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Histologic Peripheral Nerve Changes in Rats Induced by Deltamethrin
Ecotoxicology and Environmental Safety 47, 82}86 (2000) Environmental Research, Section B doi:10.1006/eesa.2000.1925, available online at http://www.idealibrary.com on
Histologic Peripheral Nerve Changes in Rats Induced by Deltamethrin Edenilson Eduardo Calore,B - Maria JoseH Cavaliere,? FlaH vio Rodrigues Puga,A Nilda Maria Perez Calore,Adriana Ruckert da Rosa,* - Ruth Weg,* - Suzana de Souza Dias,- and Regina Paes dos Santos- ? ** *Pathology Department, Veterinary Medicine School, and BPharmacology Department, Biomedical Institute, and **Public Health Faculty, SaJ o Paulo University, SaJ o Paulo, Brazil; -Pathology Section, Instituto Emn& lio Ribas, SaJ o Paulo, Brazil; ?Pathology Division, Instituto Adolfo Lutz, SaJ o Paulo, Brazil; and AToxicology Section, Instituto Biolo& gico, SaJ o Paulo, Brazil Received October 13, 1999
Nevertheless, some studies have demonstrated that these products, especially compounds with an a-cyano group, are toxic to the mammalian central nervous system (CNS) in acute intoxications (Husain et al., 1994, 1996). Two types of pyrethroid insecticide syndromes have been recognized: Type I syndrome, or T syndrome, which is caused by esters lacking the a-cyano substituent, is characterized by restlessness, incoordination, prostration, and paralysis in insects; in rats, it causes aggressive behavior, whole-body tremor, and prostration. Type II syndrome, or CS syndrome, is caused by a-cyanopyrethroids. These compounds cause intense hyperactivity, incoordination, and convulsions in insects; in contrast, rats display burrowing behavior, coarse seizures, choreoatetosis, and profuse salivation (Ecobichon, 1995). Various mechanisms have been proposed to explain the neurotoxic action of pyrethroid insecticides. Type I products a!ect sodium channels in nerve membranes, causing repetitive (sensorial and motor) neuronal discharge and prolonged negative afterpotential. The presynaptic nerve terminals (of the CNS and peripheral ganglia) are the most sensitive sites. Type II pyrethroids cause a persistent depolarization and frequency-dependent conduction block in sensorial and motor axons as well as prolonged repetitive "ring of sensorial ends and muscle "bers (Joy, 1994). Monoamine oxidase activity increases and NA>, K>-ATPase activity decreases were demonstrated in various regions of the CNS in rats intoxicated with the cyanopyrethroid deltamethrin (Husain et al., 1996). Studies of the toxicity of pyrethroid insecticides in the central and peripheral nervous system of mammals are generally concerned with the biochemical, pharmacological, and physiological e!ects of these products (Takahashi and Le Quesne, 1982; Narahashi et al., 1995). Morphological studies are scarce and the observed changes are discrete. Husain et al. (1996) described dendritic degeneration of Purkinje neurons of the cerebellar region of rats treated
Synthetic pyrethroid insecticides have been used in the last two decades largely because of their high activity as an insecticide and low mammalian toxicity. Some studies have demonstrated that these products, especially compounds with an a-cyano group, are toxic to the mammalian central nervous system (CNS) in acute intoxications. However, morphological studies are scarce. In the present work the histopathologic changes of the sciatic and tibial nerves of rats submitted to acute intoxication with the cyanopyrethroid deltamethrin were studied. For 3 consecutive days male Wistar rats received by oral gavage deltamethrin at a dose of 45 mg/kg body wt. On the 4th day fragments of sciatic and tibial nerves were studied by transmission electron microscopy (TEM) and teasing of individual nerve 5bers. In addition, another group of rats were allowed to recover until the 10th day. Teasing of nerves of animals sacri5ced on the 4th day revealed myelin ovoids, which are indicative of axonal damage. TEM demonstrated rare degenerated axons completely 5lled with organelles, in particular mitochondria, and with electron-dense lamellar bodies that resemble myelin 5gures. In addition, great cytoplasmic vacuolization caused by proliferation and dilation of the rough and smooth endoplasmic reticulum and Golgi apparatus was observed in some Schwann cells. No lesion was found 7 days after discontinuation of the treatment (group 2). Since these histologic changes are transitory and scarce, the question arises: Are they related to the changes in NAⴙ, Kⴙ-ATPase activity or Naⴙ channels caused by pyrethroid compounds? 2000 Academic Press Key Words: nerve; neuropathy; insecticides; pyrethroids; toxicity; deltamethrin.
