Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Podsumowanie

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

Streszczenie

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Wprowadzenie

Argon plasma coagulation (APC) is a widely used instrument in abdominal surgery for more than three decades^1,2. It is a standard technique for the achievement of secondary hemostasis after major hepatectomy by sealing the liver cut surface to prevent later hemorrhages³. Plasma coagulation is a specialized form of radiofrequency electrocautery, which delivers the electrical energy through an arc of ionized gas. Providing monopolar electrothermal hemostasis, this noncontact technique has the advantage of preventing the electrode to stick to the tissue⁴. The ionized gas beam is automatically directed to the area of the lowest electrical resistance and is turned away when resistance rises due to desiccation to other areas not yet desiccated. This produces a uniform limited depth of coagulation^5,6. Factors influencing the coagulation effect are the activation time, the power setting of the coagulation device and the distance from the probe to the tissue. Helium is another carrier gas, which can be used for plasma coagulation⁷. Recent clinical studies concentrated on clinical outcomes rather than histological and functional findings^3,8,9, while experimental studies focused on in vitro investigations¹⁰ or experiments on isolated perfused organs¹¹.

The underlying protocol allows the study of the effects of plasma coagulation in a large animal model close to the clinical application using standard human equipment on pigs: Microcirculation is assessed noninvasively by a laser Doppler flowmeter and spectrophotometer, which is a standard clinical tool for this indication^12,13. Temperature changes during coagulation are monitored with an infrared thermometer and a thermographic camera. The depth of coagulation is measured on histological hematoxylin/eosin stained sections after harvesting of tissue samples. For the comparison with other means for secondary hemostasis, burst pressure experiments are performed. In contrast to previously described techniques¹⁴, these are conducted on explanted organs to exclude blood pressure related effects. In addition to the described investigations on the local effects of plasma coagulation, standard blood tests can also be undertaken in the porcine model.

Protokół

Rules governed by German legislation for animal studies as well as Principles of Laboratory Animal Care (National Institutes of Health publication ed. 8, 2011) were followed. Official permission is granted from the governmental animal care office (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Recklinghausen, Germany).

1. Animals

1. Use female German landrace pigs (weighing 25-30 kg) housed in open cages.
2. Use 5 animals per group (argon and helium).
3. Allow the animals to acclimatize to the surroundings for at least one week before the experiments. Fast animals for 24 h prior to surgery with free access to water.

2. Anesthesia

1. Premedicate the animals with an intramuscular injection of ketamine (15 mg/kg body weight [BW]), xylazine (10 mg/kg BW), and atropine (0.1 mg/kg BW) 10 min before induction of anesthesia.
2. Peripheral venous access is established by placement of a 22-gauge cannula into an ear vein.
3. Induce general anesthesia by i.v. injection of propofol 2 mg/kg body weight.
4. Place the animal in the supine position and perform a longitudinal skin incision in the jugular groove over a length of 2 cm. Locate vein through the blunt preparation of the subcutaneous tissue. Insert cannula, then Seldinger wire.
5. Retract cannula and insert 14 Fr. catheter over the guide wire. Retract guide wire. Connect catheter to the extension and fixate the catheter by a strap or a suture.
6. Pull out the tongue and insert straight laryngoscope. Use the tip of the laryngoscope to pull down the epiglottis. Insert the tube through the vocal cords. Place cuff under glottis and inflate.
7. Ventilate with 40% oxygen at 20-26 breaths/min and a tidal volume of 10 ml/kg to keep the end-tidal partial carbon dioxide tension between 36 and 42 mm Hg.
8. Maintain anesthesia with isoflurane at a concentration of 1-1.5% and fentanyl at a concentration of 3-4 µg/kg/h.
9. Supply Ringer's lactate solution at an initial rate of 4 mL/kg/h, and increase after laparotomy to a constant infusion rate of 8 mL/kg/h.

