- Email: [email protected]
Effect of parabiosis of young and old rats on aortic calcification and collagen cross-links
Exp. Geront. Vol. 2, pp. 201-207. Pergamon Press 1967. Printed in Great Eritain
EFFECT AORTIC
OF PARABIOSIS CALCIFICATION
OF YOUNG AND AND COLLAGEN
OLD RATS ON CROSS-LINKS
Z. HRI~ZA and V. HLAVX~KOVX
Department of Pathology, N.Y. University School of Medicine, New York, U.S.A.
(Received 16 May 1967)
ONE OF the characteristic aging changes is the increase of Ca salts in the aortic wall. To a smaller degree Ca rises in other soft tissues too. In accordance with this accumulation of Ca in soft tissues with aging, after 45Ca administration in vivo the isotope uptake is greater in most soft tissues in old animals (Freydberg-Lucas and Verz~ir, 1957). The reason for this greater Ca deposition in old soft tissues is not clear; neither is the mechanism of calcification itself (for ref. see Yu and Blumenthal, 1963). Slower Ca incorporation into the bones of old animals plays probably a role in calcification of soft tissues, because externally administered Ca disappears faster from the blood of younger animals (Anderson and Tomlinson, 1966) and this can increase 45Ca incorporation into the soft tissues in old animals. We studied the role of internal environment in the age changes in calcification in young and old animals connected by parabiosis. At the same time we asked how this parabiotic connection will influence the speed of aging of collagen in young and old animals. METHODS Male rats, originally Wistar, fed standard diet were used in all experiments. These animals were highly inbred and their immunologic tolerance was always tested. Parabiosis of young and old animals was performed according to McCay et al. (1957) and Lunsford et al. (1963). Rats were joined at the shoulderblades by wire and their gluteal muscles, abdominal muscles, subcutaneous tissue and skin were sewed together in three layers. After 1 week, the connection of circulation was always completed and was verified by an injection of a dye into one animal. 4~Ca was injected intraperitoneally as CaC12 in a dose of 20 /xC/100 g body wt. Parabiotic animals were injected separately. Serum, collagen fibres from the tails and aortas (rinsed) were collected 1, 3, 6, 12, 24, 48 and 72 hr after the injection, in parabiotic animals only after 24 and 48 hr. Samples were digested in a mixture of perchloric and nitric acid (1:2) on a sand bath after addition of 1 mg CaC12 as a carrier. Ca was isolated according to Norris and Lawrence (1953) by precipitation with ammonium oxalate. The radioactivity was determined with Geiger-Mfiller counter with a thin end window (1-1.2 mg/cm2). Results were expressed in activity/1 mg wt. of the fibre or aorta (cpm/mg) or cpm/ml of serum. Ca in serum was estimated on a flame-photometer. Chemical contraction and relaxation of collagen fibres which is an indirect indicator of the amount of cross-links in collagen was estimated according to Chvapil and Hrflza 201
202
z. HRI~ZAAND V. HLAVACKOVA
(1959). Piece of lead weighing 50 mg was inserted to one collagen fibre 6 cm long and contraction and relaxation of the fibre was then measured for 2 hr in 2.5 M NaClO4 solution in a 25°C water bath. RESULTS (1) ~lging changes in 45Ca incorporation into collagen fibres and aortas Freydberg-Lucas and Verz~ir (1957) described that 48 hr after 45Ca injection radioactivity of the aorta and collagen fibres increased more in the older animals. We have studied the same phenomenon using more time intervals, i.e. 1, 3, 6, 12, 24, 48 and 72 hr after the injection. Figure 1 shows that the radioactivity was greater, in older animals, at all time intervals in the collagen fibres and the same holds for the aorta (Fig. 2). 15
\ \ E
5
i
,'>
..u
48
72
I 24 hr
FIG. 1. 45Ca incorporation into the collagen fibres of young (2'5 months full line) and old (16 months interrupted line) animals, 1-24 hr after isotope administration. Means __ S.E., all differences are significant. Ordinate: imp × 1000/rag/rain. (2) Changes of serum Ca levels with aging In order to explain greater incorporation of 45Ca into collagen fibres and aortas in older animals in vivo we have measured 45Ca levels in serum at several intervals after the isotope administration. Figure 3 shows that 45Ca level in serum was greater in older animals. This led to the estimation of Ca levels in blood of young and old animals. In the first experiments, we found in 2 months old rats 10.3 __+0-3 per cent Ca and in 21 months old animals 11.8 ! 0.27 mg per cent, P < 0.Ol. Results of the second experiment where three animal groups were used are shown in Fig. 4. Ca level in serum apparently increases with age. (3) Effect of parabiosis on 45Ca incorporation into the aorta In the next experiment we tried to equalize 45Ca level in the blood by parabiotic connection of a young and an old rat. Young (2-5 months) and old (13.5 months) rats were connected by parabiosis for 2.5 months. After 1 week, their vessels grew through and their circulations mixed. At the end of the experiment, 45Ca incorporation into the aortic wall of these animals and controls of the same age was measured 24 and 48 hr after the administration of the isotope. Figure 5 shows that 45Ca incorporation into the aorta went down in the old parabiotic rat in comparison with the old control, and incorporation
EFFECT OF PARABIOSIS OF RATS IN AORTIC CALCIFICATION
203
I I I I
<
~. .__.
