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Effects of the liver homogenate obtained following partial hepatectomy on cirrhosis of the liver
Exp. Path., Bd. 11, S. 215-218 (1975) Institute of Human Anatomy, Medical University, Tirgu-Mure~, Romania (Head: Prof. Dr. T. MAROS, Corresponding Member of the Academy of Medical Sciences)
Effects of the liver homogenate obtained following partial hepatectomy on cirrhosis of the liver By 1. SERES-STURM, T. MARaS, O. LAKATOS, M. SERES-STURM, E. MODY and V. BLAZSEK With 2 figures (Received April 29, 1975) Key-words: partial hepatectomy; liver regenerate; lyophilized homogenate; collagenolytic effect; rat
Summary The authors studied the influence of liver homogenates as a whole and collected at various intervals after partial hepatectomy on cirrhosis of the liver. The lyophilized homogenates were administered over a period of 6 weeks to various groups of albino rats pretreated with CClt for a period of 6 months. The normal liver homogenate did not influence the histological and biochemical picture of the hepatocirrhosis. The material collected 48 hours after partial hepatectomy causes a moderate stimulation of the mechanisms of parenchymatous regeneration. 7 days after partial hepatectomy (in the postmitotic period) the hepatic regenerate shows a biological effect with lysis of collagen fibres and protection of parenchymatous cells. In the present study we have investigated the influence of liver homogenates after partial hepatectomy on advanced cirrhotic lesions of the liver. The present literature contains only few data on this (2, 3, 4, 6, 7, 9, 10, 12).
Material and methods Healthy male albino rats with an average body weight of 150 g were used. They were injected Lm. 0.1 to 0.2 ml/100 g body weight of a mixture containing CClt + Oleum Helianthi twice per week for a period of 6 months; all rats were given 48 doses of the toxic substance. 10 rats were sacrificed at the end of the 6th month. The material collected from this group served as a standard for comparing of the data obtained from the surviving rats which were continued to receive a weekly toxic maintenance dose of cirrhosis (t. m. d. = 0.1 ml CClt /100 g body weight) or a lyophilized liver homogenate (L. 1. h.) as follows: Group I (17 rats) were given the t. m. d. once a week + normal L.1. h. Group II (20 rats) were treated with the t. m. d. + L. I. h. collected from partially hepatectomized rats, according to the technique of HIGGINS and ANDERSON (8), 48 hours (mitotic period) after operation. Grou p I II (20 rats) were given the t. m. d. + L.1. h. prepared from the residual liver at 7 days (postmitotic period) after partial hepatectomy. For lyophilization the liver was removed under aseptic conditions and homogenized in an isotonic solution of sterile saccharose. After centrifugation (3,500 x g/20 min), the supernatant was lyophilized and preserved in ampoules. This was used for the preparation of the 10 % isotonic solutions doses of which of 0.2 ml/lOO g (20 mg fresh liver/lOO g) body weight were injected intramuscularly in 3 doses per week. The treatment lasted 6 weeks, during which the animals were given a total of 18 doses of L. 1. h. The surviving rats of the 3 groups were sacrificed by decapitation. The following analyses were used: a) Histological processing of the liver fragments, after fixation in a 10% sol. of for mol and Carnoy's solution, staining with HE according to Szekely-Goldner's method for connective tissue and Sudan III stain. b) Determination of the HO-proline concentration in the liver by spectrophotometric means according to NEUMAN'S (11) method; the equivalent of collagen being obtained by multiplication of the data with the coefficient of 7.46.
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c) Spectrophotometric determination of the liver DNA with the aid of the diphenylaminic method using AGRELL'S (1) equation. d) Dosage of the activity of the serum aminotransferases (SGOT and SGPT) according to BERGMEYER and BERNT'S (5) method.
