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Experimental Neurology 158, 37–46 (1999)Article ID exnr.1999.7102, available online at http://www.idealibrary.com on
Age-Dependent Acrylamide Neurotoxicity in Mice: Morphology,Physiology, and Function
Miau-Hwa Ko,*,† Wen-Pin Chen,* Shoei-Yin Lin-Shiau,‡,§ and Sung-Tsang Hsieh*,¶,1
*Department of Anatomy, ‡Department of Pharmacology, §Department of Toxicology, and ¶Department of Neurology, College of Medicine,National Taiwan University, Taipei, 10018 Taiwan; and †Department of Anatomy, China Medical College, Taichung, 40421 Taiwan
Received November 23, 1998; accepted March 26, 1999
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Acrylamide intoxication produces peripheral neu-opathy characterized by weakness and ataxia in bothumans and experimental animals. Previous studiesn animals of different ages and species indicate thathe longest and largest nerves are affected earlier withhe major pathology in the terminal parts of axons, i.e.,istal axonopathy. However, several issues have re-ained elusive; for example, what are the earliest
athological changes? An equally intriguing questions whether younger animals are more susceptible tocrylamide than older animals. To address these is-ues, we compared the vulnerability to acrylamide of- and 8-week-old mice. These mice were intoxicatedith acrylamide in drinking water (400 ppm). The
equence of intoxication could be categorized intohree stages. In the initial stage, there was no visibleeakness or ataxia. The only noticeable changes wereoor performance on the rota-rod test and swelling ofotor nerve terminals. Obvious weakness and ataxia
f hindlimbs developed gradually (here designated ashe early stage). The weakness and ataxia progressedt variable speeds in mice of different ages, and eventu-lly the forelimbs (quadriparesis) were affected in theate stage. Each stage appeared earlier in 3-week-old
ice than in 8-week-old mice (7.1 6 1.1 vs 15.6 6 4.0ays, P F 0.01 for the early stage; and 15.3 6 2.1 vs1.7 6 6.0 days, P F 0.01 for the late stage). The progres-ion of neurological deficits was also faster in theounger mice (7.2 6 1.8 vs 16.3 6 4.2 days, P F 0.01).athological changes in the distal parts of motor nerves
nnervating hindfoot muscles were evaluated by com-ined cholinesterase histochemistry and immunocyto-hemistry for neuronal markers to demonstrate motorerve terminals and neuromuscular junctions simulta-eously. In the initial stage, there was axonal swelling
n motor nerve terminals. As acrylamide intoxicationontinued, axonal swelling extended into junctionalolds and into the intramuscular nerves, which re-
1 To whom correspondence should be addressed at Department ofnatomy, College of Medicine, National Taiwan University, 1 Jen-Aioad, Section 1, Taipei, 10018 Taiwan. Fax: 886-2-2391-5292. E-mail:
37
ulted in Wallerian-like degeneration. Our results indi-ate that younger mice show a much higher susceptibil-ty to acrylamide intoxication, and pathologicalhanges precede neurological symptoms.1999 Academic Press
Key Words: acrylamide; neurotoxicity; nerve conduc-ion; neuromuscular junction; axonal degeneration.
INTRODUCTION
Acrylamide produces weakness and ataxia in bothumans and experimental animals (10, 13, 20, 23).arly manifestations of acrylamide intoxication appear
n the hindlimbs. The neurological deficits eventuallyrogress to forelimbs with quadriparalysis. Impair-ent of motor system, failure of cerebellar connection,
nd degeneration of large sensory nerves all contributeo neurological manifestations of acrylamide intoxica-ion. Among these systems, motor nerves have beenxtensively characterized (3, 26). Pathologically, theongest and the largest axons in both peripheral andentral nerves are the most susceptible ones, and theajor changes are in the most distal parts of axons (24,
5). Characteristic findings include accumulation ofeurofilaments and organelles followed by Wallerian-
ike degeneration (6, 16, 21). Axonal swelling affectingeuromuscular junctions contributes to the develop-ent and progression of weakness after intoxication
11). However, there is only limited literature correlat-ng behavioral abnormalities with early pathologicalndings and their evolution (8, 33).In many aspects, distal filamentous pathology and
erve degeneration after acrylamide intoxication mimicarious kinds of peripheral nerve disorders, and acryl-mide neuropathy provides an experimental system totudy degeneration and regeneration of axons (35). Asany novel therapeutic strategies are under way for
rials on peripheral nerve degeneration, a multidisci-linary approach with quantification of various param-ters will be important for evaluating potential thera-
eutic strategies, such as neurotrophins (1, 18, 19).0014-4886/99 $30.00Copyright r 1999 by Academic Press
All rights of reproduction in any form reserved.
