Abstract
Background/Aim: Owing to new oncotherapy modalities, the importance of an R0 resection decreased in the last decade; however, liver metastasis of colorectal cancer significantly decreases survival. Furthermore, to prevent cardiovascular disease, more and more patients are treated with anti-ischemic drugs, which may influence oncologic treatments in such patients. This study aimed to examine the effect of Trimetazidine on liver surface injury after spray diathermy. Materials and Methods: We performed standard liver resections with resection-margin spray coagulation in 36 rats on both liver lobes. In all procedures, a Pringle maneuver was performed on the right lobe, while on the left lobe, no vessel occlusion was applied. Half of the animals were on Trimetazidine therapy. In 12 animals, histologic samples were taken immediately after operation, while 12 animals were terminated 1 week later, and the remaining 12 animals 3 weeks later. After standard HE staining, histologic analysis was performed. Results: When diathermy was used, a coagulation zone appeared. Destruction was slightly wider in case of Trimetazidine therapy (745.75 vs. 680.04 μm). In cases of 1-week-surviving animals, a necrotic zone was observed under the coagulated tissue, and a fibrotic zone appeared after 3 weeks. In TMZ medicated animals, the destruction zone was significantly thinner (645.08 vs. 893.76 μm; p<0.001) and the necrosis zone showed the same difference (2,430.05 vs. 3,238.45 μm; p<0.001). Conclusion: Administration of Trimetazidine can reduce the extent of thermic necrosis. Furthermore, a great effort should be applied to achieve R0 resection in patients on anti-ischemic therapy.
Colorectal cancer (CRC) is a frequent malignancy. Based on international data, colorectal cancer (CRC) is the third most common type of cancer and the fourth most common death cause worldwide. Based on the data of WHO in 2019, malignancies are a leading cause of death in all examined countries, more than 1,8 million new cases and 906,000 CRC associated deaths were registered in 2020 (1). Global burden is projected to grow to more than 2,2 million new cases and 1,1 million deaths by 2030 (2). The 5-year-long survival chance in advanced stages is very poor (about 30%). Distant organ, especially liver metastases are frequently present (30-60%). About 15-25% of patients have synchronous liver metastasis at the time of CRC diagnosis; further 30% become metastatic within the next 2 years (3-5). Without treatment, the life expectancy of patients with colorectal liver metastasis (CRLM) is 6-9 months (6). The only curative method is resection, still the recurrence rate is around 70-80% (7). Parenchyma sparing technique and optimal surgical margins are important objectives, the later having a leading role in patient survival. The width of resection margin is considered important, but still there is no consensus on its exact size. Some studies found, that an R1 resection (microscopically positive resection margin) has no prognostic role in recurrence, while others reported that an R1 resection is only acceptable in RAS wild-type patients responsive to chemotherapy (8, 9). In general, R0 resection is strongly suggested in CRLM cases, regardless of the administration of the modern chemotherapy protocols (10). Large and multiple metastasis can limit resections, as an excessive resection can lead to life threatening liver failure.
To facilitate an appropriate resection with sufficient remnant liver tissue, the parenchyma-sparing technique became widely used for liver metastasis resection. This may provide better disease-free survival and fewer complications, however, the need for re-resection due to recurrence is more frequent (11). The rate of R1 resections may reach 10-30% also by experienced hepatobiliary surgeons. While R0 resection remains standard, R1 resection coupled with multimodal approaches has also become acceptable (12). In the case of an extended radicality, increased blood loss is risk factor for postoperative complications (13). Vascular occlusion may cause severe ischemia/reperfusion (I/R) injury, with structural and functional damage to hepatocytes (14, 15). Diathermy used in “spray mode” can control bleeding, but on other hand, tissue destruction appears. Using diathermy in animal studies, a 3-4 mm deep coagulation zone has been described similarly to argon beam coagulation (16). In another study, this coagulation zone was deemed to be useful in cases of R1 resections, as it could wipe out the liver surface, containing possibly living tumor cells (17).
Trimetazidine (TMZ) is a widely used anti-angina and anti-ischemic drug. It is often used to reduce I/R injury in many organs. For the retina, TMZ has an advantageous effect on lipid peroxidation, it stimulates AMPK and ERK signaling pathways in cardiac myocytes. TMZ has an antioxidant effect on the testicles, and fosters a higher antioxidant level in the kidneys. TMZ pretreatment decreases injury and increases regeneration even in pedicled skin flaps and liver tissue. Furthermore, it can decrease the risk of steatotic liver failure after transplantation. In addition, treatment with TMZ during the vascular clamping technique decreased cell death. In these cases, TMZ elevates liver ATP content, and during reperfusion enhances the levels of reduced glutathione in the liver tissue, which may prevent hepatic I/R injury (13, 18-25).
