ENVIRONMENTAL

RESEARCH

Pathological

11,

40-5 1 (1976)

Changes in the Nervous System following in Utero Exposure to Halothane

LOUIS W. CHANG, Departments

of Pathology

ALDEN

W. DUDLEY,

JR., AND JORDAN KATZ

and Anesthesiology, University Wisconsin 53706

of

Wisconsin,

Madison,

Received April 25, 1975 Tissue samples from the cerebral cortex of rats which had been exposed in utero to 10 ppm halothane (8 hours/day/5 days/week throughout pregnancy) were studied electron microscopically. In the neonatal animals, focal weakening and out-pouching of the nuclear envelope in many neurons were observed. Leakage of nuclear material and myelin-figure formation also occurred at the site of the weakening, Vacuolation of the Golgi complex of the neurons was a frequent finding. Similar changes of the Golgi complex was also found in the neurons of the IOO-day-old animals. Moreover, residual degenerative changes were still observable in the neurons of animals 100 days after the exposure. The present investigation indicates that trace amounts of halothane may still be hazardous to the nervous system ofthe developing fetus and such pathological effects may be long lasting.

INTRODUCTION

Despite the popularity of halothane as an anesthetic agent, increasing concern and attention has been generated concerning the possibility that halothane may constitute an occupational hazard for pregnant operating room personnel (Whitcher et al., 1971; Corbett, 1972, 1973; Jenkins, 1973; A.S.A. Scientific Panel, 1974). When compared to general duty nurses and other physicians, a significantly higher abortion rate was found among anesthetists and anesthetic and operating room nurses (Vaisman, 1967; Vskrog and Harvald, 1970; Cohen et al., 1971; Corbett, 1972). It has been indicated that headaches, irritability, and other disturbances of the central nervous system were found in anesthetists following prolonged exposure to poorly ventilated operating rooms (Vaisman, 1967; Tyrrell and Feldman, 1968; Leuczet al., 1970). Various investigators (Porter, 1972; Adam, 1973; Bruce, 1974; Quimbyet al., 1974) also indicated that exposure to subanesthetic concentrations of anesthetics produced deficits in behavioral functions of both human subjects and experimental animals. Ultrastructural changes in the nervous system (Quimby et al., 1974; Chang et al., 1974) and in the fetal liver and kidney (Changet al., 1975 b and c) after chronic exposure to low levels of halothane have also been reported. The present investigation is designed to study the effects of halothane on the developing nervous system following in utero exposure. MATERIALS

AND METHODS

Sprague-Dawley rats were used in this experiment. After conception, eight young female rats were housed in specially designed chambers at the University of Wisconsin Biotron where the environmental conditions could be carefully manipulated. Halothane was introduced to the chambers with the fresh air supply via a Draegger vaporizer. The concentration of halothane was monitored with gas 40

EFFECT

OF

HALOTHANE

ON

DEVELOPING

NERVOUS

41

SYSTEM

chromatography. To simulate the actual working conditions of personnel in the operating room, the animals were exposed to 10 ppm halothane 8 hrlday and 5 days/week throughout the pregnancy. At term, the pregnant rats were transferred to a halothane-free chamber where the pups were delivered. An equal number of pregnant rats was used as controls which were housed in adjoining halothane-free chambers throughout the pregnancy and delivery. Four randomly chosen pups from each litter were killed by decapitation within 24 hr after birth. Tissue samples from the neonatal brain (cerebral cortex) were carefully obtained and were fixed in 2.5% phosphate buffered glutaraldehyde (Fisher Scientific) (final osmolality was approximately 450 mosmole.) for 2 hr and postfixed in 1% osmium tetroxide for 1 hour. Three to four pups from each litter were allowed to mature to 100 days. These animals were then killed by intracardial perfusion with saline followed by 2.5% phosphate buffered glutaraldehyde. Tissue samples from the cerebral cortex of three animals were also obtained for electron microscopy. The tissue samples were then dehydrated with graded ethanol and embedded in Epon. Thin sections were cut with a DuPont diamond knife on an LKB Ultrotome III automatic ultramicrotome. The tissue sections were examined with an RCA EMU-3G electron microscope. RESULTS

No remarkable changes were observed in the cerebral cortex of the neonatal or mature animals at the light microscopic level. By means of electron microscopy, however, significant pathological changes were found. Weakening of the nuclear envelope (membrane) of many neurons was observed (Fig. 1). These weakened nuclear membranes would bulge focally to form

FIG. 1. Cerebral indicating weakening

cortex, neonatal of the nuclear

rat. Note membranes.

the focal out-pouching of the nuclear N (nucleus). x 31,500.

envelope

(+)