,
20
L II
\ ~
x
.I
I
I
I
24
48
I 72
hr
FIG. 2. 4sCa i n c o r p o r a t i o n into t h e aorta in y o u n g ( - - - - ) a n d o l d (- - -) a n i m a l s . in Fig. 1. A l l d i f f e r e n c e s are significant, P <0"01 e x c e p t 24 hr.
D e s c r i p t i o n as
22 I I I
o.
E
t4
I
\
24
48
72
hr
FIG. 3. 45Ca l e v e l s in s e r u m of y o u n g ( ) a n d o l d (- - -) rats. D e s c r i p t i o n as in Fig. 1. All d i f f e r e n c e s are s i g n i f i c a n t ( P < 0 . 0 2 - 0 . 0 0 1 ) . S o m e S.E. are s m a l l e r t h a n t h e circles.
into the aorta of the young parabiotic rat increased in comparison with the young control, but there still remained some difference in the old and young parabiotic animal.
(if) Effect of parabiosis on collagen aging It is doubtful whether the old and young structures in parabiotic rats can be influenced by the mixed blood of these animals, if the internal environment has any influence on the speed of growing old. The aging of structures can be best measured by the amount of cross-links in collagen molecule. In our experiments, we used an indirect indicator
204
z.
HRUZA
AND
V.
HLAVA~KOVA
14--
T
1
12E
o
c.)
tO - -
I
1
J
8
3
13
Months
FIG. 4. Ca, mg per cent 4- S.E. in rats of different age. The difference between 8 and 13 months is not significant.
\
\
4. 12-
\
{~.~
\
\
\ \
~
"~ Ol d c o n t r o l
E
4
i
_j
24
48
hr 5. 45Ca incorporation into the aorta 24 and 48 hr after isotope injection. Old control © - - - ©, old parabiotic • - - 0 , young parabiotic • 0 , young control O ©. Differences are significant except young parab.-y, contr, after 24 hr and young and old parab, after 48 hr. FIG.
of the amount of cross-links in collagen, chemical contraction and relaxation of collagen fibres. In this technique, most characteristic for aging changes is the relaxation period. Collagen fibres from old animals relax m u c h less (for details see Chvapil and Hrflza, 1959). Rat's fibres from the parabiotic experiments described in the previous paragraph were used in an experiment on chemical relaxation. At the end of the experiment, the rats were 6 and 16 m o n t h s old. Results are s h o w n in Fig. 6. Contraction and relaxation of control collagen fibres taken from 6 and 16 m o n t h s old animals was distinctly different, fibres from the young animals relaxing m u c h more. There is no difference in contraction and relaxation between the y o u n g control and young parabiotic animal, but significant differences were found between the old control and old parabiotic animal. Collagen fibres of old animals "grew old" significantly slower during parabiosis with the young animal.
