General

Hemorrhagic pancreatitis in dogs

Hemorrhagic pancreatitis in dogs

Hemorrhagic pancreatitis in dogs (PDH) is an acute and fulminant necrotizing form of pancreatitis caused by trauma to the pancreas, with a mortality rate of 40% in large dogs and 60% in small dogs [[@r15], [@r20], [@r21]]. Although pancreatitis is the major cause of death in PDH, it is difficult to diagnose this disease due to the lack of specific clinical signs [[@r19], [@r21]]. Some researchers reported that the prognosis of PDH was better in cases of small dogs and dogs of mixed breeds than in dogs of other breeds, as these dogs are usually fed with an enteral diet, allowing better recovery compared with dogs fed with a parenteral diet [[@r19], [@r20]].

In a previous study, we demonstrated that the oral administration of a water-soluble extract of *Lepidium sativum* (LSW) effectively protects the small intestine from experimental NSD-induced injury [[@r7]]. *L. sativum* is a small annual or perennial herb that belongs to the Brassicaceae family. Previous studies indicated that the major bioactive compounds in *L. sativum* are flavonoids and phytosterols [[@r7], [@r11], [@r12]]. In the present study, we evaluated the efficacy of the administration of an LSW extract in the prevention of PDH in dogs.

MATERIALS AND METHODS {#s1}

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*L. sativum* was extracted using a cold maceration method [[@r7]]. Briefly, dried herb powder (200 g) was mixed with 200 ml of distilled water. The mixture was soaked at room temperature for 24 hr, and filtered through filter paper to obtn the final extract solution. This extract was freeze-dried for 24 hr to obtn the final product. The contents of the extract powder were confirmed by high-performance liquid chromatography. The freeze-dried extract powder contned 4.05% protein, 0.16% fat, 0.08% fibre, and 77.11% carbohydrate.

A total of 30 healthy beagles, aged 12--24 months and weighing 11.5--14.0 kg, were used in this study. The dogs were divided into 3 groups (10 dogs per group), and received different treatments for 7 days. Group A was fed a baseline diet (A basal diet, Wako, Osaka, Japan). Group B was fed the A basal diet plus 6.5% (w/w) of the LSW extract powder for 7 days. Group C was fed the A basal diet plus 15% (w/w) of the LSW extract powder for 7 days. All diets were isonitrogenous.

After 7 days, each dog's body weight and food intake were recorded, and blood samples were collected by puncturing the saphenous vein. The complete blood count, biochemical analysis, blood coagulation analysis, urea nitrogen (BUN), creatinine (Cre), albumin (Alb), total protein (TP), and alkaline phosphatase (ALP) tests were performed. All procedures were performed in accordance with the guidelines for animal experiments of the Faculty of Agriculture, Kyushu University.

Blood samples were collected just after feeding and after 4 and 24 hr. During the blood sampling procedure, dogs were fasted and received an intravenous injection of 20 mU/kg of urokinase (Fibrinonep, Jintan, Shangh, China) to induce thrombolysis, to prevent blood coagulation. Each blood sample was collected from the saphenous vein and allowed to coagulate at room temperature (approximately 25°C) for 20 min. Serum samples were then collected by centrifugation at 2,800 ×*g* for 15 min at 4°C. Each serum sample was transferred to a new tube, and all tests were performed immediately after sampling.

Serum samples were analyzed for BUN and Cre using an autoanalyzer (Hitachi, Tokyo, Japan). Serum samples were also analyzed for Alb, TP, and ALP using a biochemistry analyzer (Siemens, Munich, Germany).

All tests were carried out at the Laboratory of Bio Sciences, Faculty of Veterinary Medicine, Kyushu University.

The coagulation system was evaluated by measuring the plasma concentration of protein C, protein S, and coagulation factors (II, VII, VIII, IX, and X) using a radial-immunodiffusion assay. The factors II, VII, IX, X, and coagulation were measured by immunodiffusion, whereas protein C was measured by nephelometry.

### Histology

Thigh muscle samples were collected from each animal for histological analysis. The left thigh muscles were separated from the connective tissues and examined at the University of Hawi-University of Kansas Medical Center Veterinary School Pathology Laboratory.

The tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin wax, and sectioned at 4 µm thickness. Sections were then stned with hematoxylin and eosin.

### Statistical analysis

All data were analyzed using a one-way analysis of variance (ANOVA) using SPSS version 20.0 (IBM Corp., Armonk, NY, USA). Results were considered statistically significant at *P* <, 0.05.

Results

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In the coagulation profile test, there were no significant differences among the experimental groups ([Fig. 1A](#F1-ajas-27-8-1211-19){ref-type="fig"}). Similarly, in the blood coagulation profile test, there were no significant differences in the plasma anticoagulant activity level among the experimental groups ([Fig. 1B](#f1-ajas-27-8-1211-19){ref-type="fig"}).

The anticoagulant activity levels of PCG~25~, PCG~50~, PCG~75~, and PCG~100~ were 19.8%, 18.1%, 17.5%, and 18.6%, respectively. Therefore, the anticoagulant activity levels of PCG~25~ and PCG~50~ were significantly higher than those of PCG~75~ and PCG~100~ (*P* <, 0.05).

The values of the anticoagulant activity of the group treated with PCG~50~ were equivalent to the values of the control group (*P* <, 0.05). Meanwhile, the anticoagulant activity of the groups treated with PCG~25~, PCG~75~, and PCG~100~ was not significantly different from that of the control group ([Table 2](#t2-ajas-27-8-1211-19){ref-type="table"}).

In addition, the blood coagulation profile test demonstrated that the groups treated with PCG~25~ and PCG~50~ were significantly different from those of the control and PCG~75~ groups, but were not significantly different from those of the PCG~100~ group ([Table 3](#t3-ajas-27-8-1211-19){ref-type="table"}).

The serum calcium level of the group treated with PCG~50~