Acta Neuropathol (1995) 89:17-22 �9 Springer-Verlag 1995

A. Takahashi �9 M. Mizutani �9 C. Itakura

Acrylamide-induced peripheral neuropathy in normal and neurofilament-deficient Japanese quails

Received: 28 January 1994 / Revised: 21 Feburary 1994, 8 July 1994 / Accepted: 11 July 1994

Abstract Morphological effects of acrylamide (AC) on the peripheral nerves in normal and neurofilament (NF)- deficient (Quv) Japanese quails were investigated. AC (100 mg/kg) was injected intraperitoneally every other day. Af- ter the birds manifested neurological signs, they were necropsied (after 10 - 21 AC injections) and the sciatic and tibial nerves were examined. In both normal and Quv qualis, AC produced axonopathy with a distal-proximal progression. In AC-intoxicated normal quails, the nerve fiber pathology was characterized by typical Wallerian-like degeneration, consisting of axonal degeneration, myelin breakdown, macrophage migration, Schwann cell prolif- eration and regeneration of nerve fibers. Ultrastructurally, AC-induced NF accumulation was detected in the axon of myelinated nerve fibers. In AC-intoxicated Quv qualis, axonal degeneration with accumulation of membranous organelles occurred; however, sequential events of Wal- lerian-like degeneration were not as prominent as in AC- intoxicated normal qualis. These results demonstrated that NF-deficient Quv quails are sensitive to neurotoxic ef- fects of AC. On the other hand, the different pathology of AC-intoxicated normal and Quv qualis indicates the pres- ence or absence of NFs influences the appearance and ex- tent of AC axonopathy.

Key words Acrylamide �9 Peripheral neuropathy Neurofilament �9 Pathology �9 Quail

A. Takahashi Toxicology Research Laboratories, Japan Tobacco Inc., Hatano, Kanagawa 257, Japan

M. Mizutani Nippon Institute for Biological Science, Kobuchizawa, Kitakomagun, Yamanashi 409-16, Japan

C. Itakura (N~) Department of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan Tel.: 011-706-5191; Fax: 011-71%3701

Introduction

Acrylamide (AC) monomer, neurotoxic to man and ani- mals, causes "central.-peripheral distal axonopathy" [9, 16, 19, 20, 23]. An early AC-producing pathology is mul- tifocal axonal swellings in the distal region of long and large-caliber nerve fibers [16, 21-23, 25]. The axonal swelling results from neurofilament (NF) accumulation at the proximal side of the node of Ranvier [21-23]. In the later stage of axonopathy, the affected nerve fibers un- dergo Wallerian-like degeneration, including degenera- tion of axons and myelin, glial cell reactions and regener- ation of nerve fibers [16, 19, 22, 25].

Quv quails are a mutant strain of Japanese quails, that show congenital axonal hypotrophy resulting from NF de- ficiency in both the central and peripheral nervous systems [14, 29]. Morphometrically, a smaller number number of large-caliber myelinated nerve fibers and greater number of small-caliber myelinated nerve fibers were demon- strated in their peripheral nerves [30]. These characteris- tics in Quv have revealed an important function of NFs as a determinant of axonal caliber. On the other hand, the mu- tant shows no severe neuronal degeneration associated with NF deficiency. These facts suggest that NFs are not essential for the maintenance or survival of nerve cells. Immunohistochemical [28] and molecular biological [15] studies have suggested that the lack of visible NFs in Quv quails is caused by the defective expression of NF-L pro- teins due to point mutation in the NF-L gene. The loss of functional NF-L proteins results in a defective NF assem- bly even in the presence of NF-H and NF-M proteins.

We previously reported that [3, [3"-iminodipropionitrile (IDPN) induced no effects in neurons of Quv qualis [13]. Our another study on acrylamide-induced central nervous system lesions demonstrated that AC produced membra- nous organelle accumulations in terminal and preterminal axons of Quv quails [26]. The different responses of their neurons to AC and IDPN intoxication suggest a difference in the primary site of action of these toxic substances. This report describes the peripheral nerve lesions in AC- intoxicated normal and Quv quails.

