Ethylene oxide neuropathy in rats

Journal of the Neurological Sciences, 1986, 74:215-221

215

Elsevier JNS 2674

Ethylene Oxide Neuropathy in Rats Exposure to 250 ppm A. Ohnishi 1, N. Inoue 2, T. Y a m a m o t o 1, Y. Murai l, H. Hori 3, I. T a n a k a 3, M. K o g a 3 a n d T. A k i y a m a 3 ~Department of Neurology, 2Environmental Toxicology and 3Environmental Health Engineering, University of Occupational and Environmental Health, Kitakyushu 807 (Japan)

(Received 22 November, 1985) (Revised, received 11 February, 1986) (Accepted 11 February, 1986)

SUMMARY In Wistar rats subjected to a 6-h exposure to ethylene oxide at the concentration of 250 parts per million once, 5 times a week for 9 months, histopathologic studies of myelinated fibers of the proximal sural, distal sural and peroneal nerves and of the fasciculus gracilis at the 5th thoracic and 3rd cervical segments of the spinal cord were performed to observe whether ethylene oxide of such a concentration would lead to degeneration of primary sensory neurons. Throughout the study, no definite abnormality of the gait or posture was observed in both control and test rats. Qualitative histologic studies disclosed preferential distal axonal degeneration of myelinated fibers in both sural nerves and gracile fascicles in the test rats, although the extent of the distribution and the severity of the degenerative findings were variable. Such findings are consistent with mild axonal degeneration found among patients suffering from ethylene oxide toxicity. Therefore, in rats, exposure to 250 ppm ethylene oxide produces centralperipheral distal axonal degeneration of primary sensory neurons.

Key words: A x o n a l degeneration - Ethylene oxide - M o r p h o m e t r y - N e u r o p a t h y - Primary sensory neuron

Address for correspondenceand reprints: Dr. A. Ohnishi,Departmentof Neurology,Universityof Occupational and EnvironmentalHealth, Kitakyushu807, Japan. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (BiomedicalDivision)

216 INTRODUCTION

Ethylene oxide (EO) (Glaser 1979; Landringan et al. 1984) is widely used as a sterilising agent for equipment and supplies, particulary in hospitals and factories. Clinical polyneuropathy after occupational exposure to EO has been reported (Gross et al. 1979; FineUi et al. 1983; Kuzuhara et al. 1983; Kova~ et al. 1984; Zampollo et al. 1984; SchrOder et al. 1985). Recently, we reported the first animal model of EO-induced neuropathy, at a concentration of 500 parts per million (ppm). This neuropathy was characterized by central-peripheral distal axonal degeneration of primary sensory neurons (Ohnishi et al. 1985). In this communication, we describe the morphometric pathologic findings of myelinated fibers in hindleg nerves and in the fasciculus gracilis of rats intoxicated with EO at a concentration of 250 ppm, a dose comparable with the concentration of EO which induces neuropathy in humans (Gross et al. 1979; Kuzuhara et al. 1983; Kova~ et al. 1984; Schr0der et al. 1985). MATERIALS AND METHODS

Twenty-one male Wistar rats weighing about 250 g were grouped into 3. Seven served as the test animals subjected to 6-h exposure to EO at a concentration of 250 ppm 5 times a week for 9 months. Exposure systems were similar to those described (Tanaka et al. 1983), except for a generator and a monitor system for concentrations in a chamber. In this study, EO gas was generated from a cylinder containing 20Yo EO and 80 ~o carbon dioxide. The EO gas was mixed with the fdtered room air and then was introduced into the exposure chamber. The EO gas concentration in the chamber was sampled by syringe every hour during the exposure and was measured by flame ionization detector gas chromatography. Another 7 rats, exposed to filtered room air introduced into the chamber, were pair-fed and served as end-controls. The remainder served as onset-controls and were given no treatment. After completion of the exposure period, the test rats were killed, in pairs with the end-control rats by perfusing 3Y/o glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), under deep pentobarbital anesthesia. The distal sural, proximal sural and peroneal nerves were processed for teased fiber preparations and Epon-embedded sections in both tests and end-controls. They were processed only for Epon-embedded sections, in case of onset-controls. The transverse Epon-embedded sections of the fasciculus gracilis at the 3rd cervical (C3) and 5th thoracic (T5) levels were prepared. Morphometric evaluation ofmyelinated fibers of the teased fiber preparations and Epon-embedded sections was systematically performed as described (Dyck 1975; Ohnishi and Ikeda 1980). Student's t-test was used for statistical evaluation.