3. Surgery and Plasma Coagulation

1. Place the animal in a supine position on a standard surgical table.
2. Disinfect skin by applying a standard surgical disinfectant (2-Propanol 45 g /100 g, 1-Propanol 10 g / 100g, Biphenyl-2-ol 0.2 g / 100 g) with a surgical swab for 3 times.
3. Perform a wide midline laparotomy from the xiphoid process to the pubis with a scalpel and install surgical retractors.
4. Switch on the plasma coagulation device, open argon or helium gas bottle, depending on the used carrier gas. Adjust gas flow to 3 L/min. Select coagulation device output power as desired.
   NOTE: Both noble gases, Argon or Helium, can be used for plasma coagulation. Coagulation effects are comparable. See reference⁷ for details.
5. Perform plasma coagulation on the left liver lobe as previously described⁷. Use a titanium mold (square aperture 1 x 1 cm²) to standardize the coagulation zone. Coagulate for 5 s with a probe distance of 1 cm. The coagulations with different power settings can be performed side by side with a short distance between the coagulations of 5 mm (Figure 1).
6. For harvesting of the liver, divide all ligamentous connections to the liver. Isolate and divide the hepatic pedicle above the superior duodenal flexure leaving long portions of portal vein and common bile duct. Divide the caval vein above and below the liver and retrieve the organ.
7. After harvesting the liver, the pigs were euthanized by i.v. administration of 0.16 g/kg BW pentobarbital.
8. For the burst pressure experiments, resect half of the left medial liver lobe with sharp scissors. Plasma-coagulate the cut surface (100W output power) or seal the cut surface with fibrin sealant (Figure 2).

4. Microcirculation Measurement

NOTE: Laser Doppler spectroscopy can determine blood flow in tissue through measuring the Doppler shift caused by the movement of erythrocytes. The Laser signal correlates with the number of moving erythrocytes. Laser Doppler spectroscopy is in clinical use (e.g. transplant medicine) and has been validated multiple times¹⁵.

1. Switch on the laser Doppler flowmeter and spectrophotometer. Use a flat probe.
2. Take baseline measurements for flow and velocity. Save or note the values.
3. Perform coagulation as described under 3.5.
4. Place the flat probe on coagulation sites and measure flow and velocity. Again, save or note values.
5. Repeat for all power settings of the coagulator device.

5. Temperature Measurement

1. Switch the system on (thermographic camera, notebook, and infrared thermometer) and let it run for at least 1 h before performing measurements.
2. Adjust focus and view frame on the thermographic camera on the coagulation site. Infrared sequences can be detected with the spatial resolution of 1024 x 768 pixels with a temperature resolution bigger than 20 mK. Take into account, that the region of coagulation and the surrounding tissue — affected by heat transfer — is located in the middle of the view.
   NOTE: It should include as many pixels of the frame as possible for an optimal spatial resolution.
3. Record coagulation process with the plasma coagulator on the liver surface with the thermographic camera over a 2-min period.
4. Analyze image sequences with the thermography analysis software: Define regions of interest.
   NOTE: Software calculates the course of corresponding mean temperature over time.

6. Coagulation Depth Measurement

1. Harvest the left medial liver lobe with sharp scissors.
2. Excise the coagulation sites with 1 cm thickness. Cut into 3 mm thick longitudinal segments for further processing.
3. Fix tissue samples at 4 ° C overnight with neutral 10% buffered formalin. Heat paraffin 2 °C over the melting point and embed slices. Process overnight.
4. Perform Hematoxylin/Eosin staining.
   1. Deparaffinize and hydrate the tissue by subsequently dipping in 2x Xylene, 100% ethanol (EtOH), 95% EtOH, 70% EtOH, deionized H₂O for 2 min each.
   2. Stain the tissue sample with Meyer's Hematoxylin solution for 3 min.
   3. Now rinse in tap water for 5 min.
   4. Stain the tissue with Eosin solution for 3 min.
   5. Rinse in 2x EtOH 95% and then Xylene for 3 min each. Mount with the standard mounting medium.
5. Switch the system on (microscope connected camera, imaging software). View all sections with 40X magnification.
6. Take an image of an object micrometer at a magnification of 40X. Press Recalibrate button in the Objectives window. Select manual calibration. Draw a line on the micrometer image of 100 µm. Enter 0,1 mm in the dialog box and press OK.
7. Select Length in the View>Analysis Controls>Annotations and Measurements window. Measure from the liver surface to the coagulation margin with the mouse. Export or note the result. Repeat measurement on another location on the same slide.
   NOTE: Coagulation depth can easily be differentiated from the normal liver tissue by the sharp margin between the normal hepatocyte cords and the zone of necrosis with shrunken cytoplasm, pyknotic nuclei, and hemorrhage zones.
8. Calculate mean out of two measurements.