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Materials and methods Table 1 Clinical signs in AC-intoxicated normal quails (AC acry- lamide

Quails and intoxication protocol

Normal Japanese quails (hybrid) were purchased from a commer- cial farm. Quv quails were maintained in the Nippon Institute for Biological Science (Yamanashi, Japan). Nine normal and nine Quv quails aged 10-16 weeks were used in this study. Quails were housed and handled in accordance with the recommendations of the "Guide for the care and use of laboratory animals" (NIH publi- cation No. 86-23, revised 1985). They were divided into four groups: an AC-injected normal quail group (n = 6), a saline-in- jected normal quail group (n = 3), an AC-injected Quv quail group (n = 6), a saline-injected Quv quail group (n = 3). The birds were injected intraperitoneally either with l% AC (Kanto Chemicals, Japan) dissolved in 0.9% saline at a dose of 100 mg/kg every other day or with an equivalent volume of 0.9% saline. During the ex- periment, daily examination of the physical and neurological con- dition was carried out. The sacrifice time was determined accord- ing to the severity of clinical signs (Tables 1 and 2).

Time preparation

The quails were anesthetized with pentobarbitai and perfused through the left ventricle with a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 rain. The sciatic and tibial nerves between the sciatic foramen and knee joint were removed and dissected into the following four seg- ments, each about 1 cm in length: the proximal sciatic nerve (im- mediately distal to the sciatic foramen), the distal sciatic nerve (2 cm distal to the sciatic foramen), the proximal tibial nerve (2 em proximal to the knee joint), the distal tibial nerve (immediately proximal to the knee joint). The specimens were stored in a mix- ture of 2% paraformaldehyde and 2.5% glutaraldehyde overnight at 4~ and post-fixed in 2% buffered osmic acid for 2 h at 4 ~ C. They were then embedded in epoxy resin so as not to lose the proximo-distal direction. Epon-embedded semithin sections were stained with toluidine blue. Ultrathin sections were double-stained with uranyl acetate and Reynold's lead citrate, and examined with a JEOL JEM-100 SX electron microscope.

For teased fiber preparations, the proximal sciatic and distal tibial nerves were fixed by immersion in 3% glutaraldehyde in 0.05 M phosphate buffer (pH 7.4) for 10 min and post-fixed in 1% buffered osmic acid for 2 h at 4 ~ C. Tissues were immersed in 70% glycerin for 24 h and stored in 100% glycerin at room temperature. Carefully separated nerve fibers were examined with a light mi- croscope.

Results

Clinical signs

AC inject ion times and main clinical signs are shown in Tables 1 and 2. Both AC-intoxicated normal and Quv quails init ial ly manifested an abnormal posture like a dog sitting on its hind legs, reluctance to walk and slight inco- ordinat ion with an up and down movemen t of the legs. These symptoms were observed earlier in AC-intoxicated Quv quails (after 2 -4 AC injections) than in AC-intoxi - cated normal quails (after. 7 -9 AC injections). Three AC- intoxicated normal quails (nos. 1-3) and three AC-intoxi- cated Quv quails (nos. 1, 3 and 6) were necropsied soon after they had difficulty in walking or were unable to walk. One Quv quail (no. 2) was necropsied after 12 AC injections due to deteriorating physical condition. In other

Quail First neuro- Total no. of Neurological signs no. logical signs a AC injections at termination

(days of age) (days of age) of treatment

I b 8 (86) 10 (90) Difficulty in walking 2 b 7 (84) 10 (90) Difficulty in walking 3 9 (104) 14 (110) Lateral recumbency 4 8 (86) 17 (104) Unsteady gait 5 7 (112) 21 (140) Unsteady gait 6 9 (132) 22 (158) Unsteady gait

aThe number of AC injections at the onset of the neurological signs bTeased fiber preparations were examined

Table 2 Clinical signs in AC-intoxicated Quv quails

Quail First neurol- Total no. of Neurological signs no. ogical signs a AC injections at termination

(days of age) (days of age) of treatment

I b 2 (74) 10 (90) Lateral recumbency 2 b 3 (76) 12 (94) Unsteady gait 3 2 (116) 14 (140) Difficulty in walking 4 3 (90) 15 (114) Unsteady gait 5 4 (99) 21 (133) Unsteady gait 6 4 (99) 21 (133) Difficulty in walking

a The number of AC injections at the onset of the neurological signs b Teased fiber preparations were examined

quails, the unsteady gait persisted up to the t ime of sacri- fice. Body weight reduct ion was observed in some quails after they started to manifest neurologcial symptoms.

