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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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 .......

Representative Results

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 (<.......

Discussion

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

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
Acetaminophen (Biocetamol)EG PharmaceuticalsNo specific Catalog Number (Local Procurement)
Alkaline Phosphatase Kit (DEA)Coral Clinical System, IndiaNo specific Catalog Number (Local Procurement)
Automated analyserTulip, Alto Santracruz, IndiaScreen Maaster 3000Biochemical analyser for liver functional test
Betadine (Povidon-Iodine Solution)Win-Medicare; IndiaNo specific Catalog Number (Local Procurement)
Biological safety cabinet (Class I)Kartos international; IndiaNo specific Catalog Number (Local Procurement)
Bright Field MicroscopeOlympus, JapanLX51
Cefotaxime (Taxim®)AlKem; Indiacefotaxime sodium injection, No specific Catalog Number (Local Procurement)
Cell StrainerSigma; USCLS431752
Collagenase Type IGibco by Life Technologies17100-017
Cotton BudsPure Swabs Pvt Ltd; IndiaNo specific Catalog Number (Local Procurement)
Drape SheetJSD Surgicals, Delhi, IndiaNo specific Catalog Number (Local Procurement)
DPX MountantSigma; US6522
Eosin Y solution, alcoholicSigma; USHT110132
ForcepsMajor Surgicals; IndiaNo specific Catalog Number (Local Procurement)
Gas Anesthesia SystemUgo Basile; Italy211000
GlucoseHimedia, IndiaGRM077
Hair removing cream (Veet®)Reckitt Benckiser, IndiaNo specific Catalog Number (Local Procurement)
Hematoxylin Solution, Mayer'sSigma; USMHS16
Heparin sodium saltHimedia; IndiaRM554
Hyaluronidase From Sheep TestesSigma; USH6254
I.V. Cannula (Plusflon)Mediplus, IndiaRef 1732411420
Insulin SyringesBD; USREF 303060
Isoflurane (Forane®)Asecia QueenboroughNo B506Inhalation Anaesthetic
Ketamine (Ketamax®)Troikaa Pharmaceuticals Ltd.Ketamine hydrochloride IP, No specific Catalog Number (Local Procurement)
Meloxicam (Melonex®)Intas Pharmaceuticals Ltd; IndiaNo specific Catalog Number (Local Procurement)
Micro needle holders straight &
curved
Mercian; EnglandBS-13-8
Micro needle holders straight &
curved
Mercian; EnglandBS-13-8
MicrotomeHisto-Line Laboratories, ItalyMRS3500
Nylon ThreadMighty; IndiaNo specific Catalog Number (Local Procurement)
ParaformaldehydeHimedia; IndiaGRM 3660
Percoll®GE Healthcare17-0891-01
Refresh Tears/Eyemist GelAllergan India Private Limited/Sun Pharma, IndiaP3060No specific Catalog Number
RPMIHimedia; IndiaNo specific Catalog Number (Local Procurement)
ScalpelMajor Surgicals; IndiaNo specific Catalog Number (Local Procurement)
ScissorsMajor Surgicals; IndiaNo specific Catalog Number (Local Procurement)
SGOT (ASAT) KITCoral Clinical System, IndiaNo specific Catalog Number (Local Procurement)
SGPT (ALAT) KITCoral Clinical System, IndiaNo specific Catalog Number (Local Procurement)
Shandon Cryotome E CryostatThermo Electron Corporation; USNo specific Catalog Number
SucroseSigma; USS0389
Surgical Blade No. 22La Medcare, IndiaNo specific Catalog Number (Local Procurement)
Surgical BoardLocally madeNo specific Catalog Number (Local Procurement)
Surgical White Tape3M India; India1530-1Micropore Surgical Tape
SuturesEthicon, Johnson & Johnson, IndiaNW 5047
Syringes (1ml, 26 G)Dispo Van; IndiaNo specific Catalog Number (Local Procurement)
Trimmer (Clipper)PhilipsNL9206AD-4 DRACHTEN QT9005
Weighing MachineBraunNo specific Catalog Number (Local Procurement)
William's E MediaHimedia; IndiaAT125
Xylazine (Xylaxin®)Indian Immunologicals LimitedSedative, Pre-Anaesthetic, Analgesic and muscle relaxant

References

  1. Polson, J., Lee, W. M. AASLD position paper: the management of acute liver failure. Hepatology. 41, 1179-1197 (2005).
  2. Chung, R. T., et al. Pathogenesis of liver injury in acute liver failure. Gastroenterolo....

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