Interference Effects Of Lymph Plasma On Endotoxic Shock In Rats And Its Mechanisms

Endotoxic shock (ES) is akind of common serious diseases in clinic.It is one of the important causes of death and the mortality increases yearby year. Lipopolysaccharide (LPS) plays an essential role in itspathogenesis, which come from Gram-negative bacteria. Although theencouraging progress has been made in the treatment of some shock, thedeath rate of ES is still high. The aim of the study is to investigate the newmethod in ES therapy. The ES model of rats were duplicated byintravenous injection with LPS. The blood pressure, micro-local blood flowperfusion volume, functional and structural organs and survival time wereobeserved to reveal interference effects of lymph plasma on ES, andmoreover the changes of mesenteric microcirculation (adherent leukocytes,micro- vascular diameter, blood flow condition, lymphatic contractility)were observed and levels of intercellular adhesion molecule-1 (ICAM-1),P-selectin in plasma and homogenate, and the activities of myeloperoxidase(MPO) and Na+-K+-ATPase in homogenate were determined to explore theinterferernce mechanisms of lymph plasma on ES, which provided newideas for the clinic to treat septic shock.90 SPF-class male Wistar rats were randomly divided into threegroups: control group, model group and lymph plasma group (n=30). Allrats were anesthetized with 1% sodium pentobarbital (50mg·kg-1·bw, im)and anticoagulated with venous injection of heparin (420 U·kg-1·bw). Thenthe above animals were operated on neck. The right common carotid arterywas cannulated to record mean arterial pressure (MAP) continuously withbiological signal acquisition system, the left jugular vein were cannulatedto perepare for infusing drugs and taking blood. After stabilizaion for 30minutes, the LPS solution was infused (15mg·kg-1, Sigma) via the left jugular vein into model group and lymph plasma groups, normal salinereplaced LPS in the same moment in control group. After 15 minutes,lymph plasm was infused in lymph plasma group with ZCZ-50 automaticpump and injection machine at the speed of 0.5 ml per minute. The amountof lymph plasma was one fifteenth of whole blood volume whichcalculated by 7.4% of body weight. In control and model groups, lymphplasmwas replaced by normal saline.Each group above was divided into three subgroups: ten rats of thefirst subgroup were used for observing blood flow perfusion volume ofkidney and liver in 360 minutes after LPS challenge with laser Dopplerflowmetry of Perimed company. At the end of the experiment, thefiberoptic probe and canulas were removed, the wounds were sewed uplayer by layer, and than the survival time of rats were observed from themoment of infusion with LPS to death. If the survival time was more than24 hours, the rats were considered long-term life. Ten rats of the secondsubgroup were used for observing mesenteric blood microcirculation ofmesentery near the ileum lower by Olympus microcirculatory microscopeand video record system for 360 minutes after LPS challenge. The indexesobserved included the changes of mesenteric microvascular diameter,leukocyte adhesion and blood flow condition. Ten rats of the third subgroupwere used for observing mesenteric lymphatic microcirculation. Thechanges of lymphatic spontaneous contractile frequency, maximumcontractile diameter, maximum diastolic diameter, stillstand state diameterand contractile parameters were observed after administration with LPS. At180 minutes and 360 minutes after LPS (or corresponding liquid) infusionrespectively, blood and lung, kidney, liver tissues of fixed position weretaken from the two subgroups, which were used for observing mesentericmicrocirculation. The tissues were prepared for homogenate of 10 percentand made into paraffin section to HE staining, which tissues at 360 minutesafter LPS challenge. The blood urea nitrogen (BUN), creatinine (Cre), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) inplasma and the P-selectin, ICAM-1 in plasma and homogenate, and theactivities of MPO and Na+-K+-ATPase in homogenate were determined.The wet/dry (W/D) ratio of lung, kidney and liver were calculated, as wellas to observe the morphologic changes of lung, kidney and liver tissues.The results showed that every index observed in experiment had nosignificant difference between three groups before administration (P>0.05).In control group, the MAP, micro-local blood flow perfusion volume ofkidney and liver, the mesenteric microvascular diameter and blood flowcondition maintained constant level throughout the experiment, so did themaximum contractile diameter, maximum diastolic diameter, stillstand statediameter, spontaneous contractile frequency and three contractileparameters of IndexⅠ, IndexⅡand LD-Index (P>0.05). The adherentleukocyte in mesenteric venule was no found and only a few of adherentleukocytes were observed along the time of observation in control group.In model and lymph plasma groups, the MAP, kidney and livermicro-local blood flow perfusion volume significantly decreased and themesenteric arteriolar diameter contracted, the blood flow condition sloweddown and the number of leukocytes adherent to venular wall increased,maximum contractile range and three contractile parameters of mesentericmicro-lymphatic decreased after LPS challenge for 15 minutes. All indexeshad no significant difference between the two groups (P>0.05), but therewas significant difference compared with control group (P<0.01, P<0.05).The lymph plasma group was prevailed over model group on these indexesafter infusion with lymph plasma.Although the MAP recovered after the degression at the moment ofadministration with LPS in model and lymph plasma groups, the modelgroup were still significantly lower than that of control group (P<0.01,P<0.05) and had been decreasing continually since 180 minutes after LPSinfusion. The MAP remained a normal level during 120~240 minutes in lymph plasma group. From infusion with lymph plasma to 90 minutes andsince 330 minutes to the end, that of lymph plasma group was obviouslyhigher than that of the model group (P<0.05, P<0.01).With the MAPrecovering, the kidney and liver micro-local blood flowperfusion volume also recovered, and that of kidney in two groups werehigher than that of control group (P<0.05, P<0.01). Those of livermaintained normal level. In model group, blood flow perfusion volume ofkidney was reduced to a normal level from 45 to