Effects of vitamin B12 on isolated tubal torsion with ischaemia/reperfusion injury: evaluation with histological and biochemical parameters

Introduction

Isolated tubal torsion (ITT) is characterised by the fallopian tube rotating around itself without the ipsilateral ovary. The incidence is 1 in 1,500,000 adults [1], and it appears less frequently in children and adolescents [2]. Predisposing factors are defined intrinsically and extrinsically for adults. Among these, intrinsic factors are hydrosalpinx, haematosalpinx, tubal neoplasm and abnormal tubal peristalsis; extrinsic factors are paraovarian or paratubal masses, postoperative adhesions, pregnancy, or tumour increase in uterine dimensions [3, 4] and sudden changes in body position [5]. Predisposing factors in children are congenital malformations such as congenital müllerian duct anomalies, long mesosalpinx, and hydatid cysts of Morgagni [6].
A complete rotation of the tubal tissue results in arterial, venous, and lymphatic obstruction [7]. This rotation primarily threatens venous and lymphatic outflow. Accordingly, tubal oedema develops. With the deterioration of arterial nutrition in the next stage, arterial ischaemia and necrosis develop [8].
ITT requires urgent surgical intervention. The current procedure is to detorsion the torsioned tissue, even if it is cyanotic [9]. The detorsioned tissue provides arterial feeding and starts the healing process. Although the return of arterial nutrition removes ischaemic damage, it initiates a new process that can lead to further complications and damage to the tubal tissue. This process is called reperfusion injury.
When ischaemic damage (I) occurs, the main pathophysiological process is an imbalance resulting from the energy requirement. Aerobic metabolism is deactivated, and related damage occurs. Many pathophysiological factors play a role in reperfusion injury (R). These are mainly events such as lipid peroxidation, protein dysfunction, cellular cytoskeletal disruption, cellular integrity disruption, and DNA damage. Free oxygen radicals (FOR) that develop as a result of these events, and cytokines and chemokines involved in the pathways, contribute to the process [10]. In mammals, there is a continuous loop between methionine synthase or betaine homocysteine methyl transferase (BHMT) enzymes, which are essential amino acids, and homocysteine. In this way, accumulation of homocysteine is prevented in the environment. It is known that homocysteine accumulates in many organs such as heart, brain, and kidney and causes ischaemia/reperfusion (I/R) damage [11–13].
The antioxidant properties of vitamin B12 are due to the stimulation of methionine synthase activity [14], direct reaction with reactive oxygen and nitrogen types, protective effect thanks to the production of glutathione [15], and the effect of signal molecules on modification [16]. Vitamin B12 and its derivatives (cobalamins) have been reported to exhibit significant antioxidant activity at pharmacological concentrations and provide significant cellular protection against oxidative stress [17]. However, there is no definitive information about the effectiveness of vitamin B12 on I/R damage developing in ovarian tissue.
The aim of this study was to determine the histopathological and biochemical effects of vitamin B12 on I/R injury in the rat isolated tubal torsion/detorsion model.

Material and methods

The experiments in this study were carried out in accordance with the National Institutes of Health animal research guidelines and were approved by our Animal Ethics Committee (Ethics Committee no. 2019/063).

Animals and experimental protocol

In our study, 28 Wistar albino female rats weighing between 250 and 280 g, 10–12 weeks old, were used in four groups, with seven animals in each group. No procedures were performed for seven days for the adaptation of animals in each group in cages. Rats were housed at 22 ±20°C room temperature during the adaptation and experiment period, in rooms with 12 h light/12 h dark light cycle, with feed and water add-libitum. The live weights of the animals were determined at the beginning and at the end of the study, which would take a total of 4 h. The applications applied to the groups were as follows:
– Group 1 (Sham group) (n = 7): after localising the adnex with the right and left uterine horns the abdominal wall was closed with 4-0 nylon su-tures. Two hours later, the tubes were removed by relapa-rotomy. Then intra cardiac blood was taken to study oxi-dative stress parameters.
– Group 2 (torsion) (n = 7): to create tubal torsion, vascular clamps were placed approximately 1 cm below the adnexal structure of the right and left tubing and the incision line was closed with 4-0 nylon. Two hours later, relaparotomy was performed and blood and tissue samples required for the study were taken as described in group 1.
– Group 3 (torsion + detorsion) (n = 7): after 2 h of tubal torsion, vascular clamps were opened by relaparotomy and the incision line was closed with 4-0 nylon sutures. Reperfusion was allowed for 2 h. After the reperfusion phase, relaparotomy was performed and blood and tissue samples required for the study were taken as described in the group 1.
– Group 4 (torsion + detorsion + vitamin B12) (n = 7): after 2 h of tubal torsion, vascular clamps were opened by relaparotomy and the incision line was closed, and a 4 mg/kg dose of B12 was given intraperitoneally and reperfusion was allowed for 2 h. After the reperfusion phase, relaparotomy was performed and blood and tissue samples required for the study were taken as described in group 1. Decapitation was applied to the rats in all groups at the end of the experiment period, and the tissues mentioned in the project were taken and stored in suitable environments.

Histopathological evaluation

After the experiment, the uterine of the anaesthetised rats were removed and fixed in 10% formaldehyde solution. Then the tissues were routinely inserted and passed through the series, embedded in paraffin blocks, 4–5 μm sections were taken and stained with haematoxylin-eosin. The prepared preparations were examined under a Leica DM500 microscope and semi-quantitatively evaluated and photographed (Leica DFC295) (Figure 1).

