Assessement by laser Doppler of the peripheral tumour perfusion after radiofrequency for colorectal liver mestasis - experimental study

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Assessement by laser Doppler of the peripheral tumour perfusion after radiofrequency for colorectal liver mestasis - experimental study

C.G. Precup, D. Gonganau-Nitu, R.R. Scurtu, G. Dindelegan, A. Biro, O. Soritau, C. Crisan, O. Serban, G. Pufu, C.
Original article, no. 1, 2010
* Depart of Surgery no 1, Univ of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj-Napoca, Romania
* Depart of Surgery no 1


Introduction
The colorectal cancer (CCR) is one of the most frequent malignant conditions of the past two decades (1). In Romania, a recent study reported an increase in the CCR occurrence by 74.5%, which are 23.57 new cases in 100000 inhabitants (2). About 26-44% of the patients with CCR have ganglion metastases at the time/moment of the diagnosis and about 50% of these will recur in the first 5 years from the treatment of the primitive tumor, the hepatic metastasis representing 40-80% of the total of these recurrences (3).
The resection of the hepatic metastasis used to be considered until recently the only efficient therapy way (4). However, there are limitations to this type of approach: the presence of more than four metastases, bilobare metastasis, and the need to leave enough hepatic parenchyma after resection to ensure an efficient hepatic function after the surgery (5). Nowadays, radiofrequency is the most widely used therapeutical method for in situ destruction of the CCR hepatic metastases which cannot be resected (6). However, up to 40% of patients display recurrences of the lesions and 12 % of these are on the same spot of the treatment in the first year following therapy, recurrence rate being proportional with the size of the lesion (7). The high rate of local recurrences in tumours with a diameter of more than 3 cm is cause by the high variability and poor reproductibility of the ablation area (8).
The RF ablation efficiency is dependent on distance up to the radiofrequency probe and moreover is dependent on the tissue vascularisation type. The blood flow can undertake and dissipate the heat and herebyon the edge of radiofrequency coagulation area can persist viable tumour cells and even tumoural vessels (9).
Laser-Doppler flowmetry (LDF) has proved to be a simple and well established technique for monitoring the microcirculatory blood flow in different organs. The LDF advantages over other techniques are:the ease to use, noninterference with blood flow, and continuous and real-time measurements with a high degree of spatial resolution (10, 11, 12).
It has been shown that the LDF signal reflects changes in whole organ liver perfusion despite its small interrogation volume (11, 13).
Laser Doppler techniques were used to quantify changes in hepatic blood flow during RF ablation and it has been showed that there is a correlation between blood flow and heat retention in tissues when induced coagulation necrosis was performed (14).
The aims of the study was to evaluate microcirculatory blood flowusing lasser doppler for hepatic tissue and peripheral tumour perfusion after radiofrequency and the influence of temperature increasing at 42°C on local tissular perfusion in the same spots. We sought to evaluate whether peripheral perfusion of hepatic tissue is lower than the peripheral perfusion after heating the tissue at 42 °C in the same place (i), whether peripheral perfusion of tumor is greater than the peripheral perfusion of normal liver (ii), whether the peripheral perfusion of tumor is lower than the peripheral perfusion after heating the tumor at 42 °C in the same place (iii) and whether peripheral perfusion of normal liver, heated normal liver, normal liver tissue after heating, tumor, heated tumor, and tumor after heating is higher than the peripheral perfusion of post RF tumor in the same experimental conditions (iv).

