Effects of acrylamide on subcellular distribution of elements in rat sciatic nerve myelinated axons and Schwann cells

238

Brain Research, 608 (1993) 238-246 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 18702

Effects of acrylamide on subcellular distribution of elements in rat sciatic nerve myelinated axons and Schwann cells Richard M. LoPachin, Carolyn M. Castiglia, Ellen Lehning and Albert J. Saubermann Department of Anesthesiology, Medical School, SUNY at Stony Brook, Stony Brook, NY 11794-8480 (USA) (Accepted 3 November 1992)

Key words." Acrylamide; Neurotoxicity; Nerve cell; Schwann cell; Electron probe X-ray microanalysis; Element; Axon

Electron probe X-ray microanalysis was used to determine whether experimental acrylamide (ACR) neuropathy involves deregulation of subcellular elements (Na, P, S, C1, K, Ca and Mg) and water in Schwann cells and small, medium and large diameter myelinated axons of rat sciatic nerve. Results show that in proximal but not distal sciatic nerve, ACR treatment (2.8 mM in drinking water) was associated with an early (15 days of exposure), moderate increase in mean axoplasmic K concentrations (mmol/kg) of medium and small diameter fibers. However, all axons in proximal and distal nerve regions displayed small increases in dry and wet weight contents of axoplasmic Na and P. As ACR treatment progressed (up to 60 days of exposure), Na and P changes persisted whereas proximal axonal K levels returned to control values or below. Alterations in mitochondrial elemental content paralleled those occurring in axoplasm. Schwann cells in distal sciatic nerve exhibited a progressive loss of K, Mg and P and an increase in Na, CI and Ca. Proximal glia displa~,ed less extensive elemental modifications. Elemental changes observed in axons are not typical of those associated with cell injury and might reflect compensatory or secondary responses. In contrast, distal Schwann cell alterations are consistent with injury, but whether these changes represent primary or secondary mechanisms remains to be determined.

INTRODUCTION Occupational and experimental exposure to acrylamide (ACR) monomer produces central and peripheral nerve damage classified as a distal axonopathy 27,28. We have recently reported that experimental ACR neuropathy is associated with characteristic changes in the elemental composition and water content of myelihated axons and Schwann cells from rat tibial nerve 16'17. Electron probe X-ray microanalysis (EPMA) was used to measure both water content and concentration ( m m o l / k g dry or wet weight) of free plus bound elements (i.e. total elements, Z > 10) in selected morphological compartments (myelin, axoplasm). These previous E P M A studies demonstrated that both subchronic (2.8 mM in drinking water for up to 60 days) and subacute (50 m g / k g / d a y intraperitoneal x 5 or 10 days) exposure to ACR caused a progressive injury-typic loss of internodal axoplasmic K, CI, and Na regulation in subpopulations of medium and small diameter axons. In swollen axon regions which are characteristic of ACR neuropathy, axoplasm and mitochondrial areas

exhibited a complete decompartmentalization of elements and water ~6. Analysis of Schwann cells from tibial nerve of ACR intoxicated rats revealed a temporally-dependent loss of cytoplasmic Na, K, P, C1, Mg and water regulation t7. We hypothesize that the observed injury-typic changes in elemental composition (e.g. loss of K and gain in Na, CI) of tibial nerve myelinated fibers ~6 are a mechanistically relevant component of ACR neurotoxicity. Axonal degeneration associated with ACR exposure is confined mainly to distal tibial nerve regions with relatively few morphological alterations occurring in sciatic nerve 27-29. If the corresponding elemental changes in tibial nerve are mechanistically relevant they should exhibit a restricted regional distribution and, thus, parallel characteristic proximodistal neuropathologic modifications. Sciatic nerve axons should express either no change in elemental composition or changes that differ in direction a n d / o r elements involved. In the present study, the elemental content of axons and Schwann cells was determined in sciatic nerve. Results indicate that ACR-induced changes in

Correspondence: R.M. LoPachin, Department of Anesthesiology, Medical School, SUNY Stony Brook, Stony Brook, NY 11794-8480, USA. Fax: (1) (516) 444-2907.

