Congestive Preconditioning by Portal Venous Clamping: A Novel Preventive Concept for Small Intestinal Congestive Re-Outflow Injury

Abstract

Background

Congestion of the small intestine, which induces mucosal damage via apoptosis, is a common pathophysiological change in hepato-biliary-pancreatic surgery, including liver transplantation. Small intestinal mucosal damage can trigger postoperative complications via various pathways. Therefore, we investigated the efficacy of intermittent portal venous clamping, which we named congestive preconditioning (CPC), for decreasing congestive re-outflow injury (CRoI) of the small intestine.

Material/Methods

Small intestinal CRoI was induced in rats by clamping the portal vein (PV) for 30 min, followed by re-outflow. The CPC group underwent 3 episodes of 5 min PV clamping and 5 min re-outflow before CRoI. The Control group underwent CRoI after 30 min simple laparotomy without any preconditioning. Survival and histological changes in the small intestine after CRoI were analyzed. The histological changes were compared CPC group to Control group using a scoring system that expressed histopathological severity. Also, small intestinal apoptosis and expression of apoptosis-related genes were analyzed by immunohistochemistry.

Results

The survival rate of the CPC group was significantly higher than Control group. Histological scoring of small intestinal damage was significantly lower in the CPC group after 6 h of CRoI. Expression of anti-apoptotic gene, Bcl-xL was significantly increased, but pro-apoptotic gene caspase-3 and apoptotic cells did not differ significantly between the CPC group and Control group.

Conclusions

Induction of CPC for small intestinal CRoI was effective, which suggests that an anti-apoptotic pathway is involved in the beneficial mechanism.

Background

Congestion of the small intestine is a common pathophysiological change induced by portal vein (PV) blood flow interruption in hepato-biliary-pancreatic (HBP) surgery. The most severe small intestinal congestion usually occurs during the anhepatic period of liver transplantation surgery or PV reconstruction in HBP malignancies. A previous experimental study demonstrated that congestive re-outflow injury (CRoI) of the small intestine induces mucosal injury via apoptosis [1,2]. Therefore, CRoI of the small intestine should be avoided whenever possible. In cases without major portosystemic shunt, the efficacy of temporary portocaval shunt for avoiding small intestinal congestion has been recognized in clinical settings [3,4]. In addition, CRoI of the small intestine is reduced by administration of caspase inhibitor, which acts by decreasing mucosal apoptosis [5].

We have previously reported induction of an anti-apoptotic effect in the small intestinal mucosa by ischemic preconditioning (IPC), in the case of ischemia–reperfusion injury (IRI) in the small intestine [6]. Therefore, we anticipated that IPC might be effective against CRoI via preventing mucosal apoptosis, similar to IPC for IRI. However, application of IPC to the small intestine is complicated during liver transplantation and hepatectomy, because exposing the root of the superior mesenteric artery (SMA) is burdensome in these procedures. Therefore, we hypothesized that preconditioning by intermittent clamping of the portal vein (PV) would be effective for CRoI of the small intestine, which we named congestive preconditioning (CPC). In this study, we investigated the efficacy of CPC for CRoI of the small intestine.

Material and Methods

Animals

Male Wistar rats, each weight ~250 g (Charles River Laboratories, Kanagawa, Japan), were maintained 12 h light/dark cycle, in the Animal Experiment Laboratory of Kanazawa University. The rats had free access to water and standard solid feed (CRF-1; Charles River Laboratories). Prior to the experimental surgery, the rats had been fasted for 12 h, but had free access to water. This study protocol was approved by the Committee on Animal Experimentation of Kanazawa University.

Experimental groups and survival study

The animals were randomly divided into 2 groups (n=8) for survival study. All animals were injected with 50 U/kg heparin. After heparinization, laparotomy was performed after anesthesia was induced by intraperitoneal injection of pentobarbital sodium (50 mg/kg). The PV was detached from the hepatic artery (HA) and common bile duct (CBD) at the level of the hepatoduodenal ligament. In the CPC group, 3 episodes of 5 min congestion and 5 min re-outflow of the PV (occlusion and release of PV clamping, avoiding the HA and CBD) were applied before 30 min PV clamping followed by re-outflow. PV clamping was achieved using an atraumatic vascular clip. In the Control group, the PV was subjected to 30 min clamping (avoid clumping HA and CBD) followed by re-outflow, without any preconditioning. Immediately after surgery, the rats were allowed free access to water and standard solid feed.

