Exp. Path., Bd. 10, S. 72-82 (1975) Department of Immunopathology and Histochemistry (Head: Dozent Dr. sc. med. G. GEILER) Institute of Pathology (Director: Prof. Dr. sc. med. G. HOLLE) of the Karl-Marx-University Leipzig; Department of Rheumatology (Chief Physician: Dozent Dr. sc. med. W. KEITEL), Vogelsang Hospital Gommern and Medical CHnic (Director: Prof. Dr. sc. med. J. RECHENBERGER), Medical Academy, Magdeburg
Comparative morphological studies on adjuvant arthritis induced in the extremities of rats, mice, and golden hamsters by intracutaneous injection of Freund's complete adjuvant By G. GEILER, W. REITEL and A. FRANKE With 5 figures (Received May 4, 1974) Key-words: adjuvant arthritis; test model; Freund's adjuvant; joints; synovial membrane; rat; mouse; golden hamster
Summary Forty rats, 60 mice, and 60 golden hamsters were administered complete Freund's adjuvant by intracutaneous injections in an attempt to produce the clinical picture of arthritis. 90 % of the rats, 25 % of the mice, and 40 % of the golden hamsters developed swollen extremities similar to those observed in rats affected by adjuvant arthritis. 12 animals of each of the 3 species included in this investigation, which showed macroscopically visible swellings of their extremities, were used for comparative histological studies in order to find out whether the swellings of the extremities of mice and golden hamsters are real inflammations of joints and identical with rat adjuvant arthritis. The animals were killed, at 4-day intervals, between the 8th and the 44th days from administration of the adjuvant. Whereas rats showed the full clinical picture of adjuvant arthritis, mice and hamsters exhibited only inflammatory pedal edema without florid arthritis. The involvement of the joints was confined to a minor focal reaction of the synovial membranes of small individual joints. The results obtained support the view of rat adjuvant arthritis being a species-specific reaction rather than corresponding to a species-independent, universal principle of reaction. Adjuvant arthritis (AA) induced in rats by intracutaneous injections of Freund's complete adjuvant is an immunologically induced inflammation where the immunological processes are determined by reactions associated with immunity imparted through cells. AA had heretofore been induced in rats only. Attempts by PEARSON and WOOD (1959) as weIl as by LACAPERE (1964) to produce AA in dogs, rabbits, guinea pigs, and mice proved unsuccessful. Only areport by REITEL et al. (1971) shows that it is possible to induce AA even in mice and hamsters, the average frequency reported for both mice and hamsters being of the order of 28 percent. However, AA induced in mice and hamsters shows a milder and biphasic course of disease as weIl as less severe articular symptoms and preferential involveme nt of the fore-paws as compared to AA induced in rats. Comparative morphological studies have been made in an attempt to find out whether this form of "arthritis", which was clinically diagnosed from swollen extremities, corresponds to real arthritis and may be considered to be identical with AA induced in rats. The object of these studies has been to find an answer to the question as to whether AA in rats is a species-specific reaction or a species-independent and general form of reaction.
72
Q:i
-J
+ + +
(+ )
++ ++ ++
days after injection of the adjuvant. Gradiation of results: (( +» very slight
+
+
+
+
(+ ) +
distind
++
++
(+ ) + ++
+++ +++ +++ +
+
+++ +++ +++ +
+ +++
strong
+
++ ++ ( +) + +++ ++ + +++ +++ +++ ( +)
(+ ) ++ (+ ) + +++
R,
28 T
++ +
R6
24 T
++ ++
++ ++ (+) (+ ) ++
R5
20 T
+++
+
+ ++
++
++ +++
+++ +++ +++ (+ )
+ + (+ ) + +++
++ +
R9
36 T
very strong
+++ +++ +++ (+ )
+ ++ (+) + +++
++ +
Rs
32 T
-t-
+ +
++ + ++ ( +)
+ + + + +
++ +
~o
40 T
+
-1-+ +++
+ + (+)
(+ )
(+ ) + +
(+ ) ++ ++ ++ ++
(+ )
+ (+ )
R12
44 T
(+ )
++ +
Rn
40 T
The gradiations of results given in the table represent average values of the results obtained for two or more extremities. The positions without entry represent negative findings.
