REGENERATION HYDROCORTISONE

A. A.

OF

THE

RABBIT

AFTER

SPLEEN

INJECTION

UDC 612.6.03:612.411-06:615.357.453

Mezhlumyan

The action of h y d r o c o r t i s o n e on the rabbit spleen and the regenerative p r o c e s s e s arising after cessation of administration of the hormone were studied. During prolonged administration of hydroeortisone the absorptive function of the reticuloendotheliat s y s t e m is d e p r e s s e d and atrophy of the spleen develops, as shown by a reduction in the weight and size of the organ. Most of the splenic follicles are converted into r a n d o m c l u s t e r s of lymphocytes. P y r o n i n o philic cells disappear f r o m the white pulp. After c e s s a t i o n of the action of the hormone the a n i m a l ' s g e n e r a l condition i m p r o v e s , the development of the atrophic changes in the spleen is halted, the splenic follicles are r e s t o r e d , and the n u m b e r of pyroninophitic cells in them i n c r e a s e s . The use of a r e g e n e r a t i o n stimulator under these conditions a c c e l e r a t e s r e c o v e r y of the body weight of the animal, r e v e r s a l of the atrophy of the spleen, and r e s t o r a t i o n of its normal structure. KEY WORDS: spleen; h y d r o c o r t i s o n e ; stimulation of regeneration

After r e s e c t i o n the rabbit spleen not only does not r e g e n e r a t e , but it even shows signs of atrophy [4]. The w r i t e r showed previously that injection of ethanolamine after h e m i r e s e c t i o n of the spleen causes the development of a special form of regeneration hypertrophy, c h a r a c t e r i z e d by an i n c r e a s e in length of the organ [6]. It was important to discover how the ability of the rabbit spleen to r e g e n e r a t e is manifested after changes caused by the action of pathological f a c t o r s , both under o r d i n a r y conditions and during the action of a r e g e n eration stimulator [5, 7]. Of the factors causing a pathologicaI state of the lymphoid organs, the choice fell on h y d r o c o r t i s o n e . According to data in the l i t e r a t u r e , this substance causes atrophy of lymphoid organs [2, 3] and leads to a d e c r e a s e in the RNA concentration in the mitochondria of the spleen [1]. The study of r e g e n e r a t i v e p r o c e s s e s in the rabbit spleen after atrophy induced by h y d r o c o r t i s o n e is of definite interest because, whereas the absence of r e g e n e r a t i o n after resection could be to some extent explained by the high c o m p e n s a t o r y powers of the other lymphoid organs, after injection of h y d r o c o r t i s o n e these powers are limited, so that new conditions are c r e a t e d for the development of r e g e n e r a t i o n of the spleen and for the associated use of a regeneration stimulator. EXPERIMENTAL

METHOD

Experiments were c a r r i e d out on 45 rabbits of the Rex breed, s t r i c t l y selected by weight (1500.0 + 65.0 g). On alternate days all the experimental animals received injections of h y d r o e o r t i s o n e (from Richter) in a dose of 2 mg/kg. The rabbits of group 1 were killed on the 30th day of administration of the hormone and the rabbits of group 2 30 days after the last injection of the hormone. The animals of group 3, starting f r o m the 30th day after the last injection of h y d r o c o r t i s o n e , were given intraperitoneal injections of colamine h y d r o chloride (ethanolamine) in a dose of 10 m g / k g on alternate days for 30 days. Intact rabbits s e r v e d as the control for each group. In the c o u r s e of the observations on the animals the Congo Red index was determined periodically. This test consists essentially of c o l o r i m e t r i c calculations of the rate of disappearance of i n t r a venously injected Congo Red f r o m the blood s t r e a m , which depends on the functional state of the reticuloendotheliat s y s t e m (RES). The m a t e r i a l was subjected to the usual histological analysis, s o m e sections were Department of Histology and Pathological Anatomy, E r e v a n Z o o v e t e r i n a r y Institute. ( P r e s e n t e d by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated f r o m Byulteten' t~ksperimental'noi Biologii i Meditsiny, Vol. 84, No. 12, pp. 722-725, D e c e m b e r , 1977. Original a r t i c l e submitted April 20, 1977. 1764

