## 4 Discussion
Therefore, our further investigations focused on the morphological changes and altered oxidative status of RBCs, since RBCs play an important role in microcirculation and they are vulnerable to a hypergly environment. We found that the diameter of RBCs increased and abnormally shaped RBCs occurred more often in diabetics compared to controls, in line with other studies [11; 34]. We observed eccentricocytes, hemolyzed RBCs, and codocytes in larger proportions in diabetic smears. The flexibility of RBCs is very important for their free passage through capillaries, transporting oxygen or carbon monoxide, and this property is highly influenced by the cell shape [35]. On the other hand, these abnormal shapes, namely, those of eccentricocytes, hemolyzed RBCs, and codocytes, may be caused by oxidative stress [19; 36; 37]. An eccentricocyte is a special form of RBC in which Hb is shifted to one side of the cell because of the oxidative damage to the cell membrane and cytoskeleton [38]. Increased hemolysis was described in the pathomechanism of some oxidative-stress-related diseases, such as diabetes mellitus, sepsis, or sickle cell disease [19; 39; 40; 41]. Codocytes also occur in diseases accompanied by oxidative stress, such as diabetes mellitus, beta-thalassemia, or autism spectrum disorders [11; 36; 42]. In insulin-treated diabetics, we observed that the occurrence of eccentricocytes and hemolyzed RBCs was similar to that of controls. However, codocytes were present in insulin-treated groups, and their frequency was one-third of that observed in the diabetic samples. The highest occurrence of other RBC morphologies, namely acanthocytes, ovalocytes, dacrocytes, and schistocytes, was found in smears of insulin-treated diabetic rats compared to the controls and/or diabetics. This result is in line with earlier findings that exogenously administered insulin may be ineffective in some aspects of diabetes-related alterations [43; 44; 45]. Insulin is a drug that can induce immune-mediated hemolysis, which may explain the increased proportion of schistocytes in insulin-treated diabetic rats [46]. The complete blood count test suggests platelet abnormalities in both acute and chronic diabetic rats, in addition to reverted diabetic rats. However, orthorhombic events are seen frequently in diabetic patients [47]. Earlier findings about paradoxical results of in vivo and in vitro studies highlight possible compensatory mechanisms in long-lasting diabetes [48]. To gain insights into the antioxidant status of RBCs, we determined the GSH and GSSG level and the ratio of GSH to GSSG in RBC populations. Furthermore, summarizing the data from acute and chronic studies, we obtained a time-response curve of the antioxidant capacity of diabetic RBCs. The doubled value of both the GSH and GSSG is not only a sign of a prompt antioxidant response to STZ injection, but also indicates a balanced free radical/oxidant pool. In contrast, 10 weeks after STZ injection, the decreased levels of GSH and GSSG may indicate some kind of adaptation to the system, along with the exhaustion of the defense response. This reduction in GSH and GSSG levels in the chronic experiment is in agreement with other studies [49; 50]. [PERSON] et al. [50] investigated the GSH pathway involving the GSH level, GSHPx, and GR in RBCs of well-controlled and poorly controlled type 1 diabetic patients versus healthy subjects at the baseline and after 2 h of incubation with H\({}_{2}\)O\({}_{2}\). The greatest decrease for all investigated components was in poorly controlled diabetic patients at baseline. The 2 h incubation of RBCs with H\({}_{2}\)O\({}_{2}\) impaired the GSH pathway in all groups, and it was most pronounced in the poorly controlled diabetic group. However, in our system, the fact that the ratio of oxidized and reduced glutathione did not change significantly in acute and chronic samples may indicate that the activities of the crucial enzymes such as GSHPx and GR were unchanged. There are several limitations to our study.