T slight
+ + + +
+ ++ + + + + + + (+ )
++ ++ + (+ ) ++ ++
Large joints Small joints Synovialitis Edema Fibrin Polymorph. leucocytes Lymphocytes Lymphatic nodules Plasmacytes Proliferation of histiocytes Hyperplasia of synoviocytes Giant cells Destructive pannus Destruction of cartilage Destruction of bone Periostitis Myositis Dermatitis Scars of synovial membrane Desmal ankylosis Osteal ankylosis Osteal dis organisation Scars of muscles S cars of skin
(+ ) ++ ++ + ++ ++ +
R4
+ ++ ++ ++ ++ ++ + (+) + (+ )
Ra 16 T
R2 16 T
8T
12 T
R1
rats
Table 1. Comparison of the histological findings in the extremities of rats, mice and golden hamsters by intracutaneous injection of Freund's complete adjuvant
""'"
-J
Large joints Small joints Synovialitis Edema Fibrin Polymorph. leucoeytes Lymopheytes Lymphatic nodules Plasmaeytes Proliferation of histioeytes Hyperplasia of synovioeytes Giant cells Destruetive pannus Destruetion of cartilage Destruetion of bone Periostitis Myositis Dermatitis Sears of synovial membrane Desmal ankylosis Osteal ankylosis Osteal disorganisation Sears of muscles Sears of skin
Table 1, continuation
8T
MI
mice
(+ )
(+)
(+)
« +))
++
( +) (+)
( +)
(+)
M4
20 T
(+)
(+ )
M3
16 T
« +))
« +))
« +))
M2
12 T
(+ )
« +))
( +)
M5
24 T
(+)
(+)
(+ )
(+ )
28 T
M6
(+ )
(+ )
(+)
(+ )
28 T
M7
(+ )
32 T
MB 36 T
M9 40 T
M10
« +))
« +))
« +))
44 T
Mn
« +))
« +))
« +))
44 T
M12
~
Large joints Small joints Synovialitis Edema Fibrin Polymorph. leucocytes Lymphocytes Lymphatic nodules Plasmacytes Proliferation of histiocytes Hyperplasia of synoviocytes Giant cells Destructive pannus Destruction of cartilage Destruction of bone Periostitis Myositis Dermatitis Scars of synovial membrane Desmal ankylosis Osteal ankylosis Osteal dis organisation Scars of muscles Scars of skin
hamsters HI Hz 8T 12 T
(( +))
(+ )
(( +))
Ha 12 T
+ +
(+ )
( +)
(+)
(+ )
(+)
(( +))
( +)
H6 24 T
(+)
( +)
(( +))
(( +))
(+)
Hs 24 T
(( +))
H( 20 T
( +)
( +)
H7 28 T
( +)
( +)
( +)
( +)
Hs 32 T H IO 36 T
(( +))
(( +))
((+)) ((+))
(+ )
((+)) ((+))
((+)) ((+))
H9 32 T
(( +))
(( +))
Hn 40 T
((+))
H I2 44 T
Materials and methods 60 female mice (Agnes-Blum strain; average weight at the commencement of the test: 25 g; age: 6 weeks), 60 fern ale golden hamsters (l\fesocricetus auratus; average weight at the start of testing: 40 g; age: 6 weeks), and 40 female tras (Wistar strain; average weight at the beginning of the test: 110 g; age: 6 weeks) were given 0.1 ml of Freund's complete adjuvant intracutaneously, the inj ection being made into the skin of the base of the tail. 25 % of the mice, 40 % of the hamsters, and 90 % of the rats developed swollen extremities similar to those observed on rats with AA. 12 animals of each of the species included in this program were selected for histological studies. Animals from all of the 3 groups were killed, at 4-day intervals, between the 8th and 44th days from injection of the adjuvant. At least 2 extremities were examined in each of the cases. Without exception, those extremities which on microscopic examina ti on showed the largest swellings were selected. The extremities fixed in neutral formalin were decalcified with the use of Chelaplex, totally embedded in paraffin, sectioned, and stained using hematoxylin-eosin, van Gieson's stain, and PAS. In order to obtain comparable results, all of the cases examined in this study were evaluated by a standard scheme which is shown in table 1.