217 RESULTS

General observations The mean body weights + standard deviations in the onset-control, end-control and test groups were 254 + 6, 591 + 62 and 545 + 65 at the time of killing. There were no significant differences in body weight between end-control and test groups. In no rat was there an awkward or ataxic gait throughout the experimental period.

Findings in teased fiber preparations The nature of the myelinated fiber degeneration was axonal with linear rows of myelin ovoids. The mean frequency of myelinated fibers with axonal degeneration (Table 1) of proximal and distal sural, and peroneal nerves was greater in test than in end-control. The difference was significant only for the peroneal nerve. It was greater in distal than in proximal sural nerves, but the difference was not significant.

Light-microscopic findings of Epon-embedded preparations Peripheral nerve Myelin ovoids were occasionally seen in distal sural and peroneal nerves. Table 2 shows the mean values with standard deviation for the numbers per nerve and median diameter of myelinated fibers in proximal sural, distal sural, and peroneal nerves. Total myelinated fiber numbers per nerve were similar among onset-control, end-control, and tests for all 3 nerves. Small myelinated fiber numbers per nerve were significantly greater than those of end-control and significantly smaller than those of onset-control for all 3 nerves. On the other hand, large myelinated fiber numbers per nerve were significantly smaller than those of end-control and significantly greater than of onset-control for distal sural and peroneal nerves. The median diameters of myelinated fibers were significantly smaller than those of end-control and greater than those of onset-control for all 3 nerves.

TABLE 1 FREQUENCY (~) OF MYELINATEDFIBERS SHOWING AXONAL DEGENERATION Sural nerve

Peroneal nerve

Proximal

Distal

End-control (n = 7)

0.3 + 0.5a

0.4 + 0.8

0.9 + 0.7

Test (n = 7)

0.9 _+1.2

2.1 + 2.8

3.3 + 2.3b

a Mean + standard deviation. b P < 0.01.

218 TABLE 2 NUMBERS PER NERVE AND MEDIAN DIAMETERS OF MYELINATED FIBERS IN PROXIMAL SURAL, DISTAL SURAL AND PERONEAL NERVES

M y e l i n a t e d fiber n u m b e r s ( # / n e r v e )

Total

Small

Large

Median diameter (#m)

Proximal sural nerve

Onset-control End-control Test

(n = 7) (n = 7) (n = 7)

1 0 5 0 + 83 a 909 + 129 984 + 173

(n = 7) (n = 7) (n = 7)

9 2 0 + 71 867 + 110 869 + 152

(n = 7) (n = 7) (n = 7)

1820 + 134 1866 + 108 1813 + 157

684_+ 61 353 + 47 466 _+ 35 b'c

366 + 81 555 + 137 518 + 149 d

4.4 + 0.2 6.3 _+ 0.8 5.3 + 0.6 g,h

273 + 34 579 + 96 429 + 100 e,f

4.3 + 0 . 1 6.6 + 0.6 5.1 _+ 0.3 g.h

794 + 104 1272 + 102 1049 _+ 144 e"f

4.7 + 0.3 7.2 + 0.4 5.4 + 0.6 g,h

Distal sural nerve

Onset-control End-control Test

647+ 288 + 440 +

58 73 92 b,c

Peroneal nerve

Onset-control End-control Test

a b ¢ d e f g h

1026 + 106 595 + 94 805 + 115 b'c

M e a n + s t a n d a r d deviation. p < 0.008 (0.008-0.0001) vs end-control. P < 0.002 (0.002-0.0001) vs onset-control. p < 0.02 vs onset-control. p < 0.03 (0.03-0.0004) vs end-control. P < 0.002 (0.002-0.0001) vs onset-control. P < 0.03 (0.03-0.0004) vs end-control. p < 0.005 ( 0 . 0 0 5 - 0 . 0 0 0 1 ) v s onset-control.

Posterior column In test rats, myelin ovoids with variable frequencies in each rat were seen at both C3 (Fig. 1) and T5 levels of the fasciculus gracilis. The frequency of such abnormality was greater in test than in end-controls, and tended to be greater at the C3 than at the T5 level. The mean density of myelinated fibers (Table 3) was similar between end-controls and test rats at the T5 level. It was about 15~ less in test rats than in end-controls, but the difference did not reach a statistical significance. DISCUSSION

In our study, Wistar rats exposed to EO for 6 h at a concentration of 250 ppm once, 5 times a week for 9 months, showed axonal degeneration of myelinated fibers, in both peroneal and sural nerves and of the fasciculus gracilis, although an awkward or ataxic gait was not evident throughout the study. With regard to the distribution of pathologic findings in test rats, myelinated fibers showing axonal degeneration were more frequently found in the distal than in the

