Chronic exposure to tramadol induces cardiac inflammation and endothelial dysfunction in mice

Tramadol is an opioid extensively used to treat moderate to severe pain; however, prolonged therapy is associated with several tissues damage. Chronic use of tramadol was linked to increased hospitalizations due to cardiovascular complications. Limited literature has described the effects of tramadol on the cardiovascular system, so we sought to investigate these actions and elucidate the underlying mechanisms. Mice received tramadol hydrochloride (40 mg/kg body weight) orally for 4 successive weeks. Oxidative stress, inflammation, and cardiac toxicity were assessed. In addition, eNOS expression was evaluated. Our results demonstrated marked histopathological alteration in heart and aortic tissues after exposure to tramadol. Tramadol upregulated the expression of oxidative stress and inflammatory markers in mice heart and aorta, whereas downregulated eNOS expression. Tramadol caused cardiac damage shown by the increase in LDH, Troponin I, and CK-MB activities in serum samples. Overall, these results highlight the risks of tramadol on the cardiovascular system.


Materials and methods
Animals and experimental design. The experimental design was approved by the Institutional Animal Care and Ethics Committee of the Faculty of Medicine, Assiut University. All procedures were performed in accordance with the relevant guidelines and regulations of the Institutional Review Board. This study has been performed in accordance with the ARRIVE guidelines.
Twenty C57BL/6 mice, eight weeks old, were purchased from the Assiut University animal house. They were housed in clean, suitably ventilated cages at 25 ± °C with a 12/12 h light/dark cycle and had free access to water and food. They were acclimatized to their environment for 1 week before starting the experiment.
Mice were divided into two groups (n = 10) each: Sample size was calculated using G*Power 3 software 35 . A calculated minimum sample of 20 mice (divided into 2 equal groups) was needed to detect an effect size of 0.3 in the variance of creatine kinase myocardial band (CK-MB) level and/or lactate dehydrogenase (LDH), with an error probability of 0.05 and 80% power. On expecting 20% attrition, the final sample size will be 20 animals per group (n = 20). Group 1: included mice that received physiological saline solution for 4 successive weeks and serving as control. Group 2: had mice that received tramadol (Tramadol tablets, each contains 225 mg tramadol hydrochloride, Mina-pharm, Egypt) orally by gastric intubation in a dose of 40 mg/kg body weight 36 dissolved in saline daily for 4 successive weeks.
At the end of the experiment, all mice were sacrificed. Heart and aorta tissue were harvested and divided into two parts. One part was snap-frozen then stored at − 80 °C for qPCR, and the other part was prepared as appropriate for histological studies. Blood samples were collected in dry centrifuge tubes. Sera were separated off and frozen at − 20 °C till further use.
Histological analysis. For light microscopy, specimens from the left ventricular myocardial and thoracic aorta of all mice were obtained; they were fixed in 10% neutral buffer formalin, dehydrated in ascending grades of ethanol, cleared in xylene, infiltrated and embedded in paraffin, and sectioned at 5 μm thickness. Sections were stained with hematoxylin and eosin (H&E) for general histological evaluation or Masson's trichrome stain for the demonstration of collagen fibers according to previously published methods 37 . Immunohistochemistry. Paraffin sections of cardiac muscle and aorta (5 μm) were mounted on positively charged glass slides with poly-l-lysine and stained with TNF-α (P300A, Thermo scientific, USA), eNOS (PA3-031A, Thermo scientific, USA) and VEGF (PA1-21796, Thermo Scientific, USA) according to the manufacturer's protocol. Briefly, paraffin sections were deparaffinized in xylene, rehydrated with descending grades of ethanol, and boiled in citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidases were blocked by incubation with 3% hydrogen peroxide at 37 °C for 10 min. Sections were incubated with primary antibodies overnight at 4 °C. After washing, sections were incubated with donkey polyclonal secondary antibodies conjugated with horseradish peroxidase (1:5000) (Thermo Scientific, USA) and for 1 h at room temperature. The reaction was detected using 0.05% diaminobenzidine. Sections were counterstained with Mayer's hematoxylin, mounted, and examined under a Leica microscope (Wetzlar, Germany) equipped with a digital camera. We omitted the primary antibodies during the staining of some of our slides for negative control.
Scanning electron microscopic (SEM) examination. Aortic sections were cut and opened longitudinally to expose the luminal surface. They were rinsed gently with phosphate buffer saline (PBS) to remove surface debris and immersed in a fixative solution of 2% glutaraldehyde and 1% formaldehyde in 0.1 M phosphate buffer pH 7.2, for 2 h. After fixing, tissue sections were dehydrated in a series of alcohols, and liquid carbon dioxide was used to dry the specimens. Dried specimens were mounted on aluminum stubs, fixed in place with colloidal silver, and sputter-coated with gold 38 . A JEOL (J.S.M-5400LV; Japanese Electron Optic Laboratory) was used to view the specimens. Photographs were taken at 15 kV at the Electron Microscopy Unit, Assiut University. Morphometric analysis. Morphometric data were acquired from images of random fields and analyzed by blind histopathologists using ImageJ version 1.52 k (http:// imagej. nih. gov/ ij/). The thickness of the tunica intima, tunica media, and myocardial fiber diameter was assessed as previously reported 40,41 . Additionally, mitochondrial aspect ratio (AR), an indicator for mitochondrial fragmentation, was calculated as the ratio of the major axis/minor axis of the mitochondria. Further, the number of TNF-α, eNOS, and VEGF positive cells were counted.

