Effect of Anti-TNFα Therapy by Infliximab against Pancreatic Tissue Damage in Severe Acute Necrotizing Pancreatitis

1. Introduction

Nowadays, it is difficult to find a more complicated inflammatory disease of the abdominal organs in its pathogenesis than acute pancreatitis (AP). This pathology is included in the group of diseases united by the features of medical care, called “acute abdomen”, consistently occupying 2-3 places in this group along with acute cholecystitis. According to the World Health Organization, AP affects between 200 and 800 people per one million of the world’s population. According to the State Statistics Committee, the incidence of AP in the Russian Federation ranges from 36 to 40 cases per 100,000 population. The prevalence of destructive forms is currently 15-20% of all cases, from 75 to 80% of the diseases that are called abortive or edematous. The development of pancreatic tissue destruction is a life-threatening complication with a mortality rate of over 80% Goodchild, Chouhan ( 1 ). The extension of the necrotizing process in the pancreas causes ischemic damage, which leads to activation of acute inflammation ( 2 - 4 ). One of the most important factors in the treatment of patients with AP is the early diagnosis of the disease. Mild pancreatitis is easy to treat; however, treatment for AP includes intensive care. Access to and observation of the pancreas is not possible without surgery, and imaging observations may not provide sufficient information to the physician ( 5 ). There are also inherited and chronic forms of the disease that can have irreversible effects throughout one’s life. Patients often suffer from pain and malnutrition and are more likely to have a higher risk of pancreatic cancer ( 6 ).

Conservative treatment is an approach to treat some ailments, such as low back pain, neck pain, and spinal diseases using non-surgical treatment options, such as physiotherapy, medication, and injections ( 7 ). The development of conservative therapy methods requires research and the creation of methodological complex approaches to understanding the place and role of certain pharmacological targets ( 8 - 13 ).

One of the treatment methods is the application of synthetic antibodies that have an inhibitory effect on pancreatic enzymes. Therefore, anti-protein drugs are expected to prevent necrotic changes in the pancreas and reduce mortality (7). The application of antimediator and antimetabolites treatment is one of the most promising directions in the correction of inflammatory pathological processes ( 11 , 14 - 17 ).

The tumor necrosis factor-alpha (TNF-alpha) is one of the initial mediators of inflammation which has recently been proven to be involved with systemic inflammation in pancreatic necrosis. Norman, Fink ( 18 ) showed that tumor necrosis factor gene expression occurred locally during AP and that large amounts of TNF were produced within the pancreas with continuous levels higher than those in serum. The overall increase in TNF concentration in tissue and serum was directly related to the severity of pancreatic damage and inflammation, and they stated that intrusive macrophages played the largest role in this process ( 18 ). It was found that the concentration of TNF-alpha in the blood increased notably after the induction of AP ( 19 ).

Today, the study of the possible effects of a new group of drugs (e.g., monoclonal antibodies) that can trigger inflammation is of great interest. Some studies have suggested the effectiveness of monoclonal antibodies (TNF-alpha) in AP. However, in the analysis of these research works, it was revealed that the effects of this drug were investigated on “non-severe” models of AP with low mortality, which is most often represented in the foreign literature as mild necrotizing pancreatitis ( 20 ). Therefore, the question of extrapolating the results of these studies to clinical conditions is debatable.

The effect of infliximab on the lethality, volume, and nature of pancreatic lesions was studied in severe necrotizing ductal pancreatic necrosis.

2. Materials and Methods

The experiment was performed on females white Wistar rats (n=30) in a similar weight range (200-250g). All studies were performed in compliance with the rules of humane treatment of animals. Rats without external signs of the disease have been selected for this study. These rats have passed the quarantine regime and were kept in standard conditions.

The animals were divided into the following groups:

• Group (n=6): Control value
• Group B (n=6): A 24h mortality study was conducted on acute destructive pancreatitis in this group.
• Group C (n=6): The simulation of acute severe necrotic pancreatitis was performed along with the study of the volume of the pancreatic lesion for a period of 6 h from the moment of modeling.
• Group D (n=6): In this group, the effect of infliximab at a dose of 60 mkg/kg was studied on mortality in severe destructive pancreatitis for a period of 24 h from the moment of modeling.
• Group E (n=6): In this group, the effect of infliximab (at a dose of 120 mkg/kg) on the volume of pancreatic lesions in severe destructive pancreatitis was studied for a period of 6 h from the moment of modeling.

All manipulations were performed under general anaesthesia by intraperitoneal injection of Zoletil at a dose of 60 mg/kg, as well as chloral hydrate at a dose of 125 mg/kg.

The model was performed under general anesthesia. Intraperitoneal injection of Zoletil at a dose of 50 mg/kg, together with chloral hydrate at a dose of 125 mg/kg was carried out after the surgical field was treated with antibiotic solutions. Subsequently, the abdominal cavity was opened layer by layer. A loop of the duodenum was pulled into the wound. The main papilla of the duodenum was found during transillumination.

