Effect of the Sorbent Enterosgel on the Tissue Microregion of Liver and Regional Lymph Nodes in Rats with Chronic Toxic

Ischenko I.Yu. Effect of the sorbent “Enterosgel” on the tissue microregion of liver and

regional lymph nodes in rats with chronic toxic hepatitis / pp. 61-64

UDC 612.428:616.36-002:612.014.462.9

I.Yu. Ischenko, S.V. Michurina

State Institution Scientific Research Institute of Clinical and Experimental Lymphology, Siberian Branch of the Russian Academy of Medical Sciences, Novosibirsk

Combined action of carbon tetrachloride and ethanol causes a mixed pattern of pathologic changes in liver. The changes in the regional lymph nodes are due to the overall effect of immunosuppressive action of carbon tetrachloride and immunostimulating influence of ethanol. At the end of recovery period, there was an increase in the functional activity and intensity of recovery processes in the parenchymatous cells of liver. The state of capillary-connective tissue compartment remains strained. Paracortex suppression, stimulation of B-dependent regions and drainage function evidence the strained state of hepatic lymph nodes. The use of the sorbent "Enterosgel" in the recovery period has a corrective effect on the lymph nodes, which favors the fastest recovery of liver. However, the regression of fibrous process in the tissue liver microregion was observed only in the part of experimental animals, which evidences the necessity for continuation of this study.

Key words: hepatitis, lymph nodes, sorbent

The environmental pollution with various technogenic wastes and violent spread of hepatitis viruses lead to the growth in the number of chronic diffuse liver diseases of different etiology. Alcohol remains one of the widespread factors of liver damage. The syndrome of endogenous body intoxication caused by the cytolysis of hepatic parenchymatous cells and accumulation of toxic products in the pericellular region result in the disturbance in the cell metabolism and weakening of regulatory and adaptive functions of organs and systems purifying the internal environment, viz., liver and lymphatics.

Therefore, the efferent methods including enterosorption gain increasingly wider acceptance in the liver pathology of different genesis. The idea of using enterosorption consists in the prevention of toxin entering to lymph and blood and fixation of auto- and exotoxins at the surface of sorbent followed by their elimination from intestine. Thus, by undertaking the outflow of toxic products from tissues and reducing body intoxication with the highly toxic lymph, the sorbent interrupts the formation of pathogenic scheme of endotoxemia [2].

Purification of the internal environment requires continuous multilevel control. In such case, liver is the first barrier system carrying out detoxication at the body level. The regional lymph nodes perform drainage-detoxication function toward liver.

Taking into consideration mentioned above, the aim of the present work was to perform histologic and morphometric studies (at the light-optical level) of tissue microregion of liver and regional lymph nodes in rats with the experimental model of chronic toxic hepatitis using the sorbent “Enterosgel” in the recovery period.

The medication “Enterosgel” is polyorganosiloxane gel-like enterosorbent. It adsorbs the toxic substances and products of incomplete metabolism with molecular weight of 70 to 100 Dalton from intestinal contents and through membranes from blood plasma [6].

Methods

The eugamic male rats Wistar with a weight of 50-180 g were used in the experiment. The animals was received from the vivarium of Institute of cytology and genetics of the Siberian Branch of the Russian Academy of Sciences. The model of experimental chronic toxic hepatitis (CTH-28) was produced for 28 days by administration of a 25% oily solution of carbon tetrachloride through a gastric tube in a dose of 0.1 mL per 100 g of body weight twice a week 30 minutes before feeding and 5% ethanol in the form of drink (instead of water) daily ad libitum [5]. After termination of toxic exposure, Enterosgel (EG) was administered for 28 days (recovery period) (every day through a tube in a dose of 1 g per 1 kg of body weight 1 hour before feeding) (group CTH28+EG28). The animals received no sorbent in the recovery period (group CTH28+28) served as the reference group.

