209 Studies on the Thiolacetic acid method for Histochemical Demonstration of Cholinesterase and Its Distribution in Muscles Takao MURO, Hitoshi TANABE, Satoshi UEDA, Masanori, TOMONAGA and Masanori UONO From the third Department of Internal Medicine (Chief: Prof. S. Okinaka), Faculty of Medicine, University of Tokyo Introduction Since Gomori's histochemical method (1948)1) for cholinestestese (ChE) using high fatty acid choline ester as substrate had been reported, Koelle's thicholine method (19492), 19503), 19514)), Ravin, Zsou & Seligman's azo-dye method (1951)5), Barrnett & Seligman's (1951) and Holt & Withers' (1952)7) indoxyl methods were successively reported. Thereafter, Crevier & Belanger (1955)8) reported a new histochemical method for ChE using thiolacetic acid as substrate. The principle of this method was based on Wilson's researches9) in which thiolacetic acid was hydrolysed by AChE at an acid pH with the formation of acetic acid and hydrogen sulfide. Practically it consisted of an incubation with thiolacetic acid and lead nitrate, and liberated hydrogen sulfide at ChE active sites was precipitated as dark brown lead sulfide. Later on, this method was modified by Csillik & Savay10,11) and recently by Wachstein, Meisel &Falcon12). Csillik & Sdvay chiefly investigated about motor end-plate by their modified method and protargol impregnation as counterstaining. In this report, we studied the distribution of ChE activity in several organs or tissues by the thiolacetic acid method as comparing with the thiocholine method. Further, by that method, the distribution of ChE in muscle of several species was investigated. 210 Materials and Methods The materials from dogs, rats, mice, birds (cock and sparrow), frogs, goldfish and crayfish, and autopsied and biopsied human muscles were used. Fresh frozen sections were cut about 20-30ƒÊ thick. The Wachstein's modifica- tion12) and the Gomori's modification13) were employed for thiolacetic acid method and for thiocholine method respectively. The investigation of motor end-plate was carried out chiefly using the the thiolacetic acid method. Results The Wachstein's modification of thiolacetic acid method gave the most accurate results, probably due to the fact that by this method the constant final pH of incubation medium is obtained. 1) Thiolacetic acid method as comparing with thiocholine method : The several organs or tissues such as central nervous system, lung, liver, ecrine glands of paw, end-plates of muscles and arteries in dogs were investigated using above- mentioned two methods. As to general distribution of ChE in the central nervous system, there was no difference between two methods, but the activity of neuropils of nerve cells did not appear clearly by the thiolacetic acid method. In the lung, the thiolacetic acid method revealed high ChE activity in bronchial epithelium and alveolar wall, but the thiocholine method showed it in smooth muscle of bronchial wall in the form of network. The lung was one of the organs in which there are distinct differences between the localizations of the activity shown by these two methods. In the liver we did not found any differences between them. In the eccrine gland, the activity was revealed in the body of the gland and in the nerve fibers around it by the thiocholine method (Fig.1), but the former acti- vity was lost after pretreating with DFP. By the thiolacetic acid method the activity was found only in the body of the gland and was not demonstrated in the nerve fibers (Fig.2). In the end-plates of skeletal muscles, the thiolacetic acid method was very useful for the demonstration of subneural apparatus (SNA). The ChE activity of muscle end-plates was so high that the diffusion artifact was often found by thiocholine method, wheareas by the thiolacetic acid method there was hardly seen diffusion artifact. The thiocholine method could demonstrate the activity on the wall of the arteries of some organs, which was hardly demonstrated by the the thiolacetic acid method. From these observations, it was considered that the major part of the activity demonstrated by the thiolacetic acid method was due to pseudo ChE or non specific esterase. However the distribution pattern of AChE in the sites where its activity was very high such as end-plates of muscle, was accurately demonstrated by this method without any diffusion artifacts ; because it is probably due to low sensitivity for AChE of this method. 2) The distribution of ChE activity in the end-plates of muscles : The distri- bution pattern of ChE in the end-plates or size and form of SNA varied according to species. It appeared in diffuse network in the fish (Fig.3), in linear form 211 arranged in pallisade like form in the frog.