Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.
Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.
The size of the liver remnant limits the resectability of liver tumors.1 In general, when less than 25% liver tissue is left behind, the patient is at increased risk of death from acute liver failure due to the lack of metabolic function for the entire organism ("too small for size syndrome").2 This post-hepatectomy liver failure is the most devastating complication after liver resection. Therefore clinicians have tried to induce liver regeneration prior to resection of the liver by manipulating the flow of the portal vein.3 It was found that, once the portal vein is occluded, the remaining part with portal vein flow starts to grow at a slow rate, and can thereby increase up to 60% in size.4 Surgical ligation5 or interventional portal vein occlusion have both been clinically established.4 The increase in volume and function of the liver is reliable, but the growth rate of the liver after portal occlusion is only about one fifth compared to the growth of the remnant liver after partial hepatectomy.6
The time necessary for the liver to grow is weeks to months even though the liver can regenerate at a much faster rate after resection. As such, the liver is the only organ that grows back to normal function after removal of a part of it.7 A novel procedure inducing liver regeneration at a similar pace as after partial hepactectomy was developed by a group of surgeons who discovered that adding a transection between the occluded and the non-occluded part of the liver induces liver hypertrophy at the same growth rate as after liver resection, but prior to resection.9 The procedure initiates rapid hypertrophy of 80% within a week in the future liver remnant, which allows the resection of extensive, primarily unresectable, liver tumors within a week. The procedure was called "Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS" and became rapidly popular across the world.10 Multiple reports supported an expansion of the resectability of borderline resectable liver tumors achieved by the new technique,11 while the complex surgical procedure was also criticized for its high complication rate.12,13
The development of a rodent and also large animal models of slow and rapid hypertrophy has been attempted since the publication of ALPPS in 2012 to allow a better histological characterization and understanding of the mechanisms and to test drug effects on the different growth rates of liver tissue in animals. The first animal model developed was a rat model. In this model, rapid hypertrophy after parenchymal transection between the right and the left part of the median lobe accelerated regeneration of the right median lobe.14 A different model was introduced later in the mouse. In this model the left lateral lobe was resected and the portal vein branches to every lobe of the liver except the left median lobe were tied.15 In the meantime, large animal models of ALPPS in pigs have been described as well.16
For the study of physiological mechanisms like flow changes and pressure in the portal vein, perfusion and oxygenation of liver tissue, the rat model is superior to the model of ALPPS in mice. Another advantage of the rat over the murine model is that in the rat model there is no necessity for a resection of the left lateral lobe,15 which may contaminate the effects of liver resection with those of ALPPS. The rat model in contrast does not reduce the liver cell mass. A pig model uses the right posterior lobe as the growing lobe, but the pig liver is highly lobulated. Therefore, it is difficult to create a transection plane in the already thin tissue bridge between the right posterior and the right anterior lobe. In contrast, the median lobe in rats consist of two parts that are separately supplied by a portal vein each and a parenchymal transection plane can easily be created between the two using microsurgical techniques. The availability of small animal computer tomography (CT) and/or magnet resonance imaging (MRI) allows the very exact quantification of volumetric growth between portal vein ligation alone and portal vein ligation and the added transection, which is important for the validation of any rapid liver hypertrophy model.
The protocol presented here describes the surgical technique and procedures used for volumetric validation and physiological characterization of the model of slow and rapid hypertrophy after portal vein ligation and portal vein ligation with transection, respectively, in rats.
All experiments in this protocol were approved by the Veterinary Authorities of the Canton of Zürich, Switzerland (number 60/2014). Furthermore, all experimental steps were performed in strict compliance with the Guidelines on Experiments with Animals by the Swiss Academy of Medical Sciences (SAMS) and Guidelines of the Federation of European Laboratory Animal Science Associations (FELASA).