Animal keeping, feeding, care and their removal from the experiment were carried out in accordance with the requirements of “The rules for performing works using experimental animals” (Appendix to the order No. 755 of the Ministry of Health of USSR from 12.08.1977). The animals were decapitated under the ether narcosis. The liver samples and regional lymph nodes of liver situated in the hepatoduodenal band were taken for the light-optical and morphometric investigations. The liver pieces and lymph nodes were fixed in Telesnitsky’s solution. The histological material was performed according to the standard procedure, Mayer’s hematoxylin-eosin staining and Van Gieson’s stain on the connective tissue.

For the morphormetric analysis of tissue microregion of liver and structure-functional regions of lymph nodes, the stereologic method of punctated count using a morphometric mesh was applied [1]. Statistical data processing was performed using Student’s t-test. The differences between values to be compared were considered as statistically significant at p<0.05.

Results

The morphologic study of tissue microregion of the liver of animals with the model of chronic toxic hepatitis (CTH-28) showed considerable abnormalities corresponding to the combined effect of ethanol and carbon tetrachloride: change in the balk structure of hepatic lobes, pronounced polymorphism of parenchymatous cell, fat and ballooning degeneration of hepatocytes, karyorhexis, karyolysis, hepatocyte necrosis, especially, in the center-lobular regions. The signs of disturbed blood and lymph circulation, such as disappearance of habitual sinusoid network, expansion of lymphatic clefts in the regions of hepatoportal tracts, and appearance of sinuosity of central veins were found in the CTH-28 rats. Around the central veins, there was accrementition of the fibrous tissue. In the regions of hepatoportal tracts, we observed the appearance of inflammatory infiltrates with the predominant content of lymphocytes (Photo 1).

The above-described abnormalities did not allow performing the morphometric analysis that would adequately reflect the processes occuring in the tissue microregion of liver upon CTH-28. The damages are characterized by the center-lobular localization. The absence of significant changes in the regional lymph nodes (Table 2) was appear to be the result of overall effect of immunosuppressive action of carbon tetrachloride [7] and immunostimulating influence of ethanol [3, 8]. In addition, the drainage function of lymph nodes strengthened.

28 days after termination of toxic exposure (CTH28+28), in the tissue microregion of liver, we observed the appearance of powerful connective-tissue bands forming false lobes and, in the regions of hepatoportal tracts, we found out infiltrates with the predominant content of lymphocytes. However, no hepatocyte polymorphism and degeneration were virtually observed (Photo 2). The parenchymatous cells had a round shape and contained the weakly basophilic cytoplasm and nuclei with the sharp correct outlines. In the nuclei of hepatic cells, we found one or two nucleoluses; heterochromatin was positioned near the nuclear membrane in the form of lumps.

An increase in the volume density of nuclei and nucleoplasmic ratio in hepatocytes and a growth in the portion of diplokaryocytes among them were established by morphometry (Table 1), which evidence the enhancement of functional activity and intensity of recovery processes in the parenchymatous hepatic cells. According to V.P. Kaznacheev and M.Ya. Subbotin, the progress of recovery processes is usually accompanied by a high functional strain of capillary connective-tissue structures [4]. Reconstruction of usual sinusoid network and almost 2-fold increase in their volume density, a growth in the number and volume density of Kupffer’s cells in the animals of group CTH28+28 compared to the control apparently reflects the strained condition of capillary connective-tissue compartment of the tissue liver microregion.

Table 1

Results of morphometry for the tissue liver microregion in animals (M±m)

Indices

Control

Group CTH28+28

Group CTH28+EG28

V of hepatocyte cytoplasm гепатоцитов

84.3±0.37

76.78±0.71*

75.09±0.47*

V of of hepatocyte nuclei

9.71±0.23

10.73±0.32*

11.18±0.38*

Nucleoplasmic ratio

0.12±0.003

0.14±0.005*

0.15±0.006*

N of hepatocytes

30.08±0.8

24.1±0.59*

28.15±0.78**

N of binuclear hepatocytes

0.84±0.16

3.25±0.2*

2.6±0.27*

V of Kupffer’s cells

1.78±0.13

4.42±0.28*

4.47±0.26*

N of Kupffer’s cells

7.32±0.42

15.15±0.73*

15.05±0.65*

V of sinusoids

4.21±0.26

8.07±0.43*

9.27±0.36*.**

Note: V (%) is volume density; N (%) is number density, i.e., the number of cells per area unit; the differences are significant (p<0.05): * – with control, ** with the group CTH28+28.