239 sciatic nerve axonal elemental composition are not 'injury-typic'. This confirms the regional nature of the injury-typic tibial nerve findings and, therefore, provides further evidence that such distal changes in elemental content are a specific and selective effect of ACR. Furthermore, when sciatic nerve data are considered in conjunction with those from tibial nerve 16, it is clear that ACR intoxication disrupts the normal proximodistal distribution of elements 12.

differential response to injury, Ca sequestration) and gross structural (e.g. size, shape and orientation) criteria 12'laAs, and is designated as 'mitochondrial area '16. The extraaxonal space (EAS, Fig. 1B) is defined as the area lying outside myelinated axons and includes analysis of extracellular fluid, collagen and, possibly, small unmyelinated axons 12. Microprobe analyses of Schwann cell cytoplasm and myelin were also performed. Morphological compartments were visualized in dehydrated sections using scanning transmission electron microscopy (STEM). Morphological criteria used to identify analytical compartments in frozen sections were confirmed in a previous parallel study of conventionally fixed control rat sciatic nerve 12.

Microprobe analysis MATERIALS AND METHODS

Animals and treatments Animal use procedures were in accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the local Animal Care Committee. Male Sprague-Dawley rats (276-300 gin) were divided into control and treatment groups (n = 4/group). Animals were housed in smooth bottom plastic cages at 22_+ I°C with a 12 h light/dark cycle. Food (Purina rat chow) and water were available ad libitum. Animals were intoxicated with ACR (Bio-Rad Laboratories, electrophoretic grade, 99% pure) via oral ingestion using an exposure level of 2.8 mM in drinking water. The oral dosing protocol used in this study was designed to produce low dose, subchronic ACR intoxication. This protracted dosing regimen allows better temporal separation of behavioral and functional consequences compared to more acute dosing paradigms ~6. Water consumption and the development of neurotoxicity were assessed daily (for specific details see refs. 11 and 16). The length of oral ACR exposure was determined by the appearance of specific indices of progressive neuromuscular dysfunction (e.g. foot splaying, hopping gait). The appearance of these indices, and therefore the times of sciatic nerve sampling, corresponded to approximately 15, 22 and 30 days of oral ACR exposure 16'~7. Another treatment group was exposed to ACR for 53 days which represents the average onset of hindlimb muscle paralysis. These rats were treated for an additional 7 days and sciatic nerve samples were collected at 60 days. The control group consisted of pooled data from age-matched rats (n = 2/time point) sacrificed at the same time as the 15 and 60 day oral treatment groups. Preliminary studies showed that no statistical differences were detected when results from these control subgroups were compared.

Cryomicrotomy and electron probe X-ray microanalysis The methodologies for cryomicrotomy and electron probe X-ray microanalysis (EPMA) have been published extensively 5'22'z3 and, therefore, only a brief description of these methods is provided. At the experimental times indicated above, treated and control rats were anesthetized (i.p. ketamine, 44 mg/kg and xylazine, 5 mg/kg) and unilateral segments (approximately 5 mm in length) of proximal (distal to the sciatic notch) and distal (proximal to the fossa poplitea) sciatic nerve were rapidly removed (see Fig. 4). Nerve samples were frozen immediately in melting freon 22 and then stored in liquid nitrogen until analyzed. Samples were sectioned (500 nm nominal thickness) on a cryomicrotome at an ambient cryochamber temperature of -55°C. Unstained, unfixed, frozen, hydrated sections were then transferred under vacuum to the cold stage (-185°C) of an AMRay 1400 scanning electron microscope. The electron microscope was equipped with a Tracor Northern energy dispersive detector and pulse processor which was connected to a PC based multichannel analyzer for collection and processing of X-rays. In sciatic nerve cryosections, myelinated axons were classified as either small (internal diameter < 5 /xm), medium or large ( > 10 p.m) diameter fibers. Axons were divided according to size based on the selective vulnerability hypothesis of Spencer and Schaumburg 28 which suggests that larger diameter myelinated axons are more sensitive to the actions of neurotoxicants. The mitochondrial compartment has been identified according to both functional (e.g.