1. Animal Husbandry, Operating Room Equipment and Instruments, Anesthesia
2. Start of Surgery
3. Portal Vein Ligation (PVL)
4. Portal Vein Ligation with Transection (PVL+T)
5. Intraoperative Measurement of Portal Vein Pressure and Volume Flow
6. Final Steps of Surgery
7. Liver Volumetry in Rats Using Small Animal CT
The two different surgical procedures portal vein ligation (PVL) and PVL with transection (PVL+T) result in distinctly different growth kinetics. PVL induces moderate volume increase within 3 days, whereas in PVL+T a much larger right median lobe (RML) can be seen (Figure 5). This can be verified by daily volumetry. The volume of the RML roughly doubles within 3 days in PVL, while it triples in PVL+T.17
Flow measurements in the portal vein reveal a stable flow, despite the reduced flow area in the PLV and also the PVL+T (see Figure 3A in Schadde et al.17). This suggests that the entire portal blood volume flow is directed through approximately 26% of the previous liver parenchyma, thereby causing "portal hyperflow".
Pressure measurement inside the portal vein reveals an acute increase of the mean portal vein pressure from 5 mmHg to 9 mmHg in PVL and PVL+T (see Figure 3B in Schadde et al.17). This is likely a result of the portal hyperflow. Transection alone does not result in an acute portal pressure increase, since the volume of liver tissue is not reduced.17 Repeated measurements show that the pressure increase remains stable for 24 h.17
Figure 1: Anesthesia. (A) After flushing a box with isoflurane (5 vol%) to induce anesthesia for the animal, isoflurane is vaporized (600 mL oxygen/min) for maintenance of general anesthesia. (B) The animals are fixated on a sterile operating surface under an operating microscope. Please click here to view a larger version of this figure.
Figure 2: Sterile Equipment. (A) Sterile moist sponges for exposure and retraction. (B) Wire mini-tractors connected to rubber bands. (C) Bipolar forceps, Potts scissors; curved microforceps, straight microdissectors are used for dissection of the portal vein branches. (D) Larger scissors, needle holders and needles are used for opening and closure. Please click here to view a larger version of this figure.
Figure 3: Dissection. (A) The sides of the abdomen and the xiphoid are retracted with 3-0 silk sutures after midline laparotomy. (B) By retracting the stomach, small and large bowel laterally the gastroduodenal ligament in rats and the hepatic hilum can be exposed. (C) In portal vein ligation (PVL), the portal vein branch to the right lobes (1), the left lateral lobe (2) and the caudate lobes (3) are ligated with 6-0 silk ties (left). In PVL with transection (PVL+T), additionally, the ischemic demarcation line between right median lobe (RML) and left median lobe (LML) is transected (right). (D) The photograph shows the distinct demarcation line between LML and RML (left). This line is followed for the transection using a bipolar microforceps for PVL+T (right). Please click here to view a larger version of this figure.
Figure 4: Intraoperative Measurement of Portal Vein Pressure and Volume Flow. (A) During the procedure, a needle pressure transducer is inserted into the exposed portal vein. (B) The 2 mm volume flow probe is used to measure volume flows in the main portal vein. Please click here to view a larger version of this figure.
Figure 5: Liver Volumetry. The liver increases more in size after PVL+T than after PVL alone. The figure shows daily volumetry performed on digital imaging and communications in medicine (DICOM) files obtained with a rat micro CT scanner and using the public domain imaging platform. While the difference is obvious on axial images, quantitative volumetry as performed in a previous report17, shows a significant difference in growth kinetics. Please click here to view a larger version of this figure.
This protocol presents an animal model of ALPPS with its rapid hypertrophy induced by PVL+T, that roughly doubles volume increase within 3 days compared to PVL alone.17 The right middle hepatic lobe is used as a model for the growing liver lobe because the middle hepatic lobe is one contiguous parenchymal mass supplied by two separate portal veins to its left and to its right side, as shown in Figure 1 in a recently published work.17 Compared to other reports, the model offers some advantages. Anatomically, the choice of the middle lobe best represents the human liver as one contiguous parenchymal mass that allows for transection and thereby obliteration of collateral flow. The branching of the portal vein to the left median lobe (LML) inside the left lateral lobe (LLL) allows for a combined ligation of LML and LLL.
