Morphological features of the wall of common bile duct under the conditions of experimental opioid exposure

The morphological condition of the bile ducts remains one of the most important problems of modern medical science. In order to obtain an analgesic effect in patients with acute cholangitis, opioids are often used. However, information on the effectiveness of opioids in the treatment of pathological conditions of the bile ducts is contradictory. The rapidly progressive destruction of the intrahepatic bile ducts associated with the use of narcotic agents has been described. Further study of the effect of opioids on the structural organization of the common bile duct is relevant. In order to establish the morphological state of the common bile duct under conditions of long-term opioid exposure, a study was performed on 24 sexually mature white male rats, aged 3.5-5.0 months and weighing 180-200 g, which were injected intramuscularly with Nalbuphine for 6 weeks. The study material is represented by histological specimens of the common bile duct of white rats. The "Aver Media" computer system was used to photograph microspecimens. The "ImageJ" computer program was used to measure the diameter of the lumen and the wall thickness of the common bile duct. After 2 weeks of Nalbuphine administration to white rats, plethora of wall microvessels and a significant increase in the longitudinal diameter of the lumen of the common bile duct were observed. After 4 weeks of the experiment, the common bile duct was dilated, the transverse and longitudinal diameters of its lumen almost doubled, pathological changes in its wall had all the signs of inflammation. In the later stages of the experiment (introduction of Nalbuphine for 6 weeks), the pathological changes increased and manifested by destructuring the wall of the common bile duct, disorganization of cholangiocytes, thinning of the cell layer due to detachment of cholangiocytes, polymorphism of their nuclei, destruction of intercellular junctions, stratification of its own plate, vacuolar dystrophy of the muscular membrane "varicose" expansion of venules, significant smooth muscle hyperplasia of arterioles, the presence of perivascular lymphocytic infiltrates in the duct wall.


Introduction
The morphological state of the bile ducts remains one of the most pressing problems of modern medical science [5,6,13,17]. Damage to the bile ducts often poses a serious threat to the patient's life [4,24,26]. In 12-30% of patients operated on for cholecystitis or bile duct damage, scars and, accordingly, scar strictures of the common bile duct are formed in the postoperative period [2]. Therefore, especially many works are devoted to the tactics of surgical treatment of bile duct pathology [3,7,9,34].
Studying histological changes in the common bile duct after long placement of a metal stent in animals, inflammation, fibrosis and trauma of the epithelium, an increase in the number of lymphoid follicles, a high degree of neutrophilic infiltration, the presence of ulcers of the mucous membrane were observed [22]. Recent advances in tissue engineering make it possible to create an intrahepatic bile duct tree. It has been proposed to induce the formation of bile ducts using encapsulated immortalized epithelial cells of the mucous membrane of the bile ducts of mice using decelularized hydrogels of the extracellular matrix of the liver [23]. Some works are devoted to the therapeutic treatment of bile duct pathology [11,14,35]. Successful treatment of biliary outflow difficulties was achieved by combination therapy, which included the introduction of drugs into the portal vein and common bile duct [21].
Opioids, in particular morphine, are often used to obtain an analgesic effect in patients with acute cholangitis [12]. However, in the professional literature there are only a few works devoted to the study of the mechanism of action of opioids [8]. Information on the effectiveness of opioids in the treatment of pathological conditions of the bile ducts is often contradictory. The rapidly progressive destruction of the intrahepatic bile ducts associated with the use of narcotic agents has been described [20]. Among the nonobstructive etiologies of bile duct dilation, opioid consumption predominates [10].
The above determined the purpose of our study -to establish the features of the morphological state of the common bile duct under conditions of six weeks of opioid exposure in the experiment.