EFFECT
OF
PARABIOSIS
OF
RATS
IN
AORTIC
CALCIFICATION
205
Youngcontrol ~
//
4
i7
/
..f
g
~
parablotic Old porabiotic
. _ yoo, o,
::::::-? jf
.-+-" ..---4": o ~,I
2
--
I 30
I 60
I 90
i20
rain
Fin. 6. Effect of parabiosis on chemical contraction and relaxation of collagen fibres. Groups as on Fig. 5. Differences between old control and old parabiotic are significant as described in the figure (P <). Some of the S.E. are smaller than the circles. DISCUSSION Isotopic Ca disappears slower from the blood of old animals. This was shown up to 16 weeks by Owada et al (1962) and Anderson and Tomlinson (1966) and in older animals in our present experiments. This slower disappearance of 4sCa from the blood of older animals is probably due to slower turnover of Ca in the bones in older animals. The total serum Ca level is slightly but significantly higher in older animals too. We have confirmed and enlarged the experiments of Freydberg-Lucas and Verz4r (1957) that 4~Ca incorporation in vivo into the aorta and collagen fibres increases with aging. Freydberg-Lucas and Verz4r measured the radioactivity 48 hr after administration of the isotope and our experiments showed that this difference can be observed and is sometimes greater at different times after isotope injection. Difference in 4sCa incorporation into the collagen fibres with aging were greater in our experiments. It is a question whether higher Ca and 4~Ca serum level alone can be responsible for greater 45Ca incorporation into the soft tissues. It is probably not so, because in the aorta and in the collagen fibres, there is more Ca in older animals. This Ca is being exchanged with the Ca in blood and if there is more Ca in the tissue and turnover of Ca is not changed, more of the isotopic Ca must enter the tissue. We can suppose that Ca and 4sCa blood levels in old and young animals connected by parabiosis are the same because their circulation is connected. In the old parabiotic rats 4sCa incorporation into the aorta decreased and 4sCa incorporation in the young parabiotic animals increased in comparison with intact animals of the same age. This can be interpreted as implying that Ca content in the aorta decreased in the old animals and increased in the young animals, but the duration of the experiment was not satisfactory to equalize Ca levels in the aorta in both animals, because there was still a difference in 4sCa incorporation into the aorta in the old and young parabiotic rat. Our experiments with parabiosis are not yet complete because we have not yet been able to estimate the amount of Ca in soft tissues in the parabiotic rats. Our experiments with chemical contraction and relaxation of collagen fibres of parabiotic animals showed that the fibres of old parabiotic animals grew old more
206
Z. HRUZA AND V. HLAV/~KOV,~
slowly in parabiosis, but aging of young parabiotic animals was not influenced. We may suppose that, e.g. the amount of cross-linking agents in the blood decreased in the parabiotic animals in comparison with the old controls. This can explain why the old parabiotic animals grew old slower. It is more difficult to explain why the young parabiotic animals did not grow old faster if the level of crossdinking agents was somewhere in between the levels in young and old animals. If the theory of the amount of cross-linking agents is true, our results with the young parabiotic animals can be taken to mean either that the level of cross-linking agents does not increase in the parabiotic animals over their level in the y o u n g animals (young animals could remove the extra cross-linking agents from old parabiotic partners) or that the local conditions in the collagen of young animals are so different that the greater amount of cross-links does not increase, despite greater amount of cross-linking agents. This second explanation might be due, e.g. to faster collagen metabolism of young animals. REFERENCES ANDERSON,J. and TOMLINSON,R. W. S. (1966) J. Physiol. 182, 664. CHVAPm, M. and HR6ZA, Z. (1959) Gerontologia 3, 241. FREYDBE~G-LucAS, V. and VERZAR,F. (1957) Gerontologia 1, 195. HLAVA6KOVA,V. and HR6ZA, Z. (1967) Exp. Geront. 2, 209. HROZA, Z., HLAVA6OVA,V. and BABICK~,A. (1967) Exp. Geront. 2, 191. LUNSFORD,W. R., McCAv, C. M., LUPIEN,P. J., POPE, P. E. and SPERLING,G. (1963) Gerontologia 7,1. McCAY, C. M., POPE, F., LUNSFORD,W., SPERLING,G. and SAMBHAVAPOL,P. (1957) Gerontologia 1,7. NORRIS, W. P. and LAWRENCE,B. J. (1953) Anal. Chem. 25, 956. OWADA, K. and OSmRO, N. (1962) Osaka City MedicaIJ. 8, 121. Yu, S. Y. and BLUMENTHAL,H. T. (1963) J. Gerontol. 18, 119. S u m m a r y - - ( 1 ) Serum Ca levels increase with aging. After 45CaClz injection, the levels of 45Ca estimated after 1, 3, 6, 12, 24, 48 and 72 hr are much higher in the serum, collagen fibres and aortas of the old (16 months) than young (2 months) rats. (2) Young (3.5 months old) and old (13'5 months) inbred rats were parabiotically connected for 2-5 months. Already after 1 week the circulation of these animals fuses. After 45CAC12injection, incorporation of the isotope into the aortic wall was decreased in old parabiotic rats and increased in young parabiotic rats. (3) Aging of collagen fibres as measured by chemical contraction and relaxation was slowed down in old parabiotic rats, but did not change in the young parabiotic rat. R6sum6--(1) Les niveaux de Ca du s6rum s'61~vent avec l'~ge. Apr6s injection de 4~CaC12, les niveaux de 45Ca ~valu6s apr~s 1, 3, 6, 12, 24, 48 et 72 heures sont beaucoup plus 61ev~s dans le s6rum, les fibres collag+nes et les aortes des rats ~g~s (16 mois) que des rats jeunes (2 mois). (2) Des rats endogames jeunes (3,5 mois) et vieux (13,5 mois) furent reli6s entre eux par parabiose pendant 2,5 tools. La circulation de ces animaux fusionne d6j~ apr~s 1 semaine. Apr~s injection de 45CAC12, l'incorporation de l'isotope fi la paroi aortique diminua chez les rats parabiotiques ~g6s et augmenta chez les rats parabiotiques jeunes. (3) Le vieillissement des fibres collag~nes, tel qu'il est mesur6 par contraction et relaxation chlmiques, rut ralenti chez les rats parabiotiques ~g~s, mais ne rut pas modifi6 chez les rats parabiotiques jeunes.
