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Apoptosis Post Microwave Ablation Of The Liver; Does It Change With Power?

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Apoptosis is a type of the delayed or indirect cellular responses that happen after microwave ablation. It helps eradicate the few cancer cells that might survive the applied heat during cancer ablation. The extent of its expression is yet to be defined. Aims:We investigated whether the ablation power made any difference to the expression of apoptosis in the ablated and normal areas. Method:Ablations with 50W, 70W & 90W powers were created in three ex vivo perfused porcine livers. Biopsies were collected from the lesions and were assessed with Haematoxilin-Eosin and immunohistochemistry (Caspase 3 and M30) looking for apoptosis in each zone (central necrotic zone “CNZ”, Transitional zone “TZ” and Normal surrounding zone “NZ”). Statistical analysis was performed using AVOVA and t-test. Results:None of the CNZ showed expression of Caspase-3. In the TZ, there was significant difference between 50W and 90W (P=0.009), but not between 50W and 70W (p=0.8) or between 70W and 90W (P=0.4). In the NZ, a highly significant difference was noted between 50W and 90W (P=0.003), a significant difference between 50W and 70W (P=0.01), but not between 70W and 90W (P=0.06). For M30, no expression of M30 was noted in all necrotic zones. A significant difference was noted between 50W and 90W (P=0.02).

There were no significant differences between 50W and 70W (P=0.4) or between 70W and 90W (P=0.07). Conclusion:Increasing power enhances apoptosis in the ablated areas. This response can be an adjunct for eradicating cancer cells that might escape heat in the ablated zones.Introduction: With an enduring focus on minimally invasive techniques in surgery, ablative therapies have been of significant clinical interest in recent years for their potential utility in the treatment of early-stage tumours in-situ. Such ablation modalities have included ethanol ablation, cryoablation, laser ablation, focussed ultrasound (FUS), RFA and more lately, microwave ablation (MWA) [1]. Delivery of localised thermoablative therapy in the clinical setting is performed under image guidance, with usually US or CT based methods used to guide the deposition of energy at a site of specific therapeutic benefit, usually a tumour [2]. The supraphysiological temperature rise of the surrounding tissues derived from this energy deposition causes changes at a molecular, cellular and macro-histological level, resulting initially in a coagulative necrosis of tissues in the immediate vicinity of the ablative energy. Further afield, away from the centre of ablation and adjacent to cell necrosis occurs a zone of transition between necrosis and intact cells, where a delayed and indirect apoptotic response of a variable degree arises. This apoptotic response is of further therapeutic benefit in the treatment of neoplastic cells and the prevention of tumour recurrence [3].

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With MWA being the latest ablation modality explored, work has been done to further characterise and compare the physical properties of this technique with other ablation modalities. MWA occurs at characteristic frequencies falling between 300MHz and 300GHz, laying between radio wave and infrared on the electromagnetic spectrum. The delivery of thermal energy in microwave ablation is based on the alternation of polar electric charge on water (H2O) molecules in the local tissues causing agitation of, and thereby friction between, these H2O molecules generating heat. The frequency with which the charge on H2O molecules alternate depends on the frequency of microwave energy used, but is of the order of 2-5 x 109 times per second [4].On review of the literature to date by the authors, current in-vivo and ex-vivo work on MWA has focussed on characterising its tumour ablation potential and ability to cause cell necrosis with regards to different MWA variables. These have included the wavelength of microwave energy used, with studies comparing wavelengths of 915MHz and 2.45GHz, energy application time and power used in watts, with studies comparing powers of between 45W to 60W, and 60W to 180W [5]. In this study, we wanted to find out if apoptosis correlated with the applied MWA power. To our knowledge, no similar experiments have been published in the literature.Materials and methods: Liver Procurement:Three white pigs weighing between 45 and 60 kg were humanely sacrificed in accordance to the Home Office regulations.