42

CHANG,

DUDLEY

AND

KATZ

“pouches” or “pockets” (Fig. 1 and 2). Extrusion or leakage of the nuclear material (chromatin) into these pockets (Fig. 2 and 3) was frequent. Formation of myelin-figures by the nuclear membrane also occurred at the weakened loci (Fig. 3 and 4). Severe dilatation and vacuolation of the Golgi complex were observed in many neurons (Fig. 5). Myelin-figure formation was also observed in some cellular process (Fig. 6). Occasional cell death was also found in the neonatal cortex; however, this is not a prominent finding. Since these findings were not observed in the control animals, they are not believed to be artefacts. Degenerative changes in the cerebral cortex were still observable in the lOOday-old animals. One of the most prominent findings was the vacuolation of the Golgi complex (Fig. 7). Accumulation of cytoplasmic debris and lysosomes could be observed within some nerve cells and macrophages (Fig. 8 and 9). Degenerative neurons which contained giant lysosomes and vacuolated Golgi complex were occasionally observed (Fig. 10 and 1 I). DISCUSSION

In the past few years, an increased occurrence of congenital anomalies, spontaneous abortion, and involuntary infertility has been observed in association with anesthesia, the probable cause being prolonged exposure to anesthetic gas (Vaisman, 1967; Askrog and Harvald, 1970; Cohen et al., 1971; Whitcher et al., 1971; Corbett, 1972; Gotell and Sundell, 1972; Jenkins, 1973; Corbett, 1973, 1974). Pathological lesions in the fetal liver and kidney after in utero exposure to trace

FIG. 2. Cerebral cortex, pocket (4). N (nucleus).

neonatal rat. x 46,500.

Extrusion

of the nuclear

material

(chromatin)

into the nuclear

43

.

*

.

,

:.

Flc.. 3. Cerebral cortex, neonatal rat. Myelin-figure formation by the nuclear membranes (-) at the weakened loci. %46,500.

FIG. 4. Cerebral cortex, neonatal rat. Extensive formation of myelin-figure membranes of a cortical neuron. ~45,000.

(-)

by the nucleal

44

CHANC,

DUDLEY

AND

liA?‘%

Frc. 5. Cerebral cortex, neonatal rat. Severe dilatation and vacuolation ofthe Golgi complex (G) of a cortical neuron. x63.000.

EFFECT

OF

HALOTHANE

ON

L;E\‘ELOPING

FIG. 6. Cerebral cortex, neonatal rat. Myelin-figure ~65,000.

NERVOUS

formation (-)

SYSTEM

within

45

a cellular process

46

CHAKG,

DUDLEY

AND

KATZ

FIG. 7. Cerebral cortex, lOL%day-old rat. Vacuolation ofthe Golgi complex (G) of a cortical neuron. N nucleus. ~63,000.

47

48

CHANG,

DUDLEY

43’1)

KA’I-7.

FIG. 9. Cerebral cortex, IOO-day-old rat. Accumulation of lysosomes (Ly). lipid globules (L) and cellular debris presumably within a macrophage. x45.000.

EFFECT

OF

HALOTHASE

ON

DEVELOPING

NER\‘OUS

SYSTEM

FIG. 10. Cerebral cortex, lOO-day-old rat. A degenerative neuron (NJ which appeared electron dense. Note the indistinguishable border between the nucleus and cytoplasm vacuolated Golgi complexes (G) and swollen mitochondria (M). x 31,000.

49

to be extremely and the large

amounts of halothane have also been reported (Dudley et nl., 1974; Chang ef (II., 1975b, c). Despite all of the statistical indications and experimental findings that halothane could be hazardous to fetal development, there have been no detailed investigations on the pathological effects upon the nervous system of in utero exposure to this anesthetic agent. The transport system of cell membranes may be altered by halothane (Andersen and Amaranath, 1973). It has also been indicated, at least in the kidney epithelial cells, that halothane exerts its pathological effects mainly on the biological membrane system and organelles (Change? al., 1975a). Therefore, the lesions produced by halothane on the nuclear membrane of the neurons is not totally surprising. Similar myelin-figure formation and rupturing of the nuclear membrane in the renal

Fig. chromatin

11.

Cerebral and

containing

cortex,

IOO-day-old giant

lysosomes

rat. (1.~).

A degenerative

neuron

(N)

showing

clumping

of the

Y 3 1.000.

interstitial cells following chronic exposure ot halothane have also been reported (Chang et ul., 1975a). Vacuolation of the neuronal Golgi complex was a prominent finding in both the neonatal and loo-day-old animals. Similar changes of the Golgi complex were also observed in the cortical neurons of adult animals following chronic exposure to halothane (Chang et al., 1974). However, the signiticance of these changes is still not understood. The effects of halothane on the nervous system seem to be enduring ones as degenerative changes in the neurons were still observable in the IOO-day-old animals which were raised in halothane-free chambers since birth. Quimby and co-workers (1974) have reported learning deficits in rats which were exposed to halothane during early life, further supporting the concept that halothane may exert an undesirable influence on the developing nervous system. Further study on this area is being planned in hope of elucidating the pathogenetic mechanism of halothane on the nervous system.