Light microscopic f indings

Teased fiber preparations

Paranodal axonal swellings with myel in abnormalit ies (re- traction, foldings, ovoid formations) were observed in both AC-intoxicated normal and Quv quails. Nerve fiber de- generat ion was seen on rare occasions in AC-intoxicated normal quails. These f indings were prominent in the dis- tal tibial nerve of both AC-intoxicated normal and Quv quails.

Semithin sections

All AC-intoxicated normal quails showed Wallerian-l ike degeneration, preferentially affecting the distal tibial nerve with retrograde progression. The degenerative nerve fiber showed increased axoplasmic density with myel in fold- ings, f ragmentat ions and breakdown (Fig. 1). Loss of large-caliber myel inated nerve fibers and increased num- bers of endoneurial cells were associated with nerve fiber degeneration. Many mitotic figures of Schwann cells were

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Fig. 1 Transverse (A, B) and longitudinal (C) semithin sections of distal tibial nerve of normal quails. A Saline-injected control quail. B, C Quail given 14 AC injections. Increased axoplasmic density and myelin breakdown are present. Lipid-laden macrophages are seen (arrowheads) (AC acrylamide). Toluidine blue. A, B x 780; C x 640

present in a normal quail given 10 AC injection. Migrat- ing macrophages were seen in the endoneurial spaces, and sometimes invaded into degenerating axons. Regeneration o f tiny nerve fibers was prominent in normal quails given more than 14 AC injections. Quv quails given more than 12 AC injections showed an increase in the axoplasmic density and irregular myelin foldings (Figs. 2). However, other changes observed in AC-intoxicated normal quails were not remarkable.

Fig. 2 Transverse (A, B) and longitudinal (C) semithin sections of distal tibial nerve of Quv quails. A Saline-injected control quail. Note myelinated nerve fibers with a smaller caliber in Quv quail as compared with those in normal quail. B, C Quail given 14 AC in- jections. Irregular myelin foldings are observed. Toluidine blue. A, B x 780, C x 800

Electron microscopic findings

Nerve fibers

In AC-intoxicated normal quails, NFs were increased in number and irregularly arranged in axons o f large-caliber myelinated nerve fibers (Fig. 3). Accumulat ions of dis- torted organelles and amorphous materials, and complete

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loss of axoplasmic organelles were observed in degener- ating axons. Myelin breakdown was associated with axo- nal degeneration (Fig. 4). Myelin foldings with indenta- tions or occlusions of the adjacent axons were also ob- served. Increased endoneurial cells were recognized as proliferating Schwann cells (Fig. 4) and migrating macro- phages.

The macrophages appeared either pre-phagocytic with empty vacuoles or phagocytic with lipid droplets and myelin debris. Clusters of regenerative nerve fibers con- sisted of unmyelinated nerve fiber bundles and tiny myeli- nated nerve fibers. Some unmyelinated axons had a one- to-one relationship with Schwann cells.

In AC-intoxicated Quv quails, axons of myelinated nerve fibers had increased number of mitochondria and smooth endoplasmic reticula. Accumulations of membra- nous profiles and glycogen granules were sometimes seen in axons (Fig. 5). There were also degenerative axons with empty axoplasm or broken organelles and associated myelin abnormalities. The affected myelin showed irregu- lar foldings within Schwann cell basal lamina; however, they maintained mostly intact lamellar structures without appreciable destruction (Fig. 6).