Material and Method
Animals
Fifteen male Wag/Rij rats weighing 250-300 g were used (Charle River, Germany). The animals had free acces to food and water. The weight of the animals was measurated before tumour induction and before radiofrequency treatment. All animals were housed in the Individual Ventilated Cages (Tecniplast, Italy) in the animal facility of the University of Medicine and Pharmacy Iuliu Hatieganu Cluj-Napoca. The University Ethic Committee approved the study design.
Cells culture and liver metastasis model
The colon adenocarcinoma cell line CC531s was used for tumor inoculation. The tumour is moderately differentiated and synergic to Wag/Rij rats. Tumor cells were cultured in RPMI 1640 supplemented with L Glutamine 2 mM (Gibco, Grand Island, NY USA) 10% heat inactivated fetal calf serum, penicillin 100Uml and streptomycin 0.1 mg/ml.
Tumour cells were harvested with solution of EDTA and Trypsin 0,25% in HBSS (Sigma, St. Louis, MO, USA) and washed three times with PBS. Using trypan blue exclusion test the cells were adjusted to a susspension of 1 x 106 viable cells/ml of PBS. For liver tumour induction 0,1 ml of tumour cells suspension (1 x 105 viable tumour cells) was injected in two sites subcapsulary in the liver left lobe.
Radiofrequency protocol
When the tumour was 1 cm diameter, 21 days after tumour incoculation (15), the right tumour was destroyed using radiofrequency (RIT® generator and Starbust SDE electrode, AngioDynamics® INC USA). The RF probe was inserted 1 cm in to the tumour and the radiofrequency was applied for 2 minutes at 80°C with the set-up power at 20W. The tumour in the left lobe was kept untreated as a negative control.
Laser-Doppler flowmetry
Liver surface perfusion was measured by LDF using a Periflux System 5000 flowmeter (Perimed AB, Sweden) with a thermostatic laser doppler probe (PF457) secured with a selfadhesive probe holder (12). LDF results were expressed in arbitrary perfusion units (PU).
Perfusion was measured in normal liver, on the tumour before and after radiofrequency treatment. Perfusion was recorded for 2 min initial, 2 min after the tissue was heated at 42°C and 2 min after the initial temperature was reached again.
Data acquisition was performed using PeriSoft software (Perimed AB, version 2.5).
Before data aquisition the calibration procedure was performed. Briefely, the tip of the LDF probe was immersed in the microsphere suspension and Brownian motion of the latex particles provided the standard LDF signal.
Data analysis
All calculations were performed with SPSS for Windows Version 17.0 (SPSS Inc. Chicago, IL, US). All values are given as mean ± standard deviation. The hypotheses of the study were tested with T-Test for dependent variable.

Results
Twenty-one days after tumour inoculation, animals were anesthetized and tumour was macroscopically examined. One animal did not develop any macroscopic lesion in liver and on three animals we found peritoneal tumours and ascitis. All four rats were excluded from study. The rest of 11 animals had lesions only in liver with average diameter of 1,22 cm (SD=0,26).
After radiofrequency treatment one animal died due to an inefficient thermic insulation of the liver from the surrounding organs, especially pericardium. Other five deaths were recorded perioperative because of termal lesions on the small bowel.
The peripheral perfusion measurement was not possible after RF in three tumours, and in two tumours, and two normal tissue liver due to tissues rough surfaces and inadequate probe adherence.
Peripheral perfusion of theliver tissue (M=110.1256, SE = 5.91) was smaller than peripheral perfusion of normal liver tissue heated at 42°C (M=116.2567, SE=5.8, t(8) = 2.821, p =.022)(Fig. 1A).
When the peripheal perfusion of tumour (M=163.45, SE=41.06) was compared with the perfusion in the same spot but heated at 42°C (M=152.04, SE=33.94) there was no statistical differences (t(8)=-1.19, p=.268) with a decrease of perfusion after heating at the measurement site (Fig. 1B).
After RF treatment the perfusion of the tumour margins (M=19.2, SE=4.5) was lower then peripheral perfusion of the liver (M=105, SE=6.76), (t(5)=9.96, p=.000)(Fig. 1C -1A).
The peripheral perfusion of the tumor (M=164.25, SE=52.8) was higher than the perfusion of normal liver (M=107.25, SE=6.19) but with no statistic segnificance [t(6)=-1.176, p=.284]. (Fig. 1D)
The peripheral perfusion of the liver heated at 42°C (M=112.9, SE=6.82) was higher than the peripheral perfusion of the tumor after RF heated at 42 °C (M=16.8, SE=3.83, t(5)=12.18, p=.000) (Fig. 1E).
If the basal peripheral perfusion of the liver after heating (M=99.77, SE=11.56) was compared with the basal peripheral perfusion of the tumor post RF and after heating (M=16.06, SE=4.06) it showed statistical significance [t(5) = 6.25, p=.002] with a clear drop of tissue perfusion (Fig. 1F).
There was a decrease in marginal perfusion of the tumour after RF treatment (M=19.39, SE=3.75) when compared with the basal peripheral perfusion of the tumor (M=137.77, SE=33.86) [t(6)=3.34, p=.016] (Fig. 1D).
The peripheral perfusion of the tumor heated at 42°C (M=133.77, SE=30.41) was higher than the peripheral perfusion of the tumor after RF heated at 42°C (M=18.36, SE=3.63, t(6)=3.8, p=.009) (Fig. 1D-1E).
After RF treatment the basal peripheral perfusion of the tumor (M=19.94, SE=4.43) was lower then the basal peripheral perfusion of tumor after heating (M=132.55, SE=32.1), [t(6)=3.41, p=.014] (Fig. 1D-1F).

