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Delayed onset of distal axonal neuropathy in primates after prolonged low-level administration of a neurotoxin.

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BRIEF COMMUNICATIONS
Delayed Onset of Distal
Axonal Neuropathy in
Primates After Prolonged
Low-Level Administration
of a Neurotoxin
H. H. Schaumburg, M.D.+SJ. C. Arezzo, PhD,"?
and P. S. Spencer, PhDP
Short-latency somatosensory evoked potentials were recorded from surface electrodes overlying peripheral
nerve, spinal cord, and cortex in 4 monkeys during prolonged intoxication with low levels of acrylamide. A
fifth animal served as a longitudinal control subject.
Slowing of the response across the spinal-medullary
junction was a reliable sign, manifest only after prolonged exposure. Associated morphological changes
were preterminal accumulation of axonal neurofilaments without synaptic disruption in the gracile nucleus. The induced alterations in the latency of shortlatency somatosensory evoked potentials and in axon
morphology were reversible after 7 months of recovery.
The extreme delay in onset of subtle neurological dysfunction (940 days) following administration of a presumed safe level of acrylamide suggests that permissible
levels of human exposure to toxins of this type should be
reassessed.
Schaumburg HH, Arezzo JC, Spencer PS. Delayed
onset of distal axonal neuropathy in primates after
prolonged low-level administration of a neurotoxin.
Ann Neurol 1989;26:576-579
Toxic axonal neuropathies are heralded by early reversible dysfunction in distal ends of long sensory axons in the dorsal columns terminating in gracile nuclei.
A previous electrophysiological and morphological
study of primate neuropathy utilized relatively large
(10.0 mg/kg) doses of acrylamide and demonstrated
changes in the latency and amplitude of the shortlatency somatosensory evoked potential (SLSEP) gen-
From the Departments of #Neurology, ?Neuroscience, and fPAhology (Neuropathology) and the Institute of Neurotoxicology, Albert
Einstein College of Medicine, The Bronx, NY, and the $Center for
Occupational Disease Research, Oregon Health Science University,
Portland, OR.
Received Dec 15, 1988, and in revised form Mar 2, 1989. Accepted
for publication Mar 4, 1989.
Address correspondence to Dr Schaumburg, Departmenr of Neurology, G-9 Forchheimer Building, Albert Einstein College of
Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.
erated within the rostral dorsal columns El}. These
changes occurred within 15 days after the administration of acrylamide was started and preceded alterations
of peripheral nerve conduction and light microscopic
evidence of pathological changes in the gracile nucleus.
Since occupational and environmental injury from
neurotoxins frequently follows prolonged low-level
exposure, the present study sought to determine the
consequences of long-term exposure of primates to
acrylamide at low levels, below the reported no-effect
dose of 3.0 mg/kg [Z].
Materials and Methods
Five adolescent male monkeys (Macacafuscicdatis) with immature canine teeth weighing between 2.0 and 4.5 kg were
used. Four animals received subcutaneous injections of an
aqueous solution of 99% pure acrylamide, 6 days a week, in
the following regimens: 0.5 mg/kg for 1,128 days, 1.0 mg/kg
for 1,064 days, 2.0 mg/kg for 900 days, and 3.0 mglkg for
902 days with recovery for 2 14 days. A fifth animal received
daily injections of an equivalent dose of normal saline solution. SLSEP data were recorded using subdermal electrodes
overlying the sural and common peroneal nerves, the cauda
equina, the lumbar and sacral regions of the spinal cord, and
the cervical region of the contralateral cortex, following
stimulation of the peroneal nerve at the head of the fibula in
animals anesthetized with pentobarbital sodium (25 mg/kg).
Two to four thousand responses were averaged for each
spinal recording site; 200 to 400 were processed for each
peripheral nerve and cortical site. Data were initially recorded to a noncephalic reference (contralateral wrist), but
computer subtraction was utilized to enhance and isolate the
P2 component of SLSEP at the junction of the dorsal columns and the medulla. Details of electrophysiological recording have been reported previously { 11. Data in the present study were obtained for each monkey prior to treatment
and thereafter at 2- or 3-month intervals throughout the
course of acrylamide administration.
For morphological studies all monkeys were deeply anesthetized with ketamine and pentobarbital sodium containing
heparin, and 4% paraformaldehyde was perfused through
the aortic arch for 30 seconds followed by 5% glutaraldehyde for 10 minutes, each fixative in a 0.1 M phosphate
buffer. In each animal, central nervous system (CNS) tissue
was sampled at 25 consecutive 1-mm sections of the dorsal
column nuclei commencing at the middle of the first cervical
level and extending through a level 6 mm rostral to the area
postrema. The five levels of the dorsal column nuclei designated by Rustioni and colleagues [3] as A, B, C, D, and E
were identified in each animal. Tissue was immersed for 1
to 3 hours in Dalton chrome osmium solution, dehydrated
stepwise, immersed in propylene oxide, and infiltrated with
epoxy resin. One-micrometer-thick sections, cut from hardened epoxy resin blocks, were stained with toluidine blue
and examined by light microscopy. Thin sections from each
of the five levels for every animal were stained with uranyl
acetate followed by lead citrate, and were examined with the
electron microscope.
