ulnaires, ainsi que celle de biopsies des muscles appendiculaires distaux, s'a- vererent normales, chez toutes les chiennes. L'examen microscopique du.
Inability to Experimentally Produce a Polyneuropathy in Dogs Given Chronic Oral Low Level Lead J.E. Steiss, K.G. Braund and E.G. Clark*
revela eleve, chez les quatre chiennes experimentales, et sa valeur moyenne Electromyographic examinations atteignit respectivement 1,15; 2,18; were performed at various times over a 1,13 et 1,72 ,mol/L. Les auteurs ne 40 week period in four mature dogs noterent aucun signe clinique d'intoxireceiving chronic oral low doses of cation par le plomb. Deux des lead acetate and a control dog receiv- chiennes experimentales presenterent ing sodium acetate. Blood lead levels cependant une anemie regeneratrice. in the four dogs were elevated (mean Ni l'electromyographie, ni les etudes values 1.15, 2.18, 1.13 and 1.72 ,umol/ de velocite de la conduction nerveuse liter). No clinical signs of lead intoxi- ne revelerent l'evidence d'une polyneucation were present. Two dogs had ropathie. L'excitation de preparations evidence of a nonregenerative anemia. de flbres nerveuses de segments proNeither needle electromyographic nor ximaux et distaux des nerfs tibiaux et nerve conduction velocity studies ulnaires, ainsi que celle de biopsies des showed evidence of a polyneuropathy. muscles appendiculaires distaux, s'aTeased nerve fiber preparations of vererent normales, chez toutes les proximal and distal segments of the chiennes. L'examen microscopique du ulnar and tibial nerves and muscle cerveau, des reins et du foie des deux biopsies of distal appendicular mus- chiennes soumises a un examen cles were normal in all dogs. Light necropsique ne revela aucune anormicroscopic examination of the brain, malite. En conclusion, I'administrakidneys and liver revealed no abnor- tion experimentale de faibles doses malities in the two dogs necropsied. In buccales de plomb inorganique a des conclusion, a polyneuropathy was not chiennes, sur une periode de 40 produced experimentally in dogs semaines, ne produisit pas de ingesting low doses of inorganic lead polyneuropathie. for up to 40 weeks. Mots cles: plomb, polyneuropathie, Key words: Lead, polyneuropathy, chien, electromyographie. dog, electromyography. ABSTRACT
INTRODUCTION
RESUME, Cette expfrience s'etalait sur une periode de 40 semaines et elle consistait a effectuer des examens d'electromyographie, chez quatre chiennes adultes et croisees qui recevaient de faibles doses orales d'acetate de plomb, ainsi que chez une chienne temoin a qui on donnait de l'acetate de sodium. Le taux sanguin de plomb se
Dogs are susceptible to various polyneuropathies of inherited or acquired origin (1,2). Lead is known to cause peripheral nerve damage in certain species (3). Veterinary neurologists have considered lead poisoning in the differential diagnosis of canine polyneuropathy (4,5). However, peripheral nerve lesions have rarely been described in lead poisoned dogs and,
when present, appeared to be mononeuropathies in animals which may also have had clinical signs of lead encephalopathy (6,7). In the present study, chronic low level inorganic lead intoxication was produced in four adult dogs. They were examined for histopathological and electrophysiological evidence of a polyneuropathy. The aim was to determine whether mature household dogs ingesting small amounts of inorganic lead are likely to suffer peripheral nerve damage in the absence of overt signs of lead encephalopathy.