There have been various rat and one murine model for ALPPS developed and described.18 Many rat models19,20 and the only mouse model15 described requires a liver mass reduction. Mass reduction may trigger enhanced hypertrophy independently. This is in contrast to our17 and similar rat models.14,21,22,23 The advantage of a murine model may be the use of genetically modified animals. Alternatively to these small animal models, a pig model has been developed,16 using a similar surgical technique as in humans and resulting in similar growth rates. Despite the fact, that this probably most closely resembles the human surgical procedure, small animal models may still be very important as they are faster, are easier to perform, allow larger number of animals per group and are less costly.
Physiologically, the rat model allows the study of blood flow, collateralization and oxygenation more easily than a mouse model. This study is the first to use the same methodology used clinically in humans to assess volume changes in the liver, namely CT volumetry. In contrast to some other studies, which do not include a control group (e.g. in a pig model of ALPPS24) yet assume ALPPS growth kinetics, we validated the hypertrophy by comparing PVL with PVL+T. While under PVL the RML doubles in within 3 days, PVL+T increases hypertrophy significantly. This acceleration of hypertrophy after 3 days by the added transection exactly mirrors the human condition, where a 46% volume increase has been reported in a large meta-analysis for PVE and an 86% increase in ALPPS after 10 days in the first large ALLPS registry report of 202 patients.25 Both the rat procedure PVL+T and human ALPPS double the amount of hypertrophy achieved in their respective time frames for the two procedure types.
Critical steps within this protocol include the dissection of the portal vein branches in PVL and the transection of the median lobe in PVL +T. During the dissection of the portal vein branches, it is important to disrupt the thin layer of peritoneum covering the entire hepatoduodenal ligament and not to push the instruments against resistance. An accidental tear in the portal vein may be stopped by pressure using cotton-swabs alone, but may be irreparable and require sacrifice of the animal. In general, it must be emphasized, that animals suffering from a blood loss of more than an estimated 10% of the animal's blood volume, should be excluded from the study, since this may alter the study results and cause unnecessary suffering of the animals.
Transection of the median lobe can be achieved by careful cauterization of the liver tissue using fine silver bipolar forceps and enough saline dripping, followed by simple cutting of the cauterized tissue using a scissors. The transection ought to be stopped prior to encountering the vena cava, which runs inside of the liver in rodents, to avoid massive bleeding and air embolism into the vena cava. Entry of air into the pulmonary circulation leads to sudden cardiac arrest in rats.
Troubleshooting of this protocol may include all modifications that lead to increase physiologic stress for the animals, such as hypotension induced by oversedation, hypothermia, increased blood loss and too long operative times. This protocol may well be modified for other rodent species like mice. We have successfully performed the technique described in this protocol in C57BL/6 mice (data not shown).
A limitation of this protocol is the exclusive use of this protocol to study the mechanisms of rapid versus slow hypertrophy; therefore the second stage of the "ALPPS" procedure, the resection of all deportalized liver tissue except for the hypertrophied liver, is not described in this protocol. This resection however can easily be achieved following standard technique of liver resection in rodents, using suture ligation and then removal of the liver lobes using a scissors.
Overall, this current model allows experiments elucidating the mechanism of rapid liver hypertrophy and testing of medications and interventions. Using this model, it was recently shown that rapid hypertrophy of the RML can also be induced by portal vein ligation in conjunction with hypoxia signaling using prolyl-hydroxylase inhibitors, suggesting that hypoxia signaling my play an important role in the modulation of liver growth kinetics. Drugs like prolyl-hydroxylase inhibitors may accelerate liver regeneration and should be further tested.17,26 Future applications of this model may be that the role of conventional and novel chemotherapeutic agents may be tested and their effect on the acceleration of liver regeneration may be further elucidated. The model also offers the opportunity to study liver function using for example indocyanine green (ICG) or hepatobiliary iminodiacetic acid (HIDA) scintigraphy in rats because ultimately slow and rapid volumetric changes will have to be put in the context of functional liver regeneration since liver function is more important than liver volume.27,28 The assessments of ALPPS patients with ICG29 and HIDA scintigraphy30,31 have so far only been tested in retrospective cohort studies, but these still may be very important tools for answering the question whether volume or function changes are similar or dissimilar in rapid liver regeneration.
