Inflammation ( # 2014) DOI: 10.1007/s10753-014-0091-z
Mesenteric Lymph Duct Drainage Attenuates Acute Lung Injury in Rats with Severe Intraperitoneal Infection Yanmin Zhang,1,4 Shukun Zhang,2 and Naiqiang Tsui3
Abstract—The purpose of this study is to investigate the hypothesis that the mesenteric lymphatic system plays an important role in acute lung injury in a rat model induced by severe intraperitoneal infection. Male Wistar rats weighing 250∼300 g were randomly divided into 3 groups and subjected to sham operation, intraperitoneal infection, or mesenteric lymphatic drainage. The activity of diamine oxidase (DAO) and myeloperoxidase (MPO) were measured by enzymatic assay. The endotoxin levels in plasma, lymph, and bronchoalveolar lavage fluid (BALF) were evaluated using the limulus amoebocyte lysate reagent. The cytokines, adhesion factors, chemokines, and inflammatory factors were detected by ELISA. TLR-4, NF-kB, and IRAK-4 were analyzed by Western blotting. Compared with sham-operated rats, rats with intraperitoneal infection had increased MPO and decreased DAO activity in intestinal tissues. Mesenteric lymph drainage reduced the alterations in MPO and DAO activity induced by intraperitoneal infection. The MPO activity in pulmonary tissue and the permeability of pulmonary blood vessels were also increased, which were partially reversed by mesenteric lymph drainage. The endotoxin levels in lymphatic fluid and alveolar perfusion fluid were elevated after intraperitoneal infection but decreased to control levels after lymph drainage. No alterations in the levels of plasma endotoxin were observed. The number of neutrophils was increased in BALF and lymph in the infected rats, and was also reduced after drainage. Lymph drainage also decreased the levels of inflammatory cytokines, chemokines, and adhesion factors in the plasma, lymph, and BALF, as well as the levels of TLR-4, NF-kB, and IRAK-4 in pulmonary and intestinal tissues. The mesenteric lymphatic system is the main pathway involved in early lung injury caused by severe intraperitoneal infection, in which activation of the TLR-4 signal pathway may play a role. KEY WORDS: mesenteric lymphatic pathway; severe intraperitoneal infection; lung injury; TLR-4 signal pathway.
INTRODUCTION It is often observed in abdominal surgery that severe intraperitoneal infection induced by primary diseases, such as perforative peritonitis, severe acute pancreatitis, biliary tract infection, and intraperitoneal abscess, is frequently accompanied by sepsis and multiple organ dysfunction syndrome (MODS). This is because the intestinal dysfunction can lead 1
Intensive Care Unit, Tianjin Nankai Hospital, 6 Changjiang Road, Nankai District, Tianjin, 300100, China 2 Research Institute of Tianjin Nankai Hospital, Tianjin Nankai Hospital, 6 Changjiang Road, Nankai District, Tianjin, 300100, China 3 Department of Surgery, Tianjin Nankai Hospital, 6 Changjiang Road, Nankai District, Tianjin, 300100, China 4 To whom correspondence should be addressed at Intensive Care Unit, Tianjin Nankai Hospital, 6 Changjiang Road, Nankai District, Tianjin, 300100, China. E-mail: [email protected]
to constant intestinal bacterial translocation and endotoxemia, inducing the overactivated and uncontrolled systemic inflammation as well as remote organ injuries. Therefore, the intestinal barrier damage and the resultant intestinal endotoxin translocation are the main causes of MODS. Although the intestines play a key role in the onset, development, and prognosis of systemic inflammation and MODS, the underlying mechanisms remain poorly understood [1–4]. There is disagreement on the theory that bacteria and endotoxins are translocated through the portal system, as this theory cannot explain the clinical observation that the lung is the first affected organ in MODS [5]. In addition, considerable clinical studies have not observed “translocated” bacteria or endotoxins in the portal vein blood beyond mesenteric lymph nodes [6]. It has been shown recently that mesenteric lymphatic system is involved in the development of SIRS (systemic
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Zhang, Zhang, and Tsui inflammatory response syndrome)/MODS [7, 8]. Deitch and his colleagues suggested that, in the early stages of abdominal infection, the endotoxins and inflammatory mediators (factors) absorbed first flow into the mesenteric lymphatic ducts and lacteal vessels, and then enter into a systemic circulation via thoracic duct [9]. They found that rats with septic shock contained substances toxic to alveolar epithelial cells in their mesenteric lymphatic fluids, which were prominent within 1–3 h after infection. Baigrie et al. [10] suggested that the mesenteric lymphatic fluid could rapidly transport inflammatory mediators and cytotoxic substances. They hypothesized that the intestinal tract was the source of systemic infection and MODS. During traumatic injury, the response of the intestines is production of a series of inflammatory mediators and cytokines, followed by damage of the intestinal barrier. However, the transportation of inflammatory mediators, cytokines, and other cytotoxic substances into the thoracic ducts and systemic circulation is mediated by the mesenteric lymphatic system rather than the classical portal system. Therefore, the systemic infection and MODS can occur without translocation of inflammatory mediators or bacterial toxins in the portal system. In the present study, a severe intraperitoneal infection rat model was produced by direct intraperitoneal injection of an Escherichia coli suspension. Using this model, the protective effect of mesenteric lymphatic drainage on remote organ injury induced by intestinal injury was studied.