Figure 1

Discussion
Radiofrequency is currently the most widely used tumor ablative technique, which has been shown to be safe and feasible in patients with unresectable liver colorectal metastasis (16). Unfortunately, the reported results after RF ablation are less favorable than those recorded after surgery, with an important recurrence rate which can reach up to 40% of patients, while 12% of them were found to have recurrence at the treatment site after only one year (17). When RF ablation was performed percutaneously, the local recurrence rate, one year after the treatment, was reported to overcome 20% of patients (18).
The size of the metastasis is the most important factor that limits the efficacy of total tumor ablation by RF, with reported recurrence rates significantly higher for metastasis larger than 3 cm in diameter (19).
Up to date there are no reports on an efficient real time imagistic method that could be used to assess RF efficiency during tumour ablation. The present study used the Laser Doppler flowmetry in order to evaluate the blood flow at the edge of the tumour necrosis. The working hypothesis was that the persistence of viable blood vessels might be associated with viable tumour cells, as suggested by several authors (20). Classically, the parameter governing tissue destruction for RF ablation has been tissue heating, which induces cellular death via thermal coagulation necrosis. The volume of RF ablation is therefore governed by the temperature distribution within the tissue. The temperature distribution during ablation varies with the tumour diameter with high temperatures near the ablation probe that gradually decrease to normal at the ablation periphery (21). This supports the finding that RF-induced damage is not homogeneous throughout the ablated region (22, 23). Furthermore, evidence suggests that the peripheral margins of the tumour contain areas of normal tissue within which areas of microscopic residual tumor reside and therefore the inability to treat these areas satisfactorily with conventional RF ablation therapy alone is a leading cause of post–RF ablation residual tumor growth (24). In our study, after the RF ablation, the laser Doppler probe still recorded the presence of a blood flow at the edge of the coagulative necrosis even though there was a significantly decrease when compared to the blood flow of the surrounding normal liver parenchyma.
Goldberg et al (25) showed that local vascular perfusion interfere also with RF and reduce the volume of the tissue necrosis. Therefore, it has been suggested that reduction of blood flow by Pringle maneuvre or clamping the celiac axis, during RF application increases coagulation. The same team showed that the administration of the vasoactive pharmacologic agents could be used to reduce hepatic blood flow and, as a resultt increase RF-induced coagulation necrosis. For this study, laser Doppler techniques were used to quantify changes in hepatic blood flow (14). Their results support the findings of our study and showed that laser Doppler flowmetry allows an adequate assessment of the tumour and normal liver blood flow after RF ablation.
Based upon the exponential decrease in RF tissue heating, there is changing thermal gradient in tissues surrounding a RF electrode, and as such, a large zone of tissue is heated to temperatures that result in reversible cellular injury (26). Moderate hyperthermia recorded in the post RF necrosis zone accelerates the local blood flow and increase the microvascular permeability. This finding is suggested by the large amount of hemorrhage that we observed in tumors in this peripheral watershed area (27).
As far as we know this is the first study trying to evaluate the effects of enhanced hypertermia on the local blood flow in the normal liver and metastatic liver tumour prior and after RF treatment. In our study moderate hyperthermia (42°C) was followed by a significant improvement in the normal liver blood flow when compared to the tumour, while the blood flow at the edge of the RF zone was significantly slower than that recorded in the hypertermic tumour tissue or in the normal liver parenchyma.
Local moderate hypertermia might increase chemotherapy citotoxic effects and subsequently might improve local control of the tumour (28). The association of radiofrequency with the administration of liposomal doxorubicin has shown an increase in doxorubicin concentration in the tumour.
Therefore, assessing the local blood flow and tissue temperature during RF ablation by Laser Doppler might be useful not only for RF efficiency evaluation but also as an indication for associating adjuvant local chemotherapy.

Acknowledgment
This work was supported by research grant from the Romanian Ministry of Education, Research and Innovation, PNII Ideas Contract No 195/2007.

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