Materials and methods
The studies were performed on 24 adult white male rats weighing 180-200 g and aged 3.5-5.0 months. All experimental animals were divided into 3 series: in the first series (5 rats) studied the structural organization of the common bile duct wall of white rats after 2 weeks of Nalbuphine administration, in 2 series of experiments (5 rats) studied changes in the microstructure of the common bile duct after 4 weeks, and in 3 series of experiments (5 rats) reorganization of the wall of the common bile duct of experimental animals after 6 weeks of Nalbuphine administration. The control group included 9 white rats treated with 0.9% sodium chloride solution.
The study material is represented by histological preparations of the common bile duct of white rats. For histological examination, sections were stained with hematoxylin and eosin. The preparations were studied and photographed under microscope magnifications: x200, x400, x1000. The "Aver Media" computer system was used to photograph the micropreparations. The computer program ImageJ was used to measure the diameter of the lumen and the wall thickness of the common bile duct. For statistical processing of the received digital data the software "Excel" and "STATISTICA" 6.0 were used.

Results
After 2 weeks of administration of Nalbuphine to white rats, the structural organization of the common bile duct wall of experimental animals, as well as control animals, is mainly preserved and represented by mucous, muscular and adventitial membranes (Fig. 1). The mucous membrane of the wall of the common bile duct is lined with prismatic epithelium, which was adjacent to its own plate, formed by a layer of connective tissue. Epithelial cellscholangiocytes -had a typical structure, distinct apical, basal and lateral surfaces (the latter in contact with each other), a large oval nucleus with clear contours. In its own plate clearly visible single, but rather large glands with preserved structure. On some histological preparations of this term of experiment we found increase in number of glands in a wall of a common bilious channel, proliferation of their epithelium (Fig. 2). The muscular membrane consisted of smooth myocytes arranged in bundles with a preserved helical arrangement. The adventitial membrane is rich in  vessels, which at this time of the experiment are full-blooded (especially arterioles and capillaries, as shown in Fig. 1) and dilated.
Morphometric analysis of histological specimens revealed that the lumen of the common bile duct of white rats in cross section has an oval shape, so we considered it appropriate to measure the longitudinal diameter of the lumen of the common bile duct, which was at this time of the experiment 272.77±15.09 μm (control -126.97±3.19 μm, p<0.05), and the transverse diameter -97.36±10.86 μm (control -72.79±13.52 μm, p>0.05), wall thickness of common bile duct was 137.85±14.41 μm (control -144.77±3.91 μm, p>0.05).
A significant increase in the longitudinal diameter of the lumen of the common bile duct indicates a change in the shape of its lumen due to two weeks of Nalbuphine introduction. In addition, although the change in the wall thickness of the common bile duct after 2 weeks of the experiment is not significant, but the maximum value of this indicator in experimental animals (152.52 μm) is much larger than its maximum value in control animals (148.69 μm), which indicates about increasing the wall thickness of the common bile duct during this period of the experiment.
After 4 weeks of experimental exposure to Nalbuphine, the shape of the lumen of the common bile duct is incorrect, cholangiocytes are often without clear contours, their nuclei are large, deformed, swollen, observed epithelial protrusions, and in some places -exfoliation of epitheliocytes into the lumen of the common bile duct (Fig. 3).
The own plate is swollen, fluffy, in some places stratified, glands are destructured, shifted in the direction of an epithelial layer, in their gleams -stagnation of secretion. The bundles of smooth muscle myocytes are stratified, partially losing their helical organization. The connective tissue of the adventitial membrane is in a state of edema, the arterioles are dilated, their wall is thickened, parietal thrombi are formed in the lumens, capillaries are hyperemic, often the capillary wall is damaged, diapedetic hemorrhages are found, venules are dilated, the walls of the venules are thinned (Fig. 4). Morphometric analysis showed a pronounced dilatation of the common bile duct, the longitudinal diameter of the lumen of the duct increased to 462.98±24.15 μm (p<0.05), the transverse diameter to 275.57±18.34 μm (p<0.05), as can be seen from Figure 2. The wall thickness of the common bile duct significantly (p<0.05) increased and amounted to 217.29±18.09 μm, as shown in Figure 4.
After 6 weeks of the experiment, the wall of the common bile duct is destructed, it was often difficult to differentiate the boundaries of its membranes, the cholangiocyte layer is disorganized, formed numerous folds, in some places thinning of the epithelial layer due to detachment of cholangiocytes, cholangiocytes acquired a flattened shape, noted the polymorphism of their nuclei, the nuclei lost their location in the center of the cell, shifted to the apical surface of the cell, intercellular contacts are broken, expanded (Fig. 5).The own plate of a wall of a common bilious channel is stratified, gleams of glands are uneven, have lost the   correct form, mainly expanded, filled with contents. The bundles of smooth myocytes are separated by coarse layers of connective tissue, there is a vacuolar dystrophy of the muscular membrane, the adventitial membrane is in a state of edema, sometimes not differentiated.
When performing morphometric analysis, it was often impossible to determine the transverse diameter of the lumen of the common bile duct, because its walls collapsed (glued), the lumen of the duct was deformed, the longitudinal diameter of the lumen ranged from 294.88 μm to 386.46 μm (p<0.05), the wall thickness of the common bile duct was 176.44±26.34 μm, which is significantly more than the control animals, but significantly less than the experimental animals in the previous period of the experiment, which confirms the development of dystrophic-degenerative processes in the common bile duct wall after 6 weeks exposure to Nalbuphine. During this period of the experiment, significant changes in the vessels of the hemomicrocirculatory tract of the common bile duct, including dilation of venules, significant smooth muscle hyperplasia of the portal arterioles were also detected. It should be noted that due to plasma infiltration, sclerosis and hyalinosis, the wall of the arterioles was thickened, and perivascular edema and lymphocytic infiltrates were observed.

