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Labeling index (tritiated thymidine) of the synovium of mice at different ages
Exp. Geront. Vol. I I . pp. 205-208. Pergamon Pre~ 1976. Printed in Great Britain.
LABELING INDEX (TRITIATED THYMIDINE) OF THE SYNOVIUM OF MICE AT DIFFERENT AGES* SAM J. PILIERO and EDGAR A. TONNA New York University Dental Center and Graduate School of Arts and Science, New York, NY 10010, U.S.A. (Received 1 June 1976)
AbstractmThe H3-autoradiographic study of the cellular proliferative activity of the synovial tissue of the knee joints of aging mice reveals a significant decrease in labeling indices between 5 and 26 weeks of age. A low level persisted throughout the life span of the animal. These cellular proliferative changes exhibited by the synovium with increasing age are similar to those reported for other skeletal associated tissues. Labeling appeared to be restricted to the surface cells of the synovial lining. INTRODUCTION DESPITE the plethora o f reports on the effects o f aging on a wide variety o f tissues, only exiguous information is available to data on the effects o f aging on synovial tissue. A d vancing age is a significant factor in degenerative joint disease, although the etiology o f the osteoarthritis remains obscure. The severity o f this disease ranges f r o m simple flaking and fibrillation o f the cartilage (Grade I), to significant deformation o f the joints and m a r k e d loss o f cartilage (Grade IV). F r o m the gerontological point o f view, it c a n n o t yet be determined whether these degenerative changes will eventually occur in all individuals should they live long enough. The present research endeavor is predicated on the necessity o f recognizing the age changes associated with the synovial m e m b r a n e in n o r m a l animals. I n this initial autoradiographic study, tritiated thymidine ( H a - T D R ) was used to ascertain the labeling index changes o f the synovium c o n c o m i t a n t with aging. This information is to serve as baseline data for future cellular kinetic studies and further explorations o f the effects o f aging on synovial tissues. MATERIALS AND METHODS An aging colony of the Brooldaaven National Laboratory (BNL) inbred strain of Swiss albino mouse has been used in this study. The BNL mouse is a short-lived animal whose average life span is less than 1 year. Female mice were divided into 4 age groups, namely: 5, 26, 52 and 78 weeks of age. Each animal received a subcutaneous injection of Ha-TDR (Sp. Act. -----1.9 Ci/mmol) I'0 laCi/gm body weight at time 0. Mice were sacrificed by overetherization at 1 hr post-H3-TDR administration. The knee joints were removed from each animal by sectioning the limbs (bilaterial) at the mid-femoral and mid-tibial levels. Whole joint specimens were fixed in neutral buffer 10y. formalin and subsequently decalcified in ethylenediaminetriacetic acid (EDTA). Sagittal bistological sections were cut at 5 ku-nfrom routinely prepared paraffin blocks of the knee joints. Autoradiographs were prepared using Kodak NTB-3 liquid emulsion, an exposure time of 21 days, and development in Kodak D19. The preparations were finally stained with hematoxylin. The labeling index was determined for the synovial tissues at each age group at 1 hr post-HaTDR administration. Grain counts were made of the synovial tissues found at the anterior and posterior aspects of the saggital sections. The data were statistically evaluated using Student's-t and a confidence level of p < 0-05. *Research was supported by the Histology Department Research Fund, N.I.A. Grant AG 00314 and a National Science Foundation Grant to New York University. 205
206
S A M J . pff:ll~RO A N D E D G A R A . T O N N A
RESULTS An estimation of the magnitude of the labeling index of the population of synoviat cells at various ages is given in Table I and Fig. 1. The synodal membrane of normal B N L mice at five weeks of age revealed a maximum labeling index of 0.044. By 26 weeks of age, the
TABLE I. LABV.LINGINDEX (TRn~AT~ TH~aDn, m) OF THE SYNOVIUM OF MICE AT DIFFERENT AGES* Age (Weeks) Labelingindex 4- S.E. 5 (9)t 0.044 4- 0.015 26
(9)
0-005 4- 0.002
52 (5) 0.007 4- 0.003 78 (12) 0.006 4- 0-002 *Each value consists of an average count of 162 cells/animaL tNumber of animals. .06 -
m
.05 {
u.J . 0 4 u") +l
x I.aJ ,.-.., Z
05-
c.9 Z .J L.d rn
.02-
_.J
.01
-
.5
26 AGE
52
78
(WEEKS)
Fxo. 1. Labeling index :t: S.E. of the knee joint synovium of mice at different ages.
