Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl₄) Exposure Through an Orogastric Tube

Summary

This protocol describes a common and feasible method of inducing acute liver injury (ALI) via CCl₄ exposure through an orogastric tube. CCl₄ exposure induces ALI through the formation of reactive oxygen species during its biotransformation in the liver. This method is used to analyze the pathophysiology of ALI and examine different hepatoprotective strategies.

Abstract

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including complications such as hepatic encephalopathy and impaired protein synthesis. Appropriate animal models are vital to test the mechanism and pathophysiology of ALI and investigate different hepatoprotective strategies. Due to its ability to perform chemical transformations, carbon tetrachloride (CCl₄) is widely used in the liver to induce ALI through the formation of reactive oxygen species. CCl₄ exposure can be performed intraperitoneally, by inhalation, or through a nasogastric or orogastric tube. Here, we describe a rodent model, in which ALI is induced by CCl₄ exposure through an orogastric tube. This method is inexpensive, easily performed, and has minimal hazard risk. The model is highly reproducible and can be widely used to determine the efficacy of potential hepatoprotective strategies and assess markers of liver injury.

Introduction

The frequency of toxic insults to the liver, especially due to alcohol and drug abuse, is increasing. Acute liver injury (ALI) is associated with high mortality rates and has caused clinical concerns^1,2. Toxic injury leads to death signaling pathways in the liver, resulting in hepatocyte apoptosis, necrosis, or pyroptosis. ALI plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including complications such as hepatic encephalopathy and impaired protein synthesis^3,4. Although recent research has increased our knowledge about the physiological and pathological changes accompanying hepatic failure, it has not completely explained the pathomolecular features that affect the mechanisms of cell death. Furthermore, no medications are currently available to reverse the progressive deterioration in ALI patients. Currently, the only significantly effective treatment is liver transplantation^5,6.

In order to investigate the mechanism and pathophysiology of ALI and to test different hepatoprotective strategies, different animal models are used to induce ALI. A preferable animal model of ALI should mimic the pathological process of the disease via a reliable, validated, inexpensive and easy to apply method. Examples of experimental models include hepatotoxic agents, surgical procedures such as total or partial hepatectomy, complete or transient devascularization, and infective procedures^7,8,9. Known hepatotoxic substances include galactosamine, acetaminophen, thioacetamide, azoxymethane and CCl₄. Of these, CCl₄ is widely used although it has not yet been well characterized^10,11,12,13.

CCl₄ is an organic colorless liquid compound with a sweet smell and almost no flammability at lower temperatures. Exposure to high concentrations of CCl₄ can cause damage to the central nervous system, including deterioration of the liver and kidneys. CCl₄ induces ALI through its biotransformation in the liver, which forms reactive oxygen species. This occurs via the P450 cytochrome enzyme 2E1, forming an active metabolite and resulting in cell damage by macromolecule binding, enhancement of lipid peroxidation and disturbance of intracellular calcium homeostasis¹⁴. In addition, the CCl₄ model can be used to stimulate the astrocytes at the level of RNA synthesis¹⁵. This hepatotoxin has been administered by the intraperitoneal, intraportal, oral, and intragastric routes¹⁶.

In this protocol, we describe in detail CCl₄-induced ALI in rats via an orogastric tube. This method induces robust and reproducible ALI that can be used to investigate the pathogenesis of ALI. Determination of liver disease severity is monitored by measurement of serum glutamate-pyruvate transaminase (GPT), glutamic oxaloacetic transaminase (GOT) enzymes and total bilirubin (TB) as well as definitive histological diagnosis by hematoxylin and eosin (H&E) stained liver tissues. Exposure to CCl₄ through an intragastric access allows for a practical, inexpensive, minimally invasive method with minimal hazard risk.

Protocol

The experiments were conducted according to the recommendations of the Declarations of Helsinki and Tokyo and to the Guidelines for the Use of Experimental Animals of the European Community. The experiments were approved by the Animal Care Committee of Ben-Gurion University of the Negev.

NOTE: The CCl₄ model has been generated and used in a previous study¹⁷. The protocol timeline is demonstrated in Table 1.

1. Preparing rats for the experimental procedure

NOTE: Select adult male Sprague Dawley rats weighing 300−350 g.

1. Obtain approval for experiments from Institutional Animal Care and Use Committee (IACUC).
2. Maintain rats at room temperature (22 °C ± 1 °C), with 12 h light and 12 h dark cycles. Provide rat chow and water ad libitum.
3. Perform all experiments between 6:00 a.m. and 12:00 p.m.
4. Shave the rat and disinfect the skin with alcohol.

2. Determination of serum GOT, GPT, and TB baseline levels

1. Anesthesia
   1. Prepare a continuous isoflurane administration system to induce anesthesia. Make sure the vaporizer system is filled with isoflurane.
   2. Anesthetize the rat with 2% isoflurane. Confirm that the rat is fully anesthetized by observing the movement and pedal reflex in response to external stimuli.
      NOTE: Use 1−5% isoflurane for anesthesia induction and maintenance.
2. Cannulate the tail vein with a 22 G catheter.
3. Collect a 0.5 mL blood sample at baseline. Ensure that the blood volume retrieved does not exceed IACUC guidelines.
4. Perform blood biochemical analysis including the measurements of serum GOT, GPT and TB, as previously described¹⁸.
   NOTE: Examinations of liver enzymes and TB level were carried out in the biochemical laboratory of Soroka Medical Center. Blood samples were analyzed using a fluorescence method on a chemistry analyzer (Table of Materials).

