Fatal and non-fatal inflammatory injuries induced by different concentrations of L-arginine in Kunming rats

Abstract

To establish animal models of fatal and non-fatal pancreatitis, we randomly divided 55 Kunming rats into three groups: Group A received three intraperitoneal injections of 8% L-arginine (L-arg); Group B received three intraperitoneal injections of 10% L-arg; and Group C (the control group) received three intraperitoneal injections of saline. After the third injection, biochemical indices and pathological changes in the pancreas and lungs were evaluated. In Group A, the rats experienced peak inflammatory edema at 48 h after the injections and exhibited the highest pathological score, but the biochemical indices showed no phase-related changes. In Group B, the rats exhibited significantly increased biochemical indices at 12 h after injection and experienced peak inflammatory edema at 24 h; the pathological score was also the highest, significantly different from the observations for Groups A and C (P < 0.05), and the mortality rate was 64% over 72 h. Thus, it was inferred that L-arg could be used to establish pancreatitis models and that different concentrations lead to different lesions.

In the Western world, the incidence of pancreatitis has increased over the last 40 years in concurrence with a worldwide increase in type 2 diabetes and obesity. ¹ As various causes of acute pancreatitis exist, including biliary tract disease and alcohol consumption, many methods have been proposed to establish an animal model of pancreatitis; however, many have disadvantages such as high cost, high technical proficiency requirement, and the induction of severe trauma. In this study, we established pancreatitis models through the intraperitoneal injection of different concentrations of L-arg and detected the resulting inflammation factors to investigate the mechanism of pancreatitis.

A total of 55 healthy male Kunming rats of similar weight (± 1 g) were selected and randomly divided into three groups. Group A (n = 25) received three intraperitoneal injections of 8% L-arg (Analytic grade, Sigma-Aldrich, USA, purchased from Shanghai Huamei Biological Engineering Co., Ltd); Group B (n = 25) received three intraperitoneal injections of 10% L-arg; and Group C (n = 5) (the control group) received three intraperitoneal injections of saline. The rats in Group A appeared fatigued at 6 h after the injection, performed activities more rarely, and expressed irritability and painful emotion 12 h later; this lasted until 24 h, and at 48 h, the rats were inactive, but still alive. One case of death occurred in Group A within 48 h and one more within 72 h; the group mortality was 4%. The rats in Group B expressed irritability and painful emotion at 6 h after injection, as well as spiritual malaise, extremely painful emotion, avoidance of eating or drinking, inactivity, and messy and dull fur after 24 h. In Group B, 12 cases of death occurred within 48 h and a further 16 occurred within 72 h; the group mortality was 64%. As no cases of death were observed in Group C within 72 h, the mortality was 0.

In Group A, AMY and LPS exhibited time-dependent changes: a significant increase at 12 h, peak at 48 h, and a significant decrease at 72 h in comparison with Group C (P < 0.01); ALT, CK, and BUN remained relatively consistent. In Group B, AMY, LPS, ALT, CK, and BUN exhibited time-dependent changes: a significant increase at 12 h, peak at 24 h, and decrease at 48 h, but they remained at high levels and statistically significant differences were exhibited between Groups C and A. At each time point, AMY and LPS were higher in Group A and B than in Group C, but the levels in Group A were lower than those in Group B. ALT, CK, and BUN levels in Group B were higher than those in Groups A and C, but there were no significant differences between Groups A and C (Figure 1).

Figure 1.

Comparative analysis of different biochemical indicators at different time: (a) AMY, (b) LPS, (c) ALT, (d) CK, and (e): BUN.

Gross appearance

In Group A—at 6 h after modeling, pancreatic tissue lesions began to appear as the tension of the pancreatic capsule increased, but still exhibited clear pancreatic lobular contours; edema continued till 48 h, and the pancreatic surface appeared to contain exudate, spots, or focal necrosis, in addition to an occasional small amount of peripancreatic fluid and fat saponification spots; 72 h after injection, congestion and edema were alleviated, the pancreas became smaller, and occasional spotty bleeding or necrosis was seen. In Group B—at 6 h after injection, pancreatic tissue lesions appeared with obvious film tension, yellowish peripancreatic exudate, unclear pancreatic lobular contours, and significant swelling; at 12 h after injection, many yellow or red intraperitoneal bloody ascites can be seen; the above symptoms peaked at 24 h, together with peripancreatic organ adhesions, intestinal motility dysfunction, and even intestinal necrosis; at 48 h, pancreatic tissue appeared to have a large area of pale necrosis and various degrees of dark red blood-like changes. Endoscopy. In Group A—at 6 h after modeling, pancreatic lobules appeared with inflammatory cell infiltration and mild edema among the lobules; at 12 h, the infiltration of inflammatory cells increased; at 24 h, occasional burst and fusion of gland bubbles was seen; at 48 h, the peak of pancreatic lobular edema was reached, but the inflammatory cell infiltration was reduced, small focal coagulative necrosis can be seen, and the pathological score was the highest, which showed significant differences from Group C (P < 0.01); at 72 h, pancreatic lobular edema and neutrophil infiltration decreased, together with occasional minor spotty bleeding and a small amount of fibrous connective tissue proliferation; however, the pathological score was significantly higher than that in Group C (P < 0.01). The pathological scores began to increase and gradually aggravate by 6 h after injection, this continued till 48 h and then decreased at 72 h, which was consistent with the time-dependent pathological changes and the difference was statistically significant in comparison with Group C (P < 0.01). In Group B, inter- and inner-lobular widening, edema, and hyperemia appeared 6 h after injection, together with occasional scattered focal coagulative necrosis, no significant bleeding, and a small amount of inflammatory cell infiltration; the pathological score was higher than in Groups A and C at the same time point, and the differences were statistically significant (P < 0.01). After 24 h, the gland lobule structures appeared damaged and disordered, with severe edema, lots of inflammatory cell infiltration, extensive bleeding, diffuse acinar cell necrosis, fat necrosis, and fuzzy pancreatic structures. At this time, the pathological score of the pancreatic tissue was the highest; at 48 h, acinar cell necrosis, nuclear condensation, and occasional disappearing lobular structure were observed, but inflammatory cell infiltration and edema were more alleviated than that at 24 h; at 72 h, certain necrotic foci remained. The pathological scores began to increase and gradually aggravated 6 h after the injection, which continued to 24 h and then decreased at 48 h, which consisted of time-dependent pathological changes and the difference was statistically significant when in comparison with Groups A and C (P < 0.01). In Group C, no significant pancreatic pathological changes were observed, and the lobular gap structures were clear, with an occasional small number of inflammatory cells and no bleeding and necrosis. At 24 h, no obvious morphological changes were apparent in Groups A and C, but scattered inflammatory cell infiltration was observed. The pathological examination of Group B revealed a widened alveolar septum, thickened alveolar wall, and extensive infiltration of inflammatory cells, together with a wide range of inflammatory cell infiltration and scattered intra- and inter- pulmonary alveolar hemorrhage (Figure 2).