EFFECT OF PARABIOSIS OF RATS IN AORTIC CALCIFICATION Zusammenfassung--(1) D e r S e r u m - C a - S p i e g e l e r h 6 h t sich m i t d e m Alter. N a c h 4sCaC12- I n j e k t i o n sind die n a c h 1, 3, 6, 12, 24, 48 u n d 72 S t u n d e n b e s t i m m t e n 45Ca-Spiegel viel h t i h e r in S e r u m , K o l l a g e n f a s e r n u n d A o r t a yon alten (16 M o n a t e ) als v o n j u n g e n (2 M o n a t e ) alten R a t t e n . (2) J u n g e (3,5 M o n a t e ahe) u n d alte (13,5 M o n a t e alte) I n z u c h t r a t t e n w u r d e n ffir 2,5 M o n a t e p a r a b i o t i s c h v e r b u n d e n . S c h o n n a c h e i n e r W o c h e fliel3en die Kreisl~iufe dieser T i e r e z u s a m m e n . N a c h 4~CaC12-Injektion war die I s o t o p e n i n k o r p o r a t i o n in d e r A o r t e n w a n d v o n a h e n p a r a b i o t i s c h e n R a t t e n v e r m i n d e r t u n d bei j u n g e n p a r a b i o t i s c h e n R a t t e n e r h 6 h t . (3) Das Ahem der Kollagenfasern--gemessen durch c h e m i s c h e K o n t r a k t i o n u n d R e l a x a t i o n - - w u r d e bei alten p a r a b i o t i s c h e n R a t t e n v e r l a n g s a m t , w~ihrend es bei j u n g e n p a r a b i o t i s c h e n R a t t e n n i c h t beeinflul3t w u r d e .
P¢31OMe----(I) YpoBeHb CblBOpOTKH Ca yBemtqnaaeTc~ co cTapenneM. Floc~e anpbICKrillann~I 45CAC12 ypOBnH 45Ca, onpe~e:ienn~,le noc~ae 1, 3, 6, 12, 24, 48 rt 72 '~acoa 6bInrl ropa3~o ablme a CbIaOpOTte, KOJUIarenoabIX BOJ/OKHaX14 aopTax cTapblX (UleCTHMeCgHbIX) KpbIC, qeM MOYIO~bIX(~ByxMec~lqHbIX)KpbIC. (2) Mono~ble (3,5-Mec~wa~ie) n cTapble (13,5 -Mecnanbie) nH6pe~nbae KpbICbt 6blflH coe~[HHeHbI napa6noTnqecKH Ha 2,5 Mec~ua. Y~re ] He~lemo cnycTfl rpOBOO6pattleane aTnx )KHBOTHblX coe~HttHJIOCb, lqocYle BIIpbleKHBam4sI 45CAC12 BHe~IpeHHe H3OTOIIa B CTeHKHaOpTbI yMeltbltlHJ/OCb y cTap~,lX napa6noTnqecKHx rpbIC H yBe31ttqttJIOCby MO.rlO,/~blXnapa6HOTn~ecrnx KpbIc. (3) CTapeHrle KonJiarenoBbix BOJ'IOKOH, H3MepeHltoe coKpatueHHeM H ocJia6nettHeM, 6blno 3aMe~lnerlo B cTapbIX napa6rtOTriqecKHx rp~,lcax, no He H3MeHH.rlOCbB MOJIO~bIX napa6noTH~ecrnx rpbicax.