The autologous blood was collected in a pre-heparinised nonpyogenic container with 5000 Units of Heparin. With a minimal warm ischemic time, the livers were retrieved and were perfused with two litres of cold Soltran solution (Baxter Healthcare, Thetford, United Kingdom) via the portal vein (PV) and the hepatic artery (HA) and were then transported on ice from the abattoir to the laboratory. Circuit Preparation: The circuit was primed with the autologous heparinised blood. Cannulation of the PV, HA, bile duct, supra- and infra-hepatic inferior vena cava (IVC) was carried out during the priming. One litre of 0.9% Normal Saline solution (Baxter Healthcare, Thetford, United Kingdom) was perfused through the PV and HA to flush out the Soltran solution and remove air from the liver and the cannulae before the livers were connected to the extracorporeal circuit.Perfusion:The extracorporeal circuit (Medtronic Inc., Minneapolis, Minnesota – United States) consisted of an automatic centrifugal pump which provided the hepatic arterial flow and pressure, an oxygenator, a heat exchanger unit and a blood reservoir to simulate the venous flow and pressure (Figure- 1).

The venous blood was collected from the supra- and infra-hepatic IVC and returned to the centrifugal pump. Perfusion was carried out for six hours. To optimise the physiological condition of the system, parenteral nutrition, vasodilating prostacyclins, sodium bicarbonate, sodium taurocholate and insulin were added to the circulation.Microwave ablation:In the three livers, twenty one MWA lesions were created one hour after liver perfusion to make sure all physiological parameters have settled. The lesions were created at power s 50W, 70W and 90W (seven lesions for every power, total 21). Each lesion was created for two minutes. The microwaves were generated by a MicrosulisTissue Ablation Sulis TMV Generator (Microsulis Ltd., Denmead, Hampshire, UK) at a frequency2.45 GHz and delivered via the Accu5i applicator (Microsulis Ltd., Denmead, Hampshire, UK) which has a shaft diameter of (9mm) and a biocompatible non-stick coating. The applicator was inserted to 2 cm beneath the surface of the liver in all applications to achieve a consistent shape throughout the study.Specimens:Wedge biopsies were collected from the ablated areas including the three zones and were embedded in liquid paraffin (seven biopsies for each power; 50W, 70W & 90W, total=21). Antigen retrieval: The specimens were cut onto Vectabond slides, Dried at 37°C overnight and were then heated at 65C for 10 minutes, deparaffinised through xylene, rehydrated through graded alcohols (99% Industrial Methylated Spirits “IMS” and 95% IMS) and then rinsed in tap water for five minutes. The slides were then placed in a plastic slide rack, in a plastic dish which was then topped with 10mM Sodium Citrate (pH 6.0).