Fig.3A, B Transverse sections of myelinated fibers of normal quails. A Saline-injected control quail. B Proximal sciatic nerve of quail given 2t AC injections. Neurofilaments (NFs) increase in number with irregular arrangements (arrowheads). Microtubules tend to aggregate associated with smooth endoplasmic reticulum (arrows). A, B x 31570

Fig.4 Distal tibial nerve of normal quail given 21 AC injections. Degenerating nerve fiber loses axon with destroyed myelin. A few layers of Schwann cell processes sur- round it. Schwann cell cyto- plasm (S) and Schwann cell processes (P) contain tiny unmyelinated axons (arrow- heads) and myelin debris (arrows). x 13 175

Discussion

Previous studies on AC-intoxicated rats [16, 22, 23, 25] and cats [19, 21-23] revealed that AC induces distal ax- onopathy. A well-documented morphological change in the early stage of axonopathy is multifocal axonal swellings with NF accumulation [16, 21-23, 25]. As the axonal injury continues, the affected nerve fibers result in Wallerian-like degeneration [16, 19, 22, 25].

Fig.SA, B Transverse sections of myelinated fibers of Quv quails. A Saline-injected control quail. No visible NFs are seen in the axon. B Distal tibial nerve of quail given 21 AC injections. Mi- tochondria and smooth endoplasmic reticulum (arrow) increase in number with accumulations of glycogen granules (arrowhead). A, B x 27 300

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In the present study, AC produced distal axonopathy in the peripheral nerve of normal and Quv quails; however, AC-induced pathology of both quails was quite different. Axonal degeneration in AC-intoxicated normal quails was associated with NF accumulations in axons, and it was followed by sequential events of Wallerian-like degenera- tion. In contrast, AC-intoxiated Quv quails showed axonal degeneration with membranous organelle accumulation in axons; however, Wallerian-like degeneration was not as prominent as in AC-intoxicated normal quails.

Although NF accumulation is an early and major AC- induced axonal change, this may represent a primary site of neurotoxic action of AC or a secondary change due to other cellular injury. Recent biochemical studies on AC- intoxicated animals have showed specific binding of AC to NFs [10], altered NF transport [6], NF degradation [27] and NF phosphorylation [8], suggesting underlying mech- anism of AC neurotoxicity. However, AC-induced ax- onopathy in NF-deficient Quv quails'indicates that this occurs independently of NF accumulations. It is sug- gested that effects of AC on NFs are not a prerequisite for the initiation of AC axonopathy.

On the other hand, AC-intoxicated Quv quails showed little evidence of Wallerian-like degeneration. Wallerian degeneration in the distal stumps of the transected periph- eral nerve is a well-studied experimental model. It is initi- ated by axonal injury, and associated myelin breakdown, macrophage migration, Schwann cell proliferation and nerve fiber regeneration occur promptly.

Recent experiments using in vivo and in vitro models of Wallerian degeneration have suggested that myelin phagocytosis and myelin removal during Wallerian de- generation are critically dependent on recruitment of cir- culating macrophages [1-3, 5, 24]. It has been suggested, however, that myelin removal in small-caliber nerve fibers

Fig. 6 Distal tibial nerve of Quv quail given 14 AC injec- tions. Myelin shows irregular foldings within Schwann cell basal lamina, forming ringed loops or enclosing degenera- tive axons (asterisks). Empty axoplasm is observed (arrow). x 9350

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progresses slowly, retaining normal myel in structure, and may be accomplished by degradation within Schwann cells without participation of macrophages [5, 24]. In Quv quails, NF-defic ient axons with a small-cal iber may have some effects on the process of myel in removal, result ing in its retarded progression. In addition, AC-intoxicated Quv quails showed neither Schwann cell proliferation nor regenerat ion of nerve fibers. Recently, retarded progres- sion of Wallerian degenerat ion related to the failure to re- cruit macrophages, has been found in a mutant strain of mice, C57BL/Ola (Wld) [7, 11, 12, 17, 18]. In delayed Wallerian degeneration, Schwann cell proliferation and regeneration of nerve fibers are very poor [4, 7]. Prompt progression of Wallerian degenerat ion may require opti- mal condit ions for myel in removal to proceed.

In our previous study on AC-induced central nervous system lesions, Quv quails were shown to be sensit ive to neurotoxic effects of AC [26]. The occurrence of AC-in- duced lesions in NF-def ic ient Quv quails suggests that AC neurotoxici ty is initiated independent ly of the interac- t ion of AC with NFs.

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