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S.M. Gdanskyi, N.I. Gerasimyuk Features of Ultrastructural Changes in Hepatocytes and Sinusoids of the Liver in Rats with Concomitant Chest and Hip Trauma I. Ya. Horbachevskyi Ternopil State Medical University, Ternopil, Ukraine Abstract. In the experiment on rats inducing concomitant chest and hip trauma the features of changes in hepatocytes and hemomicrocirculatory bed of the liver were investigated using electron microscopy. The degenerative dystrophic changes in hepatocytes occurring secondary to dysfunctional adjustment of elements of hemomicrocirculatory bed were detected at early stages (within the first day) after injury. An expressed expansion of the sinusoidal lumen and filling it with blood corpuscles such as erythrocytes, lymphocytes, and neutrophils were seen in the hemomicrocirculatory bed. In the hepatocytes the nuclear membrane became wavy due to numerous protrusions and invaginations in contrast to the smooth nuclear membrane of the hepatocytes of intact animals. There was an increased number of free lysosomes in the cytoplasm. The lumen of bile capillaries located between two adjacent hepatocytes looked expanded. These phenomena were accompanied by gradual destruction of mitochondria with co-existent notable expansion of ducts and tanks of both granular and smooth endoplasmic reticulum. Such changes progressed to the 3rd day of posttraumatic period and were stabilized up to the 7th day of the experimental observation. Since the 7th day of the experiment the regenerative processes occurred continuing till 28th day with the complete restoration of the state of hemomicrocirculatory channels and the structural organization of hepatocytes. The presence of white blood cells in hepatic hemomicrocirculatory bed, especially during the early posttraumatic period may be the sign of the development of reactive changes in response to trauma with co-existent inflammatory and corresponding immune responses. Keywords: concomitant trauma, hepatocytes, microcirculation, ultrastructure Problem statement and analysis of the recent research The prevalence of concomitant chest and musculoskeletal injuries ranges from 3.4 to 11.7% [3, 4], and according to other authors reaches 20-30%. Therefore, considering the increased number of victims with serious injuries, the relevance of treatment and prevention of complications in these patients increases [7]. Moreover, case fatality rate due to concomitant trauma was found to be more than three times higher compared to that associated with other injuries [10]. In case of concomitant injuries disintegration of functional systems manifests clinical signs of multiple organ failure [9]. However, questions concerning the features of pathogenic relations between the damage and pathological changes in internal organs in severe trauma still remain unsolved [2]. The objective of the research was to determine the features of ultrastructural changes in hepatocytes and hemomicrocirculatory bed of the liver when inducing concomitant chest and hip trauma. Material and methods Experiments were conducted on 18 white outbred laboratory male rats divided into 2 groups: the control group and the experimental group. The experimental group consisted of 5 series, depending on the time of observation. Anesthesia was induced by the injection of thiopental sodium (40 mg x kg-1 of body weight). Left-sided closed pneumothorax with a broken rib associated with a fracture of the left femur was simulated. Rats were withdrawn from the experiment by total bloodletting from the heart under thiopental sodium anesthesia (60 mg x kg-1 of body weight intraperitoneally). Within the 1st, 3rd, 7th, 14th and 28th days of post-traumatic period liver tissue samples were taken for electron microscope studies. All experiments were performed in compliance with the “Rules and Guidance for Responsible Conduct of Research with Experimental Animals”. Results Inducing concomitant chest and hip trauma caused quite pronounced changes in hepatocytes and hemomicrocirculatory bed of the liver. Moreover, significant ultrastructural alterations in liver tissue appeared in the early posttraumatic period. Thus, during the first three days of experimental observation an expressed expansion of the sinusoidal lumen and filling it with blood corpuscles such as erythrocytes, lymphocytes, and neutrophils were seen in the hemomicrocirculatory bed (Figures 1, 2). As a result, endothelial cell swelling together with enlightened cytoplasm of endothelial cells developed. During the first day after the injury hepatocytes reacted to these hemodynamic changes changing the shape of their nuclei. The nuclear membrane became wavy due to numerous protrusions and invaginations in contrast to the smooth nuclear membrane of the hepatocytes of intact animals. There was an increased number of free lysosomes in the cytoplasm. The lumen of bile capillaries located between two adjacent hepatocytes looked expanded (Figure 3). On the
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third day these phenomena were accompanied by gradual destruction of mitochondria with co-existent notable expansion of ducts and tanks of both granular and smooth endoplasmic reticulum (Figure 4). The post-traumatic period from 7th to 14th days was characterized by initial stabilization of pathological changes, and even further regressive development. So, on the 7th day of the experiment micropinocytosis intensification was observed in the endothelial cells of the sinusoidal capillary walls, organelles were found close to the nuclei. Most nuclei were rounded in shape and the nuclear membrane was smooth surfaced. In hepatocytes destructive changes remained in the form of homogenization and vacuolization of the cytoplasm due to destruction of organelles and their intracellular digestion. Reduced sinusoidal endothelial cell swelling together with intensified micropinocytosis were detected on the 14th day of post-traumatic period. On this background regenerative changes in hepatocytes became visible. There was an increase in the size and number of osmiophilic mitochondria with distinct cristae. These processes occurred due to both an increase in the size and number of subcellular organelles (hypertrophy) and their new formation as evidenced by the presence of small electron-dense mitochondria. The expansion of ducts and tanks of both granular and smooth endoplasmic reticulum indicated the intensity of the functional activity of cells (Figure 5).
Figure 1. Electronogram of elements of sinusoidal capillary of rat liver 1 day after inducing concomitant chest and hip trauma. x 12,000. 1- red blood cells; 2 - neutrophil; 3 - endothelial cells.