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After acrylamide intoxication, there is a time lagefore the appearance of neurological abnormalities.ffects of acrylamide intoxication are dose-dependent
2, 10). Previous studies have mainly focused on adultnimals, and only a few observations have explored thessue of age on the susceptibility to acrylamide (5, 9,7). During maturation of the nervous system, there isn extensive reorganization of axonal structures, andhe effects of acrylamide during this process remainlusive.To address these issues, we compared neurotoxic
ffects on weaning and adult mice by a multidisci-linary approach including functional, neurophysi-logic, and pathological examinations. Specifically, wesked whether there is a difference in the onset androgression of neurological symptoms and signs be-ween 3- and 8-week-old mice after acrylamide intoxica-ion. Our findings indicate that the 3-week-old weaningice were more susceptible to acrylamide intoxication
han the 8-week-old adult mice. The difference shouldrovide insights into the mechanisms of acrylamideoxicity.
MATERIALS AND METHODS
crylamide Intoxication
Intoxication of acrylamide (Merck, Darmstadt, Ger-any) began with groups of 3-week-old (3W group) and
-week-old (8W group) male ICR mice. Animals wereoused four to five per cage in plastic boxes throughouthe experimental period. Acrylamide was added to therinking water (400 ppm). Control mice drank regularater. Experimental procedures followed the principles
n the ‘‘Use of Animals in Toxicology’’ and NIH guide-ines (‘‘Guide for the Care and Use of Laboratorynimals,’’ NIH Publication No. 86-23, 1985).
ota-Rod Test
The rota-rod test followed previous protocols (9, 14,2). Briefly, the Rota-Rod Treadmill for Mice (UGOasile, Italy) was used to measure the degree of motorysfunction in control and acrylamide-intoxicated mice.hree days prior to intoxication, mice were trained toaintain their balance on the rod at a speed of 8 rpm
or 120 s. Each day we recorded the duration duringhich the mouse stayed on the rod. There were three
rials with an interval of 20 min between trials, and theean of the three trials was used for further analysis.
lectrophysiological Studies
A nerve conduction study was performed to evaluatehe functions of peripheral nerves with the measure-ent of compound muscle action potentials (CMAP)
ollowing the modified procedure (17). At the onset of
ach stage, mice were anesthetized with 4% chloral mydrate and restrained on a warm plate to keep theurface temperature of the tail at or above 30°C. Twoairs of electrodes were inserted into the tail to recordhe CMAP with a stimulus intensity of 7 V and 10 ms.e recorded the latency to the onset of CMAP and the
mplitude of the peak of CMAP. Nerve conductionelocities were calculated and analyzed as well.