Wet weight specimen mass was measured in frozen hydrated sections by determining continuum generation rates z4. Sections were then dehydrated in the electron microscope column vacuum by raising the temperature of the cold stage from - 185°C to - 60°C for 30 min. Stage temperature was returned to -185°C for microanalysis. Compartments were selected in STEM mode and the electron beam (20 keV, 0.4 nA probe current) was rastered within anatomical boundaries of the chosen structure. X-ray spectra were collected over 100-s live counting time. Dry weight elemental mass fractions (mmol/kg dry wt.) for Na, K, CI, P, S, Ca and Mg were determined using a specially written computer program applying the Hall et al.8 method of continuum normalization s. Water content (% H20) of morphological compartments was determined by the ratio of continuum counts in the hydrated and dried states. Dry weight mass fractions could then be converted to wet weight values (mmol/kg wet weight) using the following formula: Wet weight = R xdry"Sx[ 100 -- %water/100] Where Rxdryis the dry weight mass fraction for a given element x and S x is the corresponding calibration factor derived from standard curves 3,23.

Statistics One-way ANOVA demonstrated that analyses from individual animals of a treatment group could be pooled as independent data to derive a group mean. To determine statistical differences (P < 0.05) in the variance of elemental concentrations among treated and control groups, squared deviates from within-group means were calculated and a Kruskal-Wallis test was applied between groups. A Mann-Whitney U-test (with Bonferroni correction) was used to determine differences between control and treated group (variance) data. Statistical differences (P < 0.05) among group means were determined using one-way ANOVA. Treatment-control mean differences ( P < 0.05) were assessed using Dunnett's test. RESULTS

Behavioral and morphological observations A d m i n i s t r a t i o n o f A C R (2.8 m M ) in d r i n k i n g w a t e r did not affect water consumption but did reduce normal rate of w e i g h t gain. A progressive n e u r o m u s c u l a r d e f i c i t a l s o d e v e l o p e d as a f u n c t i o n o f o r a l A C R p o s u r e 16. B a s e d ml/day,

on

a daily w a t e r

intake

ex-

o f 40_+ 1

r a t s i n g e s t e d a p p r o x i m a t e l y 7.8 m g A C R / d a y

o r 23.4 m g / k g / d a y .

A f t e r a p p r o x i m a t e l y 15 d a y s o f

c o n s u m p t i o n r a t s d e v e l o p e d m i l d f o o t splay, w h e r e a s a t 22 d a y s p r o m i n e n t

foot splay a n d gait a b n o r m a l i t i e s

(e.g. h o p p i n g , s l i g h t a t a x i a ) a p p e a r e d .

After approxi-

m a t e l y 30 d a y s o f o r a l A C R e x p o s u r e , h i n d l e g s k e l e t a l m u s c l e w e a k n e s s a n d s p a s t i c i t y w e r e e v i d e n t . R a t s exposed

to A C R

f o r a n a v e r a g e o f 53 d a y s e x h i b i t e d

240 complete hindleg paralysis. These behavioral changes associated with oral ACR exposure are similar to those reported previously 4'3°. Fig. 1A is a scanning image of an unfixed, dehydrated cryosection from control rat distal sciatic nerve. Small, medium and large diameter myelinated axons and attending Schwann cells (SC) are clearly evident. A paranodal region (PN) and cluster of unmyelinatd fibers are also indicated. Fig. 1B is a STEM image of a

A. Proximal Sciatic Axoplaam

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eo

, E 0

0

Na

P

S

CI

K

Mg

Ca

Element Fig. 2. Histogram illustrating the elemental composition of axoplasm from large, medium and small axons of proximal (A) and distal (B) sciatic nerve regions. Data are expressed as mmol element/kg dry wt. + S.E.M..

medium diameter myelinated axon from a cryosection of control distal sciatic nerve. Mitochondrial areas (M) appear as electron-opaque, ovoid, or elongated structures, depending upon the plane of section (for additional details of mitochondrial areas in frozen sections see refs. 12, 14, 15 and 16). Also depicted are the extraaxonal space (EAS), axoplasm (AXO) and myelin (My). ACR intoxication was associated with few gross morphological changes in proximal sciatic nerve. Occasional degenerating axons and swollen Schwann cells were noted in distal sciatic nerves during the latter stages of ACR exposure (micrographs not presented).

