PROGNOSIS OF LIMB MUSCULAR STRUCTURAL DISORDERS AFTER TOURNIQUET APPLICATION BASED ON THE CHANGES IN LIPID PEROXIDATION INDICATORS USING NEURAL NETWORK CLUSTERING
Authors: Selskyy Petro , Televiak Anatolii , Lutsyk Vitalii , Franchuk Valentyn , Sverstiuk Andriy , Voloshyn Volodymyr , Furdela Mykhailo 
Published: October 2024

Acute ischemia can cause a sudden decrease in limb perfusion, threatening its viability. The restoration of blood supply causes complex damage to previously ischemic tissues as well as to the tissues distant from the area of ​​ischemia, which is defined as ischemia-reperfusion injury. This condition gets significantly worse under conditions of acute blood loss. The development of new approaches for the prognosis of morphological changes in the soft tissues of the limbs and further clinical research on their correction is also relevant for civilian and military medicine, especially in martial law conditions.
The objective of the study was to establish an association between the degree of structural changes in the rat hind-limb skeletal muscles and systemic changes in the indicators of lipid peroxidation and antioxidant defense in ischemia-reperfusion injury against the background of acute blood loss to make prognosis for morphological disorders using neural network clustering.
Materials and Methods. We carried out a histological and morphometric study of skeletal muscles and measured the parameters of lipid peroxidation (content of diene conjugates, triene conjugates, TBA-reactive substances) and antioxidant defense (superoxide dismutase and catalase activity) in blood serum of 30 mature white male rats. Simulation of acute ischemia was performed by applying SWAT rubber tourniquets 5–6 mm wide on the hind limbs of rats at the level of the inguinal fold for 2 hours under thiopental sodium anesthesia. Reperfusion was induced by restoring blood circulation in the limbs of the animals after removing the tourniquet. Acute blood loss was simulated by transection of the femoral vein.
To analyze the relationship between the identified structural disorders of the hind limbs skeletal muscles and the changes in the lipid peroxidation (LPO) and antioxidant defense (AOD) indicators, as well as to predict the progression of morphological changes during the ischemia-reperfusion injury, a neural network approach was used using the NeuroXL Classifier add-on for Microsoft Excel.
Results and Discussion. In the early reperfusion period under conditions of acute blood loss, structural remodeling was manifested by edema and dystrophic-degenerative changes in skeletal muscles, with the most pronounced changes on the first day (Group III). The average diameter of the muscle fiber ((43.66±0.60) μm) in this period exceeded the corresponding indicator ((40.63±0.24) μm) of the control group by 6.94% (p˂0.01), while the similar indicator of Group II ((41.97±0.25) μm) – by 3.87% (p˂0.05). In the late reperfusion period, the remodeling of structural components of skeletal muscles was manifested as a slowing down of acute manifestations with an incomplete return to the baseline values.
We established an increase in the blood serum content of LPO products in all experimental groups in the early reperfusion period. This study confirmed the development of ischemia-reperfusion injury with activation of lipid peroxidation and a decrease in antioxidant defense in experimental animals. Thus, the diene conjugates (DC) content was the highest in the third experimental group (the first day of reperfusion), exceeding the indicator of the intact animals by 51.06% (p>0.05). The triene conjugates (TC) content also reached its maximum during this period, exceeding the control level by 1.61 times (p<0.01). One day later, the highest level of the TBA-reactive substances (TBA-RS) was observed. During the late postischemic period (Day 7 and Day 14), a gradual normalization of indicators ​​was observed; however, the content of TBA-RS was still significantly higher than that of the control group on the 14th day (p<0.005).
In all studied groups, there was a decrease in the activity of superoxide dismutase (SOD) and catalase. Blood serum catalase activity was observed at its lowest level after three days of reperfusion, exceeding the control values by 65.76% (р<0.005), while SOD content kept decreasing on Day 7 and Day 14. We observed a significant dynamic decrease of PAI in the blood serum of rats in all studied groups in the early reperfusion period (p<0.005) with a gradual increase of this indicator in the late reperfusion period, which, however, did not return to the control values ​​(p<0.005).
When predicting the development of morphological disorders in the early reperfusion period based on the results of the analysis of cluster profiles obtained due to the neural network clustering of blood serum LPO and AOD values in white rats and the morphological disorder severity in different periods of ischemia-reperfusion injury under conditions of acute blood loss, we observed the greatest significance of the combined changes in DC, TC, TBA-RS content and SOD and catalase activity, with the most significant changes being in TBA-RS and catalase values.
Conclusions. Ischemic-reperfusion injury against the background of acute blood loss was manifested by edema and dystrophic-degenerative changes in skeletal muscles during the period of ischemia and in the first hours after the restoration of blood circulation. This indicates an increased load on the organs of the homeostasis system and the systemic nature of the injury.
When predicting the development of morphological disorders in the early reperfusion period based on the results of the analysis of cluster profiles obtained due to the neural network clustering of blood serum LPO and AOD values in white rats and the morphological disorder severity in different periods of ischemia-reperfusion injury under conditions of acute blood loss, we observed the greatest significance of the combined changes in TBA-RS content and catalase activity