Histochemical and electron microscopic observations on the myoneural junctions of rats with carbon disulfide induced polyneuropathy.

M. Histochemical and electron micro scopic .obsel'Vlatiorus on the myoneural junotions .of vats with c.ambon disulfide m duced polyneuropathy. Scand. j. work environ. & health 3 {1~77) 36--42. Myoneural junctions in the .tifbialis anterior muscle of rats with clinical ISigns of polyneuropathy induced with =bon di.5ulfide w.ere studied by 1li~t and electron microscopy. Histo chemic-any demonstrable !<lcetylcholinesterose (AChE; E.C. '3,1.1.7) aclilV.ilty was dis tributed similavly in 1lhe myoneurnl jWlCtion:s of bath rt;he eXlpOSed and the coTlitIOOl1 rats. I'll both gvoups intens,e enzyme lactivity W,a5 locaJizedait ibhe level of Ithe post synaptic membrane of !the my,oneuval junction. The rpos1lsynaptic info~dings of the myoneur'al junotions of the exposed raJts appeared normal. No enzyme activmy was seen outside the WIle of <the myoneurnl jrunctionls. The uLtrastructure of the sub lSarcolemmalspace, as well 00 the postsynaptic membrnnes of the myoneural jUillclioru; of the exposed andmals, was normal. lin !the rteI1minaJ. <lxons .signs of va,vious degrees of degener:ati:on were presenJt, e.g., disappe!<lraJIlce of the Ipreter.minaJ. axoplasmd.c neurotubU'1es, partiaJ dilSa.ppeamnce of sy;naptic vesicles, appeammoe of dense bodies, and even total disappearance or destruction of <the :terminal ,axons. Synaptic cIe·fts were often widened WliJth Schwann oeM interpo&i,tion. It r1Jhus seems that systemic oarbon difsuJ.fide poisonifngpvimarily allteI1s the presynaptk Istructures of the myo neural junctiolllS, whifIe the pos.tsynaptic Slide 'l1emaJi.ns 'relattivlely Ii;n;tact, ,especiailily since the histochemicall distribution of AChE 'in myoneural junctions was normal.

It was recently shown that experimental long-term exposure to carbon disulfide (CS 2 ) by inhalation produces a peculiar type of toxic polyneuropathy with focal axonal swellings consisting of bundles of neurofilaments (20) and alterations in the nonspecific cholnesterase activity of intramuscular nerves (14). Neurophysiological investigations on exposed workers and rabbits have not only revealed a slowing of the maximal motor conduction velocities of peripheral nerves but also signs of disturbance at the level of the myoneural junction (27,28) or muscle (20). We therefore decided to study the ultrastructure of the myoneural junctions of animals exposed to CS 2 , particularly as, surprisingly, few morphological data are available on myoneural junctions in diffuse toxic polyneuropathies (1,23,24,26,29). As there is considerable evidence suggesting that acetylcholinesterase is localized at the level of the postsynaptic membrane of the myoneural junction (8,9) and that its activity is affected by denervation (4,7,30), a parallel enzyme histochemical study was also carried out.

Experimental animals and exposure to carbon disulfide
Altogether 10 adult rats of the Sprague-Dawley strain, 3 months old at the beginning of the exposure period, were studied in the present experiment. The animals were kept in stainless steel cages on metal grids without bedding material and were given commercial rat pellets (Orion Oy, Helsinki, Finland) which were removed during the exposure to CS 2 , and water ad libitum. Seven of the rats were exposed by inhalation to an air concentration of 750 ppm of CS 2 for 4 months (6 h per day, 5 days per week for the first 10 weeks, and 3 days per week thereafter). The remaining three animals, nonexposed, served as controls. For the histochemical studies material was taken from all animals, and six animals were used for electron microscopy.

Histochemical methods
'.Dhe anterior tibial muscles of the rats were unilaterally removed under ether anesthesia before perfusion (or bilaterally immediately after decapitation) and fixed at 4°C with 3.5 P/o formol-calcium (6) for 6-18 h. Frozen sections were cut at 30 /lm and washed in distilled water for 1-3 h.
Incubation was performed with freefloating sections at 37°C and at pH 6.0 for 2 h after preincubation for 20 min in a subtrate-free solution containing the inhibitor at a concentration of 10-s M.
The intensity of AChE activity was estimated from a visual comparison of the activity of the exposed muscles and that of the control ones incubated in the same medium (7).
The histochemical method used in the present study was based principally on the Gomori version (10) of the Koelle technique (17).

Electron microscopic methods
Localization and dissection of the myoneural junctions in the tibialis anterior muscle for electron microscopy were performed principally according to the method described by Engel (5).
The animals were killed under ether anesthesia by perfusion, via the left ventricle, of a solution of 1 Ofo paraformaldehyde and 1.25 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, followed by perfusion with 5 % glutaraldehyde in the same buffer (16). After dissection of the muscle pieces the fixation was continued by immersion in the 5 Ofo glutaraldehyde solution at 4°C for 2 h. Postfixation was carried out with 1 {J/o osmium tetroxide with a pH of 7.4 for 1 h, and it was followed by dehydration with ethyl alcohol. Thereafter the pieces were embedded in an Epon-Araldite mixture (21), sectioned and poststained with uranyl acetate (31) and Lead citrate (25). A Philips EM300 electron microscope was used when the sections were examined and photographed.