Results Histological findings The cirrhotic control group shows all characteristics of an advanced cirrhosis (fig. 1). Group I does not show any differences from that. In addition to the changes of group I and the controls group II shows a moderate hyperplasia of parenchymal nodules. In group III the lobular architecture appears to be relativeley well-preserved. The connective tissue much diminished as compared to the previous groups appears to be withdrawn in the porto-biliary spaces as well as in the adjacent areas of the lobules. The pseudolobules are not noticeable. Only very slight dystrophic alterations are present (fig. 2). Biochemical aspect The amount of collagen and liver DNA, expressed in mg/100 g of fresh liver, as well as the ratio between these indicators (collagen/DNA) are represented in table 1. In the same table we have also included the data regarding the activity of the serum aminotransferase. In the columns P 1 is demonstrating the situation in comparison with the healthy animals (10 rats), P 2 is expressing the relation to the cirrhotic controls. Table 1 Number Collagen of mgJI00 g fresh animals liver used
DNA Co - SGOT mg/l00 g fresh DNA I. D./ml liver
SGPT I. D./ml
Healthy
10
211.3 ± 8.5
344.5 ± 13.8
0.6
54.1 ± 1.7
17.1±1.5
Cirrhotic (6 months)
10
449 ± 13.4
377.2 ± 7.2
1.2
111.8 ± 6.0
41 ± 2.4
Group I
12
430 ± 23.6 PI = 0.01 P 2 = 0.50
360 ± 35.6 PI = 0.90 P 2 = 0.70
1.2
103.5 ± 6.2 PI = 0.01 P 2 = 0.40
41.6 ± 3.8 PI = 0.001 P 2 = 0.90
Group II
14
423 ± 21.6 PI = 0.001 P 2 = 0.30
523.2 ± 8.2 PI = 0.001 P 2 = 0.001
0.8
105.2 ± 18.4 PI = 0.02 P2 = 0.90
39.6 ± 3.4 PI = 0.001 P 2 =0.90
Group III
16
294.4 ± 38.3 PI = 0.10 P 2 = 0.01
443 ± 10 PI = 0.001 P 2 = 0.001
0.6
73.5 ± 2.4 PI = 0.001 P 2 = 0.001
24.9 ± 3 PI = 0.05 P 2 = 0.001
Discussion The 3 experimental groups have shown a variable picture, depending on the origin of the liver homogenates. Whereas the normal liver homogenate does not influence the histological and biochemical features of the 6 months cirrhosis the homogenates prepared from the hepatic regenerate are acting differently on the parenchymatous and stromal lesions. The material collected 48 hours after partial hepatectomy effectuates a moderate stimulation on the regeneration of parenchymal cells, as reflected by hyperplasia of the parenchymatous nodules and the increase of the DNA concentration within the liver cells, but does not influence the amount and disposition of the hyperplastic connective tissue. A significant
216
Fig. 1. Hepatic cirrhosis after 6 months of treatment with CCl t (control groups). Szekely-Goldner's trichrome staining, x 87.
Fig. 2. HepatIc cirrhosis (after 6 months) treated with the lyophilized liver homogenate prepared from the hepatic regenerate in postmitotic period. Szekely-Goldner's trichrome staining, x 87.
217
increase in the concentration of DNA implies the decrease of the collagen/DNA ratio. The aminotransferases are reflecting an intense activity. Contrarily the homogenate prepared from the hepatic regenerate 7 days after partial hepatectomy essentially modifies the morphological picture and the biochemical parameters of the hepatic cirrhosis. These effects are reflected by the global decrease of the hyperplastic connective tissue, the re-establishment of lobular architecture, the attenuation of the dystrophic lesions of the parenchyma. One may also note a significant decrease of collagen, a marked increase of the liver DNA and the return to normal of the collagen/DNA ratio. The activity of the aminotransferases proved to be lower than the values obtained in the cirrhotic controls without returning, however, to the normal level. It seems that the phase of 7 days (postmitotic period) following the partial hepatectomy is the optimal time for the collection of a polyvalent biological material capable of unleashing a complex effect, protective and collagenolytic, which under experimental conditions contributes fully to the regression of the lesions in the cases of advanced cirrhosis.
