The Histopathologic Effects of l-Carnitine in a Sodium Taurocholate–Induced Severe Pancreatitis Model

Severe acute pancreatitis (SP) is a life-threatening condition with a mortality rate as high as 10% to 30%.¹ Although an increasing number of treatment modalities have been developed, there is no effective treatment for SP.^2,3 In most of the cases, mortality from SP is a result of multiorgan dysfunction syndrome, which is related to mitochondrial dysfunction at the cellular level.^4,5 Recent studies have focused on identifying the pathophysiology behind the mitochondrial dysfunction, and highlight the need for an early mitochondrial-specific therapy in acute pancreatitis.^6–8

L-carnitine (LC) has an important role in the transportation of long-chain fatty acids to the mitochondrial matrix for β-oxidation to provide energy. It also takes place in the transportation of intermediate toxic compounds out of mitochondria and the prevention of lipid peroxidation, which provides the stability of mitochondria under severe oxidative stress.^9–11 The protective effects on mitochondria and the anti-inflammatory and antioxidant efficacy of LC suggest that it could also be a therapeutic option in SP.

Acute pancreatitis induced by sodium taurocholate (STC) is the most often used model of acute severe necrotizing pancreatitis in rats. To our knowledge, the protective effects of LC on pancreatic tissue in the STC-induced SP model have not been demonstrated before in the literature. The aim of the present study is to determine whether LC is a protective agent from necrosis in an STC-induced experimental SP model in rats.

Materials and Methods

Results

Serum amylase activity

Serum amylase activities of the C and LC groups were similar (mean ± SD, 641.25 ± 209 and 522.37 ± 91.42, respectively), and there was no statistical significance between the two groups (P = 0.937). Amylase activities were significantly increased in both the P and P + LC groups compared with control animals (P < 0.05). When amylase activities in the P + LC group and the P group were compared (mean ± SD, 1557.12 ± 601.10 and 1780.62 ± 505.52, respectively), there was no statistical significance between groups (P = 0.697). The amylase activities of the groups are shown in Fig. 1.

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Histopathologic findings

The pancreatic tissue of the control and carnitine groups' rats showed normal histology with light microscopy (Fig. 2). Most of the organ was made of tubuloacinar exocrine pancreas. Islets of Langerhans were placed as isolated islands within the lobules.

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The pancreas specimens of the pancreatitis group showed prominent histopathologic changes. The histologic structure of the exocrine pancreas was damaged. Acinar degeneration, inflammatory cell infiltration, parenchymal hemorrhage, and edema in the interlobular and intralobular interstitium and fat necrosis were detected (Fig. 2). Histopathologic scores of the groups are summarized in Table 1. Pancreatic damage was significantly increased in the P group compared with the C group (P < 0.05).

Table 1  Total pancreatic damage scores of the groups ^a

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In the P + LC group, the histopathologic findings of the pancreatitis were markedly reduced. Focal acinar cell degeneration was rarely seen. Parenchymal hemorrhage was also rare. Interlobular and intralobular inflammation and edema were generally mild. Few apoptotic bodies were detected (Fig. 2). The pancreatic damage score of the P + LC group was significantly lower than that of P group, but it was significantly higher those that of the C and LC groups (P < 0.05).

Discussion

Acute pancreatitis is an inflammatory disorder of the pancreas with a high mortality, up to 30%. It is one of the leading causes of gastrointestinal disease requiring hospitalization and intensive care, with an annual incidence of 13 to 45 cases per 100,000 persons in the United States.¹⁴ On the other hand, there is no specific treatment, except for symptomatic management including fluid resuscitation, gastric decompression, enteral feeding, and intravenous antibiotics. Moreover, patients with infected necrosis need surgical, radiologic, or endoscopic drainage procedures.¹⁵ Because the incidence of acute pancreatitis is increasing worldwide, many researchers are focusing on effective treatment modalities.

According to the revised Atlanta classification, severity of pancreatitis is classified as mild, moderate, or severe. Mild acute pancreatitis has no organ failure, local complications, or systemic complications. Moderately severe acute pancreatitis is defined by the presence of transient organ failure (<2 days) and local complications, and severe acute pancreatitis is defined by the presence of persistent organ failure (>2 days). This classification also identifies 2 phases of the disease, early and late.¹⁶ Patients suffering from early and persistent multiorgan dysfunction syndrome have a high risk of mortality. About 20% of patients with SP develop organ dysfunction in first 3 days, and almost half of these patients do not respond to intensive treatment.^17,18 Therefore, the present study focused on the early phase of the disease.

The experimental model of STC-induced pancreatitis in rats demonstrates acute severe hemorrhagic pancreatitis in humans.¹⁹ There are other frequently used experimental models that produce mild pancreatitis induced by different chemicals, including cerulein, l-arginine, and l-aspariginase.^20–22 However, we decided to work on an STC-induced SP model in our study because the management of SP remains a challenging problem at present. Although SP is associated with a mortality rate of 30%, mild pancreatitis is associated with a mortality rate that is less than 1%.¹

An STC pancreatitis model is a commonly used experimental model that is thought to be the model that most similarly demonstrates biliary pancreatitis clinically and histologically in humans. Histopathologic changes in the STC model, including edema, hemorrhage, inflammation, and acinar cell necrosis, can be observed within 48 hours after induction of pancreatitis. However, it has several limitations, such as long procedure time, technical difficulty of surgery, and the high death rates of experimental animals.²³ Wang et al²⁴ reported that pancreatic injection of 3.8% STC could establish an SP model with multiple organ dysfunction but with a high mortality rate of 90% in 7 days in rats. So we defined the end point time of the study as the 48th hour after induction of SP, which is a long enough period to observe the histopathologic changes but also a short enough period to observe during the experiment the rat mortality from multiorgan failure. All of the rats in our study survived until the end of the experiment.

