PERIOSTEUM IN EXPERIMENTAL LATH YRISM 699

LYNCH, K. M., AND SMITH, W. A. . 1930. J. Amer. Med. Assoc., 95, 659. MCDONALD, S. . . . . . . 1927. Brit. Med. J., 2, 1025. RATH, R. . . . . . . . . 1964. Beitr. Silikose-Forsch., 81, 1. SIMSON, F. W. . . . . . . 1928. Brit. Med. J., 1, 885. SUNDIUS, N., AND BYGDBN, A. . . 1937-38. Arch. Gewerbepath. Gewerbehyg.,

TIMMERMANS, F. D. . . . . . 1931. Zbl. Gewerbehyg., 18, 280. 8, 26.

ENZYME STUDIES OF THE PERIOSTEUM I N EXPERIMENTAL LATHYRISM

VERNON L. YEAGER', MALVA F. JOHNSON, LOWELL A. SETHER, ARLEN R. SEVERSON, RONALD R. HENDRICKSON, ELIZABETH A. FRYATT AND ROBERTA A. HUGHES Department of Anatomy, University of North Dakota, Grand Forks, U.S.A.

PLATE CCXXIV

CONNECTIVE tissue is distributed throughout the body and makes up about 30 per cent. of its bulk. Its importance is emphasised when it reacts secondarily to other diseases, as in fibrosis of the liver or kidney, or is the site of primary disease. Marfan's syndrome, a hereditary disorder of conective tissue, causes skeletal changes, dissecting aneurysms of the aorta, and loosening of the lens. These same symptoms are seen in experimental lathyrism, a disease caused by the administra- tion of beta-aminopropionitrile (BAPN).

Periosteum is one of the connective-tissue sites where lesions occur in animals suffering from lathyrism (Robinson and Bast, 1934). In animals treated with BAPN the periosteum at the insertion of the adductor longus and pectineus muscles responds early and uniformly to produce a rounded swelling in which bone forma- tion begins after 5 days in young animals (Yeager and Hamre, 1957). Periosteal lesions that are similar to those found in lathyrism have been produced by a surgical technique in which the periosteum is partially detached from the femur (Yeager and Taylor, 1965). If these two lesions, one produced through BAPN administration and the other through surgical stimulation, are produced through the same mechan- ism, they should have the same histochemical characteristics. If, however, the lesions result from different mechanisms, histochemical studies may reveal differ- ences between them.

MATERIALS AND METHODS

The 145 animals used for these studies were albino rats of the Holtzman strain. They were separately caged, given Purina Laboratory Chow and water ad libitum, and weighed daily. They ranged in age from 32 to 75 days, but most were 5 or 8 wk old at the termination of the experiments. Fifty-five rats were used as control animals; 74 were made lathyric by the addition of 150 mg. BAPN fumaratet to each 100 mi. of their drinking water: and 16 had the adductor longus-pectineus periosteal insertion partially detached surgically. The rats were killed on the 7th or 8th day after the start of BAPN treatment or surgery. Histochemical tests were done for the following enzymes : lactic dehydrogenase (Hess, Scarpelli and Parse, 1958), succinic dehydrogenase (Nachlas, Tsou et al., 1957), cytochrome oxidase (Burstone, 1959), phosphorylase (Takeuchi and Kuriaki, 1955; Eranko and Palkama, 1961), glucose-6-phosphate dehydrogenase (Balogh, Dudley and Cohen, 1961), and aminopeptidase (Nachlas, Crawford and Seligman, 1957). The animals

* Present address: Department of Anatomy, St Louis University School of Medicine,

t Generously supplied by Abbott Laboratories, North Chicago, Illinois. 1402 South Grand Boulevard. St Louis, Missouri, 63104, U.S.A.

700 VERNON L. YEAGER ET AL..

were killed with ether, and the periosteum at the insertion of the adductor longus and pectineus muscles was immediately removed from the femur, frozen with dry ice and cut at 10 p in a cryostat. Tissues of control and experimental animals were treated identically.

In addition to the above histochemical tests, serial dilutions of 0 . 1 ~ BAPN (not the fumarate) (California Corporation for Biochemical Research, Los Angeles) were used in some of the incubating media to determine whether the lathyrogenic agent could inhibit enzyme activity in vitro. Skeletal muscle was used in the inhibi- tion study for phosphorylase and succinic dehydrogenase, kidney for the lactic dehydrogenase and aminopeptidase studies.

All media were made up immediately before use and used only once. Some sections were counterstained with alizarin red S for calcium or with nuclear fast red to aid in the identification of cells. All tissues were mounted in glycerine jelly.

RESULTS All of the animals used in these experiments appeared outwardly healthy for the

duration of tile experimental period, although those that received BAPN did not gain weight at the same rate as the control animals. For all animals killed after 7 days of BAPN or surgical treatment, the periosteum at the insertion of the adductor longus and pectineus muscles was thickened. No sex differences were noted in any of the studies.