Literature 1. AGRELL, I. P. S., Nucleohistone relations in model experiments. Biochim. Biophys. Acta 186, 226 (1969). 2. BARTOK, I., Gy. DOMJAN, E. HORVATH and B. KORPASSY, Histochemical and biochemical studies of rat liver in experimental cirrhosis after partial hepatectomy. Nature 186, 397 (1960). 3. - A. TOSZEGI and I. TOROK, A cirrhotikus maj regeneratiojanak humoraIis befolyasolasa. Kiserl. Orvostud. 17, 409 (1965). 4. BAZIN, S., and A. DELAUNAY, Activite collagenasique de certains extraits hepatiques: Ses variations chez Ie rat intoxique par tetrachlorure de carbone. Ann. Inst. Pasteur (Paris) 106, 543 (1964). 5. BERGMEYER, H. U., and E. BERNT, Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim 1962. 6. FRANKLAND, D. M., and C. H. WYNN, Observations on the influence of rat-liver preparations on collagen. Biochem. J. 81, 25 (1961). 7. - - The degradation of acid-soluble collagen by rat-liver preparations. Biochem. J. 85, 276 (1962). 8. HIGGINS, G. M., and R M. ANDERSON, Experimental pathology of the liver. Restoration of the liver of the white rat following partial surgical removal. Arch. Path. 12, 186 (1931). 9. MAROS, T., and L. SERES-STURM, Regenerarea ficatului. Edit. Acad. R. S. R, Bucure~ti 1969. 10. MORRIONE, TH. G., and J. LEVINE, Collagenolytic activity and collagen resorption in experimental cirrhosis. Arch. Path. 84, 59 (1967). 11. NEUMAN, R E., Determination of hydroxiproline. In: Aminoacid Handbook (ed. by R J. BLOCK). Thomas, Springfield 1956. 12. OLSSON, R, Partial hepatectomy in experimental carbon tetrachloride induced liver fibrosis. Acta chir. scand., Suppl. 366 (1966). Author's address: Dr. sc. med. L. SERES-STURM, Dozent, Institute of Anatomy, Medical University, Str. Gh. Marinescu nr. 38, Tirgu-Mure~ (Romania).
218
Effects of the liver homogenate obtained following partial hepatectomy on cirrhosis of the liver By 1. SERES-STURM, T. MARaS, O. LAKATOS, M. SERES-STURM, E. MODY and V. BLAZSEK With 2 figures (Received April 29, 1975) Key-words: partial hepatectomy; liver regenerate; lyophilized homogenate; collagenolytic effect; rat
Summary The authors studied the influence of liver homogenates as a whole and collected at various intervals after partial hepatectomy on cirrhosis of the liver. The lyophilized homogenates were administered over a period of 6 weeks to various groups of albino rats pretreated with CClt for a period of 6 months. The normal liver homogenate did not influence the histological and biochemical picture of the hepatocirrhosis. The material collected 48 hours after partial hepatectomy causes a moderate stimulation of the mechanisms of parenchymatous regeneration. 7 days after partial hepatectomy (in the postmitotic period) the hepatic regenerate shows a biological effect with lysis of collagen fibres and protection of parenchymatous cells. In the present study we have investigated the influence of liver homogenates after partial hepatectomy on advanced cirrhotic lesions of the liver. The present literature contains only few data on this (2, 3, 4, 6, 7, 9, 10, 12).