Fig. 1. A: scanning electron micrograph of a frozen, dehydrated transverse section of control distal sciatic nerve. Small (S), medium (M) and large (L) diameter myelinated fibers are depicted. Also indicated are unmyelinated axons (UMA), a Schwann cell (SC) and a paranodal region (PN). Bar represents 10 ~m. B: scanning-transmission electron micrograph (STEM) of a medium diameter myelinated axon in a dehydrated cryosection of control distal sciatic nerve. Micrograph illustrates the morphological compartments which were analyzed; AXO, axoplasm; My, myelin; M, mitochondria; EAS, extraaxonal space (Schwann cell compartment not indicated). Bar = 1 ~m.

Electron probe X-ray microanalysis (EPMA) Control sciatic nerue myelinated axons. Fig. 2 shows the elemental composition of large, medium and small diameter myelinated fibers in control rat proximal (A) and distal (B) sciatic nerve. Axoplasmic measurements presented in this figure are of internodal axoplasm. Analysis of paranodal regions in both control and treated peripheral nerve was limited and, therefore, corresponding data are not sufficient for presentation. Regardless of nerve region, potassium was the most abundant element present in all myelinated fibers. In addition, dry and wet weight concentrations of this element were related to axon size. For example, medium proximal axons (Fig. 2A) exhibited mean ( m m o l / k g + S.E.M.) dry and wet weight K levels of 2078 + 76 and 164 + 7, respectively. In small proximal

241 TABLE I Effects of oral A C R treatment on axoplasmic elemental composition and water content o f large sciatic nerve axons Proximal sciatic

Na K P CI %water

Control (45)

× 15 (49)

× 22 (28)

× 30 (32) §

165_+12 2249±71 469±17 608 _+29 91_+ 0

266_+12" 2419_+81 597_+19 * 660 _+28 92_+ 0

233± 1 4 " 2 1 4 2 ± 117 532± 30 ~ 518 _+ 41 92_+ 0

217± 1 5 " 2 5 4 8 ± 182 a 634_+ 30 * 663 _+ 47 91_+ 1

Control (37)

× 15 (27)

× 22 (33)

× 30 (35)

181_+11 1965 _+56 479_+18 495 _+23 91_+ 0

222_+12 1848 ± 83 571_+25 * 409 ± 19 * 90_+ 0

130_+11 * 1953 + 60 589_+17 * 481 + 13 90_+ 0

240± 16 * 1886 ± 102 591_+ 22 * 442 ± 27 91_+ 1

Distal sciatic

Na K P CI %water

Elemental concentrations are expressed as mmol e l e m e n t / k g dry wt. _+S.E.M.; cell water content expressed as %water_+ S.E.M. § Days of oral A C R treatment (n = axoplasmic compartments analyzed). * Mean data significantly ( P < 0.05) different from control. a Variance of data significantly ( P < 0.05) different from control.

axons, K content was significantly less, i.e. 1427 + 50 and 125 + 5, respectively. This size dependency was also observed for dry and wet weight C1. Axoplasmic concentrations of Na, P, S and Ca were comparable in either nerve region and did not depend upon axon caliber (Fig. 2A and B). Consistent with previous E P M A studies ~2, dry and wet weight axoplasmic concentrations of K and CI decreased as a function of proximodistal distance. The average water content for all myelinated sciatic nerve axons was approximately 91%. The dry weight elemental composition of mitochondria from proximal and distal axons was similar to that of respective axoplasm (data not shown) although% water content ( + S . E . M . ) was 82% + 1%.