Many patients diagnosed with CRLM are on anti-angina medication. We aimed to investigate the effect of TMZ administration on thermic injury caused by diathermy under different liver perfusion conditions in an animal model.
Materials and Methods
In this study, 36 male Wistar rats (Charles River Breeding Laboratories - Isaszeg, Hungary) (weight 510-690 g) were used. In accordance with the Animal Welfare Act, rats were housed in a light controlled (12-h light-dark cycle) and air-filtered room in individual cages, kept at room temperature, with free access to food and water (24-hour-long fasting period before the procedures). The protocol conformed to the Guide for the Care and Use of Laboratory Animals published by the Federation of European Laboratory Animal Science Association, and was approved by local institutional committee on animal research, University of Pecs (№ BA 02/2000-16/2012).
Anesthesia was performed by an intraperitoneal injection of ketamine hydrochloride (500 mg in 10 ml solution) and diazepam (10 mg in 2 ml solution) in 1:1 ratio (0.2 ml injection for 100 g weight). After routine skin disinfection, a midline laparotomy was made.
After isolating the right and left medial lobes of the liver, 1 cm wide resections were performed on the top of the lobes on both sides. On the right side, the resection was performed using a Pringle maneuver, while the left lobes were resected without a Pringle maneuver. Resection surfaces were coagulated with monopolar electrocautery (Erbe Elektromedizin GmbH, Tübingen, Germany) in spray mode with 120-Watt energy level. At the end of the interventions, the abdominal cavity was rinsed with 35oC warm saline, and the abdominal wall was closed in two layers with absorbable sutures.
Regarding the survival, animals were divided into 3 groups, with 12 rats in each group. In the 1st group termination and histologic sampling were performed immediately after surgery, in the 2nd group after 1 week, and in the 3rd group after 3 weeks. In each group, half of the animals received TMZ medication for 1 week before the operations (10 mg TMZ for 1,000 g weight), and the other half did not (Figure 1).
Groups of examined animals. The right side was resected using a Pringle maneuver, while left lobes were resected without this maneuver.
We aimed to detect and compare the histologic changes induced by the thermic effect of diathermy between the examined groups.
Blocks were formed from all liver tissue samples and stored in 10% neutral Formaldehyde solution until histologic examination. The samples were prepared using a tissue processor device (Thermo Shandon Pathcentre, Thermo Fisher Scientific Inc., Waltham, MA, USA). A sledge microtome (5 μm, Reichert Optische Werke AG, Vienna, Austria) was used for sectioning paraffin-embedded blocks, and 10 slices were prepared from each block. The slides were stained with hematoxylin-eosin at the Department of Pathology using a carousel-type slide stainer (Thermo Varistain 24-4, Thermo Fisher Scientific Inc.). For evaluation of the histologic slices, the Pannoramic Viewer software (3DHistec Ltd., Budapest, Hungary) was used. For each slide, 20 measurements at 200× magnification were performed. To avoid interobserver error, all measurements were performed by the same experienced pathologist.
For statistical analysis the SPSS Statistics for Windows v.22 software was used (IBM Corporation, Armonk, NY, USA). All values are presented as mean±SEM. Differences were investigated by paired-sample t-test. Independent sample t-test was used to compare parameters between the groups. All p-values lower than 0.05 were considered as statistically significant.
Results
There were no complications or mortality. Examination of the histologic samples of group 1 showed that the surface of the liver tissue was completely destroyed, but normal structures were observed deeper in the liver tissue. Between these layers, spotty coagulation damage among normal cells was seen (Zone 1). The width of the zones was compared with or without TMZ medication, and with or without Pringle maneuver. In animals with Pringle maneuver, the destruction zone was wider in animals treated with TMZ than those without TMZ. The difference between TMZ and no-TMZ animals was not significant (745.75±34.40 vs. 680.04±17.90 μm; p=0.227). In the non-Pringle group with TMZ, the destructed zone was also wider, but not significantly (584.21±14.36 vs. 558.63±5.00 μm; p=0.235). Comparing the width of destruction zones in TMZ medicated rats, significantly deeper zones were observed in the Pringle group compared to the non-Pringle group (584.21±14.36 μm non-Pringle vs. 745.75±34.40 μm Pringle; p<0.001).