After finding the ampoule and the outlet of the main papilla of the duodenum, 0.5-0.6 cm was dissected from it and a needle with a diameter of 32G was used to perforate the anterior wall of the duodenum. Cannulation of the common bile duct was performed using a 36 G catheter (Figure 1).

Figure 1. Cannulated common bile duct

Insulating clips were applied to the cannula to reproduce severe destructive pancreatitis, distal to the place of duodenal duct junction, and the common bile duct so that the injected solution completely entered the pancreatic ducts.

Failure to comply with this condition may lead to the discharge of the solution into the duodenum. Subsequently, an excessive amount of the solution will enter the common bile duct which performs the function of the gallbladder in a rat, damps the excessive pressure, and accumulates a significant amount of bile. After isolation, 0.5 ml of a buffer solution containing a bile acid salt-sodium taurocholate was injected into the pancreatic ducts. The isolation clips were removed 1 min after administration which allowed the solution to penetrate deeper into the pancreatic parenchyma. At the next stage, the clips and cannula were removed from the ducts, and the abdominal cavity was sutured layer by layer completely. The ignorance about the pancreatic ducts system in rats can lead to a sharp decrease in the volume of its lesion, which is reflected in a lower mortality rate.

After the macroscopic evaluation, the pancreas, liver, kidneys, and lungs were collected for histological examination. The organs were collected, weighed, and fixated in a 10% neutral formalin solution. After fixation, the tissue areas were washed in running water, dehydrated, and poured into paraffin according to the standard procedure. Paraffin sections of the pancreas with a thickness of 5-7 mkm were prepared at standard intervals of all parts of the organ. The sections were stained with hematoxylin and eosin.

Histological processing was performed using the equipment of the company “Leica” (Germany). During the microscopic examination, the finished micro-preparations were scanned using archiving and image analysis system “MIRAX Desk”. Image analysis and morphometry were performed using the program “Pannoramic Viewer”1.15.4. Quantitative data were recorded in MS Excel software.

The volume of pancreas lesion was measured through the calculation of the ratio of the area of necrosis to the unaffected area of the pancreas, on digitized computer images that were obtained using a digital microscope in the “total scan” mode, as well as Mirax Desk and Pannoramic Viewer software (Version1.15.4). The ratio was expressed in percentage.

Infliximab (MSD Remicade) was administered 1 h before the AP modeling at a dose of 60 mkg/kg.

Statistical analysis of the obtained data was carried out using Microsoft Excel (Version 2007), and through calculating the mean of indicators, the error of the average m, and the confidence criterion (p). A p-value ≤0.05 was considered statistically significant.

3. Results

The mortality rate at the study period was one of the most accurate integral indicators to evaluate the effectiveness of a particular treatment method. It should be noted that 24 h after modeling severe pancreatic necrosis developed by researchers in the present study, the mortality rate was 100%.

In group C, a large amount of hemorrhagic effusion was localized in all parts of the abdominal cavity 6 h from the modeling of AP. The liver had marks of venous hyperemia with a smooth surface and sharpened edges of the lobes. Moreover, there was hyperemia of the spleen.

The greatest changes affected the pancreas, which was sharply edematous, with areas of hemorrhagic pancreatic necrosis. The retroperitoneal tissue was edematous. There was a hemorrhagic effusion with hyperemia of the parietal and visceral pleura in the chest. The areas of diapedesis hemorrhage were noted in the basal parts of the lungs.

Acute destructive pancreatitis was characterized by typical large-focal hemorrhagic pancreatic necrosis changes after a period of 6 h from the moment of modeling. The zone of the necrotic lesion in the duodenal part of the pancreas affected 50% of this part and was localized closer to the wall of the duodenum spreading along the duct.

The greatest destruction was noted in the gastro-splenic part of the gland in form of coagulation necrosis of acinar cells, fibrinoid necrosis of vascular walls and stromal elements, partial necrosis of acini, and severe neutrophil infiltration centrilobular with a primary lesion (Figure 1 and Figure 2).

Figure 2. Intact rat pancreas. G+E, magnification X 90

The volume of pancreatic damage was found to be 34.8%±1.2 during 6 h after the modeling.

Assessment of pancreatic damage in group D, as well as the protective effect of infliximab at a dose of 60 mkg/kg revealed that the total volume of the necrotic pancreatic lesion was estimated at 21.3%±1.4% for a period of 6 h from the moment of modeling AP. An increase in infliximab dose up to 120 mkg/kg did not lead to a significant reduction in the volume of pancreatic destruction and mortality rate which was 50% in this group (Table 1).

In the course of the work, it was revealed that the use of MAB infliximab at a dose of 60 mcg/kg led to a pronounced positive effect on the pancreatic lesion, manifested by a decrease in mortality rate up to 50% by 1 day in group D.

Positive protective effect of monoclonal antibodies to tumor necrosis factor in a dose of 60 mkg/kg 1h before modeling of acute destructive pancreatitis is manifested in the reduction of interstitial edema, a decrease in centrilobular areas of necrosis, and reduction in zones and degree of leukocyte infiltration (Figure 3 and 4).

