| Objective To explore the feasibility and effectivity of injectable gabcxate mesylatc thermo-sensitive in-situ gel (GMTI) for treatment of pancreatic trauma(PT) in animal model under the guidance of contrast-enhanced ultrasound (CEUS).Material and methods (1) According to the groups of Poloxamer-407, GM (including four concentration:1.0 mg/ml,5.0 mg/ml,10.0 mg/ml,20.0 mg/ml) and GMTI (including four concentration:1.0 mg/ml,5.0 mg/ml,10.0 mg/ml,20.0 mg/ml),25μl were absorbed and mixed with equal volume trypsin (1.0 mg/ml). Subsequently, the trypsin activity was assayed using rats trypsin enzyme-linked immunosorbent assay (ELISA) kit, and recorded at 450 nm using microplate reader. (2) 42 rats were randomly divided into seven groups (6 in each group), including control group, PT group, GM group, and GMTI group of which included 1.0 mg/ml,5.0 mg/ml,10.0 mg/ml and 20.0 mg/ml, and the grade II of pancreatic trauma model was established. Thus, GM was injected through caudal vein based on 0.3 ml/100 g in GM group, and GMTI was injected through the abdominal cavity surface of pancreas based on 0.3 ml/100 g in GMTI group, and also injected with equal volume of 0.9% normal saline through the abdominal cavity surface of pancreas in PT group. Rats were killed after 24h the treatment to collected the whole blood and pancreas. After serum separation, the curative effect of GMTI on grade II of pancreas injury was examined following with assay of amylase (AMS), C-reactive protein (CRP), trypsinogen activation peptide (TAP) using ELISA kits, and that of pancreas was sliced and stained by hematoxylin eosin (HE). (3) 42 rats were randomly divided into seven groups (6 in each group), including control group, PT group of which included 1h,6h and 24h groups, and GMTI group of which included 1h,6h and 24h groups, and the grade III of pancreatic trauma model based on main pancreatic duct dividing was established. GMTI (10.0 mg/ml) was injected through the abdominal cavity surface of pancreas based on 0.3 ml/100 g in GMTI group, and also injected with equal volume of 0.9% normal saline through the abdominal cavity surface of pancreas in PT group. After Ih,12h and 24h trauma, the serum biochemical index, including AMS, lipase (LPS), CRP, interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a), and the content of ascites were examined in control, PT and GMTI groups respectively, and that of pancreas was sliced and stained by hematoxylin eosin (HE). (4) 5 beagles underwent celiotomy to establish the grade III of pancreatic trauma model. The descending duodenum was intermittent sewed on the right side of the abdominal wall. CEUS was performed to investigate the imaging characteristics of pancreatic trauma and record the volume of ascites at Id,2d and 3d after the operation. The serum AMS, LPS, CRP, IL-6, TNF-a, WBC and urine TAP were examined using ELISA kits, and that of pancreas pathology at 3d was examined after operation. (5) 15 beagles were randomly divided into three groups (5 in each groups), including PT group, GMTI group and GM group, and underwent celiotomy to establish the grade III of pancreatic trauma model. The peri-pancreatic drainage was made under the guidance of CEUS. GM was injected through cephalic vein based on 4.625ml/10kg in GM group, and GMTI was injected through the drainage tube to the surface of pancreas based on 4.63ml/10kg under the guidance of CEUS, and also injected with equal volume of 0.9% normal saline in PT group. CEUS was performed to investigate the imaging characteristics of pancreatic trauma and record the volume of ascites at Id,2d,3d, and 7d after the operation. The serum AMS, LPS, CRP, IL-6, TNF-a, WBC and urine TAP were examined using ELISA kits, and that of pancreas pathology at 7d was examined after operation.Results (1) The trypsin activity was slightly inhibited at 1.0 mg/ml and 5.0 mg/ml in GM and GMTI group respectively (P<0.05, compare to P-407), and of which was completely inhibited at 10.0 mg/ml and 20.0 mg/ml (P<0.01, compare to P-407). (2) The expression of AMS, CRP and TAP was significantly increased in PT group (P<0.01, compare to control), and significantly decreased in GM group (P<0.01, compare to TP), and also slightly inhibited after 1.0 mg/ml and 5.0 mg/ml GMT1 treatment (P<0.05, compare to PT), and significantly inhibited after 10.0 mg/ml and 20.0 mg/ml GMTI treatment (P<0.01, compare to PT). HE results demonstrated that pancreas was uniformly distributed in control group, and of which was loose, partially dissolved, and deeply staining of nuclei in PT group. Expectedly, after gradient GMTI treatment, pancreas was gradually restored to tight distribution, slightly staining of nuclei. (3) The serum AMS, LPS, CRP, IL-6 and TNF-α in PT groups was increased with trauma time prolonging, and had a significant different compare with that of control group (P<0.01). Expected, in GMTI group, the serum AMS, LPS, CRP, IL-6 and TNF-α was decreased, especially after 24h trauma, and had a significant different compare with that of PT group (P<0.05, P<0.01). The morphological structure of pancreas was loose and acinus seriously damaged, and the nuclei were irregular hyperchromatic and inflammatory cells invasion in PT group vs control group. After GMTI treatment, the morphological structure of pancreas was restored, and the damaged acinus and inflammatory cells invasion were decreased vs PT group. (4) The traumatic regions and active bleeding were showed clearly by CEUS scanning. There were no enhancement or just lightly enhancement in the traumatic regions in both arterial phase and venous phase. The volume of ascites were increased at 1d after operation and of that decreased at 3d, and the AMS, LPS of ascites were increased. The biochemical indexes, including AMS, LPS, CRP, IL-6, TNF-α, WBC and urine TAP, were all increased from 1d to 2d after operation, and decreased at 3d (P＜0.01, P<0.05). The morphological structure of pancreas was swollen and degenerated, with focal hemorrhage, necrosis and inflammatory cells invasion. (5) There was no further expanding of traumatic regions have been found in the GMTI group by CEUS scanning. Active bleeding and abscess were found in the PT group. The volume of ascites were decreased with the time prolonging, and the AMS, LPS of ascites in GMTI group were decreased (P<0.01, compare to GM group). The serum AMS, LPS, CRP, and urine TAP in GMTI group and GM group were both decreased(P<0.01, P<0.05, compare to PT group), and no significant difference of it. The morphological structure of pancreas cells appeared necrosis and fibrosiss in all groups, and the pathological changes were more minimal in the GMTI group.Conclusion GMTI, as a novel formulation and drug delivery way, is effective to remedy PT in animal model under the guidance of CEUS. This study provided a significant reference for PT adjuvant clinical therapy, and had a potential value of minimally invasion adjuvant therapy, and suggested that the homemade GMTI application prospects in future clinical treatment. |
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