0007-4888~77/8412-1764507.50

01978 Plenum Publishing Corporation

T A B L E 1. C h a n g e s in Weight and Length of Spleen, D i a m e t e r of Splenic L y m p h a t i c F o l l i c l e s , and N u m b e r of P y r o n i n o p h i l i c C e l l s in T h e m (M • m)

Group of animals

]Number of pyroninophilic cells in Lymphoid Weight of Lengthof Diameter Oflreactiv e centers, ~ follicles withsplenic fol- I. ~ spleen, mg spleen m m . . ] under 65 under30 7nder-7-i~ out reactive ncles, /~ ] ceils per ceils per ceils per centers, 07o | field of field of field of vision vision [ t [ vision

1710,0+-0,62 55,0+0,12 Control 30th day of hjdrocorti536,0 6,41 45,0---2,11 sone adminlstration

346,8+-12,l

39,3_--4:0,63 22,2---0,45

17,3--.0,34

2i ,2-!-_0,24

14,3+-0,84

18,5--.0,68

61,1--.1,4

2t,5+0,12

39,5---_0,69

30,5---0,48

30,7---0,56

25,5---0,67

23,1--.1,15

184,7--.2,92 6,1--.0,36 30th day after end of hydrocortisone ad1359,0--.15,5 50,8+0,87 210,6--.12,3 8,5+0, l l ministration ' 30th day of stimulation of regeneration after end of hydrocortisone administration 1755,0--.14,6 56,0+-0,13 288,9-+-i3,4 20,7_0,99

Fig. 1. E x t e r n a l a p p e a r a n c e of r a b b i t s p l e e n : a) c o n t r o l ; b) on 30th day a f t e r b e g i n n i n g of h y d r o c o r t i s o n e i n j e c t i o n s ; c) 30 days a f t e r end of h y d r o c o r t i s o n e i n j e c t i o n s , d) 30th day a f t e r end of h y d r o c o r t i s o n e i n j e c t i o n s and b e g i n n i n g of a d m i n i s t r a t i o n of r e g e n e r a t i o n s t i m u l a t o r . stained by Braehet's method, the number of splenic lymphatic follicles in a field of vision was counted, their diameter was measured, and the presence of reactive centers in them and the number of pyroninophilic cells in these centers were determined. The numerical data were subjected to statistical analysis. EXPERIMENTAL

RESULTS

Throughout the period of the experiments no appreciable variation was found in the Congo Red index of the control animals (90.8 • 0.87 to 89.3 • 0.44). Under the influence of hydrocortisone it fell significantly, to 73.6 • 0.01 by the 30th day. The index rose 1 month after the end of administration of the hormone (83.2 = 2.08), but did not reach the control level. Stimulation of regeneration after administration of the hormone increased this index and brought it close to the control level. Prolonged administration of hydrocortisone caused emaciation of the animals. They became apathetic and their fur became rough and lost its sheen. However, on the 30th day of the experiment, the animals receiving hydrocortisone had increased in weight by 380.0 i 33.6 g, and 1 month after the end of its administration by 750.0 • 52.1 g (compared with 980.0 • 12.5 g in the control). The results obtained by stimulation of the animals differed considerably from those in the groups mentioned above (1140.0 • 26.0). Under the influence of the hormone the weight of the spleen on the 30th day was reduced to 536.0 ~ 6.4 mg and its l e n g t h to 45.0 • 0.1 r n m , c o m p a r e d with 1710.0 • 0.62 mg and 55.0 ~: 0.2 m m r e s p e c t i v e l y in the c o n t r o l ( T a b l e 1). The weight of the s p l e e n showed s o m e r e c o v e r y 1 m o n t h a f t e r the end of h y d r o c o r t i s o n e i n j e c t i o n s to r e a c h 1350.0 • 15.5 mg and its length 50.8 • 0.87 m m (Fig. 1). A s i g n i f i c a n t change was o b s e r v e d when

