This work was supported by the core grant received from the Department of Biotechnology, Government of India to National Institute of Immunology, New Delhi.

The procedure described below has been approved by the Institutional Animal Ethics Committee of National Institute of Immunology, New Delhi. The serial reference number of the approval is IAEC#355/14.
1. Preparation
<ol>
	<li>Prepare for the surgical procedure as described earlier by Das B et al.26.</li>
	<li>Use 6&#8211;8-week-old inbred Wistar rats with a body weight of 200&#8211;250 g.</li>
	<li>House the animals under standard animal care conditions and feed them with rat chow and libitum before and after the procedure.</li>
	<li>When performing 70% PHx, use a standard cocktail mix of ketamine hydrochloride (100 mg/kg body weight) and xylazine (10 mg/kg body weight), which is injected intraperitoneally. 
	NOTE: The type of anesthetic used can have postoperative effects on the mortality and morbidity.</li>
	<li>During the cell transplant, use inhalant anesthesia Isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane) to reduce the time of recovery of the animal after the transplantation surgery.</li>
	<li>Induce and maintain the inhalational anesthesia using a customized anesthesia system. Maintain the oxygen flow at 4 L/min. For induction use isoflurane at 4% and for maintenance use 2&#8211;3% during the surgical procedure.</li>
</ol>
2. Preoperative procedures
<ol>
	<li>Anesthetize the rat by injecting the ketamine-xylazine mixture described in step 3.1 intraperitoneally. Confirm the complete anesthetization by pinching the toe of the animal. Further procedures are carried out only when there is no pedal reflex.</li>
	<li>To prevent corneal desiccation, apply a carboxy methyl-cellulose based eye drop to both eyes.</li>
	<li>Restrain the anesthetized animal to a surgical board using white tape. Place the animal with the abdominal side facing up, ensuring the mouth is on the distal side from the person performing the operation.</li>
	<li>Remove hair from the upper right abdominal surgical area using an electric clipper.</li>
	<li>Disinfect the surgical site by three alternating scrubs of povidone iodine and 70% ethanol using sterilized cotton pads in circular motion.</li>
</ol>
3. Partial hepatectomy (PHx) to remove 70% of the mass of the liver
NOTE: Perform the entire surgical procedure under a sterile environment in a laminar flow hood. Use only sterile surgical instruments to minimize the risk of infection postsurgically. Removal of 70% of the liver mass, named 70% partial hepatectomy (70% PHx), was performed as described by C. Mitchell and H. Willenbring, 200827.
<ol>
	<li>Prior to the start of the surgery, confirm the complete anesthetization of the animal by pinching its toe. Further procedures are carried out only when there is no pedal reflex.</li>
	<li>Mark the skin to be cut just beneath the sternum, perpendicular to the xiphoid, and parallel to the ribcage.</li>
	<li>Place a sterile drape sheet having an opening of around 3 cm x 1 cm over the marked skin.</li>
	<li>Perform a transverse incision of around 2&#8211;3 cm along the marked line with a scalpel. Use surgical blade No. 22. Gently remove the attachment of the skin to the underlying muscle layer in the vicinity of the incised area using sterile moistened cotton tips.</li>
	<li>Next, make a transverse incision through the peritoneal layer just beneath the xiphoid process.</li>
	<li>With the help of two saline moistened cotton tips, expose the left lobe of the liver by applying gentle pressure on the thorax. Place one cotton tip on the diaphragmatic region of the incised portion and the other cotton tip below the incised region to lift the liver lobe up.</li>
	<li>Slip an 8&#8211;10 cm long sterile nylon thread loop (size 4&#8211;0, 0.15 mm diameter) around the exposed liver lobe. Take the loop to the base of the lobe close to the hilum with the help of microdissecting forceps or moistened cotton buds.</li>
	<li>With the help of the microsurgery needle holder and microforceps, tie the two ends of the loop, placing the knot as close to the base of the lobe as possible to constrict the blood vessel and reduce bleeding after the liver lobe is removed. Tie two additional knots on the other side.</li>
	<li>Take precaution not to tie the knot too close to the nearby blood vessels, which may otherwise cause venous obstruction (stenosis).</li>
	<li>Use microsurgery scissors to cut the tied lobe just above the knot, which leaves a discolored mass of tissue called an ischemic stump in place of the lobe. 
	NOTE: The rat liver, like those of mice, is divided into four distinct lobes: the median lobe, right lateral lobe, left lateral lobe, and caudate lobe, which represent about 40%, 20%, 30%, and 7% of the total liver mass, respectively. Any combination of these lobes can be removed to excise 70% liver mass. In the current study, the median lobe and left lateral lobes were removed.</li>