B
207
EFFECT AORTIC
OF PARABIOSIS CALCIFICATION
OF YOUNG AND AND COLLAGEN
OLD RATS ON CROSS-LINKS
Z. HRI~ZA and V. HLAVX~KOVX
Department of Pathology, N.Y. University School of Medicine, New York, U.S.A.
(Received 16 May 1967)
ONE OF the characteristic aging changes is the increase of Ca salts in the aortic wall. To a smaller degree Ca rises in other soft tissues too. In accordance with this accumulation of Ca in soft tissues with aging, after 45Ca administration in vivo the isotope uptake is greater in most soft tissues in old animals (Freydberg-Lucas and Verz~ir, 1957). The reason for this greater Ca deposition in old soft tissues is not clear; neither is the mechanism of calcification itself (for ref. see Yu and Blumenthal, 1963). Slower Ca incorporation into the bones of old animals plays probably a role in calcification of soft tissues, because externally administered Ca disappears faster from the blood of younger animals (Anderson and Tomlinson, 1966) and this can increase 45Ca incorporation into the soft tissues in old animals. We studied the role of internal environment in the age changes in calcification in young and old animals connected by parabiosis. At the same time we asked how this parabiotic connection will influence the speed of aging of collagen in young and old animals. METHODS Male rats, originally Wistar, fed standard diet were used in all experiments. These animals were highly inbred and their immunologic tolerance was always tested. Parabiosis of young and old animals was performed according to McCay et al. (1957) and Lunsford et al. (1963). Rats were joined at the shoulderblades by wire and their gluteal muscles, abdominal muscles, subcutaneous tissue and skin were sewed together in three layers. After 1 week, the connection of circulation was always completed and was verified by an injection of a dye into one animal. 4~Ca was injected intraperitoneally as CaC12 in a dose of 20 /xC/100 g body wt. Parabiotic animals were injected separately. Serum, collagen fibres from the tails and aortas (rinsed) were collected 1, 3, 6, 12, 24, 48 and 72 hr after the injection, in parabiotic animals only after 24 and 48 hr. Samples were digested in a mixture of perchloric and nitric acid (1:2) on a sand bath after addition of 1 mg CaC12 as a carrier. Ca was isolated according to Norris and Lawrence (1953) by precipitation with ammonium oxalate. The radioactivity was determined with Geiger-Mfiller counter with a thin end window (1-1.2 mg/cm2). Results were expressed in activity/1 mg wt. of the fibre or aorta (cpm/mg) or cpm/ml of serum. Ca in serum was estimated on a flame-photometer. Chemical contraction and relaxation of collagen fibres which is an indirect indicator of the amount of cross-links in collagen was estimated according to Chvapil and Hrflza 201
202
z. HRI~ZAAND V. HLAVACKOVA
(1959). Piece of lead weighing 50 mg was inserted to one collagen fibre 6 cm long and contraction and relaxation of the fibre was then measured for 2 hr in 2.5 M NaClO4 solution in a 25°C water bath. RESULTS (1) ~lging changes in 45Ca incorporation into collagen fibres and aortas Freydberg-Lucas and Verz~ir (1957) described that 48 hr after 45Ca injection radioactivity of the aorta and collagen fibres increased more in the older animals. We have studied the same phenomenon using more time intervals, i.e. 1, 3, 6, 12, 24, 48 and 72 hr after the injection. Figure 1 shows that the radioactivity was greater, in older animals, at all time intervals in the collagen fibres and the same holds for the aorta (Fig. 2). 15
\ \ E
5
i
,'>
..u
48
72
I 24 hr
FIG. 1. 45Ca incorporation into the collagen fibres of young (2'5 months full line) and old (16 months interrupted line) animals, 1-24 hr after isotope administration. Means __ S.E., all differences are significant. Ordinate: imp × 1000/rag/rain. (2) Changes of serum Ca levels with aging In order to explain greater incorporation of 45Ca into collagen fibres and aortas in older animals in vivo we have measured 45Ca levels in serum at several intervals after the isotope administration. Figure 3 shows that 45Ca level in serum was greater in older animals. This led to the estimation of Ca levels in blood of young and old animals. In the first experiments, we found in 2 months old rats 10.3 __+0-3 per cent Ca and in 21 months old animals 11.8 ! 0.27 mg per cent, P < 0.Ol. Results of the second experiment where three animal groups were used are shown in Fig. 4. Ca level in serum apparently increases with age. (3) Effect of parabiosis on 45Ca incorporation into the aorta In the next experiment we tried to equalize 45Ca level in the blood by parabiotic connection of a young and an old rat. Young (2-5 months) and old (13.5 months) rats were connected by parabiosis for 2.5 months. After 1 week, their vessels grew through and their circulations mixed. At the end of the experiment, 45Ca incorporation into the aortic wall of these animals and controls of the same age was measured 24 and 48 hr after the administration of the isotope. Figure 5 shows that 45Ca incorporation into the aorta went down in the old parabiotic rat in comparison with the old control, and incorporation
EFFECT OF PARABIOSIS OF RATS IN AORTIC CALCIFICATION
203
I I I I
<
~. .__.