1765

Fig. 2. Section through spleen of rabbits: a) control (intact spleen) ; b) on 30th day after beginning of h y d r o c o r t i s o n e injections; c) 30 days after end of h y d r o c o r t i s o n e injections; d} on 30th day a f t e r end of hydrocortisone injections and beginning of administration of r e g e n e r a t i o n stimulator. 125 • r e g e n e r a t i o n was stimulated after the end of h y d r o c o r t i s o n e injections. After 1 month the weight of the spleen was fully r e s t o r e d , to reach 1755.0 + 14.6 mg and its length was 56.0 • 0.13 mm. The splenic lymphoid follicles in the spleen of the rabbits receiving hyd~'ocortisone for 30 days were indistinctly d e m a r c a t e d f r o m the red pulp (Fig. 2). In most cases they consisted of random collections of lymphocytes, forming small islets. Reactive c e n t e r s disappeared f r o m many of the splenic follicles. The number of foltiees per field of vision remained a l m o s t unchanged under the different experimental conditions c o m p a r e d with the control, but t h e i r diameter changed a p p r e c i a b l y depending on the experimental conditions. For instance, on the 30th day of h y d r o c o r t i s o n e injections the mean d i a m e t e r of the splenic follicles was 184.7 • 2.9 p, rising to 210.6 9 12.3 p 1 month after the end of injection of the hormone c o m p a r e d with 346.8 + 12.1 p in the control. When a r e g e n e r a t i o n s t i m u l a t o r was used the i n c r e a s e in the d i a m e t e r of the splenic follicles was g r e a t e s t , and reached 288.9 9 13.4 p at the same period of observation, although it was stiI1 below the d i a m e t e r o b s e r v e d in the control animals. After 4 weeks of hormone injections the capsule and c o n n e c t i v e - t i s s u e septa of the spleen had become ~hinner and, after impregnation with silver nitrate, the argyrophilic fibers in them were c l e a r l y visible. The w a l l s of the central a r t e r i e s were thickened and often obliterated. In such c a s e s the splenic lymphoid follicles were either g r o s s l y atrophied or had lost their outlines and changed into random c l u s t e r s of deformed lymphocytes, staining dark blue with hematoxylin. Usually the reactive center was absent in such follicles. The number of these follicles was 61.1 • 1.4%. As these experiments showed, under the influence of hydrocortisone the n u m b e r of splenic lymphoid follicles containing pyroninophilic cells after 1 month of observation had fallen to 38.9% compared with 78.8% in the control. Impregnation with s i l v e r showed a c l e a r e r and denser network or argyrophilic fibers at the p e r i p h e r y of the splenic follicles. Some i n c r e a s e in the thickness of the capsule and c o n n e c t i v e - t i s s u e septa was observed. A l u m e n was found in nearly all the v e s s e l s . Most of the splenic lymphoid follicles were c l e a r l y d e m a r c a t e d from the red pulp, but at the s a m e time there were other follicles still consisting of r a n d o m clusters of lymphocytes. The number of splenic follicles without reactive centers was 30.5 • 0.48%. No signs of obliteration were found after injection of colamine hydrochloride. The splenic follicles were better formed than in the previous group. The number of splenic follicles without reactive c e n t e r s was reduced, whereas the number of follicles containing many pyroninophilie cells p e r field of vision was appreciably i n c r e a s e d , although not up to the control level. The results thus indicate that during prolonged administration of h y d r o c o r t i s o n e the Congo Red index falls, reflecting the state of the absorptive function of the RES. Under the influence of the hormone atrophy of the spleen develops, as shown by a d e c r e a s e in the weight and size of the organ, in the d i a m e t e r of the splenic