EFFECT

OF

HALOTHANE

ON

DEVELOPING

NERVOUS

SYSTEM

51

ACKNOWLEDGMENT We wish to thank Dr. R. Guillery for his valuable opinions on some of the micrographs and Mrs. F. Simanoll for the excellent technical assistance.

REFERENCES Adam. N. (1973). Effectsofgeneral anesthetics on memoryfunctionin man../. Camp. Phy.Picr/. Psychol.. 8. 294-305. Andersen, N. B. and Amaranath, L. (1973). Anesthetic effects on transport across cell membranes. Anesfhr.sio/o~~y:v.

39, 126-l 52.

A.S.A. Scientific Panel (1974). A preliminary report: The hazard of trace anesthetics in unvented operating rooms. Anesth. Rev., 1, 22-23. Bruce, D. L. (1974). Traceanestheticeffectsonperceptualandcognitiveskills.An~.sth. Rw.. I, 24-25. Cohen, E. N.. Bellville, J. W. and Brown, B. W. (1971). Anesthesia. pregnancy and miscarriage: A study of operating room nurses and anesthetics. Anrsrhesiology. 35, 343-347. Corbett. T. H. (1972). Anesthetics as a cause of abortion. Ferr. Steril.. 23, 866869. Corbett. T. H. (1973). Retention ofanesthetic agents following occupational exposure. Ane.srh. Anulg.. 52,614618. Chang. L. W., Dudley, A. W. Jr., Lee, Y. K. and Katz, J. (1974). Ultrastructural changes in the nervous system after chronic exposure to halothane. Exp. Nrun~l.. 45, 209-219. Chang. L. W., Dudley. A. W. Jr., Lee, Y. K. and Katz, J. (197Sa). Ultrastructural changes in the kidney following chronic exposure to low levels of halothane. Amer. J. Park., 78, Z-242. Chang. L. W., Dudley, A. W. Jr.. Lee, Y. K. and Katz, J. (1975b). Ultrastructural studies on the pathological changes in the neonatal kidney following in utero exposure to halothane. Em’iron. Rcr. (In press.) Chang. L. W., Lee, Y. K., Dudley, A. W. Jr. and Katz. J. (1975c). Ultrastructural evidence of hepatotoxicity of halothane in rats following in utero exposure. C’rrntrd. Anr.s~h. Sot. .I.. 22, 330-338. Dudley. A. W. Jr., Chang. L. W. and Katz, J. (1974). Ultrastructural evidence of hepatic and renal changes in neonatal rats following in utero exposure to low levels of halothane. Fed. Proc., 33,635. Gotell. P. and Sundell, L. (1972). Anesthetists exposure to halothane. Luncet. 1, 424425. Jenkins. L,. C. (1973). Chronic exposure to anesthetics: A toxic problem? Can. .4ne.~~h. .S~C,.J., 20, 104-120. Leucz, L., Nemes, C. S. and Berta, L. (1970). Psychische Belastungen und Morbid& der Anesthetisten. Third European Anesth. Conference, Prague, Abstract No. 63/02. Porter. A. L. (1972). An analytical review of the effects on non-hydrogen bonding anesthetics on memory processing. Belrar,. Bid.. 7, 291-309. Quimby. K. L., Aschkenase. L. J., Bowman. R. E.. Katz, J. and Chang, L. W. (1974). Enduring learning deficits and cerebral synaptic malformations from exposure to 10 ppm halothane. Science. 185, 625-627. Tyrrell, M. F. and Feldman, S. A. (1968). Headache following halothanc anesthesia. Brit. J. Anesth., 40.99-105. Vaisman. A. I. ( 1967). Working conditions in surgery and their effect on the health of anesthesiologists. ELsp.

Klnr.

.Jm~srh..

3. 44-49.

Vskrog. V. and Harcald. B. (1970). Teratogen effects of inhalation anesthetika. Nod. Med., 83, 49c-493. Whitcher. C. E., Cohen, E. N. and Trudell, J. R. (1971). Chronic exposure to anesthetic gases in the operating room. Ancsthe.sio/ogv, 30, 363-386.

Pathological changes in the nervous system following in utero exposure to halothane.

ENVIRONMENTAL RESEARCH Pathological 11, 40-5 1 (1976) Changes in the Nervous System following in Utero Exposure to Halothane LOUIS W. CHANG, Dep...
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