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