Results The histological resuIts are summarized in table 1. The gradations of resuIts given in the table represent average values of the resuIts obtained for 2 or more extremities. In rats, pathological conditions were not observed before the 8th day after injection of the adjuvant. However, on the 12th day after injection it is possible to observe a florid form of arthritis which is almost exclusively confined to the small pedal joints, the large joints being almost always free from inflammations. Initially, the disease picture is that of an acute serofibrinous and lympholeucocytic inflammation of the synovial membrane, which is accompanied by an inflammatory exudation into the articular lumen (fig. 1). The joint capsules, periosteum, periarticular tissue, musculature, and skin were regularly found to be involved in the process of inflammation and constitute the correlate of swollen extremities which at this time are characterized by severe inflammation. Between the 16th and 20th days after injection the inflammation of the synovial membrane resuIts in the development oi granulation tissue, the climax of which is lying between the 20th and 36th postinjection days. This destructive pannus consists of proliferating histiocytes and fibroblasts, contains a very large nu mb er of iymphocytes, usually has polymorphonuclear leucocytes, few plasma cells, whereas lymphatic nodules are found in rare cases only. The pannus fiIls the articular space, breaks through and undermines the arthrodial cartilage, and destroys large parts of both the cortical and spongy substances in the epimetaphysial region (fig. 2). It is possible to observe reactive bone formation accompanied by osteal disorganization. This may still be observed at the end of the test, i. e. after 44 days from the administration of the adjuvant (fig. 3 b). Already after the 28th day cicatrizations of the synovial membrane and pannus tissue can be observed, which lead to ankylosis and destruction of the articular bones (fig. 3a). The mice and hamsters, which usually showed macroscopically visible swollen extremities between the 16th and 32nd day after injection of the adjuvant, exhibited only very discrete changes in their respective joints. Real arthritis characterized by florid synovitis accompanied by exudation into the articular lumen, could not be observed during this experimental program. Only slight and circumscribed reactions were observed on small joints of mice after the 12th and of hamsters after the 16th day from injection of the adjuvant. Only single joints were found to be affected, and showed a slight proliferation of synovial intimal cells and of some histiocytes of the synovial adventitia. Lymphocytes are very rarely found (figs. 4 and 5). Edema, fibrin, polymorphonuclear leucocytes, and plasmocytes are not observed at all. As a pathological and anatomical correlate of clinically observed pedal sweIIings it was possible to note spongy pedal edema of the cutis and sub cutis with varying contents of lymphocytes and individual leucocytes. Pedal edema was more frequently observed in hamsters than in mice, and in some cases even the musculature was found to be slightly affected by the inflammatory edema.
76
Fig. 1. Rat R2 , after 12 days from injection of the adjuvant. a) Severe, acute, fibrinous-purnlent synovitis with exsudation into the articular lu·men. Arthrodial cartilage is intact. X 130. b) Enlarged part of the photo showing the condition of synovitis. X 420.
Discussion Adjuvant arthritis induced in rats represents the model of an easily and well reproducible, immunologically induced form of arthritis where the immunological processes are determined by reactions associated with immunity imparted through cells. A synthesis of humoral antibodies within the region of the synovial membrane and inflammatorily changed tissue of soft parts of the extremities could not be observed. Experiments made in an attempt to detect, by the use of fluorescein isothiocyanate labeled antirat globulin serum obtained from rabbits, a synthesis of antibodies in the small number of detectable plasma cells of the
77
Fig. 2. a) Rat R ö , after 20 days from injection of the adjuvant. Fully developed destructive pannus. The pannus filIs the articular space, and the articular cartilage of the lower articular bone is largely destroyed and broken through by the pannus. X 130. b) Rat R 7 , after 28 days from injection of the ad juvant. Synovial pannus broken through the cortical substance of the metaphysis and into the medullary space, this being accompanied by rapid decomposition of osseous tissue by osteoclasts. x 210.
periarticular tissue and tissue of the soft parts of the extremities of rats with typical adjuvant arthritis were unsuccessful. 12 animals, which were specifically examined by us for indications of such a synthesis, showed an entirely negative behaviour. A similarity of adjuvant arthritis to the rheumatoid arthritis in man is, therefore, limited inasmuch as immunity imparted through cells as well as humoral antibody production accompanied by the development of complement-fixing immunocomplexes are of great pathogenetic importance
78
Fig. 3. Rat R g , after 36 days from injection of the adjuvant. a) Fibrosing of the pannus which, when the articular cartilage is destroyed, completely fills the joint cavity and which has led to desmal ankylosis. X 130. b) Reactive new bone formation' and decomposition of bony tissue in the epimetaphysial region. X 210.
to the latter. With active rheumatoid arthritis it is possible to regularly detect, in the synovial membrane, a synthesis of IgA, IgG, and IgM globulins, which correlates with the aetivity of rheumatoid synovitis and derives its pathogenetic importance from the development-fixing immunocomplexes, in which antibodies mentioned above are involved (GEITLER 1971, GEILER and STIEHL 1974, literary survey by GEILER 1974). The small number of plasma eeHs in the synovial pannus of AA as weH as in the periarticular tissue of soft parts represents a morphological difference from rheumatoid synovitis. An additional difference lies in the behaviour of the synovial pannus to its surroundings, whieh, as far as the AA of
79
Fig. 4. Mouse Ma, after 16 days from injection of the adjuvant. Localized proliferation of synovial intimal cells. No exsudation into articular lumens. Articular cartilage is intact. X 210.
