SA JOURNAL OF DIABETES & VASCULAR DISEASE RESEARCH ARTICLE VOLUME 20 NUMBER 1 • JUNE 2023 5 serum to yield a concentration of 5 mg/ml and 50 mg/ml, respectively. According to the weight of each rat, the suspended drug solution was completed to 2 ml with physiological serum. All subjects were anaesthetised intraperitoneally with ketamine (40 mg/kg; Ketalar, Pfizer) and xylazine hydrochloride (4 mg/kg; Alfazyne 1, Ege Vet, Alfasan International BV). After the subjects were placed in a prone position, ECG recordings were taken from the D2 lead with needle electrodes (Fig. 1B). ECG recordings were evaluated with the Biopac MP36 system. RR and QT intervals and heart rates (HR) were measured by ECG recordings at baseline, and at the first and second hour, respectively. Heart rate was calculated as 1 500 per number of small squares between consecutive R waves. After the QT interval and HR measurements were performed, the corrected QT (QTc) was calculated with the Bazzet formula (QT/ RR1/2). QTc prolongation, measured by serial ECG, was the accepted main endpoint. In a similar previous experimental model researchers showed that the QTc difference of rats two hours after drug administration was 43 ms between the control and amitriptyline groups.12 It is generally accepted that a 20-ms change in QTc interval is significant.13 Therefore, in order to detect a difference of 20 ms in QTc interval between the groups, six rats per group would be required for a total of 24 rats to be able to reject the null hypothesis with a probability (power) of 0.8. The type I error probability associated with this test of the null hypothesis was 0.05. Statistical analysis Statistical analyses were performed with SPSS 22 (IBM Corp, Armonk, NY, USA). The mean and median QT, HR and QTc durations of all groups were calculated. The Shapiro–Wilk test was used to evaluate whether the data fitted a normal distribution. All ECG parameters at all available time points (baseline, first and second hours) were compared with repeated measurements of one-way analysis of variance (ANOVA), followed by Tukey’s or Tamhane post hoc tests. ECG parameters of all of the study groups at each time point (baseline, first and second hours) were also separately compared. Data without a normal distribution are expressed as median and interquartile range (IQR) and were compared by Kruskal–Wallis analysis (HR at first hour). Data with a normal distribution are expressed as mean ± standard deviation (SD) and were compared using ANOVA, followed by Tukey’s test for post hoc analysis (for other parameters). Differences of p < 0.05 were considered significant. Results After anaesthesia, ECG recordings of the four groups at baseline, and first and second hours were obtained and compared between Fig. 1. A. Drug administration to the animals via an oral tube. B. ECG recording of the rats from the D2 lead in a supine position with the Biopac MP36 system. A B Table 1. QT, QTc durations and heart rate for all groups at basal, first and second hour Variables Control Empagliflozin Amitriptilin Amitriptilin + empagliflozin p-value Baseline Qt (ms), mean ± SD 77.33 ± 9.02 78.33 ± 6.28 71.50 ± 5.68 73.33 ± 8.02 0.35 QTc (ms), mean ± SD 165.42 ±18.34 152.57± 11.07 163.11 ± 11.59 159.97 ±15.18 0.453 HR, mean ± SD 263.00 ± 39.38 230.00 ± 10.06 314.83 ± 42.48 287.50 ± 29.99 0.002 First hour Qt (ms), mean ± SD 73.50 ± 2.26 75.67 ± 4.27 108.67 ± 5.96A 90.33 ±5.39B < 0.001 QTc (ms), mean ± SD 166.63 ± 17.92 154.60 ± 20.43 227.45 ± 26.89A 179.40 ±17.63C < 0.001 HR, median (IQR) 335.50 (75.75) 245.00 (144.25) 248.50 (123.50) 232.50 (107.25) 0.279 Second hour Qt (ms), mean ± SD 78.17 ± 6.18 77.33 ± 7.31 106.00 ± 12.60A 87.83 ± 4.54C < 0.001 QTc (ms), mean ± SD 184.65 ± 12.86 171.63 ± 20.36 229.89 ± 19.83D 191.66 ± 10.93C < 0.001 HR, mean ± SD 335.83 ± 21.99 295.67 ± 30.54 288.67 ± 53.86 326.30 ± 36.97 0.118 Aamitiriptilin vs control < 0.001 Bamitriptilin + empagliflozin vs amitriptilin < 0.001 Camitriptilin + empagliflozin vs amitriptilin < 0.01 Dempagliflozin vs amitriptilin: 0.001.
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