3. Induction of acute liver injury in rats

CAUTION: Exposure to high concentrations of CCl₄, including absorption through vapor or skin, can have negative effects on the central nervous system and cause degeneration of the liver and kidneys. Prolonged exposure can cause coma or death.

1. Prepare a 50% solution of CCl₄ (Table of Materials) by mixing CCl₄ with olive oil as a vehicle in a 1:1 ratio.
   NOTE: The solution should be prepared according to IACUC guidelines for non-pharmaceutical grade compounds.
2. Induce hepatotoxicity in vivo by CCl₄ administration via an orogastric tube.
   1. Insert a 16 G orogastric tube (3 inches deep) through the oral cavity of the rat.
   2. Expose the rat to different doses of CCl₄ by injecting a syringe with one of the following diluted solutions into the rat’s stomach: 1 mL/kg (mild ALI), 2.5 mL/kg (moderate ALI), or 5 mL/kg (severe ALI) of the 50% solution. For the sham-operated control group, expose the rat to 5 mL/kg olive oil only.

4. Determination of serum GOT, GPT, and TB levels after 24 h

1. Anesthesia
   1. Prepare a continuous isoflurane administration system to induce anesthesia. Make sure the vaporizer system is filled with isoflurane.
   2. Anesthetize the rats with 2% isoflurane. Confirm that the rat is fully anesthetized by observing the movement and pedal reflex in response to external stimuli.
2. Collect blood samples at 24 h from CCl₄ exposure.
3. Perform blood biochemical analysis including measurements of serum GOT, GPT and TB.

5. Liver collection for histological examination

1. Euthanize the rat by replacing the inspired gas mixture with 20% O₂/80% CO₂. Ensure that CO₂ is delivered at a predetermined rate in accordance with IACUC guidelines.
2. Ensure death by checking for lack of heartbeat and confirm by a secondary method in accordance with IACUC guidelines.
3. Place the rat on a dissecting board with its dorsal surface facing down and abdomen facing up. Shave the abdomen of the rat.
4. With a scalpel, incise the full length of the ventrum skin from the anus to the chin. Separate the skin. Incise the abdominal wall with a scalpel from the anus to the xyphoid cartilage, exposing the abdominal viscera.
5. Using scissors and forceps, isolate the liver by dissecting it from its ligaments and attachments. Starting at the liver hilum, carefully perform a hepatectomy by releasing all the liver lobes from attachments. Dissect and cut away all ligaments and blood vessels.
6. Transfer the liver into a Petri dish. Fix the liver in a 4% buffered formaldehyde solution (Table of Materials) for at least 24 h.

6. Histological examination

1. Sample preparation
   1. After fixation, cut the sample into 5 μm thick slice series by microtome sectioning.
   2. Gently place the slices on glass slides with a soft brush, 1 slice per slide.
   3. Perform H&E staining as previously described¹⁹.
2. Examine the slices under a microscope at 200x magnification using a 20 mm objective lens (Table of Materials).
   NOTE: The liver sections should be graded by a specialized pathologist blinded to the treatment protocol. A score of 0 indicates no liver abnormalities, 1–2 indicates mild liver injury, 3–4 indicates moderate liver injury, and 5–6 indicates severe liver injury^20,21,22.

Representative Results

The TB, GOT, and GPT levels significantly increased 24 h after inducing ALI (more at higher CCl₄ doses) compared to sham-operated controls (p < 0.001) (Figure 1). The levels of TB, GOT, and GPT at baseline were normal and were not significantly different than sham-operated controls. At 24 h, all three interventional groups, 1 mL/kg CCl₄ (1, 1−2), 2.5 mL/kg CCl₄ (3, 3−4), and 5 mL/kg CCl₄ (4, 4−5.75), had a significantly higher histological grading score than the sham-operated control group (0, 0−0) (p < 0.05, data presented as median, 25−75% range). The H&E images of a sham-operated control (Figure 2A) and groups exposed to different CCl₄ doses (Figure 2B−D) show histopathological changes 24 h after CCl₄ exposure. Disruption of hepatocellular architecture by CCl₄ was demonstrated by a high grade of tissue injury with large fibrous septa deformation, extension of fibers, collagen accumulation, and pseudo lobe separation in liver sections (Figure 2).

Figure 1: Serum TB (A), GOT (B), and GPT (C) levels in blood samples 24 h after exposure to different CCl₄ doses compared to sham-operated controls. Blue bar: control; red bar: 24 h after CCl₄ exposure. The significance of comparisons between CCl₄-exposed rats and unexposed rats are determined using the Mann–Whitney test. A p-value of <0.05 was considered significant. Please click here to view a larger version of this figure.