ombined Cholinesterase Histochemistry andImmunocytochemistry
At various intervals after the development of behav-oral abnormalities, both intoxicated and control miceere sacrificed by intracardiac perfusion with 4% para-
ormaldehyde in 0.1 M phosphate buffer (pH 7.4). Fororphological examination, the paraformaldehyde-
xed plantar muscles were carefully removed under aissecting microscope, postfixed in the same fixativevernight, and then cryoprotected with 30% sucrose in.1 M phosphate buffer. Serial 30-µm cryostat sectionsere cut and mounted on gelatin-coated slides. Everyfth section was analyzed by cholinesterase histochem-
stry and immunocytochemistry for protein gene prod-ct 9.5 (PGP) as described previously (4). PGP is aissue-specific ubiquitin carboxyl-terminal hydrolaseresent in all types of neurons and peripheral nervebers. This method demonstrates neuromuscular junc-ions (blue) and innervating axons (brown). Briefly,fter quenching with 1% H2O2 in methanol, the sectionsere washed in 0.5 M Tris buffer, pH 7.6 (Tris), and
ncubated with 0.5% fat-free dry milk in Tris. Theections were incubated with PGP (1:1000, UltraClone,K) at 4°C overnight. After washing with Tris, theiotinylated goat anti-rabbit IgG was applied at roomemperature for 1 h. Following the avidin–biotin methodVector, Burlingame, CA), the reaction products wereetected with 0.05% 3,38-diaminobenzidine tetrahydro-hloride (DAB). The integrity of axons within neuromus-ular junctions was evaluated under a Zeiss Axiophoticroscope (Carl Zeiss, Germany) in a blinded fashion.e calculated the ratios of innervated and abnormal
euromuscular junctions (with axonal swelling) to alleuromuscular junctions.
ltrastructural Study
Following perfusion with 4% paraformaldehyde in.1 M phosphate buffer, the plantar muscles was re-oved and fixed in 5% glutaraldehyde overnight at
°C. The specimens were reacted with bromoindoxylcetate to localize neuromuscular junctions as de-cribed before (7). Blocks with neuromuscular junctionsere postfixed in osmium tetraoxide, dehydrated
hrough graded ethanol, and embedded in Epon. Semi-hin sections were stained with toluidine blue. Selectedreas with intramuscular nerves were thin-sectioned,bserved, and photographed under a Hitachi electron
icroscope.
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39AGE-DEPENDENT ACRYLAMIDE NEUROTOXICITY
tudy Design and Statistical Analysis
For each mouse, body weight, presence or absence ofeurological signs (the pattern of walking), and perfor-ance on the rota-rod test were recorded every day.he amount of water intake in each cage was measuredvery day as well. Based on the data of a preliminarytudy to determine the onset of the early and latetages, we carried out neurophysiological and pathologi-al examinations in the middle of each stage. The totalumber of mice used for pathological studies is listed inable 1. The measurement and interpretation of theesults were performed by an independent observerlinded to the information of intoxication. Data werexpressed as means 6 SD. ANOVA with post hoc testnd Student’s t test were used to compare control andntoxicated mice. Any difference with P , 0.05 wasonsidered statistically significant.
RESULTS
tereotyped Neurological Abnormalities afterAcrylamide Intoxication
Acrylamide intoxication produced stereotyped androgressive weakness and unsteady gaits. Control micealked with plantar surfaces touching the floor (Fig.A). At the start of acrylamide intoxication, the walk-ng pattern of acrylamide-intoxicated mice withoutisible neurological abnormalities was similar to thatf the control mice. This phase was designated as thenitial stage. Mice intoxicated with acrylamide devel-ped an unsteady walking pattern with abduction andxternal rotation of hindlimbs. The mice which walkedith a paralytic gait were in the early stage of intoxica-
ion (Fig. 1B). As intoxication continued, weakness andtaxia of hindlimbs became evident. Gradually, theice dragged their feet as they walked (Fig. 1C).inally the weakness of the most severely intoxicatedice included the forelimbs. When the mice had qua-
riparalysis, they were in the late stage (Fig. 1D).Both age groups of intoxicated mice showed the same
rogressive pattern of neurological abnormalities, but
TABLE 1
Number of Mice Used for Pathological Studies
Initial stage Early stage Late stage
-week-oldControl 3 (5)a 9 (5–9) 6 (13–17)Acrylamide 6 (5) 24 (5–10) 17 (13–19)
-week-oldControl 3 (7) 6 (14–27) 8 (25–41)Acrylamide 6 (7) 18 (11–27) 13 (20–43)
a
aTime of sacrifice after intoxication, in days.FIG. 1. Progressive weakness of limbs during acrylamide intoxi-ation. Compared with control mice in (A), acrylamide-intoxicatedice walked with a wide-based gait at the early stage (B). In (C),indlimb paralysis progressed and resulted in dragging of the feetlong the floor. As acrylamide intoxication continued, forelimbs were
ffected in the late stage with quadriparesis (D).