In summary, we present a well characterized and standardized model of rapid and slow liver regeneration, that will allow future studies in regenerative liver surgery.
The authors have nothing to disclose.
The authors have no acknowledgements.
Isoflurane, 250ml bottles | Attane, Piramal, Mumbai, India | LDNI 22098 | Standard vet. equipment |
Tec-3 Isofluorane Vaporizer | Ohmeda, GE-Healthcare, Chicago, IL | not available anymore | Standard vet. equipment |
Buprenorphine (Temgesic) | Indivior, Baar, Switzerland | 7680419310353 | GTIN-number |
Vitamine A ointment | Bausch&Lomp, Zug, Switzerland | 7680223980247 | GTIN-number |
Atropine sulfate 0.5mg/ml | Sintetica SA, Mendrisio, Switzerland | 7680565330045 | GTIN-number |
Microsurgery microscope | Olympus, Tokio, Japan | SZX10 | Standard vet. equipment |
Betadine | Mundipharma, Basel, Switzerland | 7680342821377 | GTIN-number |
Sponges | Carl Roth GmbH, Karlsruhe, Germany | NK83.1 | Mini-sponges |
Abdominal Wall retractors | N/A | N/A | Self-made from paper clips and Q-Tips |
3-0 silk | Ethicon, Sommerville, NJ | K872H | Standard surgical |
Scissors | World precision instruments (WPI), Sarasota, FL | 503371 | Standard microsurgical |
Adson forceps | World precision instruments (WPI), Sarasota, FL | 501244-G | Standard microsurgical |
Fine tips microforceps | World precision instruments (WPI), Sarasota, FL | 501976 | Tips need to be polished regularly |
Curved fine tips microforceps | World precision instruments (WPI), Sarasota, FL | 504513 | Essential to go around the portal vein branches |
6-0 LOOK black braided silk | Surgical Specalities Corporation, Wyomissing, PA | SP114 | Spool, precut prior to the procedure |
2-0 silk sutures | Ethicon, Sommerville, NJ | K833 | Standard surgical |
5-0 maxon sutures | Covidien, Dublin, Ireland | 6608-21 | Standard surgical |
Bipolar microforceps | Sutter, Freiburg, Germany | 780148SGS | Essential for parenchymal transection |
Q-tips small | Carl Roth GmbH, Karlsruhe, Germany | EH11.1 | Standard surgical |
Q-tips big | Carl Roth GmbH, Karlsruhe, Germany | XL54.1 | Standard surgical |
G30 needle | Terumo, Tokyo, Japan | NN-3013R | Standard anesthesia equipment |
2mm volume flow probe | Transonic Systems, Ithaca, NY | MA-2PS | Smallest available probe for HAT-311 flow meter |
Transonic flow meter | Transonic Systems, Ithaca, NY | HAT-311 Transsonic flow QC meter | One of the first generation flow flow meters for surgery |
ExiTron nano 12,000 | Miltenyi Biotech, Bergisch Gladbach, Germany | 130-095-698 | Nanomoloecular contrast medium that opacifies liver and spleen |
G26 intravenous catheter | Becton Dickinson, Franklin Lakes, NJ | 391349 | Standard anesthesia equipment |
Quantum FX MicroCT | Perkin Elmer, Waltham, MA | N/A | Standard small animal CT scanner at the institute of physiology, University of Zürich |
OsiriX 8.0 | Pixmeo Sarl, Geneva, Switzerland | N/A | Public domain software : www.pixmeo.com |