Histopathological examination, immunohistochemistry, and apoptotic index

For histopathological, immunohistochemical and apoptotic examinations, the rats were randomly divided into 2 groups (n=8). We obtained 10% formalin-fixed paraffin-embedded tissue blocks of small intestinal tissue from the rats, sacrificed 6 h after re-outflow of above mentioned experimental surgery, to stain with hematoxylin–eosin (HE). The intestinal mucosal injury was graded using the Park score [6]. Also, the slides were deparaffinized in xylene and rehydrated for immunohistochemical analysis. The immunohistochemical analysis were performed with anti-Bcl-xL antibody (dilution 1: 100; Cell Signaling Technology, Beverly, MA, USA), anti-cleaved caspase-3 antibody (dilution 1: 100; Cell Signaling Technology), and anti-ss-DNA antibody (dilution 1: 50; Immuno-Biological Laboratories, Fujioka, Gunma, Japan). Immunohistochemical staining was performed with using the commercially available Dako EnVision System Peroxidase kit (Dako, Carpentaria, CA, USA). In each antibody, immunohistochemically positive cells were counted in 10 randomly chosen fields, representing at least 1000 nuclei for quantitative analysis under 400× magnification. The results were expressed by the mean positively stained cells rate (n=8 in each group).

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using the Student’s t-test and differences with p<0.05 were considered statistically significant. Rat survival rates was analyzed by the Kaplan–Meier method, and the log-rank test was used for comparison. All statistical analyses were performed using SPSS version 23 (SPSS Inc., Chicago, IL, USA).

Results

Survival rate

In the Control group, 4 of the 8 rats died within 72 h after surgery. The 7-day survival rate of the Control group was 50%. On the other hand, the survival rate of the CPC group was 100% and the survival rate was significantly higher than Control group (p<0.025). The survival rates are shown in Figure 1. All experimental animals survived at least 6 h after small intestinal CRoI.

Figure 1.

Survival of rats in the CPC and control groups. Survival rate differed significantly between the 2 groups (p=0.025).

Histopathological examination

At 6 h after re-outflow, the Control group showed epithelial lifting along the mucosal villi with varying severity (HE staining; Figure 2A). In contrast, the CPC group showed almost normal mucosa or limited epithelial lifting (Figure 2B). In the CPC group, the small intestinal mucosal injury score according to Park classification was significantly lower than that of Control group (p<0.001; Figure 2C).

Figure 2.

Effect of CPC for CRoI of SI examined by HE staining. SI samples harvested after 6 h of CRoI, with or without CPC (A: Control, B: CPC). (C) Small intestinal mucosal injury was graded by Park score. Values are represented as mean ± standard error of the mean; n=8 in each group.

Changes in immunohistochemical activation of apoptosis-related factors after CRoI and CPC

Immunohistochemical analysis using anti-ss-DNA, anti-Bcl-xL and anti-cleaved caspase-3 antibodies was performed on small intestinal tissue obtained 6 h after CRoI. In the anti-ss-DNA study, the number of positive cells was lower in the CPC group than in the Control group, but there was no significant difference (p=0.070) (Figure 3). Also in the anti-cleaved caspase-3 study, no differences were observed between the 2 groups (p=0.718) (Figure 4). However, in the anti-Bcl-xL study, the number of positive cells was significantly higher in the CPC group (8.80±0.74%) than in the Control group (7.50±0.84%) (p=0.006) (Figure 5).

Figure 3.

Immunohistochemical analysis using anti-ss-DNA antibody (A: control, B: CPC). The number of positive cells did not differ significantly (C) (p=0.070).

Figure 4.

Immunohistochemical analysis using anti-cleaved caspase 3 antibody (A: control, B: CPC). The number of positive cells did not differ significantly (C).

Figure 5.

Immunohistochemical analysis using anti-Bcl-xL antibody (A: control, B: CPC). In the CPC group, the number of positive cells was significantly higher than in the control group (C) (p=0.006).