rats is concerned, shows highly destructive properties that were scarcely observed in case of rheumatoid arthritis. The course of AA is similar to that of arthritis mutilans with grotesque destructions of epimetaphyses especially of the small joints of extremities. The results allow an insight into the formal genesis of inflammation and also suggest that florid synovitis is the central condition of AA. This is particularly shown by the results of radioautographie studies with the use of Ha-thymidine which were made by BURSTEIN and WAKSMAN (1964) and afford a differential picture of the cellular kinetics of synovitis in case of AA. This is also confirmed by the results of studies made by GRYFE et al. (1971), which are concerned with the early phase of AA (5th to 12th days after injection of the adjuvant) and especially with the behaviour of mast cells as inductors of inflammatory reactions. Morphologically, AA has been found to be a form of arthritis of which only limited use may be made in the analysis of the pathogenetic aspects of rheumatoid arthritis. However, as an easily and weIl reproducible model it offers itself particularly to the testing of medicaments. A morphological comparison between the changes in mice and hamsters shows the possibility of inducing AA in these species (REITEL et al. 1971). However, a histological study shows that real arthritis accompanied by florid synovitis cannot be observed in both mice and hamsters during the entire observation period which was in conformity with that used for rats. The changes in the synovial membrane are very slight, circumscribed and confined to small individual joints. The morphological correlate of macroscopically deteetable pedal swellings
80
Fig. 5. Hamster H 4 , after 20 days from injection of the adjuvant. Localized proliferation of synovial intima! cells and individual connective-tissue cells in the subsynovial adventitia. No exudation into the articular lumen. The articular cartilage is intact. X 210.
is an inflammatory pedal edema which is chiefly confined to the corium and subcutis and only occasionally involves the musculature. It is in this connection that the discrete changes of the synovial membrane should be regarded as a secondary reaction only. Articular involvement comparable to AA in rats was not observed in either mice or hamsters. Therefore, the results of our studies support the view of adjuvant arthritis induced in rats being a species-specific reaction rather than corresponding to areaction principle of a speciesindependent universal character. However, examinations of parenchymatous organs showing granulomatous inflammations in rat adjuvant arthritis (adjuvant disease) will have to be made to clear up this question.
Literature BURSTEI N, N. A., and B. H. WAKSMAN, The pathogenesis of adjuvant disease in the rat. I. A bistological study of early lesions in the joints and skin. Yale J. Bio!. Med. 37, 177-194 (1964). -- The pathogenesis of adjuvant disease in the rat. II. A radioautographie study of early lesions with the use of H-thymidine. Yale J. Bio!. Med. 37, 195-203 (1964). GEILER, G., The antibody synthesis of the synovial membrane in rh eumatoid arthritis. Rheumatoid arthritis. Colloquia Geigy. Academic Press, London and New York 1971, 317-323. - Neue Erkenntnisse der Histopathologie und Immunhistologie der Rheumatoid-Arthritis. Allergie und Immunologie 1974, in press. 6 Exp. Path. Bd. 10, H. 1/2
81
-
and P. STIEHL, Zur Bedeutung der Begriffe Basisaktivität und aktuelle Aktivität in der morphologischen Beurteilung der Synovialmembran bei Rheumatoid-Arthritis. Z. Rheumaforsch. 33, 73-86 (1974). GRYFE, A., P. M. SANDERS and D. L. CrARDNER, The mast cell in early rat adjuvant arthritis. Ann. rheum. dis. 30, 24-30 (1971). KEITEL, E., W. RUDOLPH, G. KRÖNING, B. KRETSCHMAR and S. JAMBOR, UntersUl'hungen zur Adjuvansarthritis der Ratte. I. Dosierung, Zusammensetzung und Applikationsformen des Adjuvans. Z. Rheumaforsch. 28, 212-218 (1969). KEITEL, W., A. A. LOPATJONOK, R. PAMBOR, K. WILLMANN, Untersuchungen zur Adjuvansarthritis der Ratte. 11. Klinik, Serologie, Histologie. Z. Rheumaforsch. 28, 351-360 (1969). - R. WILLE, A. FRANKE and J. ZIEGLER, Adjuvant arthritis in mice and hamsters. Acta Rheum. Scand. 17, 31-34 (1971). LACAPERE, J., Notes sur la polyarthrite chronique rheumatoide experimentale du rat. Press. Med. 72, 1549-1554 (1964). PEARSON, C. M., and F. D. WOOD Studies of polyarthritis and other lesions indured in rats by injection of mycobacterial adjuvant I. Arthr. Rheum. 2 1 440-452 (1959). Authors addresses: Dozent Dr. sc. med. G. GEILER, Pathologisches Institut der Karl-Marx- Universität, 701 Leipzig, Liebigstraße 26, G.D.R., Dozent Dr. sc. med. W. KEITEL, Fachkrankenhaus Vogelsang, Rheumatologische Abteilung, 3301 Gommern, G.D.R., Dr. A. FRANKE, Medizinische Klinik der Medizinischen Akademie, 301 Magdeburg Leipziger Straße 44, G.D.R.