In the regional lymph nodes, we observed subsequent attenuation of the T-dependent region and progress of the B-dependent regions (Table 2). In the hepatic lymph nodes in the group CTH28+28 rats, the volume densities of cortex and paracortex therein (T-dependent zone) were significantly decreased compared to the control. The volume densities of lymphoid nodules, germinal centers, and cerebral bands (B-dependent zones) were increased. The volume density of sinuses in the medullary substance increased. Thus, the structural reorganizations in the lymph nodes evidence deep changes in the conditions of carrying out the immune reactions by the cellular or humoral type.

The morphological study of the tissue liver microregion in the rats received the sorbent "Enterosgel" in the recovery period (CTH28+28) allowed definition of the changes similar to those in the animals of the group without treatment: appearance of the powerful connective-tissue bands forming false lobes; virtually complete absence of hepatocyte degeneration (Photo 3); partial reconstruction of the sinusoid network with the stasis of erythrocytes being noted therein; and the presence of infiltrates with the predominant content of lymphocytes in the region of hepatoportal tracts. However, the part of the animals of this group showed the absence of the connective-tissue interlayers in the hepatic parenchyma.

An increase in the volume density of hepatocyte nuclei and nucleoplasmic ratio and a growth in the number density of parenchymatous cells and diplokaryocytes among them compared to the control were found by morphometry (Table 1), which evidence the enhancement of functional activity and intensity of recovery processes in the parenchymatous hepatic cells. The number density of all hepatocytes in the group

Photo 1. Tissue liver microregion of the rats with CTH28: fat and ballooning degeneration of hepatocytes; parenchymatous cell death; predominat damages of hepatic parenchyma in the regions around the central veins. Hematoxylin-eosin staining. Objective 20, ocular 10.

Photo 2. Tissue liver microregion of the rats with CTH28+28. Powerful connective-tissue interlayers form false lobes in the hepatic parenchyma. The absence of polymorphism of parenchymatous cells, hepatocyte degeneration, and pronounced patterns of necrosis. Hematoxylin-eosin staining. Objective 4, ocular 10.

Photo 3. Tissue liver microregion of the rats with CTH28+EG28: connective-tissue interlayers forming false lobes in the hepatic parenchyma. The absence of polymorphism of parenchymatous cells, hepatocyte degeneration, and patterns of necrosis. Van Gieson’s stain. Objective 10, ocular 10.

Fig. 4. The absence of connective-tissue interlayers in the liver of some animals from the group CTH28+EG28. Formation of usual sinusoid network. The absence of polymorphism of parenchymatous cells, hepatocyte degeneration, and patterns of necrosis. Cellular infiltration with the predominance of lymphocytes around the hepatoportal tracts. Hematoxylin-eosin staining. Objective 10, ocular 10.

Table 2

Volume densities of the structure-functional zones of the regional hepatic lymph nodes in animals (M±m)

Indices

Control

Group CTH28

Group CTH28+28

Group CTH28+EG28

Cortical/medullary indexозговой индекс

2.65±0.57

2.8±1.56

0.91±0.11

1.52±0.23

V of marginal sinus

2.83±0.41

4.3±1.54

3.41±0.84

3.0±0.8

V of cortex

71.04±4.52

62.35±13.19

46.52±3.92*

58.99±3.36

V of paracortex

53.95±3.3

45.2±6.96

26.67±2.8*

43.04±2.81*. ***

V of lymphoid nodules

10.61±2.08

16.14±4.84

16.37±3.01

12.95±1.66

V of germinative centres

2.15±0.34

1.57±0.11

3.34±0.97

2.64±0.41**

V of medullary substance

28.96±4.52

37.65±13.19

51.11±1.94*

41.01±3.36***

V of medullary bands

20.85±2.89

23.61±8.17

34.82±3.28*

28.31±1.92

V of medullary sinus

8.11±2.36

14.04±5.21

18.73±0.75

12.7±2.2***

Note: V (%) is volume density. The differences are significant (p<0.05): * – with control, ** with the group CTH28, *** – with the group CTH28+28.