1766

lymphoid follicles, and in the number of lymphocytes in the red pulp. The splenic follicles lose their configuration and change into r a n d o m c l u s t e r s of lymphocytes, and most of them lose their r e a c t i v e center. Condensation of the capsule and c o n n e c t i v e - t i s s u e s t r o m a is observed. After the end of hormone injections the absorptive function of the RES i n c r e a s e s again, the general condition of the animals i m p r o v e s , the atrophic changes in the spleen cease, the number of lymphocytes in the organ i n c r e a s e s , and the n o r m a l splenic lymphoid follicles are r e s t o r e d (reappearance of the reactive centers and an i n c r e a s e in the number of pyroninophilic cells in them). A d m i n i s t r a t i o n of a r e g e n e r a t i o n s t i m u l a t o r after the end of hormone injections appreciably a c c e l e r a t e s both the r e c o v e r y of the a n i m a l ' s general condition and r e s t o r a t i o n of the atrophied spleen. One month after the end of administration of h y d r o c o r t i s o n e , the spleen which had partially atrophied under the influence of the hormone is almost completely r e s t o r e d . If colamine hydrochloride is given under these conditions, r e g e n e r a t i o n of the organ takes place m o r e rapidly and the weight of the spleen regains the control level during the same period of 6bservation. LITERATURE 1.

2. 3.

4. 5. 6.

7.

CITED

Ya. L. Germanyuk and A. G. Minchenko, Byull. ]~ksp. Biot, Med., No. 2, 53 (1972). K. P. Zak and B. M. Khomenko, in: P r o b l e m s in Endocrinology and Metabolism [in Russian], No. 1, Kiev (1970), pp. 6 2 - 7 t . K. P. Zak and N. I. Naumenko, Vrach. Delo, No. 3, 129 (1971). L. D. Liozner, G. V. Kharlova, and B. M. Levitina, Trudy Moskovsk. Obshch. Ispyt. P r i r o d y . Otdel Biol., 2, 17 (1961). L. D. Liozner, Arkh. Patol., No. 7, 66 (1974). A. A. Mezhlumyan, Byull. Eksp. Biol. Med., No. 5, 103 (1964). D. S. Sarkisov, Arkh. Patol., No. 7, 70 (1974).

HUMORAL

MECHANISMS

OF REPARATIVE

OF REGULATION

OSTEOGENESIS

V. I. F i l i m o v , N. P . L e p e k h o v a , V. O. N e d o s p a s o v , K. S. D e s y a t n i c h e n k o , a n d V. L. T e t e r i n

UDC 612.753-06

The effect of the blood s e r u m f r o m animals with active osteogenesis on the biosynthesis of nucleic acids and protein and on mineralization of regenerating bone t i s s u e was studied in experiments in vivo and in vitro. Incorporation of labeled p r e c u r s o r s of DNA ([3H]thymidine) and protein ([~4C]proline) in the recipients was intensified and mineralization of bony callus (incorporation of 85Sr) was a c c e l e r a t e d . C o m p a r i s o n of the o r d e r of s t i m u l a tion of nucleic acid and protein synthesis suggests that the active principle of the s e r u m p r o m o t e s m o r e rapid cell proliferation in the f r a c t u r e zone. KEY WORDS: r e p a r a t i v e o s t e o g e n e s i s ; humoral regulation; radioactive isotopes

One of the aspects of the p r o b l e m of the regulation of r e p a i r p r o c e s s e s that has received the least study is the control of the natural c o u r s e of r e p a r a t i v e osteogenesis. The only information available is concerned with the stimulating e f f e c t of breakdown products of bone [4] or of the blood s e r u m of animals with f r a c t u r e s [1, 8] on this p r o c e s s . The p r e s e n t w r i t e r s [7] have also found that during the period of intensive osteogenesis the blood s e r u m of animals acquired the p r o p e r t y of stimulating f r a c t u r e healing. L a b o r a t o r y of Pathophysiology and L a b o r a t o r y of B i o c h e m i s t r y , R e s e a r c h Institute of Experimental and Clinical Orthopedics and Traumatology, Kurgan. (Presented by Academician of the A c a d e m y of Medical Sciences of the USSR N. A. Fedorov.) T r a n s l a t e d from Byulleten' t~ksperimental'noi Biologii i Meditsiny, Vol. 84, No. 12, pp. 725-727, D e c e m b e r , 1977. Original a r t i c l e submitted April 6, 1977.

0007-4888/77/8412-1767507.50

01978 Plenum Publishing Corporation

1767