82
Comparative morphological studies on adjuvant arthritis induced in the extremities of rats, mice, and golden hamsters by intracutaneous injection of Freund's complete adjuvant By G. GEILER, W. REITEL and A. FRANKE With 5 figures (Received May 4, 1974) Key-words: adjuvant arthritis; test model; Freund's adjuvant; joints; synovial membrane; rat; mouse; golden hamster
Summary Forty rats, 60 mice, and 60 golden hamsters were administered complete Freund's adjuvant by intracutaneous injections in an attempt to produce the clinical picture of arthritis. 90 % of the rats, 25 % of the mice, and 40 % of the golden hamsters developed swollen extremities similar to those observed in rats affected by adjuvant arthritis. 12 animals of each of the 3 species included in this investigation, which showed macroscopically visible swellings of their extremities, were used for comparative histological studies in order to find out whether the swellings of the extremities of mice and golden hamsters are real inflammations of joints and identical with rat adjuvant arthritis. The animals were killed, at 4-day intervals, between the 8th and the 44th days from administration of the adjuvant. Whereas rats showed the full clinical picture of adjuvant arthritis, mice and hamsters exhibited only inflammatory pedal edema without florid arthritis. The involvement of the joints was confined to a minor focal reaction of the synovial membranes of small individual joints. The results obtained support the view of rat adjuvant arthritis being a species-specific reaction rather than corresponding to a species-independent, universal principle of reaction. Adjuvant arthritis (AA) induced in rats by intracutaneous injections of Freund's complete adjuvant is an immunologically induced inflammation where the immunological processes are determined by reactions associated with immunity imparted through cells. AA had heretofore been induced in rats only. Attempts by PEARSON and WOOD (1959) as weIl as by LACAPERE (1964) to produce AA in dogs, rabbits, guinea pigs, and mice proved unsuccessful. Only areport by REITEL et al. (1971) shows that it is possible to induce AA even in mice and hamsters, the average frequency reported for both mice and hamsters being of the order of 28 percent. However, AA induced in mice and hamsters shows a milder and biphasic course of disease as weIl as less severe articular symptoms and preferential involveme nt of the fore-paws as compared to AA induced in rats. Comparative morphological studies have been made in an attempt to find out whether this form of "arthritis", which was clinically diagnosed from swollen extremities, corresponds to real arthritis and may be considered to be identical with AA induced in rats. The object of these studies has been to find an answer to the question as to whether AA in rats is a species-specific reaction or a species-independent and general form of reaction.
72
Q:i
-J
+ + +
(+ )
++ ++ ++
days after injection of the adjuvant. Gradiation of results: (( +» very slight
+
+
+
+
(+ ) +
distind
++
++
(+ ) + ++
+++ +++ +++ +
+
+++ +++ +++ +
+ +++
strong
+
++ ++ ( +) + +++ ++ + +++ +++ +++ ( +)
(+ ) ++ (+ ) + +++
R,
28 T
++ +
R6
24 T
++ ++
++ ++ (+) (+ ) ++
R5
20 T
+++
+
+ ++
++
++ +++
+++ +++ +++ (+ )
+ + (+ ) + +++
++ +
R9
36 T
very strong
+++ +++ +++ (+ )
+ ++ (+) + +++
++ +
Rs
32 T
-t-
+ +
++ + ++ ( +)
+ + + + +
++ +
~o
40 T
+
-1-+ +++
+ + (+)
(+ )
(+ ) + +
(+ ) ++ ++ ++ ++
(+ )
+ (+ )
R12
44 T
(+ )
++ +
Rn
40 T
The gradiations of results given in the table represent average values of the results obtained for two or more extremities. The positions without entry represent negative findings.