Effect of tramadol on the expression of heart injury biomarkers.
To explore the adverse effect of chronic tramadol treatment, we first assessed the serum levels of heart injury markers: CK-MB, LDH, and Troponin I (cTn-I). Mice treated with tramadol had significantly elevated serum activity of CK-MB, LDH, and cTn-I compared to control animals (p < 0.05) (Fig. 1).

Histological analysis.
We then explored the effect of tramadol on the morphology of the heart and aorta.
Microscopic examination of H&E-stained sections in the left ventricular myocardium of control mice revealed branching and anatomizing muscle fibers with central oval vesicular nuclei and acidophilic sarcoplasm. These muscle fibers were separated by narrow interstitial spaces containing fibroblasts and small blood capillaries (Fig. 2a). In contrast, left ventricular myocardial sections of mice treated with tramadol showed separation of cardiac fibers with increased interstitial spaces between them; some cardiac muscle fibers appeared pale stained while others had deeply stained sarcoplasm. Additionally, discontinuity, degeneration, and darkly stained nuclei were evident in some muscle fibers. Mononuclear cell infiltration was also seen in between affected widely spaced cardiac muscle fibers (Fig. 2b).
To confirm our observation, we measured the left ventricular cardiac muscle fibers diameter. As expected, treating mice with tramadol significantly decreased the diameter of cardiac muscle compared to control animals (p < 0.05) (Fig. 2e).
To better demonstrate collagen fibers, we stained our heart sections with Masson's trichrome stain. Control mice showed scanty collagen fibers in the interstitial spaces between muscle fibers (Fig. 2c). On the other hand, we observed a marked increase in collagen fibers in the interstitial spaces between muscle fibers of tramadoltreated mice (Fig. 2d).
On semithin sections stained with toluidine blue, the control group revealed cross striation along the length of muscle fibers, and intercalated disks also appeared as dark lines which extend transversely across the muscle fiber in an irregular manner (Fig. 3a). However, in tramadol treated group, disruption in muscle fibers, degenerative changes with pale stained areas, and congested blood vessels were detected (Fig. 3b).
To further explore the effect of tramadol on cardiac muscles, we processed our heart samples for transmission electron microscope (TEM). The sarcoplasms of control cardiomyocytes were composed of longitudinal arrays of cylindrical myofibrils. Each myofibril had myofilaments arranged in sarcomeres that extended between two Z lines (arrows). Sarcomeres were composed of dark bands (A) and two hemi light bands (I). Additionally, we observed elongated mitochondria regularly arranged in rows between the myofibrils and often mitochondrion extent the entire length of a sarcomere (Fig. 3c). In contrast, cardiomyocytes from tramadol-treated mice showed areas with disorganization of sarcomeres, fragmentation of myofilaments, and accumulated groups of circular rather than elongated mitochondria (Fig. 3d).
We measured mitochondrial aspect ratio (AR; major axis/minor axis) indicative of mitochondrial fragmentation. There was a significant decrease in AR of mitochondria of the tramadol treated group compared to the control group (Fig. 3e), indicating that the mitochondria tended to be more fragmented.
To investigate the effect of tramadol on the aorta, we first stained sections from the thoracic aorta with H&E. Regular tunica intima (TI), media (TM), and adventitia layers of the thoracic aorta were seen in sections from control mice (Fig. 4a). On the other hand, aortic sections from mice treated with tramadol revealed thickening of tunica intima and irregular arrangement of smooth muscle fibers and elastin lamella of tunica media (Fig. 4b).
The thickness of the intima and media was measured in both control and tramadol-treated groups. Statistical analysis of the measured aortal wall (intima and media) thickness showed a significant difference between both groups (p < 0.05) (Fig. 4c,d).
Then we employed a scanning electron microscope to examine the adluminal surface of endothelial cells. Sections from control mice showed aortic endothelium with relatively flattened endothelial cells. The cell outlines were easily resolved (Fig. 5a). The luminal surface of the aorta from the tramadol treated group revealed raised endothelial cells with microvillus-like projections projecting from the adluminal membranes (Fig. 5b).
We also processed our aortic sections for TEM. The tunica intima of the aorta of control mice showed a single layer of endothelial cells aligned with the internal elastic lamina (Fig. 5c). In contrast, the tramadol-treated group showed irregular alignment of endothelial cells with irregular internal elastic lamina. Endothelial cells had an irregular-shaped nucleus and rarified cytoplasm (Fig. 5d).