MATERIALS AND METHODS ANIMALS
Five adult, female, mixed breed dogs weighing 19-23 kg were housed in indoor runs and fed a commercial dog chow (Purina Dog Chow, Ralston Purina Canada Inc., Mississauga, Ontario) ad libitum. The dogs were observed daily and were weighed weekly. Lead acetate (Fisher Scientific Co., Fair Lawn, New Jersey) was given orally in gelatin capsules, once per day, five days per week (dogs 1-4) starting on week 2. The dose ranged from 1.5 to 5 mg/kg/day in order to maintain blood lead levels in the low toxic range. All dogs received the same dose, except dog 2 which received half the daily dose from weeks 7-16, inclusive. The control (dog 5) received sodium acetate (Amachem, Portland, Oregon) on an identical schedule. Dogs I and 2 were euthanized on weeks 24 and 26, respectively. Dogs 3-5 were euthanized on week 40. Clinical neurological examinations (1) were
*Department of Physiological Sciences (Steiss) and Department of Veterinary Pathology (Clark), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO and Scott-Ritchey Research Program, School of Veterinary Medicine, Auburn University, Auburn, Alabama 36849 (Braund). This research was supported in part by a grant from the Canadian Veterinary Research Trust Fund. Dr. Steiss is supported by a University Research Fellowship from the Natural Sciences and Engineering Research Council of Canada. Submitted December 3, 1984. Can J Comp Med 1985; 49: 401-404.
401
performed at the beginning of the experiment and monthly thereafter. Prior to euthanasia, samples were taken for hematology. LEAD DETERMINATION
Blood lead levels were assayed by a previously described method (8) adapted for a Unicam SP1900 Atomic Absorption Spectrophotometer. Blood was collected from the jugular vein into 10 mL heparinized Vacutainer tubes (Becton, Dickinson & Co., Rutherford, New Jersey) and stored at 4°C until assayed. Lead determinations were done weekly from weeks 1-13, biweekly from weeks 14-19 and then monthly for weeks 20-40. ELECTROM YOGRA PH Y
Needle electromyography (EMG) (Model 51 P, Rochester Electromedical Inc., Osseo, Minnesota) and motor nerve conduction velocity (NCV) calculations on the sciatic-tibial nerve were performed biweekly from weeks 1-27 and monthly from weeks 28-40, on the left side of the body, with the dogs under thiamylal anesthesia (Biotal, MTC Pharmaceuticals, Hamilton, Ontario) (18 mg/kg of body weight, IV). The method has been previously described (9). The needle EMG examination included proximal and distal muscles of the front and hind limbs plus paraspinal muscles.
nerve samples were divided longitudinally into halves. One half of each nerve sample was postfixed in 1% osmium tetroxide for 12 hours and placed in 66% glycerin for 48 hours and 100% glycerin for 24 hours for teased fiber preparation (11). At least 100 single fibers per nerve sample were teased randomly from all fascicles, without preselection for size or abnormality. The second half of each nerve sample was washed in Millonig's phosphate buffer (pH 7.3) at 4°C overnight. The samples were postfixed in 1% osmium tetroxide for one hour, washed in phosphate buffer, transferred through graded ethanol solutions and processed for embedding in epon. Semithin sections (I to 2,um) were cut transversely and stained with toluidine blue. Teased nerve fibers were histologically classified as previously described (I 1,12). In dogs 3-5, the brain, kidneys and liver were removed after euthanasia, and fixed in 10% neutral buffered formalin. Sections (6,utm) were cut and stained with H & E. Areas of the brain examined included multiple sections of medulla oblongata, pons, mesencephalon and forebrain. Additional sections of kidney were stained with the Ziehl-Neelsen method for acid-fast lead inclusions (13).
402
Il
1 6.0
DOG 3
DOG 4
° 3.0 t ;
L
o 0 m 2.0
t
0
30
20
5
10
40
20
30
40
WEEK
Fig. 1. Blood lead values (imol/liter) in four dogs fed chronic low levels of inorganic lead. Suspect levels are between 1.2-1.7 ,mol/liter; toxic levels are > 1.7 ,mol/liter. See text for lead assay procedure.