T slight
+ + + +
+ ++ + + + + + + (+ )
++ ++ + (+ ) ++ ++
Large joints Small joints Synovialitis Edema Fibrin Polymorph. leucocytes Lymphocytes Lymphatic nodules Plasmacytes Proliferation of histiocytes Hyperplasia of synoviocytes Giant cells Destructive pannus Destruction of cartilage Destruction of bone Periostitis Myositis Dermatitis Scars of synovial membrane Desmal ankylosis Osteal ankylosis Osteal dis organisation Scars of muscles S cars of skin
(+ ) ++ ++ + ++ ++ +
R4
+ ++ ++ ++ ++ ++ + (+) + (+ )
Ra 16 T
R2 16 T
8T
12 T
R1
rats
Table 1. Comparison of the histological findings in the extremities of rats, mice and golden hamsters by intracutaneous injection of Freund's complete adjuvant
""'"
-J
Large joints Small joints Synovialitis Edema Fibrin Polymorph. leucoeytes Lymopheytes Lymphatic nodules Plasmaeytes Proliferation of histioeytes Hyperplasia of synovioeytes Giant cells Destruetive pannus Destruetion of cartilage Destruetion of bone Periostitis Myositis Dermatitis Sears of synovial membrane Desmal ankylosis Osteal ankylosis Osteal disorganisation Sears of muscles Sears of skin
Table 1, continuation
8T
MI
mice
(+ )
(+)
(+)
« +))
++
( +) (+)
( +)
(+)
M4
20 T
(+)
(+ )
M3
16 T
« +))
« +))
« +))
M2
12 T
(+ )
« +))
( +)
M5
24 T
(+)
(+)
(+ )
(+ )
28 T
M6
(+ )
(+ )
(+)
(+ )
28 T
M7
(+ )
32 T
MB 36 T
M9 40 T
M10
« +))
« +))
« +))
44 T
Mn
« +))
« +))
« +))
44 T
M12
~
Large joints Small joints Synovialitis Edema Fibrin Polymorph. leucocytes Lymphocytes Lymphatic nodules Plasmacytes Proliferation of histiocytes Hyperplasia of synoviocytes Giant cells Destructive pannus Destruction of cartilage Destruction of bone Periostitis Myositis Dermatitis Scars of synovial membrane Desmal ankylosis Osteal ankylosis Osteal dis organisation Scars of muscles Scars of skin
hamsters HI Hz 8T 12 T
(( +))
(+ )
(( +))
Ha 12 T
+ +
(+ )
( +)
(+)
(+ )
(+)
(( +))
( +)
H6 24 T
(+)
( +)
(( +))
(( +))
(+)
Hs 24 T
(( +))
H( 20 T
( +)
( +)
H7 28 T
( +)
( +)
( +)
( +)
Hs 32 T H IO 36 T
(( +))
(( +))
((+)) ((+))
(+ )
((+)) ((+))
((+)) ((+))
H9 32 T
(( +))
(( +))
Hn 40 T
((+))
H I2 44 T
Materials and methods 60 female mice (Agnes-Blum strain; average weight at the commencement of the test: 25 g; age: 6 weeks), 60 fern ale golden hamsters (l\fesocricetus auratus; average weight at the start of testing: 40 g; age: 6 weeks), and 40 female tras (Wistar strain; average weight at the beginning of the test: 110 g; age: 6 weeks) were given 0.1 ml of Freund's complete adjuvant intracutaneously, the inj ection being made into the skin of the base of the tail. 25 % of the mice, 40 % of the hamsters, and 90 % of the rats developed swollen extremities similar to those observed on rats with AA. 12 animals of each of the species included in this program were selected for histological studies. Animals from all of the 3 groups were killed, at 4-day intervals, between the 8th and 44th days from injection of the adjuvant. At least 2 extremities were examined in each of the cases. Without exception, those extremities which on microscopic examina ti on showed the largest swellings were selected. The extremities fixed in neutral formalin were decalcified with the use of Chelaplex, totally embedded in paraffin, sectioned, and stained using hematoxylin-eosin, van Gieson's stain, and PAS. In order to obtain comparable results, all of the cases examined in this study were evaluated by a standard scheme which is shown in table 1.