(Fig.4), in fine arborization form in the bird (Fig.5) and ring shaped form in the mouse and rat (Fig.6). In the rabbit. dog and man (Fig.7) it was resembled to those of the mouse and rat, but it was more simple form. In the crayfish, the activity was not found in muscles. In the same species there were some differences in the distribution pattern among the muscles. In the human extraocular muscles there were large type of SNA which appeared in fine complicated arborization form (Fig.8), and small type which was densely distributed and occasionally found multiply on one muscle fiber. The specific distribution of ChE in the ocular muscles is considered to corres- pond with fine and rapid movement of eye. 3) The ChE activity in the arteries within the skeletal muscle : Although the ChE activity of the walls of the arteries within the skeletal muscle was not found in mammals, it was demonstrated in the bird (Fig.5). This suggests that in the bird there is an innervation of cholinergic vasodilators to the intra- muscular arteries. 4) The distribution of ChE activity in muscle spindle: The distribution pattern of ChE in muscle spindle also varied not only according to species but also according to kinds of muscles in same species (Fig.9,10). In the intrafusal muscle fiber, there were varing size and form in the distribution of the activity, but in general it was roughly classified into two types, one of which was as large as SNA of extrafusal muscle fiber and the other was very small and multiply distributed. The latter is probably considered as 7-end-plate. 5) The distribution of ChE activity at the myo-tendinous junction: The activity was found at the myo-tendinous junction in the frog, bird, and mammal in the fish, it was found in the myoseptum. 6) Biopsied or autopsied specimens of ill muscles: In some cases of neuromu- scular disorders such as senile muscular atrophy, periodic paralysis of extremi- ties, amyotrophic lateral sclerosis (ALS) and progressive muscular atrophy (DMP), the histochemical investigation of ChE on the biopsied or autopsied muscles was performed. In the atrophied muscle, the structure of SNA was considerably small in size, wheareas in ALS and DMP, marked deformations such as segmentation, hyperarborization or linearization besides simple shrinkage were found (Fig.11,12). In periodic paralysis of extremities, there were no significaht changes in SNA before and after provocation with glucose. Discussion For histochemical demonstration of ChE, the thiocholine method is the most sensitive and reliable one by which true- and pseudo-ChE are selectively demo- nstrated. But the procedure of this method is fairly complicated and the reagent using as substrate is considerably expensive. Further, at the site with high ChE activity such as endplates diffusion artifacts are often seen by this method, wheareas by the thiolacetic acid method, there was seen very little diffusion artifact; moreover the reagents are not so expensive. 212 Fig.1 Fig.2 Fig.3 Fig.4 Fig.5 Fig.6 From the comparative studies of the distribution pattern of ChE activity in muscles of several species, it was found that there were marked differences among distibution patterns of the activity according to species and kinds of muscles. In the smooth muscle fibers which show fine network distribution of the cholinergic nerve fibers with the high ChE activity, and, in eth skeletal muscle fibers of the fish which show diffuse network distribution of the activity, there are seen some similarities between them. In mammals the activity is concentrated in ring shaped end-plates. In the esophagus of mammals the activity is found in the nerve fibers going to end-plates14,15), wheareas it is not found in the skeletal muscle except in some baby animals. From this distribution pattern of the activity, it seems likely that the esophagus is located in the intermediate position between the smooth muscle and the skeletal muscle 213 Fig.7 Fig.8 Fig.9 Fig.10 Fig.11 Fig.12 as to the innervation and, further, there is seen an evolutional development of SNA from smooth musscle to mammalian skeletal muscle which seems to be the most differentiated type. Summary and Conclusion By the thiolacetic acid method as compared with thiocholine method, it was difficult to demonstrate the ChE activity of cholinergic nerve fibers, but at least as far as muscle end-plate was concerned that method was very suitable for demonstrating accurate localization of the activity. The distribution pattern of ChE in muscle end-plates varied according to species and kinds of muscles. The similar result was obtained about muscle spindle. The investigation of biopsied or autopsied muscles from several neuromuscular disorders, especially 214 in ALS and DMP, demonstrated marked deformations of SNA with high ChE activity. From these results the problems as to the innervation to skeletal muscles were discussed. We would like to express our thanks to prof. S. Okinaka and assist. prof. M. Yoshikawa for their constant interest and guidance in this investigation.