576 Copyright 0 1989 by the American Neurological Association
30
0.5 mg/kg
25
2.0 mg/kg
3.0 mg/kg
1.0 r n g / k g
a
*
w
z
control
2O
-J
W
s
15
I
0
10
w
a
5
5
I
0
x
0
-5
-10
I
0
0.5
1 .o
1.5
2.0
2.5
TREATMENT YEARS
Fig I. Percent changefwm baJeline in the peak latency ofthe
short-latency somatosensory evoked potential component recorded
overlying the medullary-cervicaljunction. The arrow indicates
the onset of the recovery periodfor the monkey that received 3.0
mglkg. Note the delayed onset of slowing in the monkey that
received I .O mglkg.
Results
EhctrophysioLogicuL Findings
Monkeys exposed to 3.0, 2.0, and 1.0 mg/kg developed reliable SLSEP findings, without clinical signs or
weight loss, first evident at 1.0, 1.2, and 2.6 years,
respectively (Fig 1). The nature of the initial change
was identical to that described previously [l): a decrease in the slope of the P2 component and a corresponding prolongation of the peak latency of this
response, observed in bipolar recordings across the
cervical-medullary transition zone. This recording configuration provides a sensitive index of the afferent
volley propagating in the rostral extreme of the dorsal
columns. Peripheral conduction velocity overlying the
distal part of the surd nerve and proximal part of the
peroneal nerve, as well as spinal conduction in the
more caudal segments of the spinal cord, were within
normal limits for all monkeys. The initial alterations in
latency were not associated with a reduction in compound sensory amplitude; however, continued administration of acrylamide led to both "staircasing" of the
rising phase of the P2 component and reduction in the
peak amplitude of this and subsequent SLSEP components. The SLSEP changes in the animal exposed to
3.0 mg/kg significantly improved during the recovery
3.0
3.5
period, but latency had not fully recovered by the end
of the experiment. The control animal and the monkey
receiving 0.5 mg/kg showed no consistent electrophysiological or behavioral change throughout the course
of the study.
Morphological Findings
Analysis of histological sections from the animals receiving 1.0 mg/kg and the one exposed to 2.0 mg/kg
revealed axonal changes prominent at the caudal level
(Rustioni A) of the dorsal column nuclei in both animals (Fig 2). The abnormalities were most frequent in
the gracile nucleus of the animal that received 2.0 mg/
kg and consisted of three varieties of axonal change:
Swelling was most common, dark staining and fiber
breakdown were rare. Swollen, glassy-appearing s o n s
had thinned myelin sheaths and masses of whorled
10-nm neurofilaments with occasional dense bodies,
clumps of mitochondria, and neurotubules. Dorsal column neurons and their axosomatic synapses were unremarkable.
Sections from equivalent regions of the control
animal revealed only rare myelinated fibers in the dorsal columns undergoing complete fiber breakdown.
Axonal swellings were extremely rare; one, characterized by masses of 10-nm neurofilaments, was found at
the Rustioni B level. Sections from the animal that
received 0.5 rng/kg and the recovered animals that
received 3.0 mg/kg did not differ from those of the
control animal.
Brief Communication: Schaumburg et al: Delayed Onset of Anoxal Neuropathy
577
Fig 2. Sections fmm the gracile tract (top) and nuchus (bottom)
at the Rustioni A level from the animal dosed with 2.0 mglkg.
The top illr/strateJ a cross section of a myelinated axon distended
83, masses of 10-nm neurofhmnts ( x 17,SOOj. The bottom depicts four normal-appearing axosomtic synapses ( x 16,600).
Discussion
Distal axonal neuropathy is a term that describes diseases morphologically expressed as symmetrical degeneration of distal parts of mons occurring concurrently in the peripheral nervous system and selected
tracts of the central nervous system; it is the most
frequent pattern of axonal pathological lesions in human and experimental neurotoxic injury [4-61. The
578 Annals of Neurology Vol 26 No 4
October 1989
distal ends of the rostral projections of neurons in lumbar dorsal root ganglia terminating in the gracile nucleus are among the most vulnerable loci [6]. The
pathogenesis of the neuropathy in distal regions of axons produced by chronic administration of acrylamide
is unknown; abnormalities of axonal transport are presumed to have a role in producing the accumulations
of neurofilaments [7-91.
Two cardinal findings in this study are relevant to
human degenerative disease: the recovery of the animal that received a high dose even after prolonged periods of dysfunction, and the synaptic preservation despite preterminal axonal degeneration in the animals
treated with 2.0 and 1.0 mg/kg. Although morphological studies were not done at the peak of intoxication in
the animal that received a high dose, it is certain that
preterminal accumulations of neurofilaments occurred
following such prolonged intoxication at that level of
acrylamide. A previous study of acrylamide-induced
axonal neuropathy demonstrated that abundant pretermind, multifocal axonal accumulations of neurofilaments also occur in the peripheral nervous system C41.
Taken in concert, the findings in the previous and
present studies indicate that in CNS degenerative axonal diseases, distal regions of axons may undergo
changes and subsequently reorganize without loss of
synaptic connection.