Discussion

Congestion of the small intestine is a common pathophysiological change in HBP surgery, especially in extended surgery, including liver transplantation. Congestion of the small intestine induces obvious organ injury, and CRoI induces mucosal injury via apoptosis [1,2,5]. We have previously reported the anti-apoptotic effect of by IPC in IRI [6]. Hence, we investigated the efficacy of CPC in the small intestine, which is a simpler procedure than IPC of the small intestine by SMA clamping.

In the present study, CRoI of the small intestine in the CPC group was milder than in the Control group. Park score, which indicated the degree of mucosal morphological injury, was significantly lower in the CPC than Control group. Also, the difference about number of anti-Bcl-xL-positive cells was small but significantly higher in the CPC group. These results suggest immediate induction and adequate settle down of anti-apoptotic pathway after CPC in small intestinal CRoI. Induction of an anti-apoptotic pathway is useful for small intestinal injury in various situations, including IRI and CRoI [1,5,6]. As mentioned above, induction by CPC is simple procedure than other reported surgical or pharmacological procedures.

CPC of the small intestine modulates hepatic blood flow, especially PV flow. Congestion of the small intestine is similar physiologically to PV ischemia of the liver. Therefore, CPC of the small intestine is almost similar for the liver IPC. IPC of the liver induces various beneficial effects for the liver as well as remote organs, which is reported as remote IPC for other organs [7]. Thus, repeated short-term PV flow interruption is suggested to have a synergistic effect for prevention of multiple organ injuries induced by PV flow interruption, including small intestine. This synergistic effect might be reflected in the histopathological change in the small intestine and in amelioration of survival rate in the present study.

Bedirli et al. reported that the 30-min warm ischemia of the liver in small animals has about a 50% mortality rate after 7 days of reperfusion [2]. They also reported effectiveness and usefulness of portosystemic shunting in total hepatic ischemia, which represented as small intestinal protective effects. We anticipate the pathophysiology of our models were very similar to their study. Therefore, the cause of death in our series might be triggered by small intestinal CRoI itself, and protection from CRoI might be important.

Previous studies have reported the pathophysiological changes in the liver induced by hepatic inflow occlusion [8,9]. In contrast, few studies have investigated the pathophysiological changes in the small intestine induced by hepatic inflow occlusion [1–5,10]. More detailed understanding about pathophysiological change of small intestine is important to propose innovative approach for prevention of small intestinal injury. According to our present study, CPC is a simple and effective surgical method. Therefore, further investigation is needed about this surgical procedure.

In previous studies about changes in small intestine, pathophysiological changes with CRoI and efficacy of various interventions were mainly discussed by focusing on mucosal epithelial cells [1–4]. However, it is suggested that pathophysiological changes in the mucosa in such situations are affected by changes in the mesenteric vascular endothelial cells. Congestion induces various changes in venous endothelial cells, which leads to significant vasorelaxation [11,12]. Previous study has shown the importance of NO, prostacyclin and endothelium-derived hyperpolarizing factor in venous relaxation pathways [11]. As with the systemic circulation, mesenteric venous endothelial cells are suspected to be greatly affected by congestion. Actually, PV congestion was reported to affect NO production in lipopolysaccharides (LPS) tolerance model [10]. Thus, mesenteric vascular endothelial cells, especially venous endothelial cells, are suggested to have various effective functions in CPC.

Another possible mechanism for the beneficial effect of CPC in CRoI is involvement of the pancreas in intermittent congestion. The pancreas is a delicate and sensitive organ, and is especially susceptible to injury, such as in pancreatitis [13,14]. A previous study about CRoI suggested that pancreatic enzymes are involved in the acquisition of LPS tolerance [9]. Thus, numerous mediators derived from the pancreas might also be involved in the beneficial effect of CPC in CRoI [14,15]. As with remote IPC, various organs might be involved in the efficacy of CPC [16,17]. In this study, we clarified the efficacy of CPC, with a focus on intestinal mucosa. On the contrary, effects of CPC itself and multiorgan interactions need further investigation.

Conclusions

We showed that CPC is effective in CRoI of the SI. The anti-apoptotic pathway is suggested to be involved in the beneficial mechanism of CPC. Further research is required to: (1) evaluate the contribution of splanchnic vascular endothelial cells; (2) validate more detailed mechanisms of the anti-apoptotic pathway; and (3) understand multiorgan interactions in CPC.