labeled population was seen to decrease significantly to a low labeling index of 0.005. Labeling at 52 and 78 weeks, respectively (0.007 and 0.006) was not significantly different from that observed at the 26 week period. Labeling appeared to be primarily restricted to cellsnear the surface of the synodal membranes regardless of age (Fig. 2). However, labeling was predominantly encountered at the most remote aspects of the synovial lining, e.g. the cephalad portion of the suprapatellar pouch and the caudad portion of the infrapatellar pad. N o significant differences in the population of labeled cells wcrc observed in comparisons of grain counts made from the synovial tissues found at the anterior and posterior aspects of the sagittal sections of the knee joints. Histological observations of the thin synovial lining of the knee joints of mice disclosed the appearance of two distinguishable types of synovial cells forming a compact interlacing layer, one to three cells in depth (Fig. 2). Most of the cells appeared to be macro-
e ~l~o
FIG. 2. The photomicrograph shows Ha-TDR autoradiographic labeling of predominantly surface cells of the knee joint synovium of 5-week-old mice. Most of the cells appear to be macrophagic-like forming an interlacing layer, approxnmately one to three cells in depth. Hematoxylin stained. × 735.
f.p. 206
L A B E L I N G I N D E X OF S Y N O V I U M OF A G I N G MICE
207
phagic-like cells having vacuolated granular cytoplasm in contrast to those cells found in smaller numbers which possessed a homogeneous cytoplasm with a nucleus often found in a polar position. These cells frequently exhibited cytoplasmic processes which were seen to extend along the synovial lining, paralleled with the surface. DISCUSSION It has been reported that the human fetal synovium differs little from that of the adult (Krey et al., 1971). An age correlated increase in the amount of fibrous tissue separating the synovial capillaries from the joint spaces in man has been described (Ruckes and Schuckmann, 1962). This feature was, however, not observed in the histological survey of the present study. Two types of lining cells were distinguished in the synovia of the BNL mouse. These corresponded to the A and B types which have been reported to comprise the synovia of other animals and man alike (Castor, 1960; Barland et al., 1964; Williamson et aL, 1966; Schmid, 1968; Kinsella et aL, 1969; Murane and Feagans, 1970; Cutlip and Cheville, 1973). Three distinct cell types were observed at the electron microscopic level (Barland et al., 1964). The A type cells exhibited the capacity for phagocytosis. The B type cells were adapted for protein synthesis while an intermediate type "C" cell, not recognizable via light microscopy shared the properties of the A and B type cells. The cellular proliferative activity was not high enough to allow us to differentiate the proliferative activity of the various cell types. Histologically, however, the mouse synovial membrane was found to be similar, in several respects, to that described in man (Castor, 1960; Barland et al., 1964; Williamson et al., 1966; Schmid, 1968; Kinsella et al., 1969; Murane and Feagans, 1970; Cutlip and Cheville, 1973.) In the present autoradiographic study, synovial tissue exhibited a low cellular proliferative activity with a peak labeling of ~ 4.4 % at 5 weeks of age. This value is within the range of labeling levels reported in an earlier study of the skeletal tissues of aging BNL mice (Tonna, 1961). Here the labeling was ~ 3 % for the femoral periosteum and ~ 5 % for the cells of the distal epiphyseal plate of the femur at 5 weeks of age. Furthermore, the labeling indices were significantly higher in younger mice and significantly lower by 8 weeks of age. Undoubtedly, synovial tissues fit into the same cell proliferative pattern exhibited by other skeletal associated connective tissue, since at 26 weeks, the labeling of the synovial tissues was a low of 0"5 %, a value similar to that reported for other skeletal tissues (Tonna, 1961). The observed significant reduction in labeling index values between 5 and 26 weeks of