INTRODUCTION
Synthetic pyrethroid insecticides have been used in the last two decades largely for their high activity as an insecticide and low mammalian toxicity. This low mammalian toxicity is due in part to their rapid metabolism in these animals. This work was supported by grants from FAPESP and CNPq-Brazil. 82 0147-6513/00 $35.00 Copyright 2000 by Academic Press All rights of reproduction in any form reserved.
83
DELTAMETHRIN-INDUCED NEUROPATHY
with deltamethrin. With respect to the peripheral nervous system, Dick et al. (1984) found no changes in sural and tibial nerves and in the maxillary division of cranial nerve V of rats fed permethrin for long periods. Peripheral nerve damage was described in teasing of individual nerve "bers of rats intoxicated with high doses of permethrin (Cavaliere et al., 1990). However, as that work had no ultrastructural studies, a more detailed description of nerve lesions caused by pyrethroid compounds at the ultrastructural level was necessary. The aim of the present work was to study the histopathologic changes of the sciatic and tibial nerves of rats intoxicated with the cyanopyrethroid deltamethrin by transmission electron microscopy (TEM) and teasing of individual nerve "bers. MATERIAL AND METHODS
Animals Two groups of six male Wistar rats weighing 200}250 g received by oral gavage deltamethrin dissolved in corn oil (concentration 45 mg/mL) at a dose of 45 mg/kg body wt, for 3 consecutive days. Water and food were given ad libitum. One animal in group 1 and another in group 2 died respectively on the second and third days of intoxication. Group 1. On the 4th day, under ether anesthesia, the animals were perfused with 4% paraformaldehyde (150 mL) and fragments of sciatic and tibial nerves were collected after ether anesthesia for histopathological studies. Six control animals received only a corresponding dose of corn oil.
FIG. 1. Teasing of individual nerve "bers stained with osmium tetroxide demonstrating myelin ovoids and irregularity of the myelin sheath which are indicative of axonal damage.;900.
RESULTS
Rats treated with deltamethrin exhibited clinical signs of acute intoxication including salivation, hyperactivity, incoordination, coarse seizures, choreoatetosis, and tremor from the "rst day of treatment. In group 2, these symptoms progressively disappeared from the 4th until the 10th day. Group 1 Peripheral nerve lesions were observed in three of the "ve surviving treated animals. These lesions consisted of axonal damage as observed by teasing of individual "bers (myelin ovoids and irregularities of myelin sheath, Fig. 1), as well as by electron mycroscopy. In semithin sections no in#ammatory cells or increases in the amount of connective tissue were observed.
Group 2. From the 4th to the 10th days the animals received only water and food ad libitum. On the 10th day, the animals were submitted to the same procedures as were the animals in group 1. Samples of the nerves were collected. Histopathology Fragments of the sciatic nerve (proximal) and tibial nerve (distal) 1.5 cm long were taken and divided. One fragment of each nerve (proximal and distal) was "xed in 2% glutaraldehyde for 2 h, post-"xed in 1% osmium tetroxide for 1 h, kept in glycerin, and processed for teasing of individual nerve "bers. Other fragments 0.1 cm in diameter (also of each nerve) were "xed in 2% glutaraldehyde in 0.15 M phosphate bu!er, pH 7.2, for 2 h, post"xed in 1% osmium tetroxide, dehydrated, and embedded in araldite resin. The 0.5-lm semithin sections were stained with 1% toluidine blue for light microscopy analysis. For electron microscopy ultrathin sections were obtained and stained with uranyl acetate and lead citrate.
