Inflammation in the Pancreatic Stump
The inflammatory response spreads all over the pancreatic stump. The main manifestations are as follows: Subcapsular hematoma distributes widely throughout the pancreatic stump (Figure 1A). The hematoma on the side of anastomosis goes deep into the pancreatic parenchyma, caused by the suture. Inflammatory cells, mainly the neutrophils, infiltrate pancreatic glandular lobes and interlobular structures (Figure 1B, 1C). Sporadic necrotic foci could be observed in the whole pancreatic stump (Figure 1D). Pancreatic enzymes cause homogeneous staining parts in the center of necrosis. Plasma cells, neutrophils, lymphocytes, and eosinophils could be found around the necrosis. Apoptosis is observed in the pancreatic head before POPF (Figure 1E). Apoptosis weakens or disappears after POPF happens. The whole pancreatic stump presents a state of proliferation (Figure 1F), opposite to the original prediction. The glandular lobe was in edema and disorder. The nucleus of acinar cells enlarges, and the chromatin becomes sparse. Judged from the distribution of the necrotic lesions, inflammatory cells, and hematoma, the inflammatory response in the pancreatic stump decreases gradually from the anastomosis side to the pancreatic tail (Figure 2).
Acinar-Duct Metaplasia and Chemotaxis in the Pancreatic Stump
Another vital phenomenon of inflammation in the pancreatic stump is the concentration of inflammatory cells in the ductal system. Neutrophils could be found in the main pancreatic, interlobular, and abnormal ducts formed by ADM (Figure 3A-3C). Blood red cells (RBC) are also observed in the ductal system. But the white blood cells (WBC) dominate the cells in the ducts. However, the ADM-formed duct is not suitable for the survival of blood cells. Those blood cells are destroyed in the ductal system (Figure 3C). RBCs lose membrane and become acidophilic material. WBCs are decomposed with fragmented nuclei left. ADM-formed ducts are the sites where the neutrophils and RBCs penetrate the ductal wall (Figure 3E). Chemotaxis could be found in the ductal system.
"Blood-pancreas barrier" in the pancreatic stump is destroyed. The permeability of the ductal system increases significantly. Blood cells, mainly the neutrophil cells, cross the blood vessels, enter the space between the acini and basement membrane, then cross the junctions between the acinar cells. We find a remarkable event that a neutrophil is penetrating the ADM-formed duct (Figure 3D). ADM exists in the whole PS, and its distribution shows a descending trend from the anastomosis to the tail as inflammation does (Figure 3F). Because of chemotaxis, we hypothesized that there should be reflux from the digestive tract into the ductal system.
The expression of CK19 is negative or slightly positive in the normal pancreatic parenchyma (Figure 4A-4C). In contrast, it becomes strongly positive in the pancreatic stump with POPF (Figure 4D). For CK19 is only expressed in pancreatic ducts, there should be more centroacinar cells that proliferate and turn into duct cells. The newly formed ducts are abnormal, with an interval of narrow and dilated ducts (Figure 4E, 4F). The proliferation of centroacinar cells participates in ADM, which forms new ducts and pushes the original acinar cells to one or both sides. Sometimes, acinar cells involve in the process of ADM (Figure 4G, 4H). As the pressure in the ADM-formed ducts increases, the wall of the ducts becomes thinner.
Microbial Distribution and Digestive Reflux in the Pancreatic Stump
We also tested fungus in the pancreatic stump. However, so much data could not be recognized, letting us abandon fungus as a research objective. PCR products of each specimen were visualized by agarose gel electrophoresis (Figure 5). After removing the barcode and primer, we got raw tags. Then, we obtained clean tags with high-quality sequences after excluding chimeras and short sequences. The length of most sequences is between 400-440bp. 1092 operational taxonomic units(OTUs) were obtained in PL and 531 in PZ. 26301 tags were selected randomly in each sample in PL and 31277 in PZ and then taken into count for the final analyses (Tab. 1). The OTUs increase significantly in the pancreatic stump with POPF than those in the duodena (p < 0.05) but not in the pancreatic head(p > 0.05).
Table 1. Distribution of OTUs in the jejunum, pancreatic head, and pancreatic stump
|
SampleID
|
Clean_tags
|
Final_tags
|
OTUs
|
SampleID
|
Clean_tags
|
Final_tags
|
OTUs
|
PLJB0
|
158140
|
26301
|
34
|
PZJB0
|
165233
|
31277
|
35
|
PLPB0
|
33717
|
26301
|
318
|
PZPB0
|
183971
|
31277
|
89
|
PLPB1
|
54270
|
26301
|
324
|
PZPB1
|
47561
|
31277
|
44
|
PLPB2
|
59038
|
26301
|
249
|
PZPB2
|
38512
|
31277
|
189
|
PLPB3
|
27489
|
26301
|
250
|
PZPB3
|
48707
|
31277
|
49
|
PLPB4
|
33657
|
26301
|
109
|
PZPB4
|
32320
|
31277
|
184
|
PLPB5
|
29017
|
26301
|
310
|
PZPB5
|
34351
|
31277
|
183
|
PLPB6
|
44928
|
26301
|
439
|
PZPB6
|
65726
|
31277
|
381
|
PLPB7
|
115402
|
26301
|
338
|
|
|
|
|
PLPB8
|
168118
|
26301
|
418
|
|
|
|
|
At the phylum level, the most abundant phyla in all samples in PL are Firmicutes and Proteobacteria, followed by Bacteroidetes, Actinobacteria, and Cyanobacteria. In PZ are Firmicutes, Deinococcota, Proteobacteria, Bacteroidetes, and Actinobacteria. At the genus level, the most abundant bacteria in the samples of PL are Bacillus and Bacteroides, followed by Escherichia-Shigella and Faecalibacterium, besides the unidentified part. In PZ are Bacillus and Comamonas, Stenotrophomonas, Bacteroides, Pelomonas.
The composition of bacterial DNA and the microbiome in the samples differ in the jejunum, pancreatic head, and pancreatic stump. According to the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering tree, the microbial community distribution in these two patients has a similar profile in the jejunum and the pancreatic body with POPF (Figure 6A, 6B). Meanwhile, bacterial DNA distribution in the pancreatic head without POPF is similar to that in the tail with POPF, where the inflammation is relatively slight. The bacterial distribution also has a descending trend consistent with inflammation. From the chemotaxis of inflammatory cells into the ADM-formed ducts to the unique microbial DNA distribution, it could prove that the inflammatory substances come from the ductal system. We inferred the existence of digestive reflux in the ductal system during POPF.