	<li>Carefully locate the median lobe without damaging the remaining stump of left lateral lobe. Gently pull it out of the abdominal cavity, and at the base of the lobe tie an 8&#8211;10 cm long nylon thread (size 4&#8211;0) knot as mentioned earlier. Tie two additional knots on the other side. Carefully excise and remove the tied median lobe taking all the precautions mentioned.</li>
	<li>After removing the lobes, suture the peritoneum using an absorbable chromic 4&#8211;0 suture with continuous stitches followed by skin suturing with an interrupted suture.</li>
	<li>Apply povidone iodine on the skin surrounding the sutures to prevent infection.</li>
	<li>Remove the drape sheet and remove the animal from the surgery board.</li>
</ol>
4. Postoperative care in animals
<ol>
	<li>Intraperitoneally inject the animal with a dose of 12 mg cefotaxime antibiotic in 1 mL of 5% glucose solution with a 1 mL syringe to protect it from the risk of postoperative infection.</li>
	<li>Administer a subcutaneous injection of analgesic meloxicam (1 mg/kg body weight) for pain relief after surgery and follow it up by two more doses, keeping the regimen as one dose per day.</li>
	<li>House the operated animals under standard conditions of 12 h light/dark cycle and monitor at regular intervals.</li>
</ol>
5. Injection of drug in partially hepatectomized animals to induce liver failure
<ol>
	<li>After 24 h postsurgery, when the animals have successfully recovered from 70% PHx, measure the body weight of the animal followed by the injections.</li>
	<li>Inject 750 mg/kg body weight of APAP intraperitoneally in partially hepatectomized animals 24 h after the 70% PHx following the animals&#39; successful recovery from the surgical procedure. Repeat the dose again after 24 h. 
	NOTE: Two doses of APAP are administered intraperitoneally to the animal (i.e., 24 h and 48 h post the 70% PHx procedure, respectively).</li>
	<li>At each time point after the injection of APAP, measure the body weight of the recovering animal. 
	NOTE: APAP (biocetamol) is injected in animals as a 150 mg/mL solution in 2% benzyl alcohol.</li>
</ol>
6. Transplantation of healthy hepatocytes in ALF animal models
NOTE: To study the reversibility of ALF in rats, transplant healthy syngeneic rat hepatocytes intrasplenically in the ALF-induced animals along with the 1st dose of APAP. In the current study, to provide ample time to the transplanted cells for homing and engraftment, the transplantation was done just after giving the 1st dose of APAP. Rat hepatocytes are isolated by a protocol first published by Berry and Friends et al.28 and later adapted in various other studies29,30,31 with some modifications. For intrasplenic transplantation of cells in the ALF animal model, follow the steps mentioned below.
<ol>
	<li>Place the rat into a poly (methyl methacrylate) chamber for induction of anesthesia with 4% isoflurane and 4 L/min oxygen flow for a rat of 250&#8211;350 g body weight. Check for the depth of anesthesia by the lack of pedal reflexes when pinching the toe of the animal.</li>
	<li>Place the anaesthetized rat on the surgical board such that its left lateral portion is facing up. Maintain anesthesia at 2&#8211;3% isoflurane inhalation through a suitable mouthpiece.</li>
	<li>Shave the skin on the left lateral region and sterilize it by povidone iodine solution.</li>
	<li>Make a transverse incision on the shaved region of skin.</li>
	<li>Make a 1&#8211;2 cm cut in the peritoneal layer to expose the spleen.</li>
	<li>Gently take out the spleen of the peritoneal cavity and lift it up with the help of two moistened cotton tips.</li>
	<li>Keep the cells (typically 107 per animal) to be transplanted suspended in 50 &#181;L IMDM media in a 1 mL insulin syringe with a 29 G needle.</li>
	<li>Gently pierce the needle into the spleen cortex and release the cell suspension into the spleen within 2&#8211;3 min.</li>
	<li>After the cell transplantation is completed, carefully take out the needle and dab the area of the needle puncture with a moistened cotton tip to avoid leakage of the cell suspension from the site.</li>
	<li>Close the peritoneum and skin by a 4&#8211;0 absorbable suture with continuous and discontinuous suturing respectively.</li>
	<li>Apply povidone iodine solution on the skin at the place of the sutures to prevent infection on the operated site.</li>
	<li>Intraperitoneally inject 1 mL volume of 12 mg/mL of antibiotic (e.g., cefotaxime) solution and subcutaneously inject analgesic (e.g., meloxicam) 1 mg/kg body weight to the animal as a part of postoperative care. Move the animal to a warm recovering cage.</li>
	<li>Keep the operated animal in isolation under normal conditions of 12 h light/dark cycle until the surgical wounds are completely healed. This may take 3&#8211;4 days.</li>
</ol>
7. Characterization of ALF development
<ol>
	<li>Euthanize the animals by overdose of ketamine-xylazine solution 2 h after the 2nd dose of APAP treatment and collect blood and tissue samples.</li>
	<li>Collect serum from blood for biochemical studies32.</li>
	<li>Process liver tissue samples for histological and gene expression studies33,34,35.</li>
</ol>