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ith different chronologies of onset and progression ofach stage. The onset of the early stage in the 3W groupas earlier than that in the 8W group (7.1 6 1.1 vs5.6 6 4.0 days, P , 0.01) (Fig. 2A). The late stagetarted 15.3 6 2.1 days after intoxication in the 3Wroup and 31.7 6 6.0 days in the 8W group (P , 0.01)Fig. 2B). The progression from the early stage to theate stage was faster in the 3W group than in the 8W
FIG. 2. Onset of neurological symptoms in mice of different ages.he cumulative frequency plots show the onset of the early and latetages of acrylamide intoxication in 3-week-old (3W) and 8-week-old8W) mice. The early stage was defined as mice with hindlimbaralysis (A), and the late stage as mice with quadriparesis (B). Theedians of the onset of the early stage are 7 days for the 3W group,
nd 15 days for the 8W group. By 14 days, 50% of mice in the 3Wroup were quadriparalyzed. The 8W group had a later onset of theate stage with a median of 33 days. The pattern of intoxication wasuite consistent as within 19 days all the 3W group became quadripa-alyzed. By 43 days, all the 8W mice had paralysis of both fore- andindlimbs. The progression from the early to the late stage aftercrylamide intoxication was faster in the 3W group (median 7.0 days)han in the 8W group (median 17.5 days).
roup (7.2 6 1.8 vs 16.3 6 4.2 days, P , 0.01). a
ge- and Dose-Dependent Neurotoxicity of Acrylamide
Consistent with previous observations, there was aignificant decrease in body weight in both age groupsf mice (Fig. 3). In the beginning of the experiment,ody weights of the 3W group were 11.8 6 2.1 g, andhose of the 8W group were 33.8 6 3.2 g. Control-week-old mice exhibited a progressive increase ofody weight. But the acrylamide-intoxicated mice athe late stage had growth retardation, with a bodyeight of 10.2 6 4.2 g, significantly smaller than that of
he control mice (31.1 6 2.1 g). The 8W group had a9% decrease in body weight (24.0 6 8.1 g for intoxi-ated mice and 40.1 6 1.6 g for control mice). A possibleonfounding factor for the differential onset and progres-ion of acrylamide intoxication might be a difference inhe ingestion of acrylamide.
FIG. 3. Changes in body weight after acrylamide intoxication.he graphs show body weights of mice in the 3-week-old (3W in A)roup and in the 8-week-old (8W in B) group during intoxication withcrylamide at 400 ppm. There is a significant reduction in bodyeights after acrylamide intoxication. The extent of weight reduction
s more obvious in the 3W group than in the 8W group at the end ofhe late stage (open symbols for control mice and filled symbols for
crylamide-treated mice).
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41AGE-DEPENDENT ACRYLAMIDE NEUROTOXICITY
To address this issue, we compared the daily doseDose) of acrylamide ingestion between the two groupsy measuring the daily volume (Vol) of water intakend recording the body weight (BW) of each mousevery day through the experimental period. The dailyose of acrylamide was calculated with the followingormula: Dose 5 400 ppm 3 Vol/BW. The water intakef each mouse was 2.5 6 0.3 ml/day for the 3W groupnd 7.3 6 1.5 ml/day for the 8W group (P , 0.01). Thealculated daily dose was 91.8 6 20.6 mg · kg21 · day21
or the 3W group, which was not significantly differentrom that for the 8W group at 90.8 6 10.9 mg · kg21 ·ay21 (P . 0.5).Based on the daily dose, the cumulative dose for the
arly stage was 650.1 6 145.6 mg/kg in the 3W groupnd 1417.1 6 169.9 mg/kg in the 8W group. At latetage, the cumulative dose was 1404.8 6 314.6 mg/kg inhe mice of the 3W group and 2879.7 6 345.2 mg/kg inhe mice of the 8W group.