Effects of ACR on axonal elemental composition and water content. Subchronic oral ACR exposure caused temporally-dependent changes in dry and wet weight axoplasmic levels of K, Na, P, and C1. Statistically significant alterations in elemental content were expressed as shifts in mean concentration and as increases in data variance. Non-parametric variance changes were manifest as widening of the dispersion pattern on frequency distribution curves of axoplasmic element concentrations (see ref. 16 for details). After 15 days of oral ACR exposure a significant increase in mean K content was observed in medium axons of proximal nerve regions (Fig. 3). As oral treatment continued (i.e. 22 and 30 days) K concentrations re-

T A B L E II Effects o f oral A C R treatment on axoplasmic elemental composition and water content of medium sciatic nerve axons Proximal sciatic

Na K P CI %water

Control (41)

× 15 (34)

× 22 (32)

× 30 (32) §

155±12 2078±76 487±16 546 + 25 90_+ 2

233±16" 2419_+66 * 634_+31A 605 -+ 34 91_+ 1

209± 1 5 " 2 2 2 9 + 126 a 586± 32 a 572_+ 54 91± 1

209_+ 1 5 " 1967± 102 604+ 30 * 535 _+ 27 90_+ 0

Control (33)

x 15 (24)

x 22 (36)

× 30 (39)

188±10 1783 ± 64 554_+ 13 426_+25 90+ 0

261± 1 7 " 1855 ± 111 a 681 _+ 36 * 431_+ 32 91± 0

158± 9 1839 _+62 607_+ 15 469_+ 13 90± 0

240+21" 1710 ± 68 586_+ 19 443+33 90+ 0

Distal sciatic

Na K P CI %water

Elemental concentrations are expressed as mmol e l e m e n t / k g dry wt. _+S.E.M.; cell water content expressed as %water ± S.E.M. Days of oral A C R treatment (n = axoplasmic compartments analyzed). * Mean data significantly ( P < 0.05) different from control. Variance of data significantly ( P < 0.05) different from control.

242 PROXIMAL SCIATIC POTASSIUM 20 CONTROL

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ACR x 30 days

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mmol KJkg dry wt Fig. 3. Effects of oral ACR administration on frequency distribution of axoplasmic potassium concentrations in medium axons of proximal sciatic nerve. Also shown are mean potassium content ( m m o l / k g dry wt._+SD) and its location on the abscissa and the corresponding range of concentrations (mmol K / d g dry wt.). S z indicates data variance. Bordered information denotes statistically significant (P < 0.05) difference in either mean (*) or data variance (A) compared to that of control.

turned to normal (Fig. 3). Medium axons in distal sciatic nerve did not express significant changes in mean, although after 15 days of ACR administration variance was increased significantly (Table II). In general, large axons of both sciatic nerve areas did not exhibit significant changes in axoplasmic K (Table I). In contrast, the K content of small axons in proximal nerve remained elevated over 30 days of ACR treatment, while distal axons displayed a slight loss of mean K content after 30 days (Table III). In both nerve regions, changes in axoplasmic C1 concentrations of all axons tended to parallel those of corresponding K (Tables I-III). In animals exhibiting hindlimb paralysis (60 days of ACR exposure), axoplasmic K and C1 concentrations in large axons of both nerve regions were unaffected, whereas in medium and small axons modest but significant decreases in mean levels of these elements were noted (data not shown). During the ACR exposure period, respective mitochondrial areas displayed selective changes in dry weight K and C1 concentrations which were similar in direction and magnitude to corresponding axoplasmic alterations (data not shown). Mean axoplasmic Na levels exhibited early and persistent increases in most proximal sciatic nerve axons (Tables I-III). A similar trend was noted in all distal sciatic nerve axons with the exception that after 22 days of oral ACR administration Na concentrations transiently returned to control values or below (Tables I-III). Mean P content in all proximal axons exhibited

TABLE III Effects of oral ACR treatment on axoplasmic elemental composition and water content of small sciatic nert,e axons Proximal sciatic

Na K P CI %water

Control (42)

× 15 (32)

× 22 (29)

× 30 (30) §

158+14 1427 _+50 492_+16 365+15 91_+ ()

244_+ 1 6 " 2 035 _+ 104 j 620_+ 27 * 521_+ 31a 91_+ 1

201_+15 1 615 _+65 530_+26 388+20 91_+ 1

241_+ 1 8 " 1 824 _+110 .a 642-+ 3 3 * 516_+ 30 j 90_+ 1

Control (39)

x 15 (21)

× 22 (27)

x 30 (35)