The authors have nothing to disclose.

The development of an appropriate animal model for ALF is paramount for the better understanding of pathogenesis and progression of ALF. A well characterized ALF animal model also provides the opportunity for the development and trial of new therapeutic approaches against ALF. Many attempts have been made to develop a clinically relevant model of ALF6,12,21,23,46</s...

Acute liver failure (ALF) is defined by the American Association for the Study of Liver Diseases as rapid development of acute liver injury without any prior signs of damage and is characterized by severe impairment of the synthetic, metabolic, and detoxifying functions of the liver1. ALF differs from chronic liver failure where the failure occurs as a result of liver injury caused over a long period of time and from acute chronic liver failure (ACLF), where abrupt liver damage takes place as a result of chronic liver diseases2,3,4. The only available cure for ALF is orthotopic liver transplant (OLT), or death may occur. Due to the shortage of liver donors, the rate of mortality in patients suffering from ALF is very high.
To study the potential of alternative therapeutic approaches and to better understand the pathophysiology of ALF, animal models that can reflect the ALF occurring in human beings are needed. Many of the already available ALF animal models have several shortcomings. Acetaminophen (APAP) effects are difficult to reproduce but have the closest similarities in terms of temporal, clinical, biochemical, and pathological parameters. APAP- induced animal models frequently encounter problems due to the presence of methemoglobinemia caused by the oxidation of hemoglobin by APAP and its intermediates5,6,7. Another problem is the lack of reproducibility reflected by unpredictable dose responses and the time of death. The ALF animal models produced using carbon tetra chloride (CCl4) have poor reproducibility8,9,10,11. Concavalin A (Con A) and lipoplysaccharide (LPS)-induced ALF animal models do not reflect the clinical pattern of the human disease, though they have advantages in the study of cellular mechanisms involved in autoimmune liver diseases and in the study of sepsis respectively12,13,14,15. Similarly, thioacetamide (TAA) also requires biotransformation to an active metabolite thioacetamide sulfoxide and shows species variation16,17,18,19. D-galactosamine (D-Gal) produces some biochemical, metabolic, and physiological changes similar to ALF but is not able to reflect the whole ALF pathological condition20,21,22,23. There have been very few attempts to combine two or more of these methods to develop an ALF model that is able to reflect the ALF syndrome in a better manner13. Therefore, further studies are required to develop a model that can reflect the disease parameters, has better reproducibility, and provides enough time to study the effects of a therapeutic intervention.
In the current study, an alternative ALF model in rats has been created by combining the effects of partial hepatectomy (PHx) and lower doses of a hepatotoxic reagent. APAP has a well-established role in causing liver injury5,24,25. It is a widely used analgesic and is toxic to the liver at supratherapeutic doses by forming toxic metabolites. APAP is the cause of many deaths in developed countries. Physical injury caused by partial hepatectomy initiates activation of various processes involved in inflammation as well as liver regeneration. Injection of the hepatotoxic agent APAP causes a hostile environment in the liver, preventing the proliferation of hepatocytes. This reduces the stress period on the animal, which when combined with smaller doses of hepatotoxin, leads to better reproducibility of the procedure. Therefore, using this model, a combinatory effect of two types of liver injuries has been studied. To characterize the developed ALF animal model, physiological and biochemical parameters have been studied. Successful reversibility of ALF was confirmed by transplantation of syngeneic healthy rat hepatocytes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetaminophen (Biocetamol)</td>
			<td>EG Pharmaceuticals</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase Kit (DEA)</td>
			<td>Coral Clinical System, India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Automated analyser</td>
			<td>Tulip, Alto Santracruz, India</td>
			<td>Screen Maaster 3000</td>
			<td>Biochemical analyser for liver functional test</td>
		</tr>
		<tr>
			<td>Betadine (Povidon-Iodine Solution)</td>
			<td>Win-Medicare; India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Biological safety cabinet (Class I)</td>
			<td>Kartos international; India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Bright Field Microscope</td>
			<td>Olympus, Japan</td>