Results The histological resuIts are summarized in table 1. The gradations of resuIts given in the table represent average values of the resuIts obtained for 2 or more extremities. In rats, pathological conditions were not observed before the 8th day after injection of the adjuvant. However, on the 12th day after injection it is possible to observe a florid form of arthritis which is almost exclusively confined to the small pedal joints, the large joints being almost always free from inflammations. Initially, the disease picture is that of an acute serofibrinous and lympholeucocytic inflammation of the synovial membrane, which is accompanied by an inflammatory exudation into the articular lumen (fig. 1). The joint capsules, periosteum, periarticular tissue, musculature, and skin were regularly found to be involved in the process of inflammation and constitute the correlate of swollen extremities which at this time are characterized by severe inflammation. Between the 16th and 20th days after injection the inflammation of the synovial membrane resuIts in the development oi granulation tissue, the climax of which is lying between the 20th and 36th postinjection days. This destructive pannus consists of proliferating histiocytes and fibroblasts, contains a very large nu mb er of iymphocytes, usually has polymorphonuclear leucocytes, few plasma cells, whereas lymphatic nodules are found in rare cases only. The pannus fiIls the articular space, breaks through and undermines the arthrodial cartilage, and destroys large parts of both the cortical and spongy substances in the epimetaphysial region (fig. 2). It is possible to observe reactive bone formation accompanied by osteal disorganization. This may still be observed at the end of the test, i. e. after 44 days from the administration of the adjuvant (fig. 3 b). Already after the 28th day cicatrizations of the synovial membrane and pannus tissue can be observed, which lead to ankylosis and destruction of the articular bones (fig. 3a). The mice and hamsters, which usually showed macroscopically visible swollen extremities between the 16th and 32nd day after injection of the adjuvant, exhibited only very discrete changes in their respective joints. Real arthritis characterized by florid synovitis accompanied by exudation into the articular lumen, could not be observed during this experimental program. Only slight and circumscribed reactions were observed on small joints of mice after the 12th and of hamsters after the 16th day from injection of the adjuvant. Only single joints were found to be affected, and showed a slight proliferation of synovial intimal cells and of some histiocytes of the synovial adventitia. Lymphocytes are very rarely found (figs. 4 and 5). Edema, fibrin, polymorphonuclear leucocytes, and plasmocytes are not observed at all. As a pathological and anatomical correlate of clinically observed pedal sweIIings it was possible to note spongy pedal edema of the cutis and sub cutis with varying contents of lymphocytes and individual leucocytes. Pedal edema was more frequently observed in hamsters than in mice, and in some cases even the musculature was found to be slightly affected by the inflammatory edema.
76
Fig. 1. Rat R2 , after 12 days from injection of the adjuvant. a) Severe, acute, fibrinous-purnlent synovitis with exsudation into the articular lu·men. Arthrodial cartilage is intact. X 130. b) Enlarged part of the photo showing the condition of synovitis. X 420.
Discussion Adjuvant arthritis induced in rats represents the model of an easily and well reproducible, immunologically induced form of arthritis where the immunological processes are determined by reactions associated with immunity imparted through cells. A synthesis of humoral antibodies within the region of the synovial membrane and inflammatorily changed tissue of soft parts of the extremities could not be observed. Experiments made in an attempt to detect, by the use of fluorescein isothiocyanate labeled antirat globulin serum obtained from rabbits, a synthesis of antibodies in the small number of detectable plasma cells of the
77
Fig. 2. a) Rat R ö , after 20 days from injection of the adjuvant. Fully developed destructive pannus. The pannus filIs the articular space, and the articular cartilage of the lower articular bone is largely destroyed and broken through by the pannus. X 130. b) Rat R 7 , after 28 days from injection of the ad juvant. Synovial pannus broken through the cortical substance of the metaphysis and into the medullary space, this being accompanied by rapid decomposition of osseous tissue by osteoclasts. x 210.
periarticular tissue and tissue of the soft parts of the extremities of rats with typical adjuvant arthritis were unsuccessful. 12 animals, which were specifically examined by us for indications of such a synthesis, showed an entirely negative behaviour. A similarity of adjuvant arthritis to the rheumatoid arthritis in man is, therefore, limited inasmuch as immunity imparted through cells as well as humoral antibody production accompanied by the development of complement-fixing immunocomplexes are of great pathogenetic importance
78
Fig. 3. Rat R g , after 36 days from injection of the adjuvant. a) Fibrosing of the pannus which, when the articular cartilage is destroyed, completely fills the joint cavity and which has led to desmal ankylosis. X 130. b) Reactive new bone formation' and decomposition of bony tissue in the epimetaphysial region. X 210.
to the latter. With active rheumatoid arthritis it is possible to regularly detect, in the synovial membrane, a synthesis of IgA, IgG, and IgM globulins, which correlates with the aetivity of rheumatoid synovitis and derives its pathogenetic importance from the development-fixing immunocomplexes, in which antibodies mentioned above are involved (GEITLER 1971, GEILER and STIEHL 1974, literary survey by GEILER 1974). The small number of plasma eeHs in the synovial pannus of AA as weH as in the periarticular tissue of soft parts represents a morphological difference from rheumatoid synovitis. An additional difference lies in the behaviour of the synovial pannus to its surroundings, whieh, as far as the AA of
79
Fig. 4. Mouse Ma, after 16 days from injection of the adjuvant. Localized proliferation of synovial intimal cells. No exsudation into articular lumens. Articular cartilage is intact. X 210.