For the sake of terminology, a brief description of the periosteum at the adductor longus-pectineus insertion will be given. Detailed histological descriptions can be found elsewhere (Yeager and Hamre; Yeager and Gubler, 1963). Bone forma- tion occurs slowly at this site in young normal animals. Since the periosteum consists of two layers, fibrous and cellular, and since bone formation occurs only in the deepest areas, not all cells present are in the same physiological state nor do they necessarily have the same function. Cells of the fibrous periosteum for all groups were designated fibrous periosteal cells. These appeared to be typical fibroblasts and were probably more closely related to tendon cells than to osteo- blasts. Cells of the outer portion of the cellular layer of periosteum were designated resting periosteal cells, those adjacent to bone spicules on the femur were called osteoblasts and those encased within the bone, osteocytes. The periosteum at this site was stimulated in the two experimental groups, and intramembranous bone formation occurred in the BAPN-treated animals, and both intramembranous and enchondral bone formation were present in the surgically treated animals. No cells were designated resting periosteal cells for these groups, but the terms prolifera- ting periosteal cells and pre-osteoblasts were used for the cells of the outer and intermediate areas of the cellular periosteum. Chondroblasts, chondrocytes and osteocytes were seen only in the surgically stimulated periosteum. Blood vessels and perivascular cells were also present, but were not studied.

The results of the histochemical tests are summarised in the table. The general pattern of enzyme activity was the same for lactic dehydrogenase, cytochrome oxidase, glucose-&phosphate dehydrogenase and, in 4-5-wk-old rats, succinic dehydrogenase. Osteoblasts, chondroblasts and chondrocytes showed the greatest amount of reaction product, with the precursor cells having less activity (figs. 1-3). For these enzymes the same general pattern of reaction occurred whether from BAPN or surgically stimulated periosteum. For 8-wk-old rats only traces of reaction product were seen in cells tested for succinic dehydrogenase.

The only periosteal cells giving a positive reaction with the phosphorylase technique were chondroblasts and chondrocytes (fig. 4). Muscle fibres included with periosteal sections were intensely positive for phosphorylase activity, indicating that the technique was working properly.

The pattern of aminopeptidase activity was the same as that for the dehydrogen- ase enzymes in the normal and surgically stimulated cells, but only a slight reaction was seen in cells from BAPN-stimulated periosteum.

YEAGER ET A L . PLATE CCXXIV

PERIOSTEUM IN EXPERIMENTAL LATHYRISM

FIG. I.-Periosteum and muscle from a FIG. 2.-Periosteum from a rat treated with 6-wk control rat showing succinic dehy- BAPN for 8 days, showing cytochrome drogenase activity. No counterstain. oxidase activity. Counterstained with x 32. alizarin red S for calcium to show bone

spicules (B). x 32.

FIG. 3.-Periosteum from rat 8 days after FIG. 4.-Periosteurn from rat 8 days after surgery, showing lactic dehydrogenase surgery. Only muscle and cartilage cells activity. Note cartilage (C) and bone (C) show enzyme reaction for phos- (B). No counterstain. X 32. phorylase. x 50.

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702 VERNON L. YEACER ET AL.

The control sections for enzyme reactions were negative in all cases except in the lactic dehydrogenase study, where a minimal reaction was seen in some sections. Except for intensity, the colour and distribution were the same as for slides incubated in the presence of lactic acid. The results shown in the table take into account the reaction of the control sections.

There was no inhibition of activity for phosphorylase, succinic dehydrogenase, or lactic dehydrogenase when concentrations of BAPN up to 0 - 1 ~ were included in the incubation media. Aminopeptidase activity was inhibited to a considerable extent when the incubation media contained 0.1 M-BAPN. Slight inhibition also occurred in sections incubated in O.OIM-BAPN media. No visible inhibition of activity occurred in tissues incubated in lower concentrations of BAPN.

DISCUSSION The purpose of these studies was to find whether there were differences in

enzyme activity in the two experimental groups. When similar cell types were com- pared no differences were seen between groups for any of the enzymes concerned with glucose metabolism. Karnovsky (1960) found decreased phosphorylase activity in epiphyseal plate cartilage cells of lathyric animals, but since all periosteal cells except chondroblasts and chondrocytes that were found only in surgically stimulated periosteum were negative for this enzyme, it would appear that phos- phorylase activity does not play a role in the periosteal lesions in lathyrism. Wynn and Ball (1 961) studied the succinic dehydrogenase activity in costochondral junction and liver in normal and lathyric rats and found no differences. Tonna (1958) has shown that the periosteum from 8-wk-old rats has much less succinic dehydrogenase activity than that from 5-wk-old rats. The present work supports this and further shows that stimulation by BAPN does not increase the succinic dehydrogenase activity of periosteum from 8-wk-old rats. Clemmons and Jackson (1962) and Clemmons (1964) found that both aminoacetonitrile (AAN) and BAPN inhibited cytochrome oxidase, but Smith and Orbison (1964) found that only the deterioration product of AAN had an inhibitory action on this enzyme. The results of this study tend to support the latter work since similar cells showed the same activity regardless of the group.