A previous study showed that changes in specific
SLSEP components can target vulnerable regions of
the afferent pathways and thus provide sensitive indicators of early axonal dysfunction {l}. The present
study further demonstrates the value of longitudinal
SLSEP monitoring for early detection of neuropathy in
distal regions of axons in experimental animals. Furthermore, the early electrophysiological findings appear largely reversible, even following prolonged intoxication, suggesting they reflect functional deficits
that antedate permanent structural change. The utility
of this measure in assessing human neurotoxicity is
uncertain. In humans, cervical SLSEP components following stimulation of the lower limbs are more difficult
to isolate and are generally more variable due to the
elongation of the pathways and the increased distance
from the generators [l, 101.
The extreme delay in onset of subtle neurological
dysfunction in the monkey that received 1.0 mg/kg is
relevant to human exposure to many neurotoxins.
Generally neurotoxic “safe” and “no-effect’’ levels are
determined by testing the agent of interest for a period
such as 90 days. In contrast, most occupational and
many environmental and pharmaceutical exposures to
neurotoxins are characterized by prolonged, low-level
exposure. Our findings suggest that many exposure
levels regarded as safe or conveying no effects should
be reassessed. Furthermore, such exposures may result
in a pattern of functional and structural deficit which is
similar to that produced by the relatively acute exposure, but the changes will be insidious in onset and
may be difficult to distinguish from those associated
with normal aging. In support of this assumption, our
experience has demonstrated widespread, asymptomatic nervous system disease in two groups. We have
noted that workers with subclinical occupational toxininduced neuropathy in distal regions of axons may
deny disability obvious to their spouses, and Indian
farmers display unnoticed subtle spastic paraparesis
from mild dietary lathyrism. Presumably such neurotoxic deficits were missed partly due to their slow
progress and partly due to their temporal dissociation
from the onset of exposure. Greater awareness of the
effects of long-term, low-level neurotoxic exposure is
critical, and the issues raised by the present study extend into the broad area of occupational and environmental medicine. For example, there is concern that
smelter workers experience low-level clinically inapparent intoxication from arsenic-containing ores [I 11,
that childhood cognitive impairment follows prolonged low-level environmental intoxication by lead
[12], and that medical workers may develop a subtle
dementing illness from prolonged low-level exposure
to ethylene oxide [ 131.
Supported by grants ES 02168 and NS 19611 from the US Public
Health Service.
The authors wish to thank Monica Fenton and Mona Licwak for their
technical assistance and Tina Rubano and Unda O’Donnell for help
in preparing the manuscript.
References
1. Arezto JC, Schaumburg HH, Vaughan HG Jr, et al. Hind limb
somatosensory evoked potentials in the monkey: the effects of
distal axonopathy. Ann Neurol 1982;12:24-32
2. McCollister DD, @en F, Rowe VK. Toxicology of acrylamide.
TOX~COI
Appl Phmacol 1964;6:172-181
3. Rustioni A, Hayes NL, ONeil S. Dorsal column nuclei of monkeys. Brain 1979;102:95-125
4. Schaumburg HH, Wisniewski HM, Spencer PS. Ultrastructural
studies of the dying back process. I. Peripheral nerve terminal
and axon degeneration in systemic acrylamide intoxication. J
Neuropathol Exp Neurol 1974;33:260-264
5. Spencer PS, Schaumburg HH. Central and peripheral distal axonopathy-the pathology of dying back polyneuropathies. In:
Zimmerman H, ed. Progress in neuropathology. New York
Grune and Stratton, 1976:253-295
6. Spencer PS, Schaumburg HH. Ultrastrucrure studies of the dying-back process. IV. Differential vulnerability of DNS and
CNS fibers in experimental central-peripheral distal axonopathies. J Neuropathol Exp Neurol 1977;36:300-320
7. Gold BG, Griffin JW,
Price DL.Slow axonal transport in acrylamide neuropathy: different abnormalities produced by singledose and continuous administration. J Neurosci 1985;5:17551768
8. Gold BG, Price DL, Griffinw, et al. Neurofilament antigens in
acrylamide neuropathy. J Neuropathol Exp Neurol 1988;47:
145-157
9. Miller MS, Spencer PS. Single doses of acrylamide reduce retrograde transport velocity. J Neurochern 1984;43:1401-1408
10. Aminoff MJ. Evoked potentials in clinical medicine. Q J Med
1986;59:345-362
11. Feldman RG, Niles CA, Kelly-Hayes M, et al. Peripheral neuropathy in arsenic smelter workers. Neurology 1979;29:939944
12. McMichaef AJ, Baghurst PA, Wigg NR, er al. Port pirie cohort
study: environmentalexposure to lead and children’s abilities at
the age of four years. N Engl J Med 1988;319:468-475
13. Crystal HA, Schaumburg HH, Grober E, et al. Cognitive impairment and sensory loss associated with chronic low-level ethylene oxide exposure. Neurology 1988;38:567-569
Brief Communication: Schaumburg et al: Delayed Onset of Anoxal Neuropathy 579
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