FIG. 2. Axonal damage and axonal atrophy in a myelinated nerve "ber with disorganization of neuro"laments and neurotubules. Actually, the axon was almost no longer recognizable. The myelin sheath was also partially degenerated.;6500.
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FIG. 3. Axonal atrophy and substitution of the axon by residual electron-dense bodies and organelles.;8000.
The ultrathin sections of sciatic and tibial nerves con"rmed the axonal damage involving myelinated "bers, which were present in approximately 3% of the myelinated axons of each fascicle. The neuro"laments and neurotubules were disorganized and almost unrecognizable. The myelin sheath was partially degenerated. (Fig. 2). Rarely could axonal atrophy be observed and instead of axons only residual electron-dense bodies and organelle were observed (Fig. 3). In addition, some axons were involved by disproportionately thin myelin sheaths and hyperthrophic Schwann cells (Fig. 4). These Schwann cells had great cytoplasmic vacuolization caused by proliferation and dilation of
FIG. 4. An axon involved by a disproportionately thin myelin sheath and a hyperthrophic Schwann cell. In the neighborhood there was another normal axon also involved by a hypertrophic Schwann cell.;4000.
FIG. 5. Schwann cells with great cytoplasmic vacuolization caused by proliferation and dilation of the rough and smooth endoplasmic reticulum and Golgi apparatus. The axon involved by this Schwann cell has a disorganized axoplasm.;7000.
the rough and smooth endoplasmic reticulum and Golgi apparatus (Fig. 5). Finally, rare presumptive axons were completely disorganized and "lled with organelles, in particular mitochondria, and electron-dense lamellar bodies that resemble myelin "gures (Fig. 6). Group 2 No abnormality was observed in the nerves submitted to microdissection and electron microscopy.
FIG. 6. A presumptive axon completely disorganized and "lled with organelles, in particular mitochondria, and electron-dense lamellar bodies that resemble myelin "gures. This axon is probably surrounded by a Schwann cell.;10,800.
85
DELTAMETHRIN-INDUCED NEUROPATHY
Control rats Control rats demonstrated no abnormal clinical signs and no abnormality of the sciatic and tibial nerves. DISCUSSION
Deltamethrin is a cyanopyrethroid increasingly used to treat some human and animal parasitic infections. So it is of extreme importance to characterize well all the possible collateral e!ects of this substance. In the present experimental study, fractionated doses of deltamethrin administered for 3 consecutive days (high doses) produced slight nerve damage. These lesions were observed in three of the "ve animals studied (group 1) and were characterized by axonal damage and sometimes secondary changes of the myelin sheath. The aspect of some Schwann cells (vacuolization of the cytoplasm due to proliferation and dilation of the rough and smooth endoplasmic reticulum and Golgi apparatus) suggests a particular reaction of Schwann cells to the noxious insult in an attempt to produce proteins more than a primary lesion of these cells. Rarely there was complete atrophy of the axons. It is interesting that these histologic abnormalities were not observed 7 days after discontinuation of the administration of deltamethrin. This suggests that these histologic abnormalities are scarce and can be observed only during administration of the substance. Actually, the results suggest that the majority of these changes are transitory and rapidly reversible after discontinuation of treatment. It is well known that some compounds of another group of insecticides, the organophosphates, can also be toxic to the central and peripheral nervous system. Besides clinical signs of acute intoxication these compounds can induce a so-called intermediate syndrome (Senanayake and Karalliedde, 1987) or a delayed neurotoxicity in peripheral nerves. In this delayed neurotoxicity the distal portions of the peripheral nerves are more involved than the proximal nerves. In the classic work of Cavanagh (1954) in intoxications with TOCP, besides peripheral nerve damage, changes in the anterior horn cells and lateral corticospinal tracts were described. These lesions are believed to be associated with inhibition of the so-called &&neuropathic target esterase'' (Johnson, 1982; Lotti et al., 1984; Bertolazzi et al., 1991) that su!er a process of &&aging.'' This results in a distal axonopathy of long and large-diameter axons of the spinal cord and peripheral nerves (dying-back neuropathy) which occurs several days or weeks after exposure. The morphological changes observed in the peripheral nerves of deltamethrin-induced neuropathy, here described, are di!erent from those observed in the delayed neuropathy caused by organophosphates in many aspects. In deltamethrin &&neuropathy'' the distal and proximal nerves are equally involved; the delayed neuropathy caused by