			<td>LX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Cefotaxime (Taxim&#174;)</td>
			<td>AlKem; India</td>
			<td></td>
			<td>cefotaxime sodium injection, No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Sigma; US</td>
			<td>CLS431752</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>Gibco by Life Technologies</td>
			<td>17100-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Buds</td>
			<td>Pure Swabs Pvt Ltd; India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Drape Sheet</td>
			<td>JSD Surgicals, Delhi, India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>DPX Mountant</td>
			<td>Sigma; US</td>
			<td>6522</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y solution, alcoholic</td>
			<td>Sigma; US</td>
			<td>HT110132</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Major Surgicals; India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Gas Anesthesia System</td>
			<td>Ugo Basile; Italy</td>
			<td>211000</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Himedia, India</td>
			<td>GRM077</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removing cream (Veet&#174;)</td>
			<td>Reckitt Benckiser, India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Hematoxylin Solution, Mayer&#39;s</td>
			<td>Sigma; US</td>
			<td>MHS16</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Himedia; India</td>
			<td>RM554</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase From Sheep Testes</td>
			<td>Sigma; US</td>
			<td>H6254</td>
			<td></td>
		</tr>
		<tr>
			<td>I.V. Cannula (Plusflon)</td>
			<td>Mediplus, India</td>
			<td>Ref 1732411420</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes</td>
			<td>BD; US</td>
			<td>REF 303060</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (Forane&#174;)</td>
			<td>Asecia Queenborough</td>
			<td>No B506</td>
			<td>Inhalation Anaesthetic</td>
		</tr>
		<tr>
			<td>Ketamine (Ketamax&#174;)</td>
			<td>Troikaa Pharmaceuticals Ltd.</td>
			<td></td>
			<td>Ketamine hydrochloride IP, No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Meloxicam (Melonex&#174;)</td>
			<td>Intas Pharmaceuticals Ltd; India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Micro needle holders straight &#38; 
			curved</td>
			<td>Mercian; England</td>
			<td>BS-13-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holders straight &#38; 
			curved</td>
			<td>Mercian; England</td>
			<td>BS-13-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Histo-Line Laboratories, Italy</td>
			<td>MRS3500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Thread</td>
			<td>Mighty; India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Himedia; India</td>
			<td>GRM 3660</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll&#174;</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Refresh Tears/Eyemist Gel</td>
			<td>Allergan India Private Limited/Sun Pharma, India</td>
			<td>P3060</td>
			<td>No specific Catalog Number</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Himedia; India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Major Surgicals; India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Major Surgicals; India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>SGOT (ASAT) KIT</td>
			<td>Coral Clinical System, India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>SGPT (ALAT) KIT</td>
			<td>Coral Clinical System, India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Shandon Cryotome E Cryostat</td>
			<td>Thermo Electron Corporation; US</td>
			<td></td>
			<td>No specific Catalog Number</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma; US</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Blade No. 22</td>
			<td>La Medcare, India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Surgical Board</td>
			<td>Locally made</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Surgical White Tape</td>
			<td>3M India; India</td>
			<td>1530-1</td>
			<td>Micropore Surgical Tape</td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Ethicon, Johnson &#38; Johnson, India</td>
			<td>NW 5047</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (1ml, 26 G)</td>
			<td>Dispo Van; India</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>Trimmer (Clipper)</td>
			<td>Philips</td>
			<td>NL9206AD-4 DRACHTEN QT9005</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing Machine</td>
			<td>Braun</td>
			<td></td>
			<td>No specific Catalog Number (Local Procurement)</td>
		</tr>
		<tr>
			<td>William&#39;s E Media</td>
			<td>Himedia; India</td>
			<td>AT125</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Xylaxin&#174;)</td>
			<td>Indian Immunologicals Limited</td>
			<td></td>
			<td>Sedative, Pre-Anaesthetic, Analgesic and muscle relaxant</td>
		</tr>
	</tbody>
</table>

