?i. visual evoked response, and peripheral nerve conduction velocit? in rats &er inhulation exposirre to toirrene 11000 ppm, 21 hlday, 5 dayslweek, I 2 weeks: n=6) No differences between exposed groups and controls were demonstrated (t-test. p>O.O5) Controls Mean E/ecrrnretinogrrirn Latency (ms) a- wave --._ 77 5 b-wave 70 trough 405 c-wave 1210 Amplitude CpV) a-wave 11.0 b-wave 24 I trough 3.0 c-wave 54.0 Visiicil euoked response Latency (ms) PI 36.6 N1 24.1 P2 59.2 Amplitude (pV) PI-N1 19.5 N I-P2 19.6
Exposed
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Mean
3.5 4. I 44.7 254
25 70 400 1211
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5.7 50.5 11.5 17.7
7.8 256 4.7 70.8
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flocculectomy (11). Hence the reduction in gain after toluene exposure may be due to a cerebellar disturbance. The most prominent finding after the long-term exposure to toluene was a reduced gain during optokinetic stimulation. The pathways of the accessory optic system that convey optokinetic impulses also convey impulses elicited by flash stimulation, at least in rabbits (12). No effect of the toluene exposure on the ERG and VER recordings (using single flash stimuli) was seen, indicating that no gross functional impairment of the peripheral visual system had occurred. The rats used for visual testing were. however, exposed to a somewhat tower total dose than the animals used for optokinetic testing ( 5 dayslweek for 12 weeks instead of 7 days/week for 11 weeks). In addition, Dyer et al. 14), using paired flash stimuli to test the VER, found a larger variation in the latencies of some peaks of the VER after the second flash in rats exposed to toluene. Such possible small changes in VER latencies (up to about 5 ms) can, however, not explain the reduction in gain in the vestibulo- or opto-oculomotor response. This response is built up during several seconds of continuous optokinetic stimulation. As long as the eyes are not following the optic stimulus exactly, there is a retinal slip which causes adjustment of the eye movements. A small change in conduction velocity is unlikely to affect the ability to detect a retinal slip and hence to cause a reduction in gain of optokinetic nystagmus. The observed reduction in gain is more probably explained by a disturbance of the cerebellar function, similar to that indicated during vestibular stimulation. This assumption is supported by several reports suggesting that the flocculus contributes to the regulation of the gain of optokinetic nystagmus (for review, see (13)), and the observation that gain is reduced in monkeys, rabbits and cats after flocculectomy or cerebellectomy (14). The present results. obtained after long-term exposure to toluene, partly differ from those of corresponding measurements during acute exposure. A prolonged duration of the post-acceleratory nystagmus, and of the optokinetic after-nystagmus, has been demonstrated during acute exposure to toluene (7). This finding suggests a disturbance of the velocity storage mechanism that is part of the central processing of optokinetic as well as vestibular signals. No such impairment of the velocity storage mechanism was seen 4 weeks after the end of the long-term exposure made in the present study. In other respects, the observations on long-term exposure are similar to those made on acutely exposed rats. Thus, both types of exposure cause a reduction in the gain during optokinetic stimulation. The results reported here indicate that long-term exposure to toluene causes long-lasting central, possibly cerebellar, dysfunction. These findings are consistent with a report on workers who showed reduced visual suppression after long-term exposure to another benzene derivate, styrene (15). This symptom is found also in patients suffering from chronic toxic encephalopathy, believed to be caused by exposure to organic solvents ( I 6). Cerebellar lesions due to exposure to toluene are suggested also by clinical symptoms and by computer tomography of toluene abusers (17, 18, 19, 20). The results in the present study demonstrate that the opto- and vestibulo-oculomotor systems are useful as experimental models for the understanding of mechanisms underlying chronic functional disturbances caused by organic solvents. The present experimental observations on rats agree with clinical findings on humans, supporting the interpretation that solvent exposure can cause long-lasting impairment of CNS functions.
ACKNOWLEDGEMENTS We thank Maud Hagman and Gunilla Linder for skillful technical assistance. The investigation was supported by the Swedish Work Environment Fund (grants 84-1291 and 85-0479).