7. Burst Pressure Measurement

1. Switch the system on (automatic pumps, pressure meter). Prepare the liver samples according to the step 3.7.
   NOTE: Use two parallel pumps connected via a 3-way-stopcock. The maximal pressure of 1,500 mm Hg cannot be obtained with a single pump.
2. Isolate portal vein, common hepatic artery and bile duct with scissors in the haptic pedicle. Clamp portal vein with an overholt forceps and ligate with a monofil suture 4-0. Clamp common hepatic artery an overholt forceps and ligate with a monofil suture 4-0.
3. Insert Ch-16 catheter into the common bile duct and ligate with a 2-0 silk suture. Connect the catheter to the automatic pumps, install 3-way stopcock with pressure meter (Figure 3).
4. Fill the perfusion syringe with saline.
5. Start automatic pumps with a delivery rate of 99 mL/h.
6. Monitor liver cut surface and pressure meter for leakage and record burst pressure.
   NOTE: For easier recognition of leakage, patent blue can be added to saline (2 mL patent blue + 18 mL saline). It is easier to observe burst pressure by noticing the time of loss of pressure on the pressure meter.

Wyniki

Microcirculation: Utilizing the diagnostic device for hemostasis following plasma coagulation can be demonstrated by microcirculation changes. Capillary blood flow (displayed as arbitrary units (AU)) decreases from a baseline value of 142.7 ± 76.08 AU to 57.78 ± 49.57 AU at 25 W device output power, to 48.5 ± 7.26 AU at 50 W and to 5.04 ± 1.31 AU at 100 W (Figure 4).

Dyskusje

Rodent models for liver surgery are established for a long time¹⁶. Nevertheless, large animal models offer certain advantages: no microsurgical equipment is needed as standard operative equipment for humans can be applied, surgical techniques are comparable to clinical use and standard clinical evaluation methods can be transferred to the experiments. For example, standard clinical blood tests can be carried out without the need for special laboratory test methods (Figure 10

Materiały

Name	Company	Catalog Number	Comments
Xylazine 20 mg/mL	Vetoquinol GmbH	Xylapan
Ketamine 100 mg/mL	Ceva GmbH	Ceva Ketamine Injection
Atropine 100 mg / 10 mL	Dr. Franz Köhler Chemie GmbH	Atropinsulfat Köhler 100mg Amp.
Propofol	Fresenius Kabi GmbH	Propofol 1% MCT Fresenius
Fentanyl	KG Rotexmedica GmbH	Fentanyl 0,5mg Rotexmedica
Isoflurane	Abbot GmbH	Forene 100% (V/V) 250 mL
Ringer's lactate solution	Baxter Deutschland GmbH		sodium 131mmol/l, potassium 5 mmol/l, calcium 2 mmol/l, cloride 111 mmol/l, lactate 29 mmol/l
Surgical disinfactant	Schülke & Mayr GmbH	Kodan Tinktur forte gefärbt 1l 104804
Motorized microscope	Nikon Instruments Europe	Eclipse TE2000-E
Microscope camera	Nikon Instruments Europe	Digitalsight DS-Qi1Mc
Imaging software	Nikon Instruments Europe	NIS elements Vers. 4.40
Plasma coagulator	Söring GmbH	CPC-1000
Argon gas	Linde AG	Argon 4.8
Helium gas	Linde AG	Helium 4.8
O2C	LEA Medizintechnik GmbH	O2C Version 1212	with LF-2 or LF-3 probe
Infrared thermometer	Voltcraft	VOLTCRAFT IR 260-8S
Thermographic camera	InfraTec GmbH	VarioCAM HD head 820
Thermographic analysis software	InfraTec GmbH	IRBIS 3
Mayer's Hematoxylin solution		Merck 1.09249
Eosin solution	VWR International GmbH	Merck 1.09844
Rollerpump Masterflex L/S easy Load	Cole-Parmer Instrument Company	model 7518-10
Perfusorpump	B. Braun Melsungen AG	Perfusor secura FT
Digital pressure meter	Greisinger electronic	GMH 3161
Perfusorsyringe, 50 mL	B. Braun Melsungen AG	REF 8728810 F
Perfusor line, Type IV Standard, PVC Luer lock	B. Braun Melsungen AG	REF 8722960
3-Way stopcock, Dicofix C35C	B. Braun Melsungen AG	REF 16494 C
Silk 2-0. 3 metric	Resorba	REF H5F
Vicryl 4-0 Sutupak	Ethicon	V1224H
NaCl 0.9 %	B. Braun Melsungen AG