Effect of tramadol on the expression of inflammatory cytokines.
We first assessed the effect of tramadol on the relative expression of TNF-α mRNA levels. As shown in Fig. 6a, tramadol significantly increased the relative expression of TNF-α mRNA levels compared to the control group (p < 0.05). In the aorta, tramadol also significantly increased the relative expression of TNF-α mRNA compared to control mice (p < 0.05) (Fig. 6e). We also investigated the expression of TNF-α using the immunohistochemical technique. We observed weak TNF-α immunoreactivity in the myocardium of control mice. On the contrary, tramadol administration markedly increased TNF-α signals in mice myocardium (Fig. 6i). To confirm our observation, we counted the number of TNF-α positive cells using ImageJ software. In this regard, tramadol significantly increased the number of TNF-α positive cardiomyocytes compared to untreated animals (Fig. 6j).
We next assessed the relative expression of IL-6 mRNA in the heart and aorta in response to tramadol treatment. In the heart, tramadol significantly increased IL-6 mRNA levels compared to control animals (p < 0.05) (Fig. 6b). Similarly, we observed a significant elevation of the relative expression of IL-6 mRNA in the aorta of tramadol-treated mice compared to untreated animals (p < 0.05) (Fig. 6f).  In the heart, treatment of mice daily with tramadol at a dose of 40 mg/kg for 4 weeks significantly increased the relative expression of NOX2 mRNA compared to control animals (p < 0.05) (Fig. 6c). Similarly, tramadol administration significantly elevated the mRNA levels of the NOX2 enzyme in the aorta compared to the control group (p < 0.05) (Fig. 6g). In addition, we measured lipid peroxidation in cardiac and aorta tissues through an assay of MDA levels. Results showed a significant increase in MDA levels in the heart and aorta of tramadol-treated mice compared to the control group (p < 0.05) (Fig. 6d,h).

Effect of tramadol on the endothelial function.
To further identify the mechanism of tramadolinduced cardiac and aortic injury, we assessed the expression of endothelial nitric oxide synthase (eNOS) in the heart and aorta. As shown in Fig. 7a, we investigated the expression pattern of eNOS using immunohistochemical techniques in myocardial sections. Sections from control mice showed intense eNOS reaction in capillary endothelial cells of the mice hearts. On the contrary, a marked reduction in the eNOS immunoreactivity was observed in myocardial sections from tramadol-treated mice. To confirm our immunohistochemical data, we counted the number of eNOS positive cells using ImageJ software. We observed a significant reduction in the number of eNOS positive cells in myocardial sections of tramadol-treated mice compared to untreated animals (p < 0.05) (Fig. 7b). Tramadol administration also significantly decreased the relative expression of eNOS mRNA compared to untreated animals (p < 0.05) (Fig. 7c). Similarly, we assessed the expression of eNOS in the aorta by immunohistochemistry. We observed intense eNOS immunoreaction in endothelial cells lining the aortae of control mice while marked reduction of eNOS immunoreactivity was seen in aortic sections of tramadol-treated animals (Fig. 7d, arrows) with a significant reduction in the number of eNOS positive cells in aortic sections of tramadol-treated mice compared to untreated animals (p < 0.05) (Fig. 7e). Tramadol significantly reduced the mRNA levels of eNOS in the aorta compared to control animals (p < 0.05) (Fig. 7f).
Myocardial sections were also immunostained with anti-vascular endothelial growth factor (VEGF) antibodies. As shown in Fig. 7g, we observed intense VEGF reactions in the sarcoplasm of cardiomyocytes of control mice (arrows), while tramadol administration markedly reduced VEGF immunoreactivity. We also counted VEGF-positive cells to confirm our immunohistochemical observations. We found that tramadol administration significantly reduced the number of VEGF-positive cells compared to control animals (Fig. 7h).