age is characteristic of such tissues including the synovium, and appears to represent diminished physiological demands for growth rather than age induced changes. This feature was reported in a study of respiratory enzyme changes in normal and traumatized aging rats (Tonna, 1959). Although joint growth does not take place within the confines of the joint cavity, but at the periphery of the epiphyseal plate (Tonna, 1961) which at no point is in contact with synovial tissue, the lining of the joint capsule is responsive to the overall joint size and indirectly related to joint growth. The present study, which is limited to establishment of the labeling indices of the synovial tissues concomitant with aging, cannot evaluate the effects of aging on the proliferative potential of the synovial membrane. This must be left to a future study in which the tissue is stressed via some standard form of trauma. Although, the labeling indices of the synovial tissues of mice 26--78 weeks of age were not significantly different, the effects of aging on diminishing the cell proliferative activity is anticipated, since trauma studies have clearly
208
SAM $. PII.IERO AND EDGAR A. TONNA
revealed progressively diminishing functional, as well as proliferative capacities for skeletal tissues beyond 26 weeks of age (Tonna, 1959, 1961; Tonna and Cronkite, 1962). This, however, does not imply that the tissues are not capable of responding to trauma adequately, since the cells which comprise skeletal tissues appear to be reversible postmitotics rather than fixed postmitotics (Tonna and Cronkite, 1962; Tonna, 1965). Synovial tissues are also believed to be reversible postmitotics, since the repair of synovial tissues is known to occur following surgery. The progressive effects of aging are seen as quantitative changes with time rather than qualitative alterations of the tissues (Ruckes and Schuckmann, 1962). It is held that in response to prolonged mechanical stress for over a number of years, the dormant growth potential of synovial cells is possibly reactivated with subsequent growth, setting off a chain of events which lead to the complex structural lesion of degenerative joint disease "osteoarthritis" (Sokoloff, 1969). Degenerative joint disease is a non-inflammatory disorder ofmovable joints characterized by deterioration and ablation of articular cartilage, and also by the formation of new bone at such disturbed foci. It is by no means a purely gerontologic problem. Some strains of mice (Silberberg and Silberberg, 1941; Sokoloff, 1956) exhibit a significant incidence of degenerative joint disease, whereas, the BNL Swiss albino mouse used in the present study reveals a low incidence of joint disease. The present results show that the major cell proliferative activity changes occurred early in life in response to altered functional needs and are out of phase with the time of appearance of osteoarthritis. In this regard, subsequent studies are necessary to delineate the behavior of synovial tissues to prolonged stress, and the cellular capacity of the tissue response in progressively older animals. REFERENCES BARI,AND, P., NOVIKOFF,A. B. and I-IAMbS, D. (1964) Am. J. Path. 44, 853. CA.rrOR,C. W. (1960) Arth. Rheum. 3, 140. CUTUP, R. C. and C~EVna.E,N. F. (1973) Am. J. vet. Res. 34, 45. Kn~sv.tJ.~,T. D., BAUM,J. and Zn~, M. (1969) Arth. Rheum. 13, 734. KI~Y, P. R., COHEN,A. S., SMITH,C. B. and FnWt.AND,M. (1971) Arth. Rheum. 14, 319. Mulo,r~, T. W. and Fr.AO~S, W. M. (1970) g. dent. Res. 49, 1068. Rucg.r.S, J. and SCHUCKMANN,F. (1962) Frankf. Z. Path. 72, 243. SCF.M~, F. R. (1968) J. Am. get. Soc. 16, 545. S n . ~ R O , M. and Smam~r..RG,R. (1941) Am. J. Anat. 68, 69. SOKOLOrV,L. (1956) Arch. Path. 62, 118. SOKOt.OI~,L. (1969) The Biology of Degenerative Joint Disease, pp. 24 and 63, Univ. of Chicago Press,
Chicago. TONNA,E. A. (1959) J. Geront. 14, 159. To~,~A,E. A. (1961) J. Biophys. Biochem. Cytol. 9, 813. TONNA,E. A. (1965) Clin. Orthop. 40, 57. TOrCNA,E. A. and CRONKrr~,E. P. (1962) J. Bone .Int. Surg. (A) 44, 1557. Wn.&IAMSON,N., JAMES,K., Lrlqo,N. R. and HOLT,L. P. (1966) Ann. Rheum. Dis. 25, 534.