bnormal Motor Performance on the Rota-Rod Test
There were motor deficits before the gaits of acrylam-de-intoxicated mice became abnormal. When chal-enged with the rota-rod test in the initial stage,ntoxicated mice performed poorly compared with con-rol mice. For the 3W group, the mice had difficulty inaintaining their balance on the rotating rods within
.1 6 1.7 days after intoxication. The onset of motoreficits was later in the 8W group, namely, 8.5 6 1.9ays after intoxication (P , 0.01, Fig. 4). The resultslearly suggest that abnormalities in motor functionsegan in the initial stage, although the walking pat-erns appear normal at this stage.
arly Pathological Changes in NeuromuscularJunctions
To understand pathological alterations in neuromus-ular innervation after acrylamide intoxication, wexamined the plantar muscles with combined cholines-erase histochemistry and PGP immunocytochemistry.xonal terminals were demonstrated by immunoreac-
ivity to PGP (brown reaction product) overlying neuro-uscular junctions (blue reaction by cholinesterase
taining). In control mice, intramuscular nerve bundlesonsisted of nerve fibers of similar caliber, with theirerminals branching inside neuromuscular junctionsFigs. 5A and 5E). In the initial stage, swelling of axonsppeared in the prejunctional portion before motorerves entered neuromuscular junctions (Figs. 5B andF). Axonal swelling extended distally into terminalranches (terminal swelling) and extended proximallyFigs. 5C and 5G). In the late stage, there were obviousrofiles of axonal degeneration with degenerated axonsnd myelin debris in the intramuscular nerves (Figs.D and 5H). For each stage, pathological findings were
imilar in the 3W and 8W groups. cPathologic changes of acrylamide intoxication beganrom axons before they entered neuromuscular junc-ions (here designated as prejunctional swelling), with–11% of neuromuscular junctions showing prejunc-ional swelling in the initial stage (Fig. 6). Quantifica-ion of the proportion of abnormal neuromuscularunctions indicated that axonal swelling preceded swell-ng in the terminal branches and denervation of neuro-
uscular junctions appeared only subsequently. Theotor innervation of neuromuscular junctions in the
lantar muscles was strikingly decreased after acrylam-de intoxication in both groups. In the 3W group, thereas a 24% reduction in innervated neuromuscular
unctions in the late stage with a similar degree (19%)n the 8W group.
lectron Microscopic Observations on IntramuscularNerves
We correlated pathological changes with ultrastruc-ural findings of intramuscular nerves. After acrylam-de intoxication, there was axonal degeneration in thentramuscular nerves of the plantar muscle (Fig. 7). Inontrol mice, axons of the intramuscular nerves wereell myelinated (Fig. 7A). There were profiles of axonalegeneration and shrinkage in the intramuscular nervesf acrylamide-intoxicated mice (Fig. 7B). At the ultra-tructural level, degeneration of axons, disorganizedrganelles, and disintegration of myelin sheaths be-
FIG. 4. Performance of the rota-rod test after acrylamide intoxi-ation. The plots show the evolution of motor abnormalities duringcrylamide intoxication in mice of the 3-week-old (3W, triangles) and-week-old (8W, circles) groups. Mice in the control group couldaintain balance on the rotating rod at 8 rpm for more than 120 s,
ndicated by the dashed line. Within 4 days, mice in the 3W group hadifficulty staying on the rotating rod (107 6 27 s, P , 0.01). Theotor abnormalities worsened and the mice in the 3W group failed to
erform the test by 10 days. The motor abnormalities had a laternset in mice of the 8W group. The duration of staying on the rota-rodas significantly shorter within 9 days (103 6 28 s, P , 0.01), and
he mice in the 8W group were unable to perform the rota-rod test by5 days.
ame obvious after acrylamide intoxication (Fig. 7C).