Discussion
Despite its effectiveness in managing moderate to severe pain, tramadol has been associated with several tissues damage 10,12,13 . Additionally, chronic use of tramadol was linked to increased hospitalizations due to CVD 34 . Due to the paucity of data describing its actions on the cardiovascular system, we sought to investigate the effect of chronic tramadol treatment on mice heart and aorta. Here, we showed that tramadol increased the serum levels of cardiac injury markers; creatine kinase-myocardial band (CK-MB) and lactate dehydrogenase (LDH). Additionally, we showed that tramadol induced left ventricular cardiomyocyte degeneration, disorganization of sarcomeres, fragmentation of myofilaments, increased interstitial collagen deposition, and mononuclear cell infiltration. Tramadol also increased tunica intima and tunica media thickness and disorganization of smooth muscle fibers and elastin lamella of the media. Moreover, tramadol increased heart and aorta NOX2 transcripts. Furthermore, tramadol increased heart and aorta TNF-α and IL-6 expression while decreased the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF). Our study suggested that tramadol induced heart and aorta injury through induction of oxidative stress and inflammation.
The current study revealed that tramadol administration increased the serum levels of CK-MB, Troponin I, and LDH. These are cardiomyocyte metabolic enzymes, and their serum levels are reliable indicators of myocardial damage [42][43][44] . This elevation of serum CK-MB and LDH is attributable to their leakage through damaged cardiomyocytes sarcolemma into the circulation as suggested previously 42,45 . Additionally, our histopathological results confirmed the toxic effects of tramadol on the heart and aorta in the form of discontinuity, degeneration of cardiac muscle fibers, thickening of tunica intima, and irregular arrangement of smooth muscle fibers and elastin lamella of tunica media.
Tramadol-induced tissue damage is characterized by oxidative stress that causes cardiomyocyte membrane damage through lipid peroxidation 18,46 . Malondialdehyde (MDA), a secondary lipids peroxidation product, is considered an oxidative stress intensity index 47 . NADPH oxidase 2 enzyme (NOX2) is expressed in endothelial cells and cardiomyocytes 27,48 , and it has been associated with mitochondrial dysfunction and CVD 49,50 . NOX2induced ROS production is an important contributor to cardiovascular damage 51 .
In the current study, we observed a significant elevation of NOX2 transcripts and MDA levels in the heart and aorta of tramadol-treated mice, suggesting the involvement of transcriptional regulation in tramadol-induced NOX2 expression. Our observation is in agreement with the findings of McLaughlin et al. 51 , who demonstrated that doxorubicin-induced cardiac toxicity was associated with elevated NOX2 expression. In addition, downregulation of NOX2 was associated with reduced production of ROS 43 . In the same line, in vitro experiments in cultured endothelial cells revealed that deletion of NOX2 decreased superoxide production and apoptosis upon   53,54 . Taken together, the cardiac and aortic damage observed with tramadol treatment is probably due to increased oxidative stress-mediated through NOX2 and MDA. Accumulating data suggest that the production of ROS in excess promotes inflammatory conditions. Inflammatory mediators such as TNF-α and IL-6 released during these conditions are tightly linked to pathological cardiovascular diseases, including drug intoxication 55,56 . Herein, we reported that treating mice with tramadol increased the expression of TNF-α mRNA and/or protein in the heart and aorta. We also showed that the expression of IL-6 mRNA was significantly elevated in animals treated with tramadol. Our data indicate that prolonged treatment of animals with tramadol established inflammation in the heart and aorta.
In agreement with our results, prolonged exposure to tramadol has been reported to increase cerebral expression of TNF-α and IL-6 transcripts 57 . Moreover, Anilkumar et al. 58 have demonstrated that TNF-α increased ROS www.nature.com/scientificreports/ production in endothelial cells overexpressing NOX2, suggesting the involvement of NOX2 in TNF-α-induced excess ROS production. Furthermore, treating aortic endothelial cells with IL-6 in vitro increased the expression of NOX2 and ROS production 59 . Similarly, Zhao et al. 60 showed that NOX2 inhibition decreased ROS, TNF-α, and IL-6 production. Collectively, it is likely that tramadol, through upregulation of NOX2 expression, induced oxidative stress, which triggered the release of pro-inflammatory mediators, TNF-α and IL-6. These mediators further increased ROS production and ultimately lead to heart and aortic abnormalities. Oxidative stress has been linked to the development of cardiac fibrosis characterized by the alteration of extracellular matrix components (ECM), mainly collagen contents 61 . Cardiac fibrosis increases myocardial stiffness, contributes to cardiac dysfunction, and ultimately leads to heart failure 62 . Herein, we showed increased cardiac collagen deposition in tramadol-treated mice as evidenced by Masson trichrome staining. In line with our study, Tsai et al. 63 have reported that animals treated with doxorubicin not only showed damaged cardiomyocytes but also developed myocardial fibrosis.