LC is a conditionally essential nutrient; 75% of its amount in the organism is from dietary sources, and 25% is derived from de novo synthesis from lysine and methionine amino acids in liver and kidneys.²⁵ LC is an amino acid–like molecule that serves in transporting fatty acids and producing acetyl CoA in mitochondrial matrix for β-oxidation to provide energy that is needed for metabolism. In addition, it plays a critical role in transporting intermediate toxic compounds out of mitochondria and preventing lipid peroxidation, which provides mitochondrial membrane integrity and stability under severe oxidative stress.^9–11 A recent study demonstrated that the mitochondrial damage and the disturbance of mitochondrial function in pancreas occurs after 6 hours of induction of SP with STC.²⁶ The disturbance of mitochondrial energy metabolism in pancreas is very important in the development and progression of SP. In most cases, mortality from SP is a result of multiorgan dysfunction syndrome, which is related to mitochondrial dysfunction at the cellular level. Herein, LC may ameliorate this disturbance with its anti-inflammatory and antioxidant effects on mitochondria during SP. The protective effects of LC in different models of mild pancreatitis were demonstrated previously, but protective effects of LC on pancreatic tissue in the STC-induced SP model has not been demonstrated yet in the literature.^21,22

The carnitine dose of our experiment was determined according to the previous studies with LC in order to compare the results and maintain a standardization. There are several studies evaluating the effects of LC not only in pancreatic tissue but also in other organs, with a dose of 200 mg/kg intraperitoneally.^22,27–29 In addition, intraperitoneal injection is a preferred route in many studies with experimental animals. It is commonly used in rats and mice, intraperitoneal injection administration results in a faster absorption into the systemic circulation than subcutaneous administration, it is almost equal to intravenous administration, and anesthesia is generally not required.³⁰

There was a decrease in the serum amylase activity of the LC group compared with the C group, but there was no statistical significance between the 2 groups in our study (amylase levels in the C and LC groups were 641.25 ± 209 and 522.37 ± 91.42, respectively; P = 0.937). In a study of the cerulein-induced pancreatitis model of Arafa et al²² (this study is one of the two studies that evaluate the protective role of carnitine in an experimental pancreatitis model), in parallel with our study, the amylase levels of the control and carnitine groups were similar. Also, in Kaya et al²¹ (a model of an l-aspariginase–induced pancreatitis model) there was no difference between these groups (amylase levels in the C and LC groups were 1314.9 ± 36.9 and 1333.0 ± 105.3, respectively). These results are reasonable because a significant decrease in basal amylase level after carnitine administration in a healthy individual is not an expected effect of LC.

In the present study, amylase activity was significantly increased as a result of pancreatitis in both the P and P + LC groups compared with control animals (P < 0.05). This result is in accordance with those of previous studies. When amylase activities in the P + LC group and the P group were compared, there was no statistical significance between groups. This result is in contrast with the study of Arafa et al.²² They found decreased amylase activity by 64% in LC treatment group (200 mg/kg/d for 7 days). In addition, Kaya et al²¹ reported lower amylase activities in the LC treatment group (500 mg/kg/d for 5 days), nearly approaching those of the control group. This contrast may originate from the difference in severity of the pancreatitis models between the studies and the administration of LC at a lower dose (200 mg/kg/d) for a shorter period (48 hours) in our study.

The histopathologic examination of the pancreatic specimens of the P group revealed prominent changes; thus, pancreatic damage score was significantly higher in the P group compared with the C group (P < 0.05). The histopathologic changes showing the damage of exocrine pancreas in the P group are well matched with the histopathologic findings of previous studies in the STC-induced SP model.¹⁹ In the treatment group, the histopathologic findings of the pancreatitis were markedly reduced. Focal acinar cell degeneration and parenchymal hemorrhage were rarely seen. Interlobular and intralobular inflammation and edema were generally mild. Bordalo et al³¹ demonstrated that acinar cell degeneration is the precursor of the pancreatic necrosis in the pathogenesis of pancreatitis. Therefore, it is obvious that a specific treatment of pancreatitis independent from the etiology has to prevent acinar cell degeneration. The ameliorating effect of LC on acinar cell degeneration during STC-induced SP was significant in the present study. LC improved the inflammatory injury histologically, even with a short period of administration (48 hours).

One limitation of our study was the unavailability of defining the pathophysiologic mechanism that underlies the ameliorating effect of LC on acinar cell degeneration. The present study focused on histopathologic changes only. The role of LC in mitochondrial dysfunction during SP remains unclear. From this point of view, our future research will investigate the potential healing mechanisms of LC therapy and demonstrate the biochemical and ultrastructural changes of the mitochondria of acinar cells during the course of SP.

Conclusion

This experimental study revealed that LC has a significant histopathologic protective effect on acinar cell degeneration in an STC-induced SP model in rats. This result suggests that LC administration may be an adjunctive therapy in the management of SP.