LABELING INDEX (TRITIATED THYMIDINE) OF THE SYNOVIUM OF MICE AT DIFFERENT AGES* SAM J. PILIERO and EDGAR A. TONNA New York University Dental Center and Graduate School of Arts and Science, New York, NY 10010, U.S.A. (Received 1 June 1976)
AbstractmThe H3-autoradiographic study of the cellular proliferative activity of the synovial tissue of the knee joints of aging mice reveals a significant decrease in labeling indices between 5 and 26 weeks of age. A low level persisted throughout the life span of the animal. These cellular proliferative changes exhibited by the synovium with increasing age are similar to those reported for other skeletal associated tissues. Labeling appeared to be restricted to the surface cells of the synovial lining. INTRODUCTION DESPITE the plethora o f reports on the effects o f aging on a wide variety o f tissues, only exiguous information is available to data on the effects o f aging on synovial tissue. A d vancing age is a significant factor in degenerative joint disease, although the etiology o f the osteoarthritis remains obscure. The severity o f this disease ranges f r o m simple flaking and fibrillation o f the cartilage (Grade I), to significant deformation o f the joints and m a r k e d loss o f cartilage (Grade IV). F r o m the gerontological point o f view, it c a n n o t yet be determined whether these degenerative changes will eventually occur in all individuals should they live long enough. The present research endeavor is predicated on the necessity o f recognizing the age changes associated with the synovial m e m b r a n e in n o r m a l animals. I n this initial autoradiographic study, tritiated thymidine ( H a - T D R ) was used to ascertain the labeling index changes o f the synovium c o n c o m i t a n t with aging. This information is to serve as baseline data for future cellular kinetic studies and further explorations o f the effects o f aging on synovial tissues. MATERIALS AND METHODS An aging colony of the Brooldaaven National Laboratory (BNL) inbred strain of Swiss albino mouse has been used in this study. The BNL mouse is a short-lived animal whose average life span is less than 1 year. Female mice were divided into 4 age groups, namely: 5, 26, 52 and 78 weeks of age. Each animal received a subcutaneous injection of Ha-TDR (Sp. Act. -----1.9 Ci/mmol) I'0 laCi/gm body weight at time 0. Mice were sacrificed by overetherization at 1 hr post-H3-TDR administration. The knee joints were removed from each animal by sectioning the limbs (bilaterial) at the mid-femoral and mid-tibial levels. Whole joint specimens were fixed in neutral buffer 10y. formalin and subsequently decalcified in ethylenediaminetriacetic acid (EDTA). Sagittal bistological sections were cut at 5 ku-nfrom routinely prepared paraffin blocks of the knee joints. Autoradiographs were prepared using Kodak NTB-3 liquid emulsion, an exposure time of 21 days, and development in Kodak D19. The preparations were finally stained with hematoxylin. The labeling index was determined for the synovial tissues at each age group at 1 hr post-HaTDR administration. Grain counts were made of the synovial tissues found at the anterior and posterior aspects of the saggital sections. The data were statistically evaluated using Student's-t and a confidence level of p < 0-05. *Research was supported by the Histology Department Research Fund, N.I.A. Grant AG 00314 and a National Science Foundation Grant to New York University. 205
206
S A M J . pff:ll~RO A N D E D G A R A . T O N N A
RESULTS An estimation of the magnitude of the labeling index of the population of synoviat cells at various ages is given in Table I and Fig. 1. The synodal membrane of normal B N L mice at five weeks of age revealed a maximum labeling index of 0.044. By 26 weeks of age, the
TABLE I. LABV.LINGINDEX (TRn~AT~ TH~aDn, m) OF THE SYNOVIUM OF MICE AT DIFFERENT AGES* Age (Weeks) Labelingindex 4- S.E. 5 (9)t 0.044 4- 0.015 26
(9)
0-005 4- 0.002
52 (5) 0.007 4- 0.003 78 (12) 0.006 4- 0-002 *Each value consists of an average count of 162 cells/animaL tNumber of animals. .06 -
m
.05 {
u.J . 0 4 u") +l
x I.aJ ,.-.., Z
05-
c.9 Z .J L.d rn
.02-
_.J
.01
-
.5
26 AGE
52
78
(WEEKS)
Fxo. 1. Labeling index :t: S.E. of the knee joint synovium of mice at different ages.