In group 2, a wider destruction zone was measured one week after the operation (Zone 1), and below this a coagulation necrosis was observed (tissue pattern worse than normal liver, with complete necrosis in the deepest part of the zone; Zone 2). In the deepest part, a reparation zone was identified (hypercellular connective tissue with inflammatory signs and invasion of fibrocytes; Zone 3).
In the TMZ medicated animals, Zone 1 was significantly thinner both in non-Pringle (645.08±23.71 vs. 893.76±33.69 μm; p<0.001) and Pringle groups (886.26±24.04 vs. 1,303.08±47.48 μm; p<0.001), compared to animals not treated with TMZ. Also, Zone 1 was wider in animals treated with TMZ after the Pringle maneuver than without it (745.75±34.40 vs. 584.21±14.36 μm; p<0.001).
In Zone 2, the same significant difference was measured in favor of the TMZ group. (non-Pringle: 2,430.05±52.71 vs. 3,238.45±89.84 μm; p<0.001. Pringle: 2861.41±81.76 vs. 3,531.41±61.81 μm; p<0.001).
Zone 3 was significantly thinner in the livers with Pringle maneuver after TMZ medication than those without this maneuver (77.07±2.51 vs. 111.96±3.92 μm; p<0.001). In the non-Pringle group, there was no significant difference with TMZ medication and without it (84.24±2.68 vs. 96.22±3.31 μm; p=0.017). However, the width of Zone 3 in animals treated with TMZ medication was thinner in the group with Pringle than without it. (77.07±2.51 vs. 84.24±2.68 μm; p=0.011).
Histologic results showed different zone characteristics after 3 weeks survival in group 3. Zone 1 was a young fibrotic zone where necrotic fragments were surrounded by newly formed liver tissue, with large number of cells and less collagen. Below this area in Zone 2, granulation tissue appeared. In the deepest Zone 3 large amount of collagen and a large number of cells were visible with newly formed bile ducts and vessels together with chronic inflammatory infiltration.
Similarly to Group 2, Zone 1 in TMZ medicated animals was thinner both in Pringle (1,078.17±22.12 vs. 1,272.06±59.18 μm; p=0.032) and non-Pringle groups (763.92±19.27 vs. 954.18±56.28 μm; p=0.020) than non-treated animals, however not significantly. Similarly to group 1 and 2, the Zone 1 with TMZ medication was much wider after Pringle maneuver compared to non-Pringle (1,078.17±22.12 vs. 763.92±19.27 μm; p<0.001).
Zone 2 showed the same, smaller but not significant difference in favor of TMZ medication (non-Pringle: 1,273.91±33.31 vs. 1,462.53±54.46 μm; p=0.023; Pringle: 1,770.12±25.93 vs. 1,841.39±57.89 μm; p=0.026). Zone 3 in TMZ treated animals was thinner, which was not significant without Pringle (574.50±19.01 vs. 765.55±48.22 μm; p=0.029) and significant with Pringle (469.02±13.13 vs. 570.62±14.20 μm; p<0.001) compared to the non-treated group. Zone 2 was significantly wider following TMZ treatment in the Pringle than in the non-Pringle group (1,770.12±25.93 vs. 1,273.91±33.31 μm; p<0.001), but significantly thinner in Zone 3 in the Pringle compared to the non-Pringle group (469.02±13.13 vs. 574.50±19.01 μm; p<0.001).
Zone widths in the different groups are compared in Table I, and hematoxylin-eosin staining is shown on Figure 2.
Comparison of zone widths of the different groups (mean±SEM).
Histologic examination (hematoxylin-eosin staining) of the liver tissue immediately (A; 150×), one week (B; 100×) and three weeks after resection (C; 100×). B/1 shows destruction zone (150×), B/2 coagulation necrosis (200×), and B/3 reparation zone (200×). C/1 demonstrates necrotic zone with newly formed liver tissue (150×), C/2 granulation zone (200×), and C/3 chronic inflammation with newly formed bile ducts and vessels (150×).
Discussion
Colorectal malignancies are common, and in advanced stages when metastasis occurs the survival is poor (1-5). The most frequent metastatic sites are the lungs and the liver (4, 26, 27). About 20% of patients have synchronous liver metastasis, and a further 30% develop CRLM in the next two years. Survival chance of such patients is around 6-9 months without treatment (6). Therefore, liver resection is highly suggested if feasible (6, 28-30), and also in cases of a recurrence re-resection can offer a further survival advantage (31).