Discussion
The results of research presented in the professional literature mainly indicate the negative impact of long-term use of opioids on the structural organization of organs and tissues [29]. At the heart of the development of pathological processes due to the action of narcotic drugs is often angiopathy [25,27], which is confirmed in our studies.
A characteristic feature of the effect of Nalbuphine on the wall structure of the common bile duct is its dilatation. According to the literature, opioids can cause an increase in the basic pressure and frequency of phase contractions of the sphincter of Oddi, which leads to dilation of the common bile duct [10]. The author describes an association between elevated bile duct diameter and opioid dependence even in patients without clinical symptoms, normal bilirubin and alkaline phosphatase, and no obstructive factors. On histological specimens we found nonspecific morphological changes in the components of the common bile duct, as degeneration of the epithelial layer of its mucous membrane, lymphocytic infiltration, microvascular hyperemia is characteristic of cholangitis of various etiologies. Epithelial cells of the bile duct mucosa can serve as antigens to activate the cells of the natural killer T [31]. The bile ducts dilate, cholestasis develops, and lymphocytic infiltrates appear around the bile ducts. Inflammation of the bile duct (cholangitis) is based mainly on bile stasis, which leads to its thickening, sludge and subsequent infection, or, conversely, acute inflammation of the bile ducts (cholangitis) causes impaired bile flow [18,32].
An important finding of our study is the degenerative changes in the later stages of the experiment of the muscular membrane of the wall of the common bile duct. Experiments have shown that damage to the muscular layer of the bile duct leads to the development of liver cirrhosis [16,19,28,33] and even hepatocellular carcinoma [1,30]. Significant collagen deposition, portal fibrosis, periductal edema, lymphocytic inflammation, dilatation of all bile ducts, blind end of small bile ducts, cholestasis (bile corks), increased systemic oxidative stress, and fibrosis were observed in rats after ligature was applied to the common bile duct, which eventually leads to liver cirrhosis [15,32].
In the future, subject to long-term use of opioids, the results of this study can be used in practical medicine for the diagnosis and treatment of pathological conditions of the intrahepatic bile ducts.

Conclusions
Plethora of microvessels of the wall and a significant increase in the longitudinal diameter of the lumen of the common bile duct was observed after 2 weeks of administration of Nalbuphine to white rats.
After 4 weeks of the experiment, the common bile duct was dilated, the transverse and longitudinal diameters of its lumen almost doubled, pathological changes in its wall had all the signs of inflammation.
In the later stages of the experiment (introduction of Nalbuphine for 6 weeks) pathological changes increased and manifested by destructuring of the common bile duct wall, disorganization of cholangiocytes, thinning of the cell layer due to detachment of cholangiocytes, polymorphism of their nuclei, destruction of intercellular contacts, stratification of own plate, vacuolar dystrophy of a muscular cover, "varicose" expansion of venules, expressive smooth muscular hyperplasia of arterioles, existence of perivascular lymphocytic infiltrates in a channel wall.