,
20
L II
\ ~
x
.I
I
I
I
24
48
I 72
hr
FIG. 2. 4sCa i n c o r p o r a t i o n into t h e aorta in y o u n g ( - - - - ) a n d o l d (- - -) a n i m a l s . in Fig. 1. A l l d i f f e r e n c e s are significant, P <0"01 e x c e p t 24 hr.
D e s c r i p t i o n as
22 I I I
o.
E
t4
I
\
24
48
72
hr
FIG. 3. 45Ca l e v e l s in s e r u m of y o u n g ( ) a n d o l d (- - -) rats. D e s c r i p t i o n as in Fig. 1. All d i f f e r e n c e s are s i g n i f i c a n t ( P < 0 . 0 2 - 0 . 0 0 1 ) . S o m e S.E. are s m a l l e r t h a n t h e circles.
into the aorta of the young parabiotic rat increased in comparison with the young control, but there still remained some difference in the old and young parabiotic animal.
(if) Effect of parabiosis on collagen aging It is doubtful whether the old and young structures in parabiotic rats can be influenced by the mixed blood of these animals, if the internal environment has any influence on the speed of growing old. The aging of structures can be best measured by the amount of cross-links in collagen molecule. In our experiments, we used an indirect indicator
204
z.
HRUZA
AND
V.
HLAVA~KOVA
14--
T
1
12E
o
c.)
tO - -
I
1
J
8
3
13
Months
FIG. 4. Ca, mg per cent 4- S.E. in rats of different age. The difference between 8 and 13 months is not significant.
\
\
4. 12-
\
{~.~
\
\
\ \
~
"~ Ol d c o n t r o l
E
4
i
_j
24
48
hr 5. 45Ca incorporation into the aorta 24 and 48 hr after isotope injection. Old control © - - - ©, old parabiotic • - - 0 , young parabiotic • 0 , young control O ©. Differences are significant except young parab.-y, contr, after 24 hr and young and old parab, after 48 hr. FIG.
of the amount of cross-links in collagen, chemical contraction and relaxation of collagen fibres. In this technique, most characteristic for aging changes is the relaxation period. Collagen fibres from old animals relax m u c h less (for details see Chvapil and Hrflza, 1959). Rat's fibres from the parabiotic experiments described in the previous paragraph were used in an experiment on chemical relaxation. At the end of the experiment, the rats were 6 and 16 m o n t h s old. Results are s h o w n in Fig. 6. Contraction and relaxation of control collagen fibres taken from 6 and 16 m o n t h s old animals was distinctly different, fibres from the young animals relaxing m u c h more. There is no difference in contraction and relaxation between the y o u n g control and young parabiotic animal, but significant differences were found between the old control and old parabiotic animal. Collagen fibres of old animals "grew old" significantly slower during parabiosis with the young animal.