organophosphates is irreversible and the histologic changes caused by deltamethrin, here described, were scarce and transitory. Symptoms commonly observed in workers spraying pyrethroids, especially a-cyano pyrethroids, are cutaneous paresthesia and abnormal facial sensations (Moretto, 1991; Lessenger, 1992). Except for these cases of paresthesia no other manifestation of peripheral nerve involvement has been described in human beings. With respect to the morphological changes in peripheral nerves in intoxication with pyrethroids no changes were observed by Dick et al. (1984) in rats fed permethrin for long periods. In contrast to the experiments of Dick et al. (1984), in this study toxic doses of deltamethrin (45 mg/kg, consecutively for 3 days) were used, and the clinical symptoms of intoxication with pyrethroids were easily observed even on the "rst day of intoxication. So although the LD of deltamethrin is known to be extremely variable (135 to 5000 mg/kg in rats (Wortihing and Walker, 1987) the dose used in the present work was thought to be a high dose because one animal died in the second day of intoxication. It is possible that the di!erence between these results and those of Dick et al. (1984) can be explained on the basis of the di!erent types of treatment of the experimental animals. Probably the histologic changes described here have no clinical importance in long-term use of deltamethrin. Instead, these are the pathological substratum of the transitory sensorial disturbance caused by these compounds. Since these histologic changes are transitory and scarce, the question arises: Are they related to the physiological changes in NA>, K>-ATPase activity or Na> channels caused by pyrethroid compounds, which would be related to other ionic or biochemical abnormalities, such as the content and distribution of calcium? CONCLUSION
Fractionated doses of deltamethrin administered for 3 consecutive days (high doses) produced slight nerve damage in rats. These lesions were observed in three of the "ve animals studied (group 1) and were characterized by axonal damage and sometimes secondary changes of the myelin sheath. The aspect of some Schwann cells (vacuolization of the cytoplasm due to proliferation and dilation of the rough and smooth endoplasmic reticulum and Golgi apparatus) suggests a particular reaction of Schwann cells to the noxious insult in an attempt to produce proteins more than a primary lesion of these cells. Rarely was three complete atrophy of the axons. It is interesting that these histologic abnormalities were not observed 7 days after discontinuation of the administration of deltamethrin. The results suggest that the majority of these changes are transitory and rapidly reversible after discontinuation of treatment. Probably the histologic changes described here have no clinical
86
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importance in long-term use of deltamethrin. Instead, these are the pathological substratum of the transitory sensorial disturbance caused by these compounds. REFERENCES Bertolazzi, M., Caroldi, S., Moretto, A., and Lotti, M. (1991). Interaction of methamidophos with hen and human acetylcholinesterase and neuropathy target esterase. Arch. ¹oxicol. 65, 580}585. Cavaliere, M. J., Maeda, M. Y. S., Shih, L. W. S., and Puga, F. R. (1990). Changes in myelinated nerve "bers in skeletal muscle of rats exposed to high doses of permethrin. Ecotoxicol. Environ. Saf. 3, 139}145. Cavanagh, J. B. (1954). The toxic e!ect of tri-ortho-cresyl phosphate on the nervous system, an experimental study in hens. J. Neurol. Neurosurg. Psychiatry 17, 163}172. Dick, P. J., Shimono, M., Schoening, G. P., Lais, A. C., Oviatt, K. F., and Sparks, M. F., (1984). The evaluation of a new synthetic pyrethroid pesticide (permethrin) for neurotoxicity. J. Environ. Pathol. ¹oxicol. Oncol. 5, 109}117. Ecobichon, D. J. (1995). Toxic e!ects of pesticides. In Casarett and Doull1s ¹oxicology: ¹he Basic Science of Poisons, pp. 643}690. 5th ed. McGraw}Hill, New York. Husain, R., Husain, R., Adhami, V. M., and Seth, P. K. (1996). Behavioral, neurochemical and neuromorphological e!ects of deltamethrin in adult rats. J. ¹oxicol. Environ. Health 48, 515}526.