120 { 100
60 34
'0
60 4
RESULTS
H ISTOPATHOLOGY
Biopsies of skeletal muscles (extensor carpi radialis and tibialis cranialis) and nerves (ulnar and tibial) were taken from the right side of the body at the time of euthanasia with an overdose of pentobarbital (Somnotol, MTC Pharmaceuticals, Hamilton, Ontario) intravenously. Muscle biopsies were stored in moist gauze at 40 C for two to three hours before freezing in isopentane cooled in liquid nitrogen. Sections were cut at 8,unm on a cryostat and stained for myosin ATPase, pH 4.3 and 10.0, NADH, PAS, modified Gomori trichrome and H & E (10). Fiber diameters were measured on an IBAS I image analysis system (Zeiss Canada Ltd., Toronto, Ontario). Nerve biopsies were stretched on wooden tongue depressors and stored in 10% neutral buffered formalin. After fixation for several days, the
E
ANI MALS
Despite elevated blood lead levels, no dog manifested clinical signs of lead
encephalopathy. Clinical neurological examinations were consistently normal. None of the dogs lost weight. At the time of euthanasia, dogs 1 and 2 had evidence of a nonregenerative anemia, viz. decreased packed cell volume without reticulocytosis. LEAD DETERMINATIONS
The blood lead levels in the individual dogs are shown (Fig. 1) along with the oral dosage levels (Fig. 2). The blood levels were maintained in the suspect to toxic range for dogs 1-4 most of the time, while control dog 5 was always within normal range. The mean blood levels for dogs 1-5, calculated from samples every four weeks starting at week 7 until euthanasia, were 1. 15, 2.18, 1.13, 1.72 and
tate fed over a 40 week
74 72 E 70 H 0 68 LnV 66 zD> 64 0 H 62 z 0 60 0 58
period.
-
-
-
-
z
10
20 WEEK
30
40
Fig. 3. Sciatic-tibial motor nerve conduction velocities (x ± S.E.) for dogs fed lead acetate (solid line) and control dog (dotted line). Normal range for this laboratory is 59-73 m/s.
0.07,4lmol/liter, respectively. The
initial oral lead dose was decreased from about 95 to 35 mg daily on week 5 due to the rapidly rising blood lead levels in most dogs. From then until the end of the experiment, the dosage had to be gradually increased to maintain elevated blood lead levels. ELECTROM YOGRAPHY
The needle EMG examinations revealed occasional foci of positive waves in one or two muscles in several dogs. This was not considered abnormal. There was no sustained abnormal spontaneous activity in either proximal or distal muscles suggestive of a polyneuropathy. Dog 1 had evidence of a mononeuropathy involving the left ulnar nerve, starting week 8. This was attributed to a wound sustained in a dog fight. The motor nerve conduction velocities for the sciatic-tibial nerve were consistently within normal range for this laboratory for all dogs (Fig. 3). The mean NCV ± S.E. for the lead-treated dogs showed no decrease over time. Similarly, the amplitude and duration of the evoked potentials recorded in the interosseous muscles showed no significant alterations over time (not shown). HISTOPATHOLOGY
Analysis of the muscle biopsies revealed no foci of atrophic or angular fibers that would be expected with a chronic denervating process. Fiber diameter measurements on the extensor carpi radialis for the lead-treated animals were within normal range and showed a unimodal distribution (Table I). Minimal changes were present in single teased nerve fibers (Table II). TABLE I. Fiber Diameters (psm) Measured on Myosin ATPase pH 4.3 Stained Sections of extensor carpi radials Muscle Biopsies from Dogs Fed Lead Acetate (no. 1-4) and Control Dog (no. 5) Fiber Type I IIC IIA Dog No. 47.3 ± 8.0a 44.9 ± 8.4 42.8 ± 7.0 1 39.2 ± 6.4 NDb 47.3 ± 9.4 2 41.7 6.6 ND 45.5 ±8.2 3 43.5 7.8 ND 48.6 ± 8.9 4 42.2 ±6.3 46.0 7.3 48.4 ±8.2 5 ax ± S.D., measured from 100 muscle fibers bND = none detected
TABLE II. Percentage of Abnormal Teased Nerve Fibers in Dogs Fed Lead Acetate (no. 1-4) and Control (no. 5)a Tibial Nerve Distal
Ulnar Nerve Dog No. I
2 3 4 5
Proximal 0 0 0 0
Distal 0 ND 3C 0
Proximal 0 0
0
le
Lateral Plantar
NDb ND
0
0
0
Id
IC
0
Medial Plantar 0 0 0 0
aEach value was determined by examining 105-162 teased nerve fibers
bND = not determined
CParanodal/ segmental demyelination dOvoids and balls elntercalated internode(s)