198 _+ 12 1331 _+49 539 _+ 17 359 + 15 90_+ 0

275 _+ 16 * 1529_+79 697 _+27 * 345 _+ 14 89_+ 0

140 _+ 13 * 1359_+74 588 _+ 18 359 + 17 89_+ 0

260 _+14 * 1 105_+64 * 558 _+17 289 _+13 * 90_+ 0

Distal sciatic

Na K P CI %water

Elemental concentrations are expressed as mmol element/kg dry wt. + S.E.M.; cell water content expressed as % w a t e r + S.E.M. Days of oral ACR treatment (n = axoplasmic compartments analyzed. * Mean data significantly (P < 0.05) different from control. a Variance of data significantly (P < 0.05) different from control.

243 early and persistent increases (Tables I - I I I ) . Similar changes in P were observed for large distal axons, however for small and medium fibers P levels returned to control values following initial elevations (Tables I - I I I ) . In contrast, mitochondrial areas did not exhibit changes in either Na or P. A C R intoxication was not associated with significant changes in axoplasmic levels of S, Mg, Ca (data not shown) and axonal water content (Tables I - I I I ) . After 60 days of A C R exposure, myelinated axons and corresponding mitochondria in proximal and distal sciatic nerve exhibited few changes in elemental composition (i.e. P remained elevated). Swollen axons were occasionally observed in distal sciatic nerve sections (n = 6) from rats treated with A C R for 30 days. Microprobe analysis of these damaged axons revealed a complete loss of normal elemental composition (data not shown). On both a dry and wet weight basis, large significant increases in axoplasmic Na, CI and Ca were noted in conjunction with decreased P, K and Mg. Quantitative elemental changes were statistically comparable to those associated with swollen tibial nerve fibers L6. Mean ( + S.D.) water content of swollen axons was significantly greater than that of normal medium distal sciatic fibers (i.e. 95 + 2% vs. 90 + 0%). Mitochondrial areas (n = 11) from swollen distal axons also exhibited severe elemental deregulation which was similar to that reported for affected tibial mitochondrial areas 16. Relative to control, these organelles lost K and Mg, and gained Na, C1 and Ca. Mean ( + S . D . ) water content of mitochondrial areas from swollen axons also increased significantly (i.e. 82.1 + 3.0% vs. 89.3 + 4.4%).

E l e m e n t a l c o m p o s i t i o n a n d water c o n t e n t o f S c h w a n n cells a n d myelin in sciatic nerve o f control a n d A C R -

The elemental composition of normal Schwann cells and myelin did not appear to be dependent upon corresponding axon size or nerve region. The quantitative pattern of elemental distribution in these proximal and distal sciatic nerve compartments was similar to that reported for control tibial nerve Schwann cells and myelin tT. In proximal sciatic nerve of A C R exposed rats, Schwann cell cytoplasm exhibited few changes in elemental content during the early stages of intoxication (i.e. 15 and 22 days; Table IV). However, as treatment progressed (i.e. 30 days), dry and wet weight concentrations of cytoplasmic Na and C1 increased significantly, while decreases in mean P levels were associated with increased variance (Table IV). Similar changes in these elements were noted in proximal nerve of paralyzed animals (i.e. 60 days). In addition, mean cytoplasmic dry and wet weight K levels were significantly decreased (Table IV). Changes in cytoplasmic S, Ca, Mg and water content were not evident in proximal nerve of A C R treated rats (data not shown). In distal sciatic nerve, A C R administration was associated with substantial derangement of Schwann cell elemental composition. At 22 days, data variance for Na and Ca increased significantly, while mean Mg concentrations decreased (Table IV). At later exposure times (i.e. 30 and 60 days), cytoplasmic wet and dry weight Na and CI concentrations increased, while mean K and Mg levels decreased. Also during this time, cytoplasmic Ca content returned toward normal values treated rats.