MATERIALS AND METHODS Experimental Animals Male Wistar rats of 250∼300 g were purchased from the Experimental Animal Center, Institute of Environmental Medicine, Academy of Military Medical Sciences (laboratory animal license no. SCXK), acclimatized for 1 week, fed a normal diet, and water ad libitum before the experiment. Severe Intraperitoneal Infection Model Escherichia coli samples were collected from patients with peritonitis and identified using an Automated Microbic System (VITEK-AMS). The bacteria were inoculated onto nutrient agar medium and cultured at 37 °C for 18 h. The bacteria were then collected by washing with normal saline and measured for concentration using turbidimitry. A suspension with a bacterial concentration of 2×109 cfu/ ml was prepared. The rats were randomly divided into 3 groups. The rats in the model group received an intraperitoneal
injection of E. coli (0.3 ml/100 g of the bacterial suspension). The rats in the drainage group underwent mesenteric lymph duct ligation for drainage of lymphatic fluids. The rats in the sham operation group received no other treatments except surgical operation. Sample collection was conducted 8 h later for the sham operation group and 16 h later for the model and drainage groups. Sampling of Blood, Lymphatic Fluid, BALF, and Tissues The lymphatic fluids were collected and centrifuged at 2000 rpm for 10 min at 4 °C. Aliquots of the supernatant were frozen at −80 °C. Blood was taken from the inferior vena cava, and plasma was isolated and preserved at −80 °C. In order to take the BALF, the right main bronchus was exposed and cannulated. In total, 2 ml of saline were used to lavage the bronchoalveoli. After 5 min retention, the lavage was sanctioned slowly. The BALF was centrifuged at 3000 rpm for 15 min at 4 °C. Aliquots of the supernatant were stored at −80 °C. The bilateral pulmonary lobes and terminal ileum were dissected and frozen in liquid nitrogen. Some of the pulmonary and ileum tissues were fixed with 10 % of formalin. Measurement of MPO and DAO Activity The MPO and DAO activities were determined using purchased kits (NanJing JianCheng Bioengineering Institute, China) according to the instructions. Analysis of Pulmonary Vascular Permeability The pulmonary vascular permeability was analyzed using three indices. The first one was the concentration of Evans Blue Dye (EBD) in pulmonary tissues. Briefly, 1 % of the EBD (2 ml/kg) was injected into jugular vein; and 15 min later, the pulmonary tissues were dissected and immersed in formamide solution (10 ml/g). The tissues were incubated at 50 °C for 36 h and centrifuged. The supernatant was assayed at λ 620 nm with a spectrophotometer (722). The EBD concentration per gram tissue was calculated according to the standard curve. The second method was the wet/dry lung weight ratio. The wet lung weight was obtained by weighing the lung tissue after removing any water on the tissue surface. The dry lung weight was obtained by measuring the weight of dried lung tissue after incubation in an 80 °C incubator for 48 h. The last method was water content identification. The water content was calculated using the formula: wet lung weight-dry lung weight×100/wet lung weight.