Figure 2.

Pathological findings of rat pancreas caused by different concentrations of arginine—pathological figures of group A at different time points: (a) Group C (×200), (b) Group A, 6 h after modeling (×200), (c)12 h after modeling (×200), (d) 24 h after modeling (×200), (e): 48 h after modeling (×200), (f) 72 h after modeling (×200). Pathological figures of group B at different time points: (g) 6 h after modeling (×200), (h): 12 h after modeling (×200), (i): 24 h after modeling (×200), (j): 48 h after modeling (×200), and (k): 72 h after modeling (×200).

Discussion

Although the pathogenesis of L-arg-induced noninvasive acute pancreatitis is still unclear, it causes the cascade-type release of oxygen free radicals, nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and other inflammatory mediators. ² In addition, L-arg can be converted to large amounts of NO, which indirectly interfere with energy metabolism, as well as the promotion of the tyrosine nitration of superoxide dismutase (SOD); in addition, L-arg can reduce pancreatic blood perfusion, which results in the spread of inflammation. ³ The common biochemical indices for pancreatitis and the damage of liver and kidney, such as AMY, BUN, and ALT, increased, which indicated organ dysfunction and damage. Some studies also showed that L-arg caused direct oxidative damage to the pancreas and distant organs. ⁴ In short, different concentrations of L-arg can affect the normal pancreatic physiological responses in a variety of ways, which result in varying degrees of pancreatitis, including fatal and non-fatal pancreatitis. The clinical pathological processes of typical mild acute edematous pancreatitis appear as time-dependent changes of serum AMY and LPS, which show an initial gradual increase until a peak was reached and then a gradual decrease, which was consistent with the results of Group A observed in this study. During the overall changes process, bleeding and necrosis were relatively rare and, based on to the parameters of CK, ALT, and BUN, we determined that adjacent and distant organs were not involved; however, effects on mortality were observed, which may have been related to the errors in intraperitoneal injection or other processes.

The mortality rate of acute severe pancreatitis can reach 60%–65%, and the main cause of death is pancreatitis complicated with infection and sepsis, ⁵ which has been alternatively viewed as an L-arginine-deficient state or as a syndrome caused by excess NO. ⁶ Therefore, the pathological appearance of Group B had obvious characteristics of bleeding and necrosis that resulted from edema, as well as peripheral and distant organ involvements, which was consistent with severe acute pancreatitis. ⁷ This could be differentiated from mild acute edematous pancreatitis to be used as a severe acute pancreatitis model.

In this study, the pH value of L-arg was adjusted to avoid strong alkaline-induced experimental instability; meanwhile, this study avoided using a large or small single dose (a single dose greater than 4 mg/g can cause rapid death of mice, but less than 3 mg/g can induce significant pancreatic lesions) and other adverse operations, including uneven solution concentration; based on the clinical manifestations of various degrees of pancreatitis, we performed multiple preliminary experiments so as to prepare animal models of pancreatitis with various degrees of severity using different concentrations of L-arg. Asymmetric dimethylarginine (ADMA) levels on admission correlated well with sequential organ failure assessment score since L-arginine/ADMA ratio increased significantly from day 1 to day 3 and then decreased from day 3 to day 5. ⁸

However, L-arg-induced animal models still have certain shortcomings; for example, the immunohistochemical study of its pathological slices has not yet been conducted, and research results are dependent on multiple factors, such as the total injection dose, concentration, time, or interval. The low-concentration L-arg-induced mouse model had a tendency for self-healing, so it can only currently be used for the study of short-time interventions; meanwhile, the mechanisms of L-arg-induced acute pancreatitis are unclear and should be clarified in further in-depth studies.