Material and methods Healthy male albino rats with an average body weight of 150 g were used. They were injected Lm. 0.1 to 0.2 ml/100 g body weight of a mixture containing CClt + Oleum Helianthi twice per week for a period of 6 months; all rats were given 48 doses of the toxic substance. 10 rats were sacrificed at the end of the 6th month. The material collected from this group served as a standard for comparing of the data obtained from the surviving rats which were continued to receive a weekly toxic maintenance dose of cirrhosis (t. m. d. = 0.1 ml CClt /100 g body weight) or a lyophilized liver homogenate (L. 1. h.) as follows: Group I (17 rats) were given the t. m. d. once a week + normal L.1. h. Group II (20 rats) were treated with the t. m. d. + L. I. h. collected from partially hepatectomized rats, according to the technique of HIGGINS and ANDERSON (8), 48 hours (mitotic period) after operation. Grou p I II (20 rats) were given the t. m. d. + L.1. h. prepared from the residual liver at 7 days (postmitotic period) after partial hepatectomy. For lyophilization the liver was removed under aseptic conditions and homogenized in an isotonic solution of sterile saccharose. After centrifugation (3,500 x g/20 min), the supernatant was lyophilized and preserved in ampoules. This was used for the preparation of the 10 % isotonic solutions doses of which of 0.2 ml/lOO g (20 mg fresh liver/lOO g) body weight were injected intramuscularly in 3 doses per week. The treatment lasted 6 weeks, during which the animals were given a total of 18 doses of L. 1. h. The surviving rats of the 3 groups were sacrificed by decapitation. The following analyses were used: a) Histological processing of the liver fragments, after fixation in a 10% sol. of for mol and Carnoy's solution, staining with HE according to Szekely-Goldner's method for connective tissue and Sudan III stain. b) Determination of the HO-proline concentration in the liver by spectrophotometric means according to NEUMAN'S (11) method; the equivalent of collagen being obtained by multiplication of the data with the coefficient of 7.46.
215
c) Spectrophotometric determination of the liver DNA with the aid of the diphenylaminic method using AGRELL'S (1) equation. d) Dosage of the activity of the serum aminotransferases (SGOT and SGPT) according to BERGMEYER and BERNT'S (5) method.
Results Histological findings The cirrhotic control group shows all characteristics of an advanced cirrhosis (fig. 1). Group I does not show any differences from that. In addition to the changes of group I and the controls group II shows a moderate hyperplasia of parenchymal nodules. In group III the lobular architecture appears to be relativeley well-preserved. The connective tissue much diminished as compared to the previous groups appears to be withdrawn in the porto-biliary spaces as well as in the adjacent areas of the lobules. The pseudolobules are not noticeable. Only very slight dystrophic alterations are present (fig. 2). Biochemical aspect The amount of collagen and liver DNA, expressed in mg/100 g of fresh liver, as well as the ratio between these indicators (collagen/DNA) are represented in table 1. In the same table we have also included the data regarding the activity of the serum aminotransferase. In the columns P 1 is demonstrating the situation in comparison with the healthy animals (10 rats), P 2 is expressing the relation to the cirrhotic controls. Table 1 Number Collagen of mgJI00 g fresh animals liver used
DNA Co - SGOT mg/l00 g fresh DNA I. D./ml liver
SGPT I. D./ml
Healthy
10
211.3 ± 8.5
344.5 ± 13.8
0.6
54.1 ± 1.7
17.1±1.5
Cirrhotic (6 months)
10
449 ± 13.4
377.2 ± 7.2
1.2
111.8 ± 6.0
41 ± 2.4
Group I
12
430 ± 23.6 PI = 0.01 P 2 = 0.50
360 ± 35.6 PI = 0.90 P 2 = 0.70
1.2
103.5 ± 6.2 PI = 0.01 P 2 = 0.40
41.6 ± 3.8 PI = 0.001 P 2 = 0.90
Group II
14
423 ± 21.6 PI = 0.001 P 2 = 0.30
523.2 ± 8.2 PI = 0.001 P 2 = 0.001
0.8
105.2 ± 18.4 PI = 0.02 P2 = 0.90
39.6 ± 3.4 PI = 0.001 P 2 =0.90
Group III
16
294.4 ± 38.3 PI = 0.10 P 2 = 0.01
443 ± 10 PI = 0.001 P 2 = 0.001
0.6
73.5 ± 2.4 PI = 0.001 P 2 = 0.001
24.9 ± 3 PI = 0.05 P 2 = 0.001
Discussion The 3 experimental groups have shown a variable picture, depending on the origin of the liver homogenates. Whereas the normal liver homogenate does not influence the histological and biochemical features of the 6 months cirrhosis the homogenates prepared from the hepatic regenerate are acting differently on the parenchymatous and stromal lesions. The material collected 48 hours after partial hepatectomy effectuates a moderate stimulation on the regeneration of parenchymal cells, as reflected by hyperplasia of the parenchymatous nodules and the increase of the DNA concentration within the liver cells, but does not influence the amount and disposition of the hyperplastic connective tissue. A significant
216
Fig. 1. Hepatic cirrhosis after 6 months of treatment with CCl t (control groups). Szekely-Goldner's trichrome staining, x 87.