labeled population was seen to decrease significantly to a low labeling index of 0.005. Labeling at 52 and 78 weeks, respectively (0.007 and 0.006) was not significantly different from that observed at the 26 week period. Labeling appeared to be primarily restricted to cellsnear the surface of the synodal membranes regardless of age (Fig. 2). However, labeling was predominantly encountered at the most remote aspects of the synovial lining, e.g. the cephalad portion of the suprapatellar pouch and the caudad portion of the infrapatellar pad. N o significant differences in the population of labeled cells wcrc observed in comparisons of grain counts made from the synovial tissues found at the anterior and posterior aspects of the sagittal sections of the knee joints. Histological observations of the thin synovial lining of the knee joints of mice disclosed the appearance of two distinguishable types of synovial cells forming a compact interlacing layer, one to three cells in depth (Fig. 2). Most of the cells appeared to be macro-
e ~l~o
FIG. 2. The photomicrograph shows Ha-TDR autoradiographic labeling of predominantly surface cells of the knee joint synovium of 5-week-old mice. Most of the cells appear to be macrophagic-like forming an interlacing layer, approxnmately one to three cells in depth. Hematoxylin stained. × 735.
f.p. 206
L A B E L I N G I N D E X OF S Y N O V I U M OF A G I N G MICE
207
phagic-like cells having vacuolated granular cytoplasm in contrast to those cells found in smaller numbers which possessed a homogeneous cytoplasm with a nucleus often found in a polar position. These cells frequently exhibited cytoplasmic processes which were seen to extend along the synovial lining, paralleled with the surface. DISCUSSION It has been reported that the human fetal synovium differs little from that of the adult (Krey et al., 1971). An age correlated increase in the amount of fibrous tissue separating the synovial capillaries from the joint spaces in man has been described (Ruckes and Schuckmann, 1962). This feature was, however, not observed in the histological survey of the present study. Two types of lining cells were distinguished in the synovia of the BNL mouse. These corresponded to the A and B types which have been reported to comprise the synovia of other animals and man alike (Castor, 1960; Barland et al., 1964; Williamson et aL, 1966; Schmid, 1968; Kinsella et aL, 1969; Murane and Feagans, 1970; Cutlip and Cheville, 1973). Three distinct cell types were observed at the electron microscopic level (Barland et al., 1964). The A type cells exhibited the capacity for phagocytosis. The B type cells were adapted for protein synthesis while an intermediate type "C" cell, not recognizable via light microscopy shared the properties of the A and B type cells. The cellular proliferative activity was not high enough to allow us to differentiate the proliferative activity of the various cell types. Histologically, however, the mouse synovial membrane was found to be similar, in several respects, to that described in man (Castor, 1960; Barland et al., 1964; Williamson et al., 1966; Schmid, 1968; Kinsella et al., 1969; Murane and Feagans, 1970; Cutlip and Cheville, 1973.) In the present autoradiographic study, synovial tissue exhibited a low cellular proliferative activity with a peak labeling of ~ 4.4 % at 5 weeks of age. This value is within the range of labeling levels reported in an earlier study of the skeletal tissues of aging BNL mice (Tonna, 1961). Here the labeling was ~ 3 % for the femoral periosteum and ~ 5 % for the cells of the distal epiphyseal plate of the femur at 5 weeks of age. Furthermore, the labeling indices were significantly higher in younger mice and significantly lower by 8 weeks of age. Undoubtedly, synovial tissues fit into the same cell proliferative pattern exhibited by other skeletal associated connective tissue, since at 26 weeks, the labeling of the synovial tissues was a low of 0"5 %, a value similar to that reported for other skeletal tissues (Tonna, 1961). The observed significant reduction in labeling index values between 5 and 26 weeks of