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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 diseases^2,3,4. 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 intermediates^5,6,7. 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 reproducibility^8,9,10,11. 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 respectively^12,13,14,15. Similarly, thioacetamide (TAA) also requires biotransformation to an active metabolite thioacetamide sulfoxide and shows species variation^16,17,18,19. D-galactosamine (D-Gal) produces some biochemical, metabolic, and physiological changes similar to ALF but is not able to reflect the whole ALF pathological condition^20,21,22,23. 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 injury^5,24,25. 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.

Protokół

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

1. Prepare for the surgical procedure as described earlier by Das B et al.²⁶.
2. Use 6–8-week-old inbred Wistar rats with a body weight of 200–250 g.
3. House the animals under standard animal care conditions and feed them with rat chow and libitum before and after the procedure.
4. 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.
5. 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.
6. 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 2–3% during the surgical procedure.

2. Preoperative procedures

1. 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.
2. To prevent corneal desiccation, apply a carboxy methyl-cellulose based eye drop to both eyes.
3. 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.
4. Remove hair from the upper right abdominal surgical area using an electric clipper.
5. Disinfect the surgical site by three alternating scrubs of povidone iodine and 70% ethanol using sterilized cotton pads in circular motion.

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.

1. 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.
2. Mark the skin to be cut just beneath the sternum, perpendicular to the xiphoid, and parallel to the ribcage.
3. Place a sterile drape sheet having an opening of around 3 cm x 1 cm over the marked skin.
4. Perform a transverse incision of around 2–3 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.
5. Next, make a transverse incision through the peritoneal layer just beneath the xiphoid process.
6. 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.
7. Slip an 8–10 cm long sterile nylon thread loop (size 4–0, 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.
8. 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.
9. Take precaution not to tie the knot too close to the nearby blood vessels, which may otherwise cause venous obstruction (stenosis).
10. 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.
11. 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 8–10 cm long nylon thread (size 4–0) 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.
12. After removing the lobes, suture the peritoneum using an absorbable chromic 4–0 suture with continuous stitches followed by skin suturing with an interrupted suture.
13. Apply povidone iodine on the skin surrounding the sutures to prevent infection.
14. Remove the drape sheet and remove the animal from the surgery board.

4. Postoperative care in animals

1. 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.
2. 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.
3. House the operated animals under standard conditions of 12 h light/dark cycle and monitor at regular intervals.

5. Injection of drug in partially hepatectomized animals to induce liver failure

1. After 24 h postsurgery, when the animals have successfully recovered from 70% PHx, measure the body weight of the animal followed by the injections.
2. Inject 750 mg/kg body weight of APAP intraperitoneally in partially hepatectomized animals 24 h after the 70% PHx following the animals' 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).
3. 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.

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 1^st 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 1^st 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 studies^29,30,31 with some modifications. For intrasplenic transplantation of cells in the ALF animal model, follow the steps mentioned below.

1. 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 250–350 g body weight. Check for the depth of anesthesia by the lack of pedal reflexes when pinching the toe of the animal.
2. Place the anaesthetized rat on the surgical board such that its left lateral portion is facing up. Maintain anesthesia at 2–3% isoflurane inhalation through a suitable mouthpiece.
3. Shave the skin on the left lateral region and sterilize it by povidone iodine solution.
4. Make a transverse incision on the shaved region of skin.
5. Make a 1–2 cm cut in the peritoneal layer to expose the spleen.
6. Gently take out the spleen of the peritoneal cavity and lift it up with the help of two moistened cotton tips.
7. Keep the cells (typically 10⁷ per animal) to be transplanted suspended in 50 µL IMDM media in a 1 mL insulin syringe with a 29 G needle.
8. Gently pierce the needle into the spleen cortex and release the cell suspension into the spleen within 2–3 min.
9. 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.
10. Close the peritoneum and skin by a 4–0 absorbable suture with continuous and discontinuous suturing respectively.
11. Apply povidone iodine solution on the skin at the place of the sutures to prevent infection on the operated site.
12. 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.
13. 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 3–4 days.

7. Characterization of ALF development

1. Euthanize the animals by overdose of ketamine-xylazine solution 2 h after the 2^nd dose of APAP treatment and collect blood and tissue samples.
2. Collect serum from blood for biochemical studies³².
3. Process liver tissue samples for histological and gene expression studies^33,34,35.

Wyniki

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 (<...

Dyskusje

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 ALF^{6,12,21,23,46}

Materiały

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