Figure 2. Electronogram of elements of sinusoidal capillary of rat liver in 3 days after inducing concomitant chest and hip trauma. x 12,000. 1 - cells of sinusoidal capillary lumen; 2 - endothelial cells.
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Figure 3. Electronogram of rat hepatocyte 1 day after inducing concomitant chest and hip trauma. x 14,000. 1 - the nucleus of hepatocyte; 2 - lysosomes; 3 - bile capillary; 4 - intercellular contact.
Figure 4. Electronogram of rat hepatocyte in 3 days after inducing concomitant chest and hip trauma. x 18,000. 1- nucleus; 2 - mitochondria; 3 - endoplasmic reticulum. Further follow-up to 28 day after inducing traumatic injury allowed us to establish a gradual restoration of the state of hemomicrocirculatory channels and the structural organization of hepatocytes. The lumen of sinusoidal capillaries was moderate-sized, some red blood cells and almost no white blood cells were seen inside of them. Endothelial cells were regular in size and displayed normal electron density. A moderate number of organelles was found close to the nuclei. On this background, an intense regeneration of hepatocytes was detected. Their nuclei became rounded in shape containing the diffuse euchromatin. Hypertrophied mitochondria with distinct cristae and moderately dilated ducts of the endoplasmic reticulum also indicated regenerative processes (Figure 6).
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Figure 5. Electronogram of rat hepatocyte 14 days after inducing concomitant chest and hip trauma. x 12,000. 1- electron dense mitochondria and expanded channels of the endoplasmic reticulum.
Figure 6. Electronogram of sinusoidal capillary and hepatocyte of rat in 28 days after inducing concomitant chest and hip trauma. x 16,000 1 - erythrocyte in the lumen of sinusoidal capillary; 2 - endothelial cell of sinusoidal capillary wall; 3 - hepatocyte nucleus; 4 - mitochondria and channels of the endoplasmic reticulum. Discussion Thus, the results obtained in the ultrastructural study of rats liver after inducing concomitant chest and hip trauma allowed us to define that the degenerative dystrophic changes in hepatocytes occur secondary to dysfunctional adjustment of elements of hemomicrocirculatory bed at early stages (within the first day) after injury. Such changes progress to the 3rd day of posttraumatic period and are stabilized up to the 7th day of the experimental observation. Since the 7th day of the experiment the regenerative processes occur continuing till 28th day with the complete restoration of the state of hemomicrocirculatory channels and the structural organization of hepatocytes. Functional substantiation of this data may be the fact that hepatocellular disorders are known to occur in trauma due to violation of hepatic microcirculation and manifest themselves even when blood circulating volume (BCV) decreases by 10%, when there is no significant decrease in blood pressure. It leads to a violation of all types of metabolism, high blood glucose and lactic acid levels, the development of acidosis [5, 8]. The presence of white blood cells in hepatic hemomicrocirculatory bed, especially during the early posttraumatic period may be the sign of the development of reactive changes in response to trauma with co-existent inflammatory and corresponding immune responses. It is consistent with modern ideas regarding immune responses according to which
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serious injury is characterized by biphasic neutrophil priming with initial increase in functional activity and its subsequent suppression [1, 6]. Conclusions 1. Ultrastructural dystrophic changes in hepatocytes of rats after inducing concomitant chest and hip trauma occur at early stages (within the first day) secondary to disturbed hemomicrocirculation and are present up to the 7th day of the experimental observation accompanied by a gradual restoration of the structural organization of cells. 2. Identification of lymphocytes and neutrophils in the lumen of sinusoidal capillaries in the early period after injury indicates systemic inflammatory and immune response to the traumatic injury. Perspectives for further research Further research will give experimental substantiation of direction and contents of conservative treatment in order to prevent the development of liver complications in trauma patients. References 2. Hembytskii M.M. Pathology of internal organs in trauma. Meditsina. Moscow. 1994; 256. 3. Desiateryk V.I., Mikhno S.P., Miroshnichenko V.M. et al. The place and role of thoracic trauma in the structure of polytrauma: the first experience of a specialized department. Materialy nauk.-prakt. konf., prysviach. 30richchiu kafedry torakalnoii khirurhii ta pulmonolohii KMAPO im. Shupyka. Kyiv. 2005; 18 – 21. 4. Peschanskii R.E., Tantsiura V.P., Dudyn A.M., Yaroshchak V.V. Principles of surgical treatment of patients with concomitant chest and extremities trauma. Vestnik neotlozhnoy i vosstanovitelnoy meditsiny. 2012; 13(3): 384 – 386. 5. Seleznev S.A., Khudaiberenov H.S. Traumatic disease (relevant issues). Ashkhabad. 1984; 224. 6. Kalinina N.M., Sosiukin A.E., Volohzhanin D.A., Kuzin A.A., Kniazev P.S. Trauma: inflammation and immunity. Tsitokiny i vospalenie. 2005; 1: http://www.cytokines.ru/2005/1/Art4.php. 7. Ursol H.N., Bondarchuk S.P., Davydkin V.A. et al. Treatment of chest trauma. Materialy nauk.-prakt. konf., prysviach. 30-richchiu kafedry torakalnoi khirurhii ta pulmonolohii KMAPO im. Shupyka. Kyiv. 2005; 97 – 99. 8. Ushakov B.N., Dolzhanov A.Ya. Traumatic disease. Voronezh. Moscow. 1998; 389. 9. Mazurkevych H.S., Bahnenko S.F. Shock: theory, clinic, organization of medical care in case of shock. Politekhnika. St.-Petersburg. 2004; 539. 10. Hryhorev E.H, Apartsin K.A., Kornilov N.H. et al. Epidemiology of concomitant injuries in the industrial centers of Eastern Siberia. Byulleten VSNTS SO RAMN. 2005; 3 (41): 109 – 110.