generation of a rat model of acute liver failure by combining 70% partial hepatectomy and acetaminophen

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and detoxifying functions of the liver. In most irreversible liver damage cases, orthotropic liver transplant (OLT) remains the only available treatment. To study the therapeutic potential of a treatment for ALF, its prior testing in an animal model of ALF is essential. In the current study, an ALF model in rats was developed by combining 70% partial hepatectomy (PHx) and injections of acetaminophen (APAP) that provides a therapeutic window of 48 h. The median and left lateral lobes of the liver were removed to excise 70% of the liver mass and APAP was given 24 h postsurgically for 2 days. Survival in ALF-induced animals was found to be severely decreased. The development of ALF was confirmed by altered serum levels of the enzymes alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP); changes in prothrombin time (PT); and assessment of the international normalized ratio (INR). Study of the gene expression profile by qPCR revealed an increase in expression levels of genes involved in apoptosis, inflammation, and in the progression of liver injury. Diffused degeneration of hepatocytes and infiltration of immune cells was observed by histological evaluation. The reversibility of ALF was confirmed by the restoration of survival and serum levels of ALT, AST, and ALP after intrasplenic transplantation of syngeneic healthy rat hepatocytes. This model presents a reliable alternative to the available ALF animal models to study the pathophysiology of ALF as well as to evaluate the potential of a novel therapy for ALF. The use of two different approaches also makes it possible to study the combined effect of physical and drug-induced liver injury. The reproducibility and feasibility of current procedure is an added benefit of the model.

Survival percentage in animal models of ALF 
The optimum dose of APAP to cause ALF in combination with 70% PHx was standardized as 750 mg/kg body weight. The treatment regimen started 24 h after 70% PHx, when the animals had completely recovered from surgery, and consisted of two APAP doses at 24 h intervals. Mortality was observed at the rate of 80% after the administration of the second dose of APAP, 48 h post-surgery. The survival percentage was analyzed and plotted via the Kaplan-Meier method (<...

Watch this Scientific Journal Video about Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen at JoVE.com

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

The acute liver failure animal model developed in the current study presents a feasible alternative for the study of potential therapies. The current model employs the combined effect of physical and drug-induced hepatic injury and provides a suitable time window to study the potential of novel therapies.

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and ...

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9.8K Views.  National Institute of Immunology. The acute liver failure animal model developed in the current study presents a feasible alternative for the study of potential therapies. The current model employs the combined effect of physical and drug-induced hepatic injury and provides a suitable time window to study the potential of novel therapies.

Video: Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

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.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

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 ...

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdcscid/J) mouse. 
In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdcscid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...