Husain, R., Malaviya, M., Seth, P. K., and Husain, R. (1994). E!ect of deltamethrin on regional brain polyamines and behaviour in young rats. Pharmacol. ¹oxicol. 74, 211}215. Johnson, M. K. (1982). The target for initiation of delayed neuropathy by organophosphorus esters: Biochemical studies and toxicological applications. Rev. Biochem. ¹oxicol. 4, 141}212. Joy, R. M. (1994). Pyrethrins and pyrethroid insecticides. In Pesticides and Neurological Diseases (D. J. Ecobichon and R. M. Joy, Eds.), 2nd ed., pp. 291}312. CRC Press, Boca Raton, FL. Lessenger, J. E. (1992). Five o$ce workers inadvertently exposed to cypermethrin. J. ¹oxicol. Environ. Health 35, 261}267. Lotti, M., Becker, C. E., and Amino!, M. J. (1984). Organophosphate polyneuropathy: pathogenesis and prevention. Neurology 34, 658}662. Moretto, A. (1991). Indoor spraying with the pyrethroid insecticide lambda-cyalothrin: E!ects on spraymen and inhabitants of sprayed houses. Bull. =HO 69, 591}594. Senanayake, N., and Karalliedde, L. J. (1987). Neurotoxic e!ects of orgaphosphorus insecticides. N. Engl. J. Med. 316, 761}763. Takahashi, M., and Le Quesne, P. M. (1982). The e!ects of the pyrethroids deltamethrin and cismethrin on nerve excitability in rats. J. Neurol. Neurosurg. Psychiatry 45, 1005}1011. Wortihing, R. C., and Walker, S. B. (1987). Pesticide Manual: A =orld Compendium 8th ed., pp. 234}235. British Crop Protection Council.
Histologic Peripheral Nerve Changes in Rats Induced by Deltamethrin Edenilson Eduardo Calore,B - Maria JoseH Cavaliere,? FlaH vio Rodrigues Puga,A Nilda Maria Perez Calore,Adriana Ruckert da Rosa,* - Ruth Weg,* - Suzana de Souza Dias,- and Regina Paes dos Santos- ? ** *Pathology Department, Veterinary Medicine School, and BPharmacology Department, Biomedical Institute, and **Public Health Faculty, SaJ o Paulo University, SaJ o Paulo, Brazil; -Pathology Section, Instituto Emn& lio Ribas, SaJ o Paulo, Brazil; ?Pathology Division, Instituto Adolfo Lutz, SaJ o Paulo, Brazil; and AToxicology Section, Instituto Biolo& gico, SaJ o Paulo, Brazil Received October 13, 1999
Nevertheless, some studies have demonstrated that these products, especially compounds with an a-cyano group, are toxic to the mammalian central nervous system (CNS) in acute intoxications (Husain et al., 1994, 1996). Two types of pyrethroid insecticide syndromes have been recognized: Type I syndrome, or T syndrome, which is caused by esters lacking the a-cyano substituent, is characterized by restlessness, incoordination, prostration, and paralysis in insects; in rats, it causes aggressive behavior, whole-body tremor, and prostration. Type II syndrome, or CS syndrome, is caused by a-cyanopyrethroids. These compounds cause intense hyperactivity, incoordination, and convulsions in insects; in contrast, rats display burrowing behavior, coarse seizures, choreoatetosis, and profuse salivation (Ecobichon, 1995). Various mechanisms have been proposed to explain the neurotoxic action of pyrethroid insecticides. Type I products a!ect sodium channels in nerve membranes, causing repetitive (sensorial and motor) neuronal discharge and prolonged negative afterpotential. The presynaptic nerve terminals (of the CNS and peripheral ganglia) are the most sensitive sites. Type II pyrethroids cause a persistent depolarization and frequency-dependent conduction block in sensorial and motor axons as well as prolonged repetitive "ring of sensorial ends and muscle "bers (Joy, 1994). Monoamine oxidase activity increases and NA>, K>-ATPase activity decreases were demonstrated in various regions of the CNS in rats intoxicated with the cyanopyrethroid deltamethrin (Husain et al., 1996). Studies of the toxicity of pyrethroid insecticides in the central and peripheral nervous system of mammals are generally concerned with the biochemical, pharmacological, and physiological e!ects of these products (Takahashi and Le Quesne, 1982; Narahashi et al., 1995). Morphological studies are scarce and the observed changes are discrete. Husain et al. (1996) described dendritic degeneration of Purkinje neurons of the cerebellar region of rats treated