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REFERENCES I . WHO. Environmental Health Criteria 52: Toluene. World Health Organization. Geneva, 1985. 2. Benignus VA. Neurobehavioral effects of toluene: a review. Neurobehav Toxicol Teratol 1981: 3: 407-15. -~ 3. Coscia CG, Tabaro G. Albera C et al. Alterazioni vestibolari nell’esposizione a toluene. Med Lav 1983; 74: 23-9. 4. Dyer RS, Muller KE. Janssen R, Barton CN. Boyes WK, Benignus VA. Neurophysiological effects of 30 days chronic exposure to toluene in rats. Neurobehav Toxicol Teratol 1984: 6: 363-8. 5 . Sullivan MJ, Rarey KE, Conolly RB. Ototoxicity of toluene in rats. Neurotoxicol Teratol 1989; 10: 525-30. 6. HydBn D, Larsby B, Andersson H, Odkvist LM, Liedgren SRC. Tham R. Impairment of visuovestibular interactions in humans exposed to toluene. ORL 1983: 45: 262-9. 7. Larsby B, Tham R, Eriksson B, 6dkvist LM. The effect of toluene on the vestibulo- and optooculomotor system in rats. Acta Otolaryngol (Stockh) 1986: 101: 422-8. 8. Hess BJM, Precht W, Reber A, Cazin L. Horizontal optokinetic ocular nystagmus in the pigmented rat. Neurosci 1985; 15: 97-107. 9. Johnson AC, Juntunen L. Nylkn P. Borg E, Hoglund G. Effect of interaction between noise and toluene on auditory function in the rat. Acta Otolaryngol (Stockh) 1988: 105: 5643. 10. Raphan T, Cohen B. Velocity storage and the ocular response to multidimensional vestibular stimuli. In: Berthoz A, Melvill L, Jones G, eds. Adaptive mechanisms in gaze-control. Facts and theories. Amsterdam: Elsevier Science Publisher. 1985: 12343. 11. Lannou J, Hess BJM, Precht W, Reber A. Effects of bilateral flocculectomy on the horizontal vestibulo-ocular reflex (HVOR) in the rat. Neurosci Lett 1985: Suppl 22: 275. 12. Soodak RE, Simpson JI. The accessory optic system of rabbit. 1. Basic visual response properties. J Neurophys 1988; 60: 2037-54. 13. Van Neerven J, Pompeiano 0, Collewijn H. Depression of the vestibulo-ocular and optokinetic responses by intrafloccular microinjection of GABA-A and GABA-B agonists in the rabbit. Arch Ital Biol 1989: 127: 243-63. 14. Precht W, Blanks RHI, Strata P. Montarolo P. On the role of the subprimate cerebellar flocculus in the optokinetic reflex and visual-vestibular interaction. In: Bloedel JR. Dichgans J. Precht W. eds. Cerebellar functions. Heidelberg: Springer-Verlag. 1985: 86-107. 15. Moller C, Odkvist LM, Larsby B. Tharn R. Ledin T. Bergholtz LM. Otoneurological findings in workers exposed to styrene. Scand J Work Environ Health 1990. In press. 16. Moller C, Odkvist LM, The11 J. et al. Otoneurological findings in psycho-organic syndrome caused by industrial soIvent exposure. Acta Otolaryngol (Stockhl 1989: 107: 5-12. 17. Fornazzari L, Wilkinson DA, Kapur BM. Carlen PL. Cerebellar. cortical and functional impairment in toluene abusers. Acta Neurol Scand 1983: 67: 319-29. 18. Malm G , Lying-Tunell U. Cerebellar dysfunction related to toluene sniffing. Acta Neurol Scand 1980; 62: 188-90. 19. Streicher HZ, Gabow PA, Moss AH. Kono D, Kaehny WD. Syndromes of toluene sniffing in adults. Ann Int Med 1981: 94: 758-62. 20. Lazar RB, Ho SU, Melen 0. Daghestani AN. Multifocal central nervous system damage caused by toluene abuse. Neurology 1983; 33: 1337-40. Manriscript received April 4 , 1990: mcepted Max 10. 1990
Address for correspondence: Per NylCn. National Institute of Occupational Health. S-17184 Solna. Sweden
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