with Enterosgel was greater than that in the group without treatment and reached the level in intact animals. As in the group CTH28+28, the state of capillary connective-tissue compartment remained strained, i.e., the volume and number densities of Kupffer's cells increased compared to the control and the volume density of sinusoids became increasingly higher.

Thus, the use of the sorbent “Enterosgel” in the recovery period had a protective effect, which was manifested in the recovery of the number density of hepatocytes in the tissue liver microregion up to the control level and, in the part of animals, still in the regression of fibrous process.

The study of regional hepatic lymph nodes (Table 2) showed the lymph-corrective effect of Enterosgel. The use of this sorbent promoted the growth of the volume density of cortical substance and, particularly, paracortex (T-dependent zone) compared to the group CTH28+28, caused the considerable tendency for decrease in the volume density of lymphoid nodules, germinative centres, and cerebral bands (B-dependent zones), and restored the volume density of cerebral sinuses.

Conclusion

The combined action of carbon tetrachloride and ethanol results in the progress of chronic toxic hepatitis with the manifestations of fat and ballooning degeneration of hepatocytes, hepatic cell death, disturbed blood and lymph circulations, activation of fibrogenesis around the central veins, and cellular infiltration with the predominant content of lymphocytes. No significant changes occur in the regional lymph nodes providing lymphatic detoxication of the organ itself. Apparently, this fact can be explained by the result of the overall effect of immunosuppressive action of carbon tetrachloride and immunostimulating influence of ethanol.

At the end of recovery period after termination of toxic exposure, there were the enhancement of functional activity and intensity of recovery processes in the parenchymatous hepatic cells. The state of capillary-connective tissue compartment remains strained. The structural reorganizations in the lymph nodes evidence deep changes in the conditions of carrying out the immune reactions by the cellular or humoral type.

The use of the organosilicone sorbent “Enterosgel” after termination of intoxication had the corrective effect on the regional hepatic lymph nodes and brings the ratio of structure-functional zones virtually to the control level. By undertaking the outflow of toxic products from tissues and reducing body intoxication with the highly toxic lymph, the sorption material plays the role of lymph-prosthetic mechanism [2]. Enterosgel allows one to form the conditions for normalization of the microanatomical organization of hepatic lymph nodes, which, in turn, should favour the fastest reconstruction of drainage organ, i.e., liver.

The latter is confirmed by the increase in the functional activity of parenchymatous hepatic cells, enhancement of the regenerative ability of the organ, and the regression of fibrous process therein. However, the regression of fibrous process in the tissue liver microregion was observed only in the part of experimental animals, which evidences the necessity for continuation of this stud

EFFECT OF THE SORBENT “ENTEROSGEL” ON THE TISSUE MICROREGION OF LIVER AND REGIONAL LYMPH NODES IN RATS WITH CHRONIC TOXIC HEPATITIS

I.Yu. Ischenko, S.V. Michurina

Combined action of carbon tetrachloride and ethanol causes a mixed pattern of pathologic changes in liver. The changes in the regional lymph nodes are due to the overall effect of immunosuppressive action of carbon tetrachloride and immunostimulating influence of ethanol. At the end of recovery period, there was an increase in the functional activity and intensity of recovery processes in the parenchymatous cells of liver. The state of capillary-connective tissue compartment remains strained. Paracortex suppression, stimulation of B-dependent regions and drainage function evidence the strained state of hepatic lymph nodes. The use of the sorbent "Enterosgel" in the recovery period has a corrective effect on the lymph nodes, which favors the fastest recovery of liver. However, the regression of fibrous process in the tissue liver microregion was observed only in the part of experimental animals, which evidences the necessity for continuation of this study.

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