There is no definite correlation between the status of surgical margin (R0 vs. R1 resection) and survival in the case of CRLM resection. Some studies reported higher local recurrence after R1 resection, suggesting that positive resection margin is a negative prognostic factor only in cases without perioperative chemotherapy (32). However, in the last decade a newer aspect appeared: vascular R1 resection might be acceptable when a parenchyma sparing technique is applied (33-35). Thanks to modern chemo-, and targeted oncology therapies, tumor biology might be of the same importance as the surgical margin (8, 10, 34-37). Parenchyma sparing technique became the method of choice to assure better tumor clearance with reduced blood loss and proper resection margin if achievable. This method implies a Pringle maneuver with “Kelly clamp crushing technique” and spray diathermy for a fast, safe and cheap resection (38). A further advantage of the “Kelly clamp crushing technique” is a 2-4 mm deep tissue destruction zone on the resection surface, which can be extended by an additional 3-4 mm wide region with spray diathermy (16, 39, 40). In case of extended resections, the function of the remnant liver directly influences patient survival. Depending on the patient’s liver function, up to 60-80% of liver parenchyma can be resected with a remnant functional liver approximately 20% in a liver without previous chemotherapy, 30% after chemotherapy and 40% with cirrhosis or fibrosis (41). Most recent techniques like portal vein embolization, two-stage hepatectomy, and associating liver partition and portal vein ligation are used to enhance liver hypertrophy, thereby increasing the extent of liver resection (33, 42). To reach a balance between adequate resection margin and decreased blood loss, the most frequently used technique is the portal triad occlusion, or the Pringle maneuver. Both cause ischemia/reperfusion (I/R) injury to the remnant liver tissue. To prevent it, a periodical occlusion is recommended (14, 15). Another tool to decrease bleeding is the spray mode diathermy coagulation, which forms a 3-4 mm deep thermic damage zone (similarly to argon beam coagulation) (16). This coagulation zone can be useful in case of an R1 resection, because it can destroy additional liver surface possibly containing viable tumor cells.
The widely used anti-ischemic and anti-angina agent TMZ is also suggested to reduce I/R injury on several organs (retina, heart, testicles, kidney, and even transplanted liver). Previous studies reported that TMZ administration can prevent hepatic I/R injury if vascular occlusion is applied (13, 18-24). Studies on the effect of TMZ on tumorous liver tissue, and CRLM recurrence are not yet available.
Since many of CRLM patients are also treated for ischemic heart disease using TMZ, we aimed to investigate whether the tissue destruction effect of diathermy differs if a Pringle maneuver is used, or TMZ is administered. We analyzed the short- and long-term effects using a chronic model.
Analysis of the instant consequences of diathermy showed a single coagulation zone caused by the thermic effect. The width of this destructed area was slightly but not significantly larger in cases with TMZ medication. This finding suggests, that TMZ has no instant protective effect on the depth of immediate tissue destruction following spray diathermy. After 1 week a necrotic zone appeared under the coagulation area, which became thinner at 3 weeks. In this group a fibrotic zone also appeared with collagen formation. In both groups, TMZ reduced the necrosis.
These findings suggest that the effect of TMZ is variable: immediate tissue damage is augmented (together with I/R effect of vessel clamping) and later necrosis is reduced. If a Pringle maneuver was applied, all tissue destruction zones were wider independently of TMZ application, due to the reduced blood flow (ischemic effect).
However, the most important result of this study is, that TMZ can protect remnant tumor cells on the resection surface (R1 category) increasing the risk for CRLM recurrence. In these cases, efforts for a certain R0 resection may be highly important.
Acknowledgements
The Authors would like to thank all the administrative employees of the Department of Surgery and the Department of Surgical Research and Techniques for their invaluable help. The research was funded by EFOP 3.6.1-16.2016.00004, University of Pécs. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the article.
Footnotes
Authors’ Contributions
SF, AP and AP designed the study. AV, IT and AP supervised the project. SF and AP performed data analysis. AF performed histological examination. SF, AP and AV drafted the manuscript. BN validated and revised the draft. All Authors critically revised the article and approved the final manuscript.
Conflicts of Interest
All Authors declare no conflicts of interest in relation to this study.
- Received June 28, 2022.
- Revision received July 13, 2022.
- Accepted July 14, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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