The dish was then microwaved at 80% power for 20 minutes and was then left to cool slowly in the buffer at room temperature.Immunohistochemistry assays:Immunohistochemistry for apoptosis was conducted with the primary antibodies (Cleaved Caspase-3 “Asp 175”, New England Biolabs) and (M30 CytoDeath. Bioaxxess) using the (NovoLink Polymer Detection System), Leica Microsystems. RE7140-CE. The slides were washed with buffer (Blocking Solution – TBS/3%BSA/0.1% Triton-X-100) as a primary antibody diluent. Neutralisation of the endogenous peroxidase was done by using Peroxidase Block for 5 minutes. The slides were then washed in TBS for 2 x 5 minutes, incubated with Protein Block for 5 minutes, washed in TBS for 2 x 5 minutes and were then incubated with optimally diluted primary antibody, then washed in TBS for 2 x 5 minutes, incubated with Post Primary Block for 30 minutes, washed again in TBS for 2 x 5 minutes and incubated with NovoLink Polymer for 30 minutes, washed in TBS for 2 x 5 minutes and developed peroxidase activity with DAB working solution for 5 minutes. The slides were then washed in tap water for 5 minutes and counterstaining with Mayer’s Haematoxylin was done for 30 seconds. Finally, the slides were washed in tap water for 5 minutes and were then dehydrated and mounted in DPX. Apoptotic cells were identified when there was evidence of Caspase-3 and M30 positivity.Quantitative apoptotic index:The apoptotic index was expressed as the ratio of the number of hepatic cells with Caspase-3 and M30 positivity out of the total number of nucleated cells in each field (magnification, x40) calculated after counting 5 random microscopic fields for each time point with a 19mm Whipple grid graticule lens (Pyser-SGI LTD). In each field, 100 squares were evaluated for the presence of apoptotic cells. Activated Caspase-3 hepatocytes positive for DNA fragmentation and for cytoplasmic activity for M30 were counted on the whole sections. The mean counts were expressed as a percentage of the total number of non-apoptotic cells counted in each field.Microscopic examination:Haematoxylin-Eosin staining:Signs of central coagulative necrosis with unrecognizable cell boundaries and collapsed collagen fibres were seen in the CNZ, surrounded by a transitional zone (TZ) with signs of vacuolation of hepatocytes, sinusoidal dilatation and haemorrhagic extravasation of red blood cells from the sinusoids into the liver parenchyma (Figure-2) Figure-2: HE staining of the ablated zone with different powers showing coagulative necrosis with unrecognizable cell boundaries and collapsed collagen fibres in the CNZ (central necrotic zone), vacuolation of hepatocytes, sinusoidal dilatation and haemorrhagic extravasation of red blood cells from the sinusoids into the liver parenchyma in the TZ ( transitional zone) & normal hepatocytes in the NZ (normal zone) (X50).Caspase-3 expression:None of the samples showed any expression of Caspase-3 in the CNZ with all powers. There was a strong evidence of its expression in all TZ, especially with power 70W, with less expression in the NZ (Figure-3). M30 expression:Different staining of M30 expression was noted in the transitional and normal zones ranging from weak (A), intermediate (B) to strong (C) reactions. There was more expression in the TZ (Figure-4). Statistical analysis:Caspase-3 expression:No expression of Caspase-3 was noted in all necrotic zones.

These newer ablative techniques include microwave ablation, high-intensity focused ultrasound, laser induced interstitial thermotherapy, radiofrequency ablation and cryoablation [6].With all forms of thermal ablation therapies there are three distinct zones in heat-ablated lesions: the central necrotic zone (CNZ), which is in the immediately vicinity of the application rod and which undergoes ablation-induced coagulative necrosis; a transitional zone (TZ) of sub lethal hyperthermic injury, which mostly occurs from thermal conduction of the central area that is either undergoing apoptosis or recovering from reversible injury; and the surrounding normal zone (NZ) tissue that is unaffected by ablation [7]. As well as having three distinct lesion zones in MWA, there are also three factors that determine the extent of cellular damage caused by the heat-based ablative therapy: the amount of the applied energy, the rate of energy delivery and the target tissue’s intrinsic thermal sensitivity [8].

Previous studies suggest that due to increased cellular density, fewer interstitial vascular and lymphatic channels to dissipate heat and the hypoxic/acidic tumour microenvironment, tumour tissue is more sensitive than normal tissue [9-13]. The focus of our study was to investigate whether the rate of MWA induced apoptosis in the CNZ, TZ and NZ increased when three different ablative powers were applied. In our three controlled experiments we applied 50W, 70W and 90W MWA energies directly into the porcine hepatic lesions from which we took seven biopsies each and observed cell apoptosis using M30 and caspase-3 as immunohistochemistry markers six hours post ablation.Previous MWA studies investigating the rate of apoptosis in liver cells have been published. A study by Ohno et al. measured the rate of apoptosis by Caspase-3 activity with flow cytometry in the transition zone immediately, 2, 6, 12, 24,72 and 168 hours following MWA. Their study reported peak activity at 2–6 hours with no evidence of CNZ apoptosis [14]. Another study by Bhardwaj, et al also confirmed the lack of apoptotic activity in the CNZ tissue post MWA and their study demonstrated peak Caspase 3 expression in the transition zone at 4 hours. Bhardwaj et al. hypothesized that the lack of apoptotic activity in the transition zone at time 0 was most likely due to the fact that very little energy had dissipated outside the immediate applicator and central zone and that in addition to other factors such as oedema and local haemorrhage, transition zone cells undergoing apoptosis post-ablation may also account for the increase in macroscopic ablation size observed at 4 and 24 hours [4].In our study, we too were able to confirm these findings as there was no evidence of apoptosis in the CNZ at all applied powers using Caspase -3 and M30 as apoptotic markers six hours post MWA. However, unlike previous studies, our study was able to demonstrate that there was a noticeable change in the rate of apoptosis within the TZ and NZ when the applied MWA power was increased.