EFFECT
OF
PARABIOSIS
OF
RATS
IN
AORTIC
CALCIFICATION
205
Youngcontrol ~
//
4
i7
/
..f
g
~
parablotic Old porabiotic
. _ yoo, o,
::::::-? jf
.-+-" ..---4": o ~,I
2
--
I 30
I 60
I 90
i20
rain
Fin. 6. Effect of parabiosis on chemical contraction and relaxation of collagen fibres. Groups as on Fig. 5. Differences between old control and old parabiotic are significant as described in the figure (P <). Some of the S.E. are smaller than the circles. DISCUSSION Isotopic Ca disappears slower from the blood of old animals. This was shown up to 16 weeks by Owada et al (1962) and Anderson and Tomlinson (1966) and in older animals in our present experiments. This slower disappearance of 4sCa from the blood of older animals is probably due to slower turnover of Ca in the bones in older animals. The total serum Ca level is slightly but significantly higher in older animals too. We have confirmed and enlarged the experiments of Freydberg-Lucas and Verz4r (1957) that 4~Ca incorporation in vivo into the aorta and collagen fibres increases with aging. Freydberg-Lucas and Verz4r measured the radioactivity 48 hr after administration of the isotope and our experiments showed that this difference can be observed and is sometimes greater at different times after isotope injection. Difference in 4sCa incorporation into the collagen fibres with aging were greater in our experiments. It is a question whether higher Ca and 4~Ca serum level alone can be responsible for greater 45Ca incorporation into the soft tissues. It is probably not so, because in the aorta and in the collagen fibres, there is more Ca in older animals. This Ca is being exchanged with the Ca in blood and if there is more Ca in the tissue and turnover of Ca is not changed, more of the isotopic Ca must enter the tissue. We can suppose that Ca and 4sCa blood levels in old and young animals connected by parabiosis are the same because their circulation is connected. In the old parabiotic rats 4sCa incorporation into the aorta decreased and 4sCa incorporation in the young parabiotic animals increased in comparison with intact animals of the same age. This can be interpreted as implying that Ca content in the aorta decreased in the old animals and increased in the young animals, but the duration of the experiment was not satisfactory to equalize Ca levels in the aorta in both animals, because there was still a difference in 4sCa incorporation into the aorta in the old and young parabiotic rat. Our experiments with parabiosis are not yet complete because we have not yet been able to estimate the amount of Ca in soft tissues in the parabiotic rats. Our experiments with chemical contraction and relaxation of collagen fibres of parabiotic animals showed that the fibres of old parabiotic animals grew old more
206
Z. HRUZA AND V. HLAV/~KOV,~
slowly in parabiosis, but aging of young parabiotic animals was not influenced. We may suppose that, e.g. the amount of cross-linking agents in the blood decreased in the parabiotic animals in comparison with the old controls. This can explain why the old parabiotic animals grew old slower. It is more difficult to explain why the young parabiotic animals did not grow old faster if the level of crossdinking agents was somewhere in between the levels in young and old animals. If the theory of the amount of cross-linking agents is true, our results with the young parabiotic animals can be taken to mean either that the level of cross-linking agents does not increase in the parabiotic animals over their level in the y o u n g animals (young animals could remove the extra cross-linking agents from old parabiotic partners) or that the local conditions in the collagen of young animals are so different that the greater amount of cross-links does not increase, despite greater amount of cross-linking agents. This second explanation might be due, e.g. to faster collagen metabolism of young animals. REFERENCES ANDERSON,J. and TOMLINSON,R. W. S. (1966) J. Physiol. 182, 664. CHVAPm, M. and HR6ZA, Z. (1959) Gerontologia 3, 241. FREYDBE~G-LucAS, V. and VERZAR,F. (1957) Gerontologia 1, 195. HLAVA6KOVA,V. and HR6ZA, Z. (1967) Exp. Geront. 2, 209. HROZA, Z., HLAVA6OVA,V. and BABICK~,A. (1967) Exp. Geront. 2, 191. LUNSFORD,W. R., McCAv, C. M., LUPIEN,P. J., POPE, P. E. and SPERLING,G. (1963) Gerontologia 7,1. McCAY, C. M., POPE, F., LUNSFORD,W., SPERLING,G. and SAMBHAVAPOL,P. (1957) Gerontologia 1,7. NORRIS, W. P. and LAWRENCE,B. J. (1953) Anal. Chem. 25, 956. OWADA, K. and OSmRO, N. (1962) Osaka City MedicaIJ. 8, 121. Yu, S. Y. and BLUMENTHAL,H. T. (1963) J. Gerontol. 18, 119. S u m m a r y - - ( 1 ) Serum Ca levels increase with aging. After 45CaClz injection, the levels of 45Ca estimated after 1, 3, 6, 12, 24, 48 and 72 hr are much higher in the serum, collagen fibres and aortas of the old (16 months) than young (2 months) rats. (2) Young (3.5 months old) and old (13'5 months) inbred rats were parabiotically connected for 2-5 months. Already after 1 week the circulation of these animals fuses. After 45CAC12injection, incorporation of the isotope into the aortic wall was decreased in old parabiotic rats and increased in young parabiotic rats. (3) Aging of collagen fibres as measured by chemical contraction and relaxation was slowed down in old parabiotic rats, but did not change in the young parabiotic rat. R6sum6--(1) Les niveaux de Ca du s6rum s'61~vent avec l'~ge. Apr6s injection de 4~CaC12, les niveaux de 45Ca ~valu6s apr~s 1, 3, 6, 12, 24, 48 et 72 heures sont beaucoup plus 61ev~s dans le s6rum, les fibres collag+nes et les aortes des rats ~g~s (16 mois) que des rats jeunes (2 mois). (2) Des rats endogames jeunes (3,5 mois) et vieux (13,5 mois) furent reli6s entre eux par parabiose pendant 2,5 tools. La circulation de ces animaux fusionne d6j~ apr~s 1 semaine. Apr~s injection de 45CAC12, l'incorporation de l'isotope fi la paroi aortique diminua chez les rats parabiotiques ~g6s et augmenta chez les rats parabiotiques jeunes. (3) Le vieillissement des fibres collag~nes, tel qu'il est mesur6 par contraction et relaxation chlmiques, rut ralenti chez les rats parabiotiques ~g~s, mais ne rut pas modifi6 chez les rats parabiotiques jeunes.