Biochemical examination

Fallopian tube tissue samples stored at –20°C, glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) rat GSH-Px ELISA kit for enzyme activity (Rel Assay Diagnostics, Bursa, TURKEY, lot # 201903) and rat SOD ELISA kit (Assay Rel Diagnostics, Bursa, TURKEY, lot # 201903), respectively, were read as described above at 450 nm wavelength on an ELISA reader (BioTek ELISA ELX800, BioTek Instruments, USA). The results are expressed in ng/ml.

Statistical analysis

SPSS software 22 was employed for the statistical analysis (SPSS Inc., Chicago, IL). Mean and standard deviation descriptive statistical methods were used. The differences among the four groups were evaluated by one-way ANOVA (Tukey analysis) and Kruskal-Wallis (Tamhane analysis) test. A p < 0.05 level was considered statistically significant.

Results

Histopathological findings

The tuba uterina tissue of the experimental groups stained with haematoxylin-eosin is shown in Figure 1 at different magnifications.
Tuba uterina tissues were normal in groups 1 and 4. In group 2, epithelial degeneration and abnormal epithelial proliferation, cilia loss, and vascular congestion foci were observed. Histological examination of group 3 showed increased epithelial degeneration, abnormal epithelial proliferation, and vascular congestion compared to group 2 (Figure 1).

Biochemical findings

GSH-Px values and SOD values are given in Table I. GSH-Px and SOD values were measured at the lowest level in group 2 and were increased in group 3. In group 4, these values were the highest (Figure 2). The values between the groups show statistically significant differences (p < 0.001) (Table I).
When the relationship between SOD values between the groups was analysed statistically, the values between group 1 and group 3 were significantly different (p = 0.001). In addition, SOD values between group 3 and group 4 showed a statistically significant difference (p < 0.001) (Table II).
In the comparison of GSH-Px values according to the groups, there was a significant difference between group 1 and group 3 (p = 0.034), group 3 and group 4 (p < 0.001), and between group 2 and group 4 (p = 0.012) (Table III).

Discussion

In this study, antioxidant effects of vitamin B12 were investigated in I/R damage in a rat ITT model. In histopathological examination, increased epithelial degeneration and abnormal epithelial proliferation in group 2 was indicative of vascular changes developing in torsioned tissue. In addition, epithelial degeneration, abnormal epithelial proliferation, and increased vascular congestion compared to group 2 in group 3 support the changes associated with reperfusion injury. In contrast, the histopathological data in group 4 are similar to those of group 1, which supports the positive effect of vitamin B12 on histopathological findings.
In the biochemical analysis, GSH-Px and SOD values were the lowest in group 2 and the highest in group 4 in the analysis of GSH-Px values and SOD values. This is another indicator of the antioxidant effects of vitamin B12. In the evaluation of SOD values between the groups, the SOD values in group 3 were significantly lower than those in group 1. This finding is a biochemical indicator of reperfusion injury caused by detorsion. Likewise, GSH-Px values were quite low in group 3 compared to group 1. Again, it was higher in group 4 compared to both group 2 and group 3. All these findings show the antioxidant effects of vitamin B12 and support our hypothesis. According to the authors’ knowledge, this study is the first study in which isolated tubal torsion was performed experimentally and antioxidant effects of vitamin B12 were shown in I/R damage.
The first treatment option for isolated tubal torsion is surgical detorsion. Revascularization of tissue after ITT detorsion can trigger damage known as ischaemia/ reperfusion injury. Many events that cause tissue damage and cell death occur in tissues whose vascular supply has been lost. This loss in vascular nutrition decreases intracellular ATP levels and causes the accumulation of an-aerobic metabolism products. Accordingly, the use of anaer-obic energy also increases. When reperfusion occurs, it can break the anaerobic cycle with blood coming into the tissue. However, reintroduction of oxygen and energy into the cell in which the anaerobic environment is formed can lead to cellular free radical formation, migration of cytokines and leukocytes, activation of the pro-apoptotic signal pathway, and consequently cell damage. All these processes are called ischaemia/ reperfusion injury [10].
In an in vitro study of aortic endothelial cells, cyanoco-balamin concentrations (B12 supplement form) were shown to cause a decrease in SOD levels in mitochondria and cyto-plasm [18]. In a systematic evaluation of studies on vitamin B12 and oxidative stress, vitamin B12 deficiency was shown to be associated with both increased oxidants and decreased antioxidants compared to normal vitamin B12 values [19].
Vitamin B12 may increase SOR excretion by maintain-ing glutathione (GSH) levels [20]. Antioxidant enzymes SOD and GSH-Px activities have been shown to decrease in B12 deficiency [21]. Many studies have shown that H2O2 decreas-es in vitamin B12 deficiency. SOD inhibits H2O2. It is stated that there is an accumulation of H2O2 due to SOD decrease in vitamin B12 deficiency [22–24]. In our study, the increase of SOD values in the group using vitamin B12 supports this data.
GSH is an important antioxidant. It is a substrate for the GSH-Px enzyme that catalyses the detoxification of H2O2 and other peroxides. In this way, it plays an important role in defending free oxygen radicals effectively [21]. In our study, the increase in GSH-Px activity in the rat group given vi-tamin B12 shows the antioxidant effect of vitamin B12 and supports these data.

Conclusions

As a result, it has been experimentally demonstrated that vitamin B12 reduces I/R damage in an isolated tubal tor-sion rat model. Wider clinical studies should be conducted to investigate the effectiveness of vitamin B12 in the treat-ment of I/R damage of isolated tubal torsion. According to the information of the authors, it is the first study on the effectiveness of vitamin B12 on I/R damage in ITT.

Conflict of interest

The authors declare no conflict of interest.

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