Lymphatic Pathway and Severe Intraperitoneal Infection 1.6
25
1.2
*
10
*
5
1
U/g
15
0.8 MPO
U/L
20
DAO
*
1.4
0
0.6 0.4
-5
0.2 0 sham
200μm
model+MLD
200μm
sham
200μm
model+MLD HE staining
model
model 100
Fig. 1. DAO and MPO levels and histopathological changes of ileum tissues. All results (mean ± SD) shown are representative of three separate experiments. Data are the mean ± SD of n = 8 rats in each group. Asterisk represents p
Mesenteric Lymph Duct Drainage Attenuates Acute Lung Injury in Rats with Severe Intraperitoneal Infection Yanmin Zhang,1,4 Shukun Zhang,2 and Naiqiang Tsui3
Abstract—The purpose of this study is to investigate the hypothesis that the mesenteric lymphatic system plays an important role in acute lung injury in a rat model induced by severe intraperitoneal infection. Male Wistar rats weighing 250∼300 g were randomly divided into 3 groups and subjected to sham operation, intraperitoneal infection, or mesenteric lymphatic drainage. The activity of diamine oxidase (DAO) and myeloperoxidase (MPO) were measured by enzymatic assay. The endotoxin levels in plasma, lymph, and bronchoalveolar lavage fluid (BALF) were evaluated using the limulus amoebocyte lysate reagent. The cytokines, adhesion factors, chemokines, and inflammatory factors were detected by ELISA. TLR-4, NF-kB, and IRAK-4 were analyzed by Western blotting. Compared with sham-operated rats, rats with intraperitoneal infection had increased MPO and decreased DAO activity in intestinal tissues. Mesenteric lymph drainage reduced the alterations in MPO and DAO activity induced by intraperitoneal infection. The MPO activity in pulmonary tissue and the permeability of pulmonary blood vessels were also increased, which were partially reversed by mesenteric lymph drainage. The endotoxin levels in lymphatic fluid and alveolar perfusion fluid were elevated after intraperitoneal infection but decreased to control levels after lymph drainage. No alterations in the levels of plasma endotoxin were observed. The number of neutrophils was increased in BALF and lymph in the infected rats, and was also reduced after drainage. Lymph drainage also decreased the levels of inflammatory cytokines, chemokines, and adhesion factors in the plasma, lymph, and BALF, as well as the levels of TLR-4, NF-kB, and IRAK-4 in pulmonary and intestinal tissues. The mesenteric lymphatic system is the main pathway involved in early lung injury caused by severe intraperitoneal infection, in which activation of the TLR-4 signal pathway may play a role. KEY WORDS: mesenteric lymphatic pathway; severe intraperitoneal infection; lung injury; TLR-4 signal pathway.
INTRODUCTION It is often observed in abdominal surgery that severe intraperitoneal infection induced by primary diseases, such as perforative peritonitis, severe acute pancreatitis, biliary tract infection, and intraperitoneal abscess, is frequently accompanied by sepsis and multiple organ dysfunction syndrome (MODS). This is because the intestinal dysfunction can lead 1
Intensive Care Unit, Tianjin Nankai Hospital, 6 Changjiang Road, Nankai District, Tianjin, 300100, China 2 Research Institute of Tianjin Nankai Hospital, Tianjin Nankai Hospital, 6 Changjiang Road, Nankai District, Tianjin, 300100, China 3 Department of Surgery, Tianjin Nankai Hospital, 6 Changjiang Road, Nankai District, Tianjin, 300100, China 4 To whom correspondence should be addressed at Intensive Care Unit, Tianjin Nankai Hospital, 6 Changjiang Road, Nankai District, Tianjin, 300100, China. E-mail: [email protected]
to constant intestinal bacterial translocation and endotoxemia, inducing the overactivated and uncontrolled systemic inflammation as well as remote organ injuries. Therefore, the intestinal barrier damage and the resultant intestinal endotoxin translocation are the main causes of MODS. Although the intestines play a key role in the onset, development, and prognosis of systemic inflammation and MODS, the underlying mechanisms remain poorly understood [1–4]. There is disagreement on the theory that bacteria and endotoxins are translocated through the portal system, as this theory cannot explain the clinical observation that the lung is the first affected organ in MODS [5]. In addition, considerable clinical studies have not observed “translocated” bacteria or endotoxins in the portal vein blood beyond mesenteric lymph nodes [6]. It has been shown recently that mesenteric lymphatic system is involved in the development of SIRS (systemic
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Zhang, Zhang, and Tsui inflammatory response syndrome)/MODS [7, 8]. Deitch and his colleagues suggested that, in the early stages of abdominal infection, the endotoxins and inflammatory mediators (factors) absorbed first flow into the mesenteric lymphatic ducts and lacteal vessels, and then enter into a systemic circulation via thoracic duct [9]. They found that rats with septic shock contained substances toxic to alveolar epithelial cells in their mesenteric lymphatic fluids, which were prominent within 1–3 h after infection. Baigrie et al. [10] suggested that the mesenteric lymphatic fluid could rapidly transport inflammatory mediators and cytotoxic substances. They hypothesized that the intestinal tract was the source of systemic infection and MODS. During traumatic injury, the response of the intestines is production of a series of inflammatory mediators and cytokines, followed by damage of the intestinal barrier. However, the transportation of inflammatory mediators, cytokines, and other cytotoxic substances into the thoracic ducts and systemic circulation is mediated by the mesenteric lymphatic system rather than the classical portal system. Therefore, the systemic infection and MODS can occur without translocation of inflammatory mediators or bacterial toxins in the portal system. In the present study, a severe intraperitoneal infection rat model was produced by direct intraperitoneal injection of an Escherichia coli suspension. Using this model, the protective effect of mesenteric lymphatic drainage on remote organ injury induced by intestinal injury was studied.