219

Fig. 1. Transverse section of the fascieulus gracilis at the 3rd cervical segment in end-control (A) and test (B). Myelin ovoids (some of them are indicated by white arrow heads) are frequently seen in test (B). TABLE 3 MEAN DENSITY OF MYELINATED FIBERS OF THE FASCICULUS GRACILIS Density (#/mm 2) C3

T5

End-control

(n = 7)

58914 + 6301"

47469 _+ 5458

Test

(n = 7)

49614 + 12405

45791 + 13393

" Mean + standard deviation. proximal sural nerve, indicating a preferential involvement o f the distal peripheral axon o f the l u m b a r primary sensory neuron. F u r t h e r m o r e , a mild decrease o f myelinated fiber density in the test rats c o m p a r e d with end-controls was found at the C3, b u t not at the T5 level o f the fasciculus gracilis, indicating a preferential involvement o f the distal central axon o f the l u m b a r p r i m a r y sensory neuron. Therefore, it is c o n c l u d e d that pathologic changes o f rats e x p o s e d to 250 p p m E O are c o m p a t i b l e with centralperipheral distal a x o n o p a t h y ( S p e n c e r a n d S c h a u m b u r g 1976, 1977), although the nerve cell b o d y o f the l u m b a r p r i m a r y sensory n e u r o n was n o t examined. The m e d i a n diameter o f total myelinated fibers in test rats was significantly smaller than in end-controls, but was significantly greater than in onset-controls, in all

220 3 nerves. This probably reflects retardation in growth and maturation of myelinated fibers (Sharma et al. 1985) due to EO exposure during the period from the onset to the end of this experiment. On the other hand, in the analysis of the number of myelinated fibers, the number of large myelinated fibers in test rats was significantly decreased compared with that of end-controls in the distal sural nerve. Such a significant decrease in the number of large myelinated fibers was absent in test rats compared with end-controls in the proximal sural nerve, thereby suggesting a greater retardation of growth and maturation in the distal sural nerve. Therefore it is concluded that EO induces a retardation of growth and maturation of myelinated fibers, in the presence of axonal degeneration. Among patients with neuropathy due to EO exposure, 2 developed neuropathy within 3 and 5 months exposure, respectively and they had been repeatedly exposed to EO up to several hundreds ppm (Kuzuhara et al. 1983; SchrOder et al. 1985). Therefore, the exposure of rats to 250 ppm EO is a reasonable condition for the induction of experimental EO neuropathy. The nature of degeneration of peripheral nerves in patients with neuropathy due to EO exposure is compatible with the mild axonal degeneration seen in histological studies of the biopsied sural nerve (Kuzuhara et al. 1983; Schr0der etal. 1985). Furthermore the results of nerve conduction studies of limb nerves and needle EMG examination were also compatible with mild axonal degeneration of peripheral nerves (Gross et al. 1979; Finelli et al. 1983; Zampollo et al. 1984; SchrOder et al. 1985). Thus, axonal degeneration of myelinated fibers of peripheral nerves induced in rats exposed to 250 ppm EO is consistent with the nature of degeneration of peripheral nerves in patients with neuropathy due to EO exposure. Greenberg and Swift (1982) reported that human peripheral neuropathy due to EO is a self-limited disorder with reversal of the clinical and electrodiagnostic abnormalities following removal from exposure. In our experimental animals, distal axonal degeneration was found in the fasciculus gracilis. If myelinated fibers in the fasciculus gracilis are degenerated in clinical EO neuropathy, such may not be a self-limited disorder. Careful studies of short-latency sensory evoked potentials on tibial nerve stimulation (Cracco et al. 1984) in case of human EO neuropathy should be done to rule out myelinated fiber degeneration in the fasciculus gracilis. ACKNOWLEDGEMENTS We thank S. Ishimatu, A. Sashihara, R. Kaku, and T. Kitagawa for technical help and M. Ohara for comments on the manuscript. REFERENCES Cracco, J., S. Castellsand E. Mark (! 984) Spinalsomatosensoryevokedpotentialsinjuvenilediabetes,Ann. Neurol., 15: 55-58. Dyck, P.J. (1975) Pathologic alterations of the peripheral nervous system of man. In: P.J. Dyck, P.K. Thomas and E.H. Lambert (Eds.), Peripheral Neuropathy. Vol. 1, Saunders, Philadelphia, pp. 314-319.

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