Fig. 2. HepatIc cirrhosis (after 6 months) treated with the lyophilized liver homogenate prepared from the hepatic regenerate in postmitotic period. Szekely-Goldner's trichrome staining, x 87.
217
increase in the concentration of DNA implies the decrease of the collagen/DNA ratio. The aminotransferases are reflecting an intense activity. Contrarily the homogenate prepared from the hepatic regenerate 7 days after partial hepatectomy essentially modifies the morphological picture and the biochemical parameters of the hepatic cirrhosis. These effects are reflected by the global decrease of the hyperplastic connective tissue, the re-establishment of lobular architecture, the attenuation of the dystrophic lesions of the parenchyma. One may also note a significant decrease of collagen, a marked increase of the liver DNA and the return to normal of the collagen/DNA ratio. The activity of the aminotransferases proved to be lower than the values obtained in the cirrhotic controls without returning, however, to the normal level. It seems that the phase of 7 days (postmitotic period) following the partial hepatectomy is the optimal time for the collection of a polyvalent biological material capable of unleashing a complex effect, protective and collagenolytic, which under experimental conditions contributes fully to the regression of the lesions in the cases of advanced cirrhosis.
Literature 1. AGRELL, I. P. S., Nucleohistone relations in model experiments. Biochim. Biophys. Acta 186, 226 (1969). 2. BARTOK, I., Gy. DOMJAN, E. HORVATH and B. KORPASSY, Histochemical and biochemical studies of rat liver in experimental cirrhosis after partial hepatectomy. Nature 186, 397 (1960). 3. - A. TOSZEGI and I. TOROK, A cirrhotikus maj regeneratiojanak humoraIis befolyasolasa. Kiserl. Orvostud. 17, 409 (1965). 4. BAZIN, S., and A. DELAUNAY, Activite collagenasique de certains extraits hepatiques: Ses variations chez Ie rat intoxique par tetrachlorure de carbone. Ann. Inst. Pasteur (Paris) 106, 543 (1964). 5. BERGMEYER, H. U., and E. BERNT, Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim 1962. 6. FRANKLAND, D. M., and C. H. WYNN, Observations on the influence of rat-liver preparations on collagen. Biochem. J. 81, 25 (1961). 7. - - The degradation of acid-soluble collagen by rat-liver preparations. Biochem. J. 85, 276 (1962). 8. HIGGINS, G. M., and R M. ANDERSON, Experimental pathology of the liver. Restoration of the liver of the white rat following partial surgical removal. Arch. Path. 12, 186 (1931). 9. MAROS, T., and L. SERES-STURM, Regenerarea ficatului. Edit. Acad. R. S. R, Bucure~ti 1969. 10. MORRIONE, TH. G., and J. LEVINE, Collagenolytic activity and collagen resorption in experimental cirrhosis. Arch. Path. 84, 59 (1967). 11. NEUMAN, R E., Determination of hydroxiproline. In: Aminoacid Handbook (ed. by R J. BLOCK). Thomas, Springfield 1956. 12. OLSSON, R, Partial hepatectomy in experimental carbon tetrachloride induced liver fibrosis. Acta chir. scand., Suppl. 366 (1966). Author's address: Dr. sc. med. L. SERES-STURM, Dozent, Institute of Anatomy, Medical University, Str. Gh. Marinescu nr. 38, Tirgu-Mure~ (Romania).
218