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europhysiological Abnormalities after AcrylamideIntoxication
To understand the functional significance of pathologi-al abnormalities, we performed nerve conduction stud-es on tail nerves. Acrylamide intoxication produces a
FIG. 5. Changes in motor nerve terminals after acrylamide intoxior neuromuscular junction (blue) and immunocytochemistry for th
ethods. (A–D) From 3-week-old mice; (E–H) from 8-week-old mice. Ixons of similar caliber. In the initial stage (5 days after intoxicatippeared in the prejunctional portion of motor nerves. The swelling (ntoxication for C and 15 days after intoxication for G). During the late
FIG. 6. Axonal pathology in acrylamide neurotoxicity. The graphshow the proportion of abnormal neuromuscular junctions, includingxonal swellings in the prejunctional areas (A) and terminal branchesB) with subsequent denervation (C) in different stages after acrylam-de intoxication in 3-week-old (3W) and 8-week-old (8W) mice. Theuantitation was based on sections of combined cholinesteraseistochemistry and immunocytochemistry for the axonal protein,GP 9.5, in Fig. 5. (A) Axonal swelling in prejunctional portionsegan in the initial stage (8% in the 3W group and 11% in the 8Wroup). (B) Swellings in terminal branches (terminal swelling) be-ame obvious in the early stage (14% for the 3W group and 5% in theW group). (C) In the late stage, the portions of innervated neuromus-ular junctions were substantially reduced (24% in the 3W group and9% in the 8W group). Asterisks denote a significant difference fromontrol (P , 0.05).
), neuromuscular junctions became denervated, and there were profiles
radual but significant reduction in the amplitudes ofMAP. In the late stage of the 3W group, there was a1% decrease in amplitudes of CMAP from 5.9 6 1.4 to.5 6 1.8 mV (P , 0.01, Table 2). In the 8W group, theeduction in amplitudes was 45%, from 14.2 6 4.5 to.8 6 1.3 mV (P , 0.01, Table 2).
DISCUSSION
Acrylamide intoxication provides an important experi-ental approach for studying nerve degeneration, in
articular, distal axonopathy. Exposure to acrylamideas profound influence on the nervous system, and theffect is modified by age. The major findings in thiseport add two more important aspects in understand-ng the mechanisms: (i) earlier occurrence and fasterrogression of neurological abnormalities in 3-week-oldice than in 8-week-old mice and (ii) early pathology inotor axons innervating neuromuscular junctions pre-
eding neurological symptoms (Table 3).
arly Pathological Involvement in AcrylamideIntoxication
In this report, we documented the sequence of motorbnormalities. Consistent with previous studies, theathological changes began from the most terminalarts of axons and evolved proximally (12, 33). Thenvolvement was quite early as the paralytic gait wasot evident at the initial stage, but sophisticated tests,uch as the rota-rod test, could already demonstratebnormalities of motor performance. The obvious changen the amplitude of nerve-evoked muscle action poten-ial was not apparent until the late stage of intoxica-ion. The reduction in amplitude of muscle potentials isttributed to Wallerian-like degeneration. Of course,ysfunctions of the cerebellum and proprioceptive sen-ations could also contribute to the abnormal perfor-ance after acrylamide intoxication (25).
ffects of Age on Neurological Abnormalities inAcrylamide Toxicity
This report clearly demonstrates the influence of agen vulnerability to acrylamide intoxication. Only lim-ted reports have addressed this issue (37). Suzuki andfaff found that sucking rats had a faster pace of nerveegeneration after acrylamide intoxication (27). Kaplannd Murphy observed that aging 14-week-old rats were
ion. Sections of the plantar muscles were stained with cholinesterasexonal protein, PGP 9.5 (brown), as described under Materials andontrol mice (A,E), neuromuscular junctions are well innervated withfor B and 7 days after intoxication for F), axonal swelling (arrows)ws) extended into terminal branches in the early stage (9 days after
age (14 days after intoxication for D and 35 days after intoxication for
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ore susceptible to acrylamide than were 5-week-oldats (9). These findings suggest that age is an impor-ant factor in determining the susceptibility to acrylam-de and should be taken into consideration when evalu-ting various dosing schedules (20). In the presenttudy, the daily dose of acrylamide ingestion was
FIG. 7. Electron microscopic observation of intramuscular nervesntramuscular nerve from control mouse. (B) A comparable sectiontoxication) with profiles of axonal degeneration (arrow) and shrinkntoxicated nerve (late stage, 35 days after intoxication) exhibitsissolution of myelin debris (arrows). Bar in A, B, 12 µm; C, 5 µm.