When we compared apoptosis at the TZ and NZ using caspase-3 as a marker we observed that there was a marked increase in apoptosis induction between 50W and 90W. We were also able to observe a higher peak rate of apoptosis in the TZ at 70W compared to 90W and increased apoptosis induction in the NZ at 90W compared to 50W and 70W. When we compared our biopsy samples with the different MWA ablative powers using the M30 maker, we also observed a marked increase in apoptosis induction at 90W compared to 50W in the TZ. There were less significant increases of apoptosis at 50W to 70W and 70W to 90W with the peak induction of apoptosis observed at 90W in the TZ. A common limitation shared by all ablative heat-based modalities is the dissipation of thermal energy from the ablation zone to the peripheral zone or TZ known as the heat sink effect [15].This effect primarily affects treatment efficacy within the TZ due to increased tumour tissues vasculature. Blood flowing within an adjacent blood vessel of highly vascular tumour tissue may prevent the peripheral zones reaching cytotoxic temperatures. This effect has been observed in the clinical setting, with higher rates of recurrence in tumours adjacent to large blood vessels [16]. In this study we have been able to demonstrate that by increasing the MWA power, it is potentially possible to overcome the heat sink effect by induce a larger amount of apoptosis in the TZ and NZ of liver tissue. However it must be noted that the size of the ablation zone with MWA can be harder to predict than other ablative modalities such as RFA and by increasing the power of MWA to escape the heat sink effect could potentially lead to overtreatment and damage to adjacent off-target [16]. Further study needs to be carried out to access the possible advantageous or disadvantageous nature of these outcomes.

To calculate the extent of MWA induced apoptosis in our study, we used the apoptotic index. Numerous sources of literature have pointed out that there are technical and methodological factors that can influence the determination of the apoptotic index and our study is no acceptation to this [17]. There is also no consensus on the criteria of how to define and calculate the apoptotic index. Some authors use it to denote the number of apoptotic cells per 1000 tumour cells and other studies have defined it as a percentage of apoptotic cells and bodies per all tumour cells [18, 19].

We calculated the apoptosis index as ratio of all immunohistochemically caspase-3 and M30 stained apoptotic cells out of the total number of nucleated cells in 500 high power microscopic fields. It is now a general consensus that evasion of apoptosis is a hallmark of true established cancers [20]. However some studies have discovered that inhibition of cell death is actually protective against HCC development in the liver. A study by Nakamoto, et al demonstrated in a preclinical model that anti-apoptotic treatment was preventative in development of HCC in an animal model with HBV-mediated liver cancer [21].

Another study by Pierce et al demonstrated that transgenic expression of Bcl-2 also prevented HCC in a mouse model of HCC [22]. However, other studies that examined increased induction of hepatocyte apoptosis with its associated compensatory cellular proliferation and inflammation found that this process was actually carcinogenic in the liver. For example, Vick et al. [23] demonstrated that in knockout Mcl-1 mice models there was hepatocyte genetic deletion of the potent antiapoptotic Bcl-2 protein leading to spontaneous hepatocyte apoptosis, cell turnover, inflammation, and, hepatocarcinogenesis. As our results have illustrated, with increasing MWA power there is an increase in apoptosis before a threshold is hit. The next key question to ask is; is there a balance between applying an optimum MWA power that induces a high rate of apoptosis and can be used as an adjunct with other therapies to treat malignancy, or does the increase of apoptosis enhance the rate of malignancy and protect the malignant cells to develop and cause treatment resistance? There have been numerous studies that have investigated the role of apoptosis in HCC and other hepatic malignancies with inconclusive and conflicting results [21-23]. We would recommend further studies to establish if an increase of apoptosis through ablation therapies is beneficial or detrimental to the treatment of HCC and metastatic malignancies.

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