Morphometric Methods for Examining the Liver in the Diagnosis of Acute and Chronic Alcohol Intoxication

Introduction

Currently, for forensic medicine, the problem of post- mortem diagnosis of acute and chronic alcohol intoxication using modern research methods remains relevant, which allows not only to increase the accuracy of the description of the studied phenomena, but also to objectify the morphological verification of pathological processes [1, 2, 3, 4].

Remaining basic, the qualitative assessment of pathomorphological changes in acute and chronic alcohol intoxication, and especially the liver as the main detoxifying organ, should be supplemented by quantitative studies - micromorphometry, which allows obtaining more complete information about the processes occurring in hepatocytes under the influence of alcohol and can be used in the diagnosis and prediction of the outcome of emerging pathological processes [5].

At the present stage, morphometric models of various diseases and conditions have been studied, at the same time, there are practically no morphometric data on intraorgan blood and biliary tracts, hepatocytes and their nuclei in normal conditions and in ethanol poisoning [6, 7, 8, 9].

Some of the studies studied are devoted to cyto- and karyometric parameters of the liver of experimental animals when modeling experimental alcoholic cirrhosis of the liver or in other conditions in combination with chronic ethanol intoxication [10, 11, 12]. At the same time, the authors assessed various morphometric characters, which, in our opinion, are not comparable, and sometimes contradictory.

Benyavsky MV [13] provides data on an increase in the diameters of nuclei in human liver cells with viral and alcoholic damage. A shift of the nucleus towards enlargement - 12μm and more, as well as an increase in the number of 2-nuclear hepatocytes, are characteristic of viral hepatitis, but not for alcoholic ones [14] Lebedeva EI [2016] in experimental studies, data were obtained on an increase in the average area of ​hepatocytes, especially at the stage of cirrhosis (by 1.5-1.75 times) in male and female rats, a decrease in the average number of binuclear hepatocytes and nuclear-cytoplasmic ratio (NCR) in 1 , 2 times compared to control. In humans, there were no changes in the average area of ​hepatocytes with steatosis and cirrhosis relative to the control group. At the same time, at the stage of acute hepatitis in men and women, this indicator decreased by 1.11 times. At all stages of alcoholic liver disease, the average area of ​the nuclei was lower than the control value in men and women by 1.06 times only at the stage of acute hepatitis. In men and women, the NCR indicator was 1.07 times higher than the control level (p = 0.026) only at the AC stage.

Thus, as the analysis of domestic and foreign literature shows, studies carried out in this direction are few in number, the results obtained are ambiguous, and at the moment there is no unified system for the use of morphometric liver studies, techniques and analysis of its conduct [15, 16].

This determined the goal of the work - the need for further research and the choice of parameters of the vascular and parenchymal structural components of the liver, reflecting its morphofunctional state in acute and chronic alcohol intoxication, as well as in the control group (rapid death from traumatic brain injury at the scene) for their use in as additional diagnostic markers of these conditions.

Materials and Methods

110 deaths were investigated, including 20 cases of acute ethanol intoxication and 75 cases of chronic alcohol intoxication in persons with alcohol-induced liver pathology in the form of steatosis (AS) - 30, hepatitis (AH) - 20 and cirrhosis (AC) - 25. Acute intoxication ethanol corresponded to liver damage in the form of adaptive hepatopathy (AHP).

Alcohol levels ranged from 3.2 to 5.5 ppm with acute ethanol intoxication, with chronic - in cases of steatosis - from 3.4 to 6.9 ppm, hepatitis - from 2.3 to 7.7 ppm, cirrhosis - from 0, 4 to 2.5 ppm.

The control group consisted of 15 cases of rapid death of persons from traumatic brain injury at the scene of the incident; the deceased had no exogenous alcohol in their blood.

For histological examination, we used archival paraffin blocks, from which a series of 5 µm thick sections were made and mounted on prepared slides.

For observation light microscopy and morphometric examination, histological preparations were stained with hematoxylin and eosin. Subsequently, photomicrographs of random fields of view of histological preparations were made with an OLYMPUS XC30 digital camera based on an OLYMPUS CX41 microscope (Japan) with a SWH × 10 eyepiece magnification and UPLanFL × 10, × 20, × 40 lenses (at least 10 visual fields in each histological section). Image Scope Color and cellSens 3 (Japan) computer programs for image analysis were used for morphometric data analysis. Statistical analysis with the calculation of the arithmetic mean and its error was performed using the statistical application Excel (Microsoft Office-2007) and the Statistica v. 6.

Results and Discussion

The morphometric study data are presented in Table 1. It follows from it that in acute alcohol intoxication the analyzed parameters of the hemomicrocirculatory bed statistically insignificantly differ from the control group (P (0.05). In chronic alcohol intoxication, there is a tendency (P <0.001) to change the diameters of the lumens of the central and interlobular veins, interlobular arteries and interlobular sinusoidal capillaries downward, especially in AC in comparison with the control and with other groups of CAI - AS and AH.

At the same time, there is a statistically significant increase in the diameter of the interlobular bile ducts (P <0.001), the maximum values ​of which were recorded in hepatitis and liver cirrhosis.

To assess the level of morphofunctional activity of hepatocytes, we used the measurement of the area of hepatocytes and their nuclei. The area of the cytoplasm was calculated, which was equal to the difference between the areas of hepatocytes and their nuclei. To determine the NCR, the ratio of the area of the nucleus to the area of the cytoplasm was calculated.

In acute ethanol poisoning and in alcoholic steatosis, the average area of ​hepatocytes, nuclei and cytoplasm, as well as the NCR, did not differ statistically significantly from the control group. In alcoholic hepatitis, there was a statistically significant decrease (P <0.05). The data obtained indicate significant changes in the nuclear apparatus in liver cirrhosis and indicate the activation of plastic processes in hepatocytes.>0.001) in the average area of ​hepatocytes, nuclei and cytoplasm relative to the control values ​and the steatosis group, while in alcoholic cirrhosis the average area of ​hepatocytes was higher than in the control and in other groups of chronic alcohol intoxication. In cirrhosis, high values ​of the average areas of nuclei and cytoplasm were also noted relative to other study groups. NCR indices had a statistically significant difference between the control groups and AC, AC and AC.

The results in the form of a decrease in the area of the vascular bed, cyto- and karyometric parameters obviously reflect the processes of an increase in dystrophic processes in hepatocytes and inflammatory-proliferative processes in the connective tissue, and indicate the tension of the capillary- connective tissue structures, impaired blood circulation, bile and lymph outflow in the liver in chronic alcoholic intoxication, especially with hepatitis and cirrhosis.

Conclusion

Thus, the morphometric parameters of the liver identified by us in acute and chronic alcohol intoxication differ from each other, which allows us to recommend them as additional modern diagnostic criteria for acute and chronic alcohol intoxication.

These include:

• A decrease in the lumen of blood vessels in the hemomicrocirculatory bed;
• An increase in the diameter of the interlobular bile duct;
• An increase in the karyometric indicator - NCP.

The morphometric data obtained by us can be used as a comparison in the study of the pathomorphological features of the liver and in other pathological conditions.