TABLE 2
Nerve Conduction Studies after Acrylamide Intoxication
ControlInitialstage
Earlystage
Latestage
CV (m/s)3-week-old 12.1 6 7.3 12.8 6 8.0 10.8 6 2.4 10.5 6 5.68-week-old 17.4 6 4.5 17.2 6 4.4 14.8 6 3.9 15.8 6 4.2mplitude (mV)3-week-old 5.9 6 1.4 5.6 6 0.8 5.2 6 2.0 3.5 6 1.8*8-week-old 14.2 6 4.5 13.1 6 3.3 13.6 6 3.5 7.8 6 1.3*
* P , 0.01, different from the control.
imilar in the 3- and 8-week-old mice, suggesting thathe age is a decisive factor. From weaning to youngdulthood, there are dramatic changes in the organiza-ion of the nervous system, including myelination andytoskeletal compositions. The differential susceptibil-
the plantar muscle of 8-week-old mice. (A) A semithin section of therom an acrylamide-intoxicated mouse (early stage, 15 days after
(arrowhead). (C) At the electron microscopic level, the acrylamide-organized organelles, various stages of axonal degeneration, and
TABLE 3
Progression of Acrylamide Intoxication
Initial stage Early stage Late stage
ppearance Normal Paraparesis Quadriparesisota-rod test Decreased
retention timeFailure Failure
euromuscularjunction
Prejunctionalswelling
Terminalswelling
Denervation
erve conductionstudy
No change No change Decreasedamplitude ofcompound muscleaction potential
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45AGE-DEPENDENT ACRYLAMIDE NEUROTOXICITY
ty should shed light on the possible mechanisms ofcrylamide intoxication.
echanisms for Differential Vulnerability inAcrylamide Intoxication
The neuropathological hallmarks of acrylamide in-oxication are filamentous swelling of axons in para-odal area and the terminal part of axons, distalxonopathy, or dying-back neuropathy. The contents ofxonal swelling consist of microtubules, neurofila-ents, and mitochondria. Recent observations suggest
hat microtubules, rather than neurofilaments, play anmportant role in the pathogenesis of acrylamide intoxi-ation. Compelling evidence comes from a mutanttrain of Japanese quail, Quv. The mutant quail lackeurofilaments because of a missense mutation in the
ow molecular weight neurofilament (NF-L), with pre-ature termination of NF-L synthesis. Despite the
bsence of neurofilaments, Quv is susceptible to acryl-mide as are the normal quail (15). These resultsmphasize the important contribution of microtubulesn acrylamide intoxication. Microtubules are importantn neurite elongation and are more abundant thaneurofilaments in young animals (28, 29, 36). Theompositions of microtubules, motors of microtubules,nd microtubule-associated proteins change as animalsecome mature (30, 31, 34). A recent study suggestshat acrylamide has a direct effect on kinesin-basedicrotubule motility (22). Taken together, the differ-
nce in microtubule organization and related proteinsould potentially underlie the differential vulnerabilityo acrylamide. Further investigations are required tolucidate these points. Nevertheless, the present reportstablishes an experimental system to produce motorysfunctions, which is also useful for further therapeu-ic trials.
ACKNOWLEDGMENTS
This work was supported by a grant from National Health Re-earch Institute, Department of Health, R.O.C. (DOH-88-HR-727).e thank C. C. Chu and Y. L. Hsieh for technique assistance.
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