TABLE IV Effects of A CR treatment on cytoplasmic elemental composition and water content of sciatic nerce Schwann cells Proximal sciatic

Na K P CI

Control (36)

x 22 (24)

× 30 (26)

× 60 (25) §

226 _+22 434_+18 706_+15 216 _+23

286 -+37 415_+19 661 _+35 a 185 _+24

452 _+34 * 411 _+28 651 _+35a 310 _+24 *

389 _+46 * 344_+22 * 682_+30 a 253 _+29

Distal sciatic

Na K P CI Ca Mg

Control (25)

× 22 (32)

× 30 (30)

× 60 (20)

244 + 24 454+ 18 749_+23 164+_17 2-+ 1 35± 4

275 + 39 a 405+24 703 -+20 198_+20 7_+ 3 a 22-+ 3"

421 _+46 a 339_+28 * 673 -+28 255 + 25 * 4_+ 2 a 21-+ 4*

410 + 38 ,a 370+32 * 672-+41 ~ 251 _+22 * 3-+ 1 21-+ 4*

Elemental concentrations are expressed as mmol element/kg dry wt. _+S.E.M.; cell water content expressed as %water_+S.E.M. § Days of oral ACR treatment (n = cytoplasmiccompartments analyzed) * Mean data significantly(P < 0.05) different from control. a Variance of data significantly(P < 0.05) different from control.

244 and mean P tended to decrease (Table IV). These elemental changes were not associated with statistically significant alterations in mean Schwann cell water content (data not shown). In both proximal and distal sciatic nerve regions, ACR intoxication did not cause consistent changes in myelin elemental composition or water content (data not shown). DISCUSSION We have determined the effects of oral ACR administration on the subcellular distribution of elements and water in myelinated axons and Schwann cells of sciatic nerve. This study was conducted so that tibia116'17 and sciatic nerve changes could be compared and contrasted. Such analysis was used to assess the selectivity and specificity of ACR-induced elemental changes. In general, sciatic nerve axons in both proximal and distal regions exhibited small but persistent increases in mean axoplasmic Na and P concentrations. As a function of ACR exposure, axoplasmic K content of proximal small and medium sciatic nerve fibers increased initially (i.e. 15 days) and then returned to control values (i.e. at 30 days). In contrast, the concentrations of this element in

- - SciaticNotch ~ r o x i m a l

L |

Sciatic Nerve

Sciatic Sample

Large Na P K Cl 1" 1" 1" NS

Medium Na P K Cl 1" 1" 1" 1"

al Sciatic Sample Popliteal Fossa

ge K Cl NS NS

//

Medium Na P K CI 1" NSNSNS

Tibial Branches

ibial Sample

/

Plantar Nerve

~" B ~ , ~

Large Na P K C1 . . . .

Ns NS NS NS

Medium Na P K Cl

1" NS ~

Fig. 4. S u m m a r y diagram showing relative qualitative changes in large and m e d i u m axon elemental composition as a function of proximodistal distance along the seiatic-tibial nerve axis. Illustration represents changes occurring after 30 days of oral A C R exposure. Direction of arrow indicates loss or gain of dry weight axoplasmic element ( m m o l / k g ) , NS = no significant change in either m e a n or variance. Tibial nerve changes based on data from LoPachin et al. is.

large proximal and most distal axons remained unaffected. The elemental changes that characterize sciatic nerve differ substantially from those of tibial nerve 16 (see Fig. 4). In tibial nerve of oral ACR intoxicated rats, subpopulations of small and medium diameter fibers exhibited initial (i.e. 15 days) increases in axoplasmic K concentrations which were coupled to decreases in Na levels. As ACR ingestion continued, these axons lost K and gained Na (Fig. 4). This progressive pattern of elemental alteration in tibial nerve axons is typical of cellular injury 13,16,18,31,32, and accordingly it was hypothesized ~6 that this sequence of deregulation represented a premonitory event which, in conjunction with other ACR-induced functional deficits (e.g. inhibition of axonal transport), lead to the global element and water decompartmentalization that characterize swollen and degenerating axons (see Introduction). Our observation that injury-typic elemental changes parallel the spatiotemporal development of morphological alteractions suggests that such changes are a specific component of the mechanism of neurotoxicity. Although not injury-typic, changes in elemental composition were nonetheless observed in sciatic nerve. The mechanism(s) responsible for these changes is (are) unknown but might represent repair or homeostatic reactions possibly initiated in response to distal axon compromise. This suggestion is based on the absence of morphological alterations in frozen (this study) and fixed 25 sciatic nerve sections and the observation that the magnitude and direction of corresponding Na, K and P changes are not consistent with traditional patterns of injury-induced elemental disruption in axons (vide supra). In addition, the aforementioned proposal is supported by several lines of evidence from previous E P M A studies of nerve injury and regeneration. For example, elevated axoplasmic K and P levels similar to those associated with proximal sciatic axons from ACR-treated rats have been observed in peripheral nerve axons following subchronic mixed ganglioside administration to normal rats 2°. The neuroregenerative and neurotropic properties of gangliosides are well documented 1°. Furthermore, in rats allowed to recover from oral ACR intoxication (LoPachin et al., unpublished observations), tibial nerve axons exhibited increases in dry and wet weight Na, P, C1 and K that are similar to those changes occurring in proximal axons of intoxicated animals. Evidence for a repair or homeostatic response is also provided by data from transected rat sciatic nerve where axoplasmic K and P levels were increased in medium and large diameter fibers at a time (8 and 16 h after transection) when