Tramadol-mediated myocardial fibrosis can be explained by several mechanisms. Zhang et al. 64 showed that damaged cardiomyocytes secrete factors that induce cardiac fibroblasts proliferation, expression of α-smooth muscle actin, migration, and collagen gene expression. Additionally, oxidative damage to myocardial microvascular endothelial cells has been associated with the transformation of endothelial cells to mesenchymal cells (EndMT) and increased production of ECM 63,65 . Interestingly, Huang et al. 66 reported that mice challenged with lipopolysaccharides showed increased expression of NOX2, elevated ROS levels, and development of cardiac fibrosis; these effects were reversed after NOX2 inhibition. Altogether, tramadol-induced cardiac fibrosis could www.nature.com/scientificreports/ be attributable to NOX2-induced oxidative stress, which leads to activation of cardiac fibroblasts and/or microvasculature endothelial cells to undergo EndMT and lay down ECM components. NO is a vasodilator molecule produced by endothelial cells. Dysregulation of NO characterizes endothelial dysfunction, which underlies several vascular abnormalities. This defect is due to the abnormal activity or expression of eNOS, the predominant NOS isoform in endothelial cells 67,68 , or decreased NO bioavailability as a consequence of increased ROS generation 69 . Our results showed that treating animals with tramadol significantly decreased the expression of eNOS in the heart and aorta at both mRNA and protein levels compared to control mice. Moreover, the current SEM and TEM examination support our finding as they revealed endothelium changes. The altered eNOS expression in tramadol-treated animals could be attributable to endothelial cell inflammation. This assumption is further supported by the recent work of Uthman et al. 70 , who demonstrated disturbance of eNOS expression upon treating cultured endothelial cells with TNF-α. In a similar line, Singh et al. 71 have shown that upregulation of eNOS expression reversed endothelial dysfunction induced by exposure to inflammatory stress. Further, Simplicio et al. (2017) reported that the inhibition of NOX by apocynin reversed the ethanol-induced reduction of eNOS expression and improved the endothelial function of rat-resistant arteries. This could indicate that NOX2-derived ROS and inflammation might have participated in reduced eNOS expression in our model.
In the present study, our immunohistochemical analysis showed that treating mice with tramadol reduced the expression of myocardial vascular endothelial growth factor (VEGF). Mice lacking VEGF were reported to have small heart size and abnormal coronary vasculature 72 . Moreover, direct transfer of the VEGF gene into the myocardium improved cardiac function and promoted angiogenesis in an animal model of myocardial infarction 73 .
Our results showed that tramadol treatment decreased VEGF expression. Previous research described that activation of eNOS is involved in the expression of VEGF 74 . Additionally, Chen and co-workers demonstrated that the eNOS gene delivered directly into the heart reversed cardiomyocytes' apoptosis and increased capillary density in infarcted myocardium 75 . On the other hand, Haddad et al. 76 reported that treating cultured endothelial cells with oxidized LDL increased ROS, impaired VEGF-mediated cell migration, and endothelial tubal formation. These effects were abolished upon inhibition of NOX with apocynin. These data suggest that NOX2-derived ROS compromised VEGF-mediated angiogenesis.
Interestingly, TNF-α disturbs the capacity of endothelial cells to form capillary tubes in vitro, suggesting the involvement of inflammation in impaired angiogenesis 71 . TNF-α decreases eNOS phosphorylation and hence NO production 28 . Collectively, tramadol impaired VEGF expression could be attributable to the tramadol-induced reduction of eNOS expression and/or NOX2 derived ROS, and inflammation.

Conclusion
The results of the current study confirmed the toxic effects of tramadol on the heart and aorta. Tramadol treatment induced cardiac damage demonstrated by the increased serum levels of cardiac injury markers and histopathological findings. Moreover, tramadol-induced oxidative stress, inflammation, and endothelial dysfunction. This study provides insights into the mechanisms of tramadol-induced cardiac and aortic injury.

Data availability
Data supporting the findings of this study are available from the corresponding author upon reasonable request.