Synthetic pyrethroid insecticides have been used in the last two decades largely because of their high activity as an insecticide and low mammalian toxicity. Some studies have demonstrated that these products, especially compounds with an a-cyano group, are toxic to the mammalian central nervous system (CNS) in acute intoxications. However, morphological studies are scarce. In the present work the histopathologic changes of the sciatic and tibial nerves of rats submitted to acute intoxication with the cyanopyrethroid deltamethrin were studied. For 3 consecutive days male Wistar rats received by oral gavage deltamethrin at a dose of 45 mg/kg body wt. On the 4th day fragments of sciatic and tibial nerves were studied by transmission electron microscopy (TEM) and teasing of individual nerve 5bers. In addition, another group of rats were allowed to recover until the 10th day. Teasing of nerves of animals sacri5ced on the 4th day revealed myelin ovoids, which are indicative of axonal damage. TEM demonstrated rare degenerated axons completely 5lled with organelles, in particular mitochondria, and with electron-dense lamellar bodies that resemble myelin 5gures. In addition, great cytoplasmic vacuolization caused by proliferation and dilation of the rough and smooth endoplasmic reticulum and Golgi apparatus was observed in some Schwann cells. No lesion was found 7 days after discontinuation of the treatment (group 2). Since these histologic changes are transitory and scarce, the question arises: Are they related to the changes in NAⴙ, Kⴙ-ATPase activity or Naⴙ channels caused by pyrethroid compounds? 2000 Academic Press Key Words: nerve; neuropathy; insecticides; pyrethroids; toxicity; deltamethrin.
INTRODUCTION
Synthetic pyrethroid insecticides have been used in the last two decades largely for their high activity as an insecticide and low mammalian toxicity. This low mammalian toxicity is due in part to their rapid metabolism in these animals. This work was supported by grants from FAPESP and CNPq-Brazil. 82 0147-6513/00 $35.00 Copyright 2000 by Academic Press All rights of reproduction in any form reserved.
83
DELTAMETHRIN-INDUCED NEUROPATHY
with deltamethrin. With respect to the peripheral nervous system, Dick et al. (1984) found no changes in sural and tibial nerves and in the maxillary division of cranial nerve V of rats fed permethrin for long periods. Peripheral nerve damage was described in teasing of individual nerve "bers of rats intoxicated with high doses of permethrin (Cavaliere et al., 1990). However, as that work had no ultrastructural studies, a more detailed description of nerve lesions caused by pyrethroid compounds at the ultrastructural level was necessary. The aim of the present work was to study the histopathologic changes of the sciatic and tibial nerves of rats intoxicated with the cyanopyrethroid deltamethrin by transmission electron microscopy (TEM) and teasing of individual nerve "bers. MATERIAL AND METHODS
Animals Two groups of six male Wistar rats weighing 200}250 g received by oral gavage deltamethrin dissolved in corn oil (concentration 45 mg/mL) at a dose of 45 mg/kg body wt, for 3 consecutive days. Water and food were given ad libitum. One animal in group 1 and another in group 2 died respectively on the second and third days of intoxication. Group 1. On the 4th day, under ether anesthesia, the animals were perfused with 4% paraformaldehyde (150 mL) and fragments of sciatic and tibial nerves were collected after ether anesthesia for histopathological studies. Six control animals received only a corresponding dose of corn oil.
FIG. 1. Teasing of individual nerve "bers stained with osmium tetroxide demonstrating myelin ovoids and irregularity of the myelin sheath which are indicative of axonal damage.;900.