245 small axons were exhibiting elemental and morphological changes characteristic of injury 14. Alternatively, it is possible that elemental modifications in sciatic nerve are a result of specific ACR-induced biochemical effects. Increases in nodal membrane ion permeability, polyphosphoinositide metabolism and protein phosphorylation have been reported in proximal sciatic nerve of ACR intoxicated rats L2 and might underlie expression of sciatic nerve elemental changes. In addition, development of proximal axonal atrophy secondary to decreased perikaryal neurofilament synthesis6'7, and alterations in the rate, quantity and deposition of axonally transported materials 9'26 could conceivably contribute to or be primarily responsible for ACR-induced manifestations of elemental composition in sciatic nerve. Schwann cells of rat sciatic nerve also exhibited characteristic disruptions of dry and wet weight elemental composition. In cytoplasm of distal sciatic Schwann cells, mean Na, Ca and C1 levels increased significantly while K and Mg contents decreased following 30 and 60 days of ACR exposure. These changes are injury-typic and are similar to the pattern of elemental deregulation observed for tibial nerve Schwann cells ~7. Less extensive elemental alterations were detected in Schwann cell cytoplasm of proximal nerve. Whether these elemental alterations reflect a direct Schwann cell site of action for ACR remains to be determined. Such an interpretation is suggested by selective Schwann cell modifications in the absence of respective axonal changes in distal sciatic nerve. However, if ACR affects Schwann cells directly, it is unclear why proximal sciatic nerve glia were not similarly affected. Moreover, in tibial nerve of rats recovered from ACR intoxication (LoPachin et al., unpublished observations), we have found that corresponding Schwann cells exhibit deranged elemental compositions comparable to those of distal sciatic and tibial nerve glia from ACR affected rats. These results suggest that ACR associated Schwann cell changes might be reactive or secondary. Regardless, reported modifications of Schwann cell elemental composition might be a manifestation of ACR's influence on the highly complex, reciprocal relationship that exists between axons and glial cells 19. Clearly, additional studies are necessary to decipher the neurotoxicological relevance of Schwann cell changes. In summary, the results of this study demonstrate that ACR neurotoxicity is associated with characteristic changes in elemental composition of rat sciatic nerve myelinated axons. When the spatiotemporal pattern of axonal elemental disruption along the sciatic-tibial nerve axis is considered, it is evident that ACR intoxi-

cation alters the normal proximodistal distribution of K, C1, P and Na 12 (Fig. 4). Moreover, it is clear that ACR exposure is associated with disruption of Schwann cell elemental composition. Based on the functional importance of the glial-neuronal relationship ~9, possible Schwann cell compromise might represent a confounding event which contributes to the development of ACR-induced peripheral nerve damage. Regardless, further investigation is necessary to establish the neurotoxicological relevance of altered elemental composition in myelinated axons and glial cells. Acknowledgements. The authors would like to thank Dr. Helen Badoyannis for her helpful comments and criticisms. This research was supported by NIH grants ES03830 to R.M.L. and NS21455 to A.J.S.

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