EFFECT OF PARABIOSIS OF RATS IN AORTIC CALCIFICATION Zusammenfassung--(1) D e r S e r u m - C a - S p i e g e l e r h 6 h t sich m i t d e m Alter. N a c h 4sCaC12- I n j e k t i o n sind die n a c h 1, 3, 6, 12, 24, 48 u n d 72 S t u n d e n b e s t i m m t e n 45Ca-Spiegel viel h t i h e r in S e r u m , K o l l a g e n f a s e r n u n d A o r t a yon alten (16 M o n a t e ) als v o n j u n g e n (2 M o n a t e ) alten R a t t e n . (2) J u n g e (3,5 M o n a t e ahe) u n d alte (13,5 M o n a t e alte) I n z u c h t r a t t e n w u r d e n ffir 2,5 M o n a t e p a r a b i o t i s c h v e r b u n d e n . S c h o n n a c h e i n e r W o c h e fliel3en die Kreisl~iufe dieser T i e r e z u s a m m e n . N a c h 4~CaC12-Injektion war die I s o t o p e n i n k o r p o r a t i o n in d e r A o r t e n w a n d v o n a h e n p a r a b i o t i s c h e n R a t t e n v e r m i n d e r t u n d bei j u n g e n p a r a b i o t i s c h e n R a t t e n e r h 6 h t . (3) Das Ahem der Kollagenfasern--gemessen durch c h e m i s c h e K o n t r a k t i o n u n d R e l a x a t i o n - - w u r d e bei alten p a r a b i o t i s c h e n R a t t e n v e r l a n g s a m t , w~ihrend es bei j u n g e n p a r a b i o t i s c h e n R a t t e n n i c h t beeinflul3t w u r d e .
P¢31OMe----(I) YpoBeHb CblBOpOTKH Ca yBemtqnaaeTc~ co cTapenneM. Floc~e anpbICKrillann~I 45CAC12 ypOBnH 45Ca, onpe~e:ienn~,le noc~ae 1, 3, 6, 12, 24, 48 rt 72 '~acoa 6bInrl ropa3~o ablme a CbIaOpOTte, KOJUIarenoabIX BOJ/OKHaX14 aopTax cTapblX (UleCTHMeCgHbIX) KpbIC, qeM MOYIO~bIX(~ByxMec~lqHbIX)KpbIC. (2) Mono~ble (3,5-Mec~wa~ie) n cTapble (13,5 -Mecnanbie) nH6pe~nbae KpbICbt 6blflH coe~[HHeHbI napa6noTnqecKH Ha 2,5 Mec~ua. Y~re ] He~lemo cnycTfl rpOBOO6pattleane aTnx )KHBOTHblX coe~HttHJIOCb, lqocYle BIIpbleKHBam4sI 45CAC12 BHe~IpeHHe H3OTOIIa B CTeHKHaOpTbI yMeltbltlHJ/OCb y cTap~,lX napa6noTnqecKHx rpbIC H yBe31ttqttJIOCby MO.rlO,/~blXnapa6HOTn~ecrnx KpbIc. (3) CTapeHrle KonJiarenoBbix BOJ'IOKOH, H3MepeHltoe coKpatueHHeM H ocJia6nettHeM, 6blno 3aMe~lnerlo B cTapbIX napa6rtOTriqecKHx rp~,lcax, no He H3MeHH.rlOCbB MOJIO~bIX napa6noTH~ecrnx rpbicax.
B
207