Figure 3. Severe centrilobular lesion of the pancreas with neutrophil infiltration of the necrotic zone during 6 hours from the moment of modeling AP. G+E, magnification X 400

Figure 4. Centrilobular necrosis with a moderate lesion volume when corrected with infliximab at a dose of 60 mkg/kg in the simulation of AP for a period of 6 h from the moment of the simulation. G+E, magnification X 200

4. Discussion

Biologic drugs act on a TNF molecule and are called TNF blockers or anti-TNF drugs, which are used to treat rheumatoid arthritis, pediatric arthritis, psoriatic arthritis, and ankylosing spondylitis ( 21 ). Remicade or infliximab is used to reduce the pain and swelling of certain inflammatory diseases (e.g. rheumatoid arthritis, and psoriasis) and certain intestinal diseases, such as Crohn’s disease ( 22 ). Infliximab reduces inflammation by blocking interleukin-12 and interleukin-23 (a substance made by the body that causes inflammation) ( 23 ). Although infliximab does not cure the disease, it helps reduce the symptoms of the disease. This drug can help reduce the number of plaques in plaque psoriasis, pain and swelling in arthritis, and heartburn and cramps in Crohn’s disease ( 24 ).

The development of acute destructive pancreatitis is accompanied by the disruption of the pancreatic stroma. The parietal and visceral peritoneum is edematous and hyperemic in almost all parts. There is pronounced edema of the intestinal mesentery. The application of TNF-α is one of the most effective treatments for AP. Based on the obtained results in this study, TNF-α was significantly increased in pancreatic tissue in AP, and anti-TNF-α treatment significantly improved pathological and biochemical findings ( 25 ). The disorder of pancreatic exocrine secretion is one of the important features. A significant amount of amylase is secreted during pancreatitis, indicating the release of the enzyme from damaged cells ( 26 ). The disruption of pancreatic tissue leads to an increase in lipase and cytokine levels and oxidative tissue stress ( 27 ). Therefore, infliximab has a pronounced protective effect in AP, reducing the volume of the lesion as well as the mortality rate up to 50% for a period of 24 h. It can be concluded that further research on the application of monoclonal antibodies against tumor necrosis factor-alpha will form the basis for its application in clinical conditions in severe forms of necrotizing pancreatitis ( 28 ).

The TNF drugs, such as infliximab, may be an alternative treatment for patients who do not respond to treatment with corticosteroids or immunosuppressants ( 29 ). Based on the results of some studies, the blocking of TNF leads to minimization of inflammation and limitation of pancreatic damage ( 30 , 31 ). TNF-α is responsible for the local and systemic effects in pancreatitis, and it is thought that inhibition of TNF-α can reduce local and systemic complications ( 32 ). TNF blockade in AP could reduce the complications associated with pancreatitis. Some researchers have reported that the inhibition of TNF-α solution can reduce the severity of experimental pancreatitis and mortality; however, it does not affect pancreatic vacuolation, necrosis, and inflammatory cell infiltration. Oruc, Ozutemiz ( 33 ) pointed that infliximab has anti-inflammatory effects in the model of edematous and necrotic pancreatitis which can be explained by the differences in the performance between different molecules used in the studies. Other researchers reported that a TNF-receptor fusion protein etanercept could improve the histological scores and biochemical parameters in the necrotizing Na-taurocholate pancreatitis model ( 34 , 35 ).

5. Conclusion

Based on the obtained results, it can be concluded that anti-TNF therapy with infliximab can significantly reduce the volume of pancreatic lesions in severe forms of pancreatic necrosis, which contributes to a pronounced decrease in mortality for one day from the moment of pathology reproduction.

The main findings of the current study can be summarized in the following three statements:

1. Administration of infliximab (at a dose of 60 mkg/kg) in a model of severe acute necrotizing pancreatitis helped to reduce the death rate by 50% at a period of 24 h.

2. The application of infliximab at 60 mkg/kg dose at a period of 6 h after modeling of severe acute necrotizing pancreatitis led to the reduction of pancreatic damage volume from 34.8%±1.2% in group

C to 21.3%±1.4% in group D.

An increase of infliximab dose up to 120 mkg/kg in group E did not lead to a significant reduction in the volume of pancreatic destruction, compared to group D.

Authors' Contribution

Study concept and design: S. A. A. and T. I. F.

Acquisition of data: S. A. A., T. I. F. and L. V. D

Analysis and interpretation of data: D. P. N.

Drafting of the manuscript: E. N. B.

Critical revision of the manuscript for important intellectual content: S. A. A. and T. I. F.

Statistical analysis: L. V. D.

Administrative, technical, and material support: S. A. A.

Ethics

All studies were performed in compliance with the rules of humane treatment of Belgorod State University, Russia.

Conflict of Interest

The authors declare that they have no conflict of interest.

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