All cells from lathyric animals appeared to have less aminopeptidase activity than comparable cells from normal or surgically treated animals. Although amino- peptidase was inhibited by BAPN in vitro, it is difficult to believe that BAPN could have been concentrated in high enough amounts in vivo to cause this inhibition. Even if this were the case, it would not explain why periosteal hypertrophy results, since aminopeptidase is usually believed to be active in the breakdown of protein chains. BAPN causes increased solubility of collagen, presumably by breaking intra- and inter-molecular bonds, but aminopeptidase is not known to have this function. Further studies of the role of aminopeptidase in lathyrism are in progress.

SUMMARY Lactic dehydrogenase, succinic dehydrogenase, cytochrome oxidase, phos-

phorylase, glucose-6-phosphate dehydrogenase and aminopeptidase were studied histochemically in the periosteum of normal, BAPN- and surgically-treated rats. Osteoblasts and chondroblasts showed most enzyme activity, with precursor cells having less. All periosteal cells, except cartilage cells, were negative for phosphory- lase. Eight-week-old rats had only traces of succinic dehydrogenase activity, even when stimulated by BAPN. Lathyric periosteal cells appear to have less amino- peptidase activity than similar cells from either normal or surgically stimulated periosteum. BAPN did not inhibit phosphorylase, succinic dehydrogenase or lactic dehydrogenase activity in vitro, but did inhibit aminopeptidase in concentra- tions of 0 . 1 ~ and 0 0 1 ~ .

SERUM AND PERIOSTEAL AMINOPEPTIDASE 703

This investigation was supported in part by Public Health Service Research Grant AM-04748 from the National Institute of Arthritis and Metabolic Diseases, and Grant 4C-7113 from the National Cancer Institute.

REFERENCES BALOGH, K., JR, DUDLEY, H. R., AND 1961. Lab. Invest., 10, 839.

BURSTONE, M. S. . . . . . . 1959. J. Histochem. Cytochem., 7, 112. CLEMMONS, J. J. , . . . . . 1964. Fed. Proc., 23, 390. CLEMMONS, J. J., AND JACKSON, E. B., 1962. J. Exp. Med., 115, 555.

ERjimb, O., AND PALKAMA, A. . . 1961. J. Histochem. Cytochem., 9, 585. HW, R., SCARPELLI, D. G., AND 1958. J. Biophys. Biochem. Cytol., 4, 753.

KARNOVSKY, M. J. . . . . . 1960. Lab. Invest., 9, 639. NACHLAS, M. M., CRAWFORD, D. T.,

NACHLAS, M. M., TSOU, K. C., 1957. Ibid., 5,420.

&HEN, R. B.

JR

PEARSE, A. G. E.

1957. J. Histochem. Cytochem., 5, 264. AND SELIGMAN, A. M.

DESOUZA, E., CHENG, C. S., AND SELIGMAN, A. M.

ROBINSON, J., AND BAST, T. H. . . 1934. Anat. Rec., 59,283. SMITH, D. W., AND ORBISON, J. L. . 1964. Proc. SOC. Exp. Biol. Med., 117,822. TAKEUCHI, T., AND KURIAKI, H. . . 1955. J. Histochem. Cytochem., 3, 153. TONNA, E. A. . . . . . . . 1958. J. Gerontol., 13, 14. WYNN, C. H., AND BALL, J. . . 1961. Brit. J. Exp. Path., 42, 114. YEAGER, V. L., AND GUBLER, D. . . 1963. Archs Path., 76, 158. YEAGER, V. L., AND HAMRE, C. J. . 1957. Ibid., 64, 171. YEAGER, V. L., AND TAYLOR, J. J. . 1965. Ibid., 80, 647.

SERUM AND PERIOSTEAL AMINOPEPTIDASE ACTIVITY IN NORMAL, SURGICALLY TREATED AND LATHYRIC RATS

MALVA F. JOHNSON AND VERNON L. YEAGER* Department of Anatomy, University of North Dakota, Grand Forks, U.S.A.

PLATE CCXXV

HYPERTROPHY of the periosteum at the adductor longus-pectineus insertion occurs in animals fed with beta-aminopropionitrile (BAPN) or subjected to partial surgical detachment of the insertion (Yeager and Hamre, 1957; Yeager and Taylor, 1965). Histochemical studies revealed that several enzymes had similar activities in the two lesions, but aminopeptidase activity always appeared to be less in the periosteum of rats fed with BAPN than in normal or surgically stimulated periosteum (Yeager et al., 1967). Since quantitation in enzyme histochemistry is difficult, biochemical and electrophoretic studies were made on periosteum from normal, lathyric and surgically stimulated animals.

MATERIALS AND METHODS

A total of 122 albino rats of the Holtzman strain ranging in weight from 6 to 15 wk were separately caged and given Purina Laboratory Chow. Fourteen rats

* Present address: Department of Anatomy, St Louis University School of Medicine, 1402 South Grand Boulevard, St Louis, Missouri, 63104, U.S.A.