rats is concerned, shows highly destructive properties that were scarcely observed in case of rheumatoid arthritis. The course of AA is similar to that of arthritis mutilans with grotesque destructions of epimetaphyses especially of the small joints of extremities. The results allow an insight into the formal genesis of inflammation and also suggest that florid synovitis is the central condition of AA. This is particularly shown by the results of radioautographie studies with the use of Ha-thymidine which were made by BURSTEIN and WAKSMAN (1964) and afford a differential picture of the cellular kinetics of synovitis in case of AA. This is also confirmed by the results of studies made by GRYFE et al. (1971), which are concerned with the early phase of AA (5th to 12th days after injection of the adjuvant) and especially with the behaviour of mast cells as inductors of inflammatory reactions. Morphologically, AA has been found to be a form of arthritis of which only limited use may be made in the analysis of the pathogenetic aspects of rheumatoid arthritis. However, as an easily and weIl reproducible model it offers itself particularly to the testing of medicaments. A morphological comparison between the changes in mice and hamsters shows the possibility of inducing AA in these species (REITEL et al. 1971). However, a histological study shows that real arthritis accompanied by florid synovitis cannot be observed in both mice and hamsters during the entire observation period which was in conformity with that used for rats. The changes in the synovial membrane are very slight, circumscribed and confined to small individual joints. The morphological correlate of macroscopically deteetable pedal swellings
80
Fig. 5. Hamster H 4 , after 20 days from injection of the adjuvant. Localized proliferation of synovial intima! cells and individual connective-tissue cells in the subsynovial adventitia. No exudation into the articular lumen. The articular cartilage is intact. X 210.
is an inflammatory pedal edema which is chiefly confined to the corium and subcutis and only occasionally involves the musculature. It is in this connection that the discrete changes of the synovial membrane should be regarded as a secondary reaction only. Articular involvement comparable to AA in rats was not observed in either mice or hamsters. Therefore, the results of our studies support the view of adjuvant arthritis induced in rats being a species-specific reaction rather than corresponding to areaction principle of a speciesindependent universal character. However, examinations of parenchymatous organs showing granulomatous inflammations in rat adjuvant arthritis (adjuvant disease) will have to be made to clear up this question.
Literature BURSTEI N, N. A., and B. H. WAKSMAN, The pathogenesis of adjuvant disease in the rat. I. A bistological study of early lesions in the joints and skin. Yale J. Bio!. Med. 37, 177-194 (1964). -- The pathogenesis of adjuvant disease in the rat. II. A radioautographie study of early lesions with the use of H-thymidine. Yale J. Bio!. Med. 37, 195-203 (1964). GEILER, G., The antibody synthesis of the synovial membrane in rh eumatoid arthritis. Rheumatoid arthritis. Colloquia Geigy. Academic Press, London and New York 1971, 317-323. - Neue Erkenntnisse der Histopathologie und Immunhistologie der Rheumatoid-Arthritis. Allergie und Immunologie 1974, in press. 6 Exp. Path. Bd. 10, H. 1/2
81
-
and P. STIEHL, Zur Bedeutung der Begriffe Basisaktivität und aktuelle Aktivität in der morphologischen Beurteilung der Synovialmembran bei Rheumatoid-Arthritis. Z. Rheumaforsch. 33, 73-86 (1974). GRYFE, A., P. M. SANDERS and D. L. CrARDNER, The mast cell in early rat adjuvant arthritis. Ann. rheum. dis. 30, 24-30 (1971). KEITEL, E., W. RUDOLPH, G. KRÖNING, B. KRETSCHMAR and S. JAMBOR, UntersUl'hungen zur Adjuvansarthritis der Ratte. I. Dosierung, Zusammensetzung und Applikationsformen des Adjuvans. Z. Rheumaforsch. 28, 212-218 (1969). KEITEL, W., A. A. LOPATJONOK, R. PAMBOR, K. WILLMANN, Untersuchungen zur Adjuvansarthritis der Ratte. 11. Klinik, Serologie, Histologie. Z. Rheumaforsch. 28, 351-360 (1969). - R. WILLE, A. FRANKE and J. ZIEGLER, Adjuvant arthritis in mice and hamsters. Acta Rheum. Scand. 17, 31-34 (1971). LACAPERE, J., Notes sur la polyarthrite chronique rheumatoide experimentale du rat. Press. Med. 72, 1549-1554 (1964). PEARSON, C. M., and F. D. WOOD Studies of polyarthritis and other lesions indured in rats by injection of mycobacterial adjuvant I. Arthr. Rheum. 2 1 440-452 (1959). Authors addresses: Dozent Dr. sc. med. G. GEILER, Pathologisches Institut der Karl-Marx- Universität, 701 Leipzig, Liebigstraße 26, G.D.R., Dozent Dr. sc. med. W. KEITEL, Fachkrankenhaus Vogelsang, Rheumatologische Abteilung, 3301 Gommern, G.D.R., Dr. A. FRANKE, Medizinische Klinik der Medizinischen Akademie, 301 Magdeburg Leipziger Straße 44, G.D.R.
82