Intoxication symptoms
After 4 months of exposure to CS 2 there was marked weakness in the posterior extremities of all the animals. They had considerable difficulty in moving about and were unable to support the caudal parts of their bodies adequately.

Acetylcholinesterase activity
In the control animals the zone of myoneural junctions with intense AChE activity formed an upward convex arch, the individual muscle fibers containing a single myoneural junction. The enzyme activity was localized in the postsynaptic part of the myoneural junction and thus revealed the postsynaptic infoldings clearly (figs. [1][2]. The area of AChE activity was ramified and segmented. In most of the myoneural junctions of the exposed animals the distribution of the histochemically demonstrable AChE activity was normal. Degenerative changes, Le., lowered intensity of AChE and irregular postsynaptic infoldings, were observed in only a few of the myoneural junctions. No AChE activity was seen outside the zone of the myoneural junctions.

Electron microscopy
Altogether, 13 myoneural junctions from the tibialis anterior muscle of three animals exposed to CS 2 were studied with the electron microscope. Three myoneural junctions from the control group were similarly analyzed. The subsarcolemmal ,space of the myoneural area of the CS 2 exposed rats was normal. Most of the myoneural junctions of the exposed rats had a normal postsynaptic structure. The postsynaptic membrane of the myoneural junction was typically electron dense and deeply folded and it extended as fingerlike invaginations into the subsarcolemmal space. The bottoms of these infoldings were broader and less electron dense than their necks. Amorphous material occupied the space between the axonal and muscular membranes. Degenerative changes, Le., irregularly shaped and lowered postsynaptic infoldings, were observed in only two of the myoneural junctions studied (fig. 5). In most of the myoneural junctions analyzed, the primary synaptic cleft was often irregularly widened and it contained only remnants of the amorphous surface material. In the myoneural junctions with a widened synaptic cleft there was usually interposition of Schwann cells, the side of the axon terminal facing the muscle thus being 40 separated from the postsynaptic membrane ( fig. 3).
Most axon terminals of the myoneural junctions of the exposed animals had signs of various degrees of degeneration. Those parts of the axons separated from the postsynaptic membrane by Schwann cell cytoplasm were often devoid of synaptic vesicles and mitochondria ( fig. 3). Occasional dense bodies and irregular, membranelimited spaces were present ( figs. 3 and 4). The preterminal axoplasm was also frequently devoid of neurotubules, while the number of neurofilaments had increased. Almost total destruction of the axon was observed in a few myoneural junctions, particularly in those with altered postsynaptic membranes ( fig. 5). In the cytoplasm of the teloglial cells covering the most severely affected axon terminals, signs of reactive alterations were seen, including dilatation of the cisternae of the endoplasmic reticulum and appearance of inclusions in the cytoplasm.

DISCUSSION
The present findings show that in chronic polyneuropathy induced by CS 2 inhalation various stages of degeneration of the myoneural junction occur that are not found in controls. The degenerative changes essentially consist of ultrastructural alterations of the presynaptic side of the myoneural junctions, while the postsynaptic side is only slightly affected.
Because of the supposed postsynaptic localization of AChE already mentioned, .it seems conceivable, on the basis of the present ultrastructural findings, that no major changes occur in the distribution of this enzyme in the myoneural junction. In the present study no signs of local concentration of AChE activity outside the zone of the myoneural junctions appeared which can be considered as a lack of formation of new neuromuscular contacts during regeneration of the nerve (15). Weakening of the enzyme activity was seen only occasionally in some myoneural junctions, a finding consistent with denervation (7) as evidenced by ultrastructural observation of myoneural junctions with advanced degeneration or total destruction of the terminal axons. In these myoneural junctions alterations in the postsynaptic membrane also occurred which may be attributed to denervation-like changes (18).
In most of the n€rve terminals of the exposed animals signs of advanced degeneration were observed with no signs of regeneration. The major ultrastructural alterations included an increase in the number of preterminal neurofilaments, a decrease in the number of preterminal neurotubules and terminal synpatic vesicles, the appearance of dense bodies and degenerating mitochondria into the axon terminals, and the interposition of the teloglial cells. Similar ultrastructural changes have been described in the myoneural junctions in association with acrylamide-induced neuropathy (24,29). On the other hand, no lamellated inclusions of the axoplasm of the nerve terminal, typical of tri-ortho-cresyl phosphate intoxication (23), were observed in the present study.
Of considerable interest is the role of disturbed trophic or inductive functions of the nerve (11,13,19) in the degeneration of the terminal or postsynaptic structures of the myoneural junction in polyneuropathy. There is evidence suggesting that structural changes of the axon, especially disruption of the axoplasmic neurotubules, cause inhibition of the axonal transport (12,22), and consequently the delivery of a trophic substance or substances may be inhibited (13,22). It is also believed that the so-called dying-back neuropathies result from failure of neuronal perikarya to supply the material required by their axons (2,3). CS 2 induced polyneuropathy is characterized by marked structural alterations of the axon that include the disappearance of the axoplasmic neurotubules (14,20). Therefore, it seems probable that axoplasmic transport is inhibited to some extent. On the other hand the observation of relatively few postsynaptic structural alterations in the myoneural junctions of the exposed animals indicates that disturbances of trophic functions of the nerve do not cause any marked changes during exposure conditions similar to those applied in the present study. Considerable interspecies differences apparently exist in this respect; e.g., rabbits develop severe denervation atrophy of the muscle under identical experimental conditions (20).
ACKNOWLEDGMENT This investigation was supported by a grant from the Finnish Cultural Foundation.