Figure 2: Histopathological changes in liver tissue stained with H&E after 24 h CCl₄ intoxication in various doses. (A) sham-operated control, (B) 1 mL/kg CCl₄, (C) 2.5 mL/kg CCl₄, and (D) 5 mL/kg CCl₄. Scale bar = 50 µm. The distribution of the histological outcomes was predicted by linear regression. Please click here to view a larger version of this figure.

Groups	0 hours	24 hours
Sham (15 rats)	GOT, GPT, TB baseline level	GOT, GPT, TB level
Mild ALI (15 rats)
Moderate ALI (15 rats)	CCl4 exposure for ALI groups and olive oil for sham group	Histological examination
Severe ALI (18 rats)

Table 1: Demonstration of the protocol timeline. The various groups of rats at different times include a sham-operated control group, mild ALI (exposure to 1 mL/kg CCl₄), moderate ALI (exposure to 2.5 mL/kg CCl₄), and severe ALI (exposure to 5 mL/kg CCl₄). At time 24 h, serum GOT, GPT and TB levels were measured, and histological examination was performed for all four groups.

Discussion

In this protocol, CCl₄ is used as a liver toxin to induce ALI in rats. ALI is characterized by loss of hepatic parenchyma and subsequent dysregulation of the liver’s metabolic and synthetic functions. Drugs, viruses, toxins, autoimmune diseases, metabolic diseases, and vascular disorders all induce hepatocyte death, and the subsequent inflammatory response contributes to the pathogenesis of ALI.

The initial insult to the liver leads to cytokine production, chemokine release, and subsequent infiltration of inflammatory cells into the liver. Three of the commonly tested biomarkers for ALI evaluation are GPT, GOT and TB levels. GPT and GOT are enzymes measured by activity level while TB level measures liver function by serum concentration. When elevated, serum enzyme activity levels denote injury to hepatocytes or cholangiocytes²³. Rapid spectrophotometric method was first reported in the work of Arthur Karmen in 1955²⁴, which allowed for the widespread clinical application of serum enzyme measurement. Since then, GOT and GPT measurements have also been applied to detect hepatocyte injury. GPT is used more frequently, and simultaneous GPT testing usually reveals redundant results. The increase in activity levels of GOT and GPT between the release rates and the clearance rates from injured cells can be used to measure approximately the rate of injury to the cells. When the injured liver cells cause the liver to fail in its normal activities, such as processing and removing bilirubin as bile, this indicates that the ALI is more severe.

There are several steps in the protocol that are critical and merit careful consideration. Most protocols require serum biomarker testing before and after exposure to the investigative agent, as elevations in serum enzyme concentration levels are common. However, due to fluctuations in the timing of elevated ALT, several tests should be conducted periodically to detect any elevation. In this protocol, we chose to test GOT, GPT and TB levels at baseline and 24 h after exposure to the toxin. According to recent studies, the levels of these biomarkers correlated well with the severity of ALI during this time interval¹⁷. As shown in Figure 1, levels of blood GOT, GPT, and TB were elevated in all samples 24 h after inducing ALI. This indicates that the model has quantified outcomes in a very short time interval since exposure to CCl₄. One should take into account that in severe ALI the liver loses its ability to synthesize GOT and GPT. Therefore, in these cases these enzymes may lack their predictive value as demonstrated in the literature.

Histological findings of rats exposed to CCl₄ are characterized by ballooning of cells, centrilobular necrosis and the presence of Councilman bodies²⁵. In this model there was widespread damage shown to be proportional to the dose of CCl₄ administered.

This method of inducing ALI via orogastric CCl₄ exposure has numerous advantages. It is simple, inexpensive, and with minimum hazard risk. The protocol provides significant results in a very short time interval. The model is highly reproducible and can be commonly used to determine the efficacy of potential hepatoprotective strategies and assess markers of liver injury.

It is important to note that CCl₄ mainly affects the central zone of the liver, which does not match the massive necrosis typically seen in human liver failure. Moreover, CCl₄ is not completely metabolized in the liver, and some of the nonmetabolized CCl₄ can damage other organs, including lungs and kidneys¹⁶. In addition, due to different levels of cytochrome P450 development and efficacy, there is a large variation in sensitivity depending on species and age²⁶. Despite these limitations, the method of orogastric CCl₄-induced ALI still serves as a valuable rodent model.

Materials

Name	Company	Catalog Number	Comments
22 G catheter BD Neoflon TM	Becton Dickinson Infusion Therapy AB
4% buffered formaldehyde solution	Sigma - Aldrich lab materials technologies
BD Microtainer SST TM Tubes	Becton Dickinson and Company
Carbone tetrachloride	Sigma - Aldrich lab materials technologies	CAS 56-23-5
Isofluran, USP 100%	Piramamal Critical Care, Inc
Olympus AU2700 Chemistry-Immuno Analyzer	Olympus (MN, USA)		Analysis of blood samples was done by the fluorescence method
Olympus BX 40 microscope	Olympus
RAT Feeding Needles	ORCHID SCIENTIFICS
SYRINGE SET 1 and 2 ml MEDI -PLUS	Shandong Zibo Shanchuan Medical Instruments Co., Ltd