age is characteristic of such tissues including the synovium, and appears to represent diminished physiological demands for growth rather than age induced changes. This feature was reported in a study of respiratory enzyme changes in normal and traumatized aging rats (Tonna, 1959). Although joint growth does not take place within the confines of the joint cavity, but at the periphery of the epiphyseal plate (Tonna, 1961) which at no point is in contact with synovial tissue, the lining of the joint capsule is responsive to the overall joint size and indirectly related to joint growth. The present study, which is limited to establishment of the labeling indices of the synovial tissues concomitant with aging, cannot evaluate the effects of aging on the proliferative potential of the synovial membrane. This must be left to a future study in which the tissue is stressed via some standard form of trauma. Although, the labeling indices of the synovial tissues of mice 26--78 weeks of age were not significantly different, the effects of aging on diminishing the cell proliferative activity is anticipated, since trauma studies have clearly
208
SAM $. PII.IERO AND EDGAR A. TONNA
revealed progressively diminishing functional, as well as proliferative capacities for skeletal tissues beyond 26 weeks of age (Tonna, 1959, 1961; Tonna and Cronkite, 1962). This, however, does not imply that the tissues are not capable of responding to trauma adequately, since the cells which comprise skeletal tissues appear to be reversible postmitotics rather than fixed postmitotics (Tonna and Cronkite, 1962; Tonna, 1965). Synovial tissues are also believed to be reversible postmitotics, since the repair of synovial tissues is known to occur following surgery. The progressive effects of aging are seen as quantitative changes with time rather than qualitative alterations of the tissues (Ruckes and Schuckmann, 1962). It is held that in response to prolonged mechanical stress for over a number of years, the dormant growth potential of synovial cells is possibly reactivated with subsequent growth, setting off a chain of events which lead to the complex structural lesion of degenerative joint disease "osteoarthritis" (Sokoloff, 1969). Degenerative joint disease is a non-inflammatory disorder ofmovable joints characterized by deterioration and ablation of articular cartilage, and also by the formation of new bone at such disturbed foci. It is by no means a purely gerontologic problem. Some strains of mice (Silberberg and Silberberg, 1941; Sokoloff, 1956) exhibit a significant incidence of degenerative joint disease, whereas, the BNL Swiss albino mouse used in the present study reveals a low incidence of joint disease. The present results show that the major cell proliferative activity changes occurred early in life in response to altered functional needs and are out of phase with the time of appearance of osteoarthritis. In this regard, subsequent studies are necessary to delineate the behavior of synovial tissues to prolonged stress, and the cellular capacity of the tissue response in progressively older animals. REFERENCES BARI,AND, P., NOVIKOFF,A. B. and I-IAMbS, D. (1964) Am. J. Path. 44, 853. CA.rrOR,C. W. (1960) Arth. Rheum. 3, 140. CUTUP, R. C. and C~EVna.E,N. F. (1973) Am. J. vet. Res. 34, 45. Kn~sv.tJ.~,T. D., BAUM,J. and Zn~, M. (1969) Arth. Rheum. 13, 734. KI~Y, P. R., COHEN,A. S., SMITH,C. B. and FnWt.AND,M. (1971) Arth. Rheum. 14, 319. Mulo,r~, T. W. and Fr.AO~S, W. M. (1970) g. dent. Res. 49, 1068. Rucg.r.S, J. and SCHUCKMANN,F. (1962) Frankf. Z. Path. 72, 243. SCF.M~, F. R. (1968) J. Am. get. Soc. 16, 545. S n . ~ R O , M. and Smam~r..RG,R. (1941) Am. J. Anat. 68, 69. SOKOLOrV,L. (1956) Arch. Path. 62, 118. SOKOt.OI~,L. (1969) The Biology of Degenerative Joint Disease, pp. 24 and 63, Univ. of Chicago Press,
Chicago. TONNA,E. A. (1959) J. Geront. 14, 159. To~,~A,E. A. (1961) J. Biophys. Biochem. Cytol. 9, 813. TONNA,E. A. (1965) Clin. Orthop. 40, 57. TOrCNA,E. A. and CRONKrr~,E. P. (1962) J. Bone .Int. Surg. (A) 44, 1557. Wn.&IAMSON,N., JAMES,K., Lrlqo,N. R. and HOLT,L. P. (1966) Ann. Rheum. Dis. 25, 534.