mutilated the corresponding limb. A second dog in that series had a dilated esophagus, but the vagus nerve was not examined. It was postulated that reversible esophageal paralysis found in several other immature dogs with confirmed lead poisoning was due to a vagal neuropathy. In one experimental study (7), an immature dog that received lead salts (5 mg/ kg/ day) along with a high-fat low-calcium diet experienced intermittent left hind limb tions of any nerve. Sections of the brain revealed no weakness. There was axonal degenerasignificant lesions in the dogs exam- tion in the left sciatic nerve. This dog ined. Sections of liver and kidney also convulsed. None of the other eight showed no lesions or acid-fast lead dogs in that series, which received 560 mg/kg/day lead salts, had clinical inclusions. or pathological evidence of a neuropathy. In the present study, despite eleDISCUSSION vated blood lead levels in all four dogs In laboratory animals, such as rats and anemia in two dogs, no clinical and guinea pigs, a polyneuropathy can signs of lead encephalopathy or polyresult from lead intoxication (3). The neuropathy were detected. Nerve conmost prominent pathological feature duction velocities of the sciatic-tibial is segmental demyelination. In nerves consistently remained within humans, however, lead-induced poly- normal range and diffuse spontaneous neuropathy is associated with axonal activity in the form of fibrillation (Wallerian) degeneration. Clinically, potentials or positive waves was not the disease usually manifests itself as detected. Although a few peripheral motor weakness (3). Although lead nerve lesions were seen, a similar incialters the blood-nerve barrier, result- dence of teased nerve fiber abnormaliing in endoneurial edema with ties has been reported in clinically increased endoneurial pressure, the normal mature dogs (12). Therefore, primary insult may be a direct toxic the electromyographic and histopathological findings ruled out the effect on Schwann cells (14). Numerous reports exist on the clini- possibility of a subclinical polyneurocal and pathological findings in lead- pathy. To what extent the dosage, induced encephalopathy in dogs duration of exposure and effect of (6,7,15-19). There is a report of Waller- organic vs. inorganic lead might be ian degeneration in "leg nerves" of one significant factors in producing a polydog out of 32 necropsied after acciden- neuropathy in the dog remains tal lead poisoning (6). That dog self- unknown. One wonders whether the
With the exception of the distal ulnar nerve from dog 3, in which three percent of fibers had evidence of paranodal and segmental demyelination, one percent or less of teased fibers contained abnormalities such as intercalated internodes (indicative of remyelination), myelin ovoids and balls (representative of axonal degeneration), or demyelination. No abnormalities were observed in semithin sec-
403
difficulty in maintaining a sustained toxic lead level, i.e. > 1.7 ,mol/ liter, could account for the negative results. A further trial, after establishing a protocol which yielded a consistent toxic, nonconvulsant blood lead level, would help to answer this question. Hamir et al (7) fed several dogs a commercial balanced diet plus 5 mg/kg/ day lead salts for about two months. Blood lead content did not exceed 50 ,g/dL. Dogs fed a similar dose of lead but a high-fat low-calcium diet attained higher blood lead values. In the present experiment, blood lead levels in the four dogs reached the suspect to toxic range with maintenance on commercial dog chow. There is some indication of tolerance to lead in the dogs in this study. This phenomenon has been discussed as early as 1927 (20). We had to increase the lead dosage over the duration of the experiment to maintain blood lead levels in the suspect to low toxic range. In particular, the initial dose of 95 mg lead acetate per day that was administered until week 6 produced a faster rise in blood lead levels than occurred between weeks 30 to 40 when this dosage level was again given, e.g. see Figure 1, dog 4. In conclusion, a polyneuropathy was not produced experimentally in four adult dogs ingesting low doses of inorganic lead for up to 40 weeks. Dogs may not be at high risk for polyneuropathy when ingesting small amounts of lead even for prolonged periods of time.
404
ACKNOWLEDGMENTS The authors thank Drs. D.L. Hamilton and B. R. Blakley for helpful discussion, Ms. D. Guedo and C. Hounjet for lead analyses and Ms. S. Riedlinger for typing. Special thanks to Ms. L. Milne, F. Minty, C. Moir, K. Neilsen, W. Nugent and W. Saunders (2nd year veterinary students) for assistance.
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