MATERIALS AND METHODS Experimental Animals Male Wistar rats of 250∼300 g were purchased from the Experimental Animal Center, Institute of Environmental Medicine, Academy of Military Medical Sciences (laboratory animal license no. SCXK), acclimatized for 1 week, fed a normal diet, and water ad libitum before the experiment. Severe Intraperitoneal Infection Model Escherichia coli samples were collected from patients with peritonitis and identified using an Automated Microbic System (VITEK-AMS). The bacteria were inoculated onto nutrient agar medium and cultured at 37 °C for 18 h. The bacteria were then collected by washing with normal saline and measured for concentration using turbidimitry. A suspension with a bacterial concentration of 2×109 cfu/ ml was prepared. The rats were randomly divided into 3 groups. The rats in the model group received an intraperitoneal
injection of E. coli (0.3 ml/100 g of the bacterial suspension). The rats in the drainage group underwent mesenteric lymph duct ligation for drainage of lymphatic fluids. The rats in the sham operation group received no other treatments except surgical operation. Sample collection was conducted 8 h later for the sham operation group and 16 h later for the model and drainage groups. Sampling of Blood, Lymphatic Fluid, BALF, and Tissues The lymphatic fluids were collected and centrifuged at 2000 rpm for 10 min at 4 °C. Aliquots of the supernatant were frozen at −80 °C. Blood was taken from the inferior vena cava, and plasma was isolated and preserved at −80 °C. In order to take the BALF, the right main bronchus was exposed and cannulated. In total, 2 ml of saline were used to lavage the bronchoalveoli. After 5 min retention, the lavage was sanctioned slowly. The BALF was centrifuged at 3000 rpm for 15 min at 4 °C. Aliquots of the supernatant were stored at −80 °C. The bilateral pulmonary lobes and terminal ileum were dissected and frozen in liquid nitrogen. Some of the pulmonary and ileum tissues were fixed with 10 % of formalin. Measurement of MPO and DAO Activity The MPO and DAO activities were determined using purchased kits (NanJing JianCheng Bioengineering Institute, China) according to the instructions. Analysis of Pulmonary Vascular Permeability The pulmonary vascular permeability was analyzed using three indices. The first one was the concentration of Evans Blue Dye (EBD) in pulmonary tissues. Briefly, 1 % of the EBD (2 ml/kg) was injected into jugular vein; and 15 min later, the pulmonary tissues were dissected and immersed in formamide solution (10 ml/g). The tissues were incubated at 50 °C for 36 h and centrifuged. The supernatant was assayed at λ 620 nm with a spectrophotometer (722). The EBD concentration per gram tissue was calculated according to the standard curve. The second method was the wet/dry lung weight ratio. The wet lung weight was obtained by weighing the lung tissue after removing any water on the tissue surface. The dry lung weight was obtained by measuring the weight of dried lung tissue after incubation in an 80 °C incubator for 48 h. The last method was water content identification. The water content was calculated using the formula: wet lung weight-dry lung weight×100/wet lung weight.
Lymphatic Pathway and Severe Intraperitoneal Infection 1.6
25
1.2
*
10
*
5
1
U/g
15
0.8 MPO
U/L
20
DAO
*
1.4
0
0.6 0.4
-5
0.2 0 sham
200μm
model+MLD
200μm
sham
200μm
model+MLD HE staining
model
model 100
Fig. 1. DAO and MPO levels and histopathological changes of ileum tissues. All results (mean ± SD) shown are representative of three separate experiments. Data are the mean ± SD of n = 8 rats in each group. Asterisk represents p