RESULTS
Rats treated with deltamethrin exhibited clinical signs of acute intoxication including salivation, hyperactivity, incoordination, coarse seizures, choreoatetosis, and tremor from the "rst day of treatment. In group 2, these symptoms progressively disappeared from the 4th until the 10th day. Group 1 Peripheral nerve lesions were observed in three of the "ve surviving treated animals. These lesions consisted of axonal damage as observed by teasing of individual "bers (myelin ovoids and irregularities of myelin sheath, Fig. 1), as well as by electron mycroscopy. In semithin sections no in#ammatory cells or increases in the amount of connective tissue were observed.
Group 2. From the 4th to the 10th days the animals received only water and food ad libitum. On the 10th day, the animals were submitted to the same procedures as were the animals in group 1. Samples of the nerves were collected. Histopathology Fragments of the sciatic nerve (proximal) and tibial nerve (distal) 1.5 cm long were taken and divided. One fragment of each nerve (proximal and distal) was "xed in 2% glutaraldehyde for 2 h, post-"xed in 1% osmium tetroxide for 1 h, kept in glycerin, and processed for teasing of individual nerve "bers. Other fragments 0.1 cm in diameter (also of each nerve) were "xed in 2% glutaraldehyde in 0.15 M phosphate bu!er, pH 7.2, for 2 h, post"xed in 1% osmium tetroxide, dehydrated, and embedded in araldite resin. The 0.5-lm semithin sections were stained with 1% toluidine blue for light microscopy analysis. For electron microscopy ultrathin sections were obtained and stained with uranyl acetate and lead citrate.
FIG. 2. Axonal damage and axonal atrophy in a myelinated nerve "ber with disorganization of neuro"laments and neurotubules. Actually, the axon was almost no longer recognizable. The myelin sheath was also partially degenerated.;6500.
84
CALORE ET AL.
FIG. 3. Axonal atrophy and substitution of the axon by residual electron-dense bodies and organelles.;8000.
The ultrathin sections of sciatic and tibial nerves con"rmed the axonal damage involving myelinated "bers, which were present in approximately 3% of the myelinated axons of each fascicle. The neuro"laments and neurotubules were disorganized and almost unrecognizable. The myelin sheath was partially degenerated. (Fig. 2). Rarely could axonal atrophy be observed and instead of axons only residual electron-dense bodies and organelle were observed (Fig. 3). In addition, some axons were involved by disproportionately thin myelin sheaths and hyperthrophic Schwann cells (Fig. 4). These Schwann cells had great cytoplasmic vacuolization caused by proliferation and dilation of
FIG. 4. An axon involved by a disproportionately thin myelin sheath and a hyperthrophic Schwann cell. In the neighborhood there was another normal axon also involved by a hypertrophic Schwann cell.;4000.
FIG. 5. Schwann cells with great cytoplasmic vacuolization caused by proliferation and dilation of the rough and smooth endoplasmic reticulum and Golgi apparatus. The axon involved by this Schwann cell has a disorganized axoplasm.;7000.
the rough and smooth endoplasmic reticulum and Golgi apparatus (Fig. 5). Finally, rare presumptive axons were completely disorganized and "lled with organelles, in particular mitochondria, and electron-dense lamellar bodies that resemble myelin "gures (Fig. 6). Group 2 No abnormality was observed in the nerves submitted to microdissection and electron microscopy.
FIG. 6. A presumptive axon completely disorganized and "lled with organelles, in particular mitochondria, and electron-dense lamellar bodies that resemble myelin "gures. This axon is probably surrounded by a Schwann cell.;10,800.
