THE COMPARATIVE ANALYSIS OF STRUCTURAL CHANGES IN MUSCLES OF THE HIND LIMBS AND BIOCHEMICAL CHANGES IN BLOOD SERUM OF THE RATS WITH ACUTE ISCHEMIA

Abstract

Acute limb ischemia occurs as a result of sudden decrease in its perfusion, which usually occurs during the obturation of the lumen of large arteries due to acute thrombosis or embolism either impaired of vascular patency caused by trauma or compression (including the application of hemostatic tourniquets). In case of restoration of blood supply to the previously ischemic tissues arises an ischemic-reperfusion syndrome (IRS), pathophysiological links of which are the important mechanisms of the tissue changes.

The aim of the study – to make the comparative analysis of structural changes in muscle fibers and biochemical changes in the blood serum in case of acute ischemia caused by the application of an arterial tourniquet on the hind limbs of rats.

Materials and Methods. The histological and morphometric study of the tissues of the four-head muscles of the hind limbs of 60 rats under condition of experimental acute ischemia were performed. There were 11 intact animals in the control group. Acute ischemia was caused by the application of SWAT (Stretch–Wrap–And–Tuck) rubber bands on the hind limbs of the animals. The histological investigation was carried out according to standard methods. The surface layers of the four-headed muscle of the hind limb of the rats were studied in the middle third of the thigh below the overlapping area of the tourniquet. By the morphometric examination following parameters were determined: the mean diameter of muscle fibers, the mean cross-sectional area of muscle fibers, the avera­ge diameter and the nuclei area of muscle fibers. The biochemical changes, namely the indices of total bilirubin, triglycerides (TG), cholesterol, creatinine, total protein (TP), alkaline phosphatase (AF) and the level of transaminases (ALAT, ASAT) were determined in blood serum of each group. Quantitative indicators were processed statistically.

Results and Discussion. The comprehensive analysis of the obtained data revealed that the pathological changes in the muscle tissue were aroused in the early period of the postischemic lesions and increased to the end of the 1st day. Histological examination revealed the following changes in the muscle tissue: the disorganization of structural components of the muscle cells, the reduction of the striation, edema, friability, fluffiness and rupture of the muscle fibers. The most significant edema of the muscle tissue occurred on the 1st day in the third group of rats. The average cross-sectional area of the fibers (1369.15±26.57) μm²) is 6.72 % higher than the corresponding index of the control group (P˂0.05) and the average diameter ((41.73±0.39) μm) was exceeded by 3.37 % the index of the control group (P˂0.05). The average area of the nuclei of the muscle fibers ((64.03±0.08) μm ²) in the third group was only slightly higher than the corresponding value ((63.95±0.07) μm) of the control group (P <0.05). The laboratory researches of biochemical parameters of the blood serum showed increasing activity of transaminases, AF and total bilirubin, decreasing creatinine and total protein (TB) in the early reperfusion period. In particular, in comparison with the corresponding index of the control group, the level of ALAT reached its the highest index in the first day, (89.5 % higher) (P˂0.05) and the ASAT index was the highest in the animals with 2-hour reperfusion (78.73 % higher) (Р˂0.005), the index of AF and total bilirubin reached its maximum on the 7th day (45.78% and 105.3 % higher respectively than the index of the control group) (Р˂0.005). Correlation analysis revealed a direct correlation between the parameters of the average area (s_s) of muscle fibers and the average area of their nuclei (s_n) (+0.7), the indicators of creatinine (+ 0.1), cholesterol (+ 0.4), total bilirubin (+ 0,1), ALAT (+ 0,5), AsAT (+ 0,5) and AF (+ 0,1), in addition the inverse correlation between the indices of the average area (s_s) of muscle fibers and the level of triglyceride (-0.1) of the studied groups. The similar pattern is manifested in relation to the average area of their nuclei (s_n) and mentioned biochemical indicators.

Conclusions. The results of remodeling of the muscle fibers confirm the development of ischemic-reperfusion syndrome in experimental animals. During the investigation of the skeletal muscles of the hind limbs of the white rats after acute ischemia caused by the application of the tourniquet was found that the remodeling of the muscle fibers was mainly manifested by edema, homogenization of sarcoplasm, violation of linear arrangement of nuclei in the myocytes and leukocyte infiltration of endo- and perimizia, myolysis with the destruction of the sarcolemma and release of nuclei into the intercellular space. The reactive lesions were developed in the microvessels of all segments and maximally expressed to the end of the 1st day. Changes in biochemical indices were most pronounced in the animals of the early period model of postischemic lesions (2^nd hour and 1^st day). In this period, in the blood serum of the experimental rats, a statistically significant increasing in the content of total bilirubin level, rising the transaminases level, increasing average AF activity, and decreasing in the content of TP and creatinine were observed. Changes in biochemical indices of blood homeostasis were developed synchronously with morphological, that proofs a direct correlation between the change in the average area of muscle fibers (and their nuclei) and changes in the blood serum content of the total bilirubin, cholesterol, creatinine, total protein (TB), alkaline phosphatase (AF) and transaminase levels (ALAT, ASAT).