85
DELTAMETHRIN-INDUCED NEUROPATHY
Control rats Control rats demonstrated no abnormal clinical signs and no abnormality of the sciatic and tibial nerves. DISCUSSION
Deltamethrin is a cyanopyrethroid increasingly used to treat some human and animal parasitic infections. So it is of extreme importance to characterize well all the possible collateral e!ects of this substance. In the present experimental study, fractionated doses of deltamethrin administered for 3 consecutive days (high doses) produced slight nerve damage. These lesions were observed in three of the "ve animals studied (group 1) and were characterized by axonal damage and sometimes secondary changes of the myelin sheath. The aspect of some Schwann cells (vacuolization of the cytoplasm due to proliferation and dilation of the rough and smooth endoplasmic reticulum and Golgi apparatus) suggests a particular reaction of Schwann cells to the noxious insult in an attempt to produce proteins more than a primary lesion of these cells. Rarely there was complete atrophy of the axons. It is interesting that these histologic abnormalities were not observed 7 days after discontinuation of the administration of deltamethrin. This suggests that these histologic abnormalities are scarce and can be observed only during administration of the substance. Actually, the results suggest that the majority of these changes are transitory and rapidly reversible after discontinuation of treatment. It is well known that some compounds of another group of insecticides, the organophosphates, can also be toxic to the central and peripheral nervous system. Besides clinical signs of acute intoxication these compounds can induce a so-called intermediate syndrome (Senanayake and Karalliedde, 1987) or a delayed neurotoxicity in peripheral nerves. In this delayed neurotoxicity the distal portions of the peripheral nerves are more involved than the proximal nerves. In the classic work of Cavanagh (1954) in intoxications with TOCP, besides peripheral nerve damage, changes in the anterior horn cells and lateral corticospinal tracts were described. These lesions are believed to be associated with inhibition of the so-called &&neuropathic target esterase'' (Johnson, 1982; Lotti et al., 1984; Bertolazzi et al., 1991) that su!er a process of &&aging.'' This results in a distal axonopathy of long and large-diameter axons of the spinal cord and peripheral nerves (dying-back neuropathy) which occurs several days or weeks after exposure. The morphological changes observed in the peripheral nerves of deltamethrin-induced neuropathy, here described, are di!erent from those observed in the delayed neuropathy caused by organophosphates in many aspects. In deltamethrin &&neuropathy'' the distal and proximal nerves are equally involved; the delayed neuropathy caused by
organophosphates is irreversible and the histologic changes caused by deltamethrin, here described, were scarce and transitory. Symptoms commonly observed in workers spraying pyrethroids, especially a-cyano pyrethroids, are cutaneous paresthesia and abnormal facial sensations (Moretto, 1991; Lessenger, 1992). Except for these cases of paresthesia no other manifestation of peripheral nerve involvement has been described in human beings. With respect to the morphological changes in peripheral nerves in intoxication with pyrethroids no changes were observed by Dick et al. (1984) in rats fed permethrin for long periods. In contrast to the experiments of Dick et al. (1984), in this study toxic doses of deltamethrin (45 mg/kg, consecutively for 3 days) were used, and the clinical symptoms of intoxication with pyrethroids were easily observed even on the "rst day of intoxication. So although the LD of deltamethrin is known to be extremely variable (135 to 5000 mg/kg in rats (Wortihing and Walker, 1987) the dose used in the present work was thought to be a high dose because one animal died in the second day of intoxication. It is possible that the di!erence between these results and those of Dick et al. (1984) can be explained on the basis of the di!erent types of treatment of the experimental animals. Probably the histologic changes described here have no clinical importance in long-term use of deltamethrin. Instead, these are the pathological substratum of the transitory sensorial disturbance caused by these compounds. Since these histologic changes are transitory and scarce, the question arises: Are they related to the physiological changes in NA>, K>-ATPase activity or Na> channels caused by pyrethroid compounds, which would be related to other ionic or biochemical abnormalities, such as the content and distribution of calcium? CONCLUSION
Fractionated doses of deltamethrin administered for 3 consecutive days (high doses) produced slight nerve damage in rats. These lesions were observed in three of the "ve animals studied (group 1) and were characterized by axonal damage and sometimes secondary changes of the myelin sheath. The aspect of some Schwann cells (vacuolization of the cytoplasm due to proliferation and dilation of the rough and smooth endoplasmic reticulum and Golgi apparatus) suggests a particular reaction of Schwann cells to the noxious insult in an attempt to produce proteins more than a primary lesion of these cells. Rarely was three complete atrophy of the axons. It is interesting that these histologic abnormalities were not observed 7 days after discontinuation of the administration of deltamethrin. The results suggest that the majority of these changes are transitory and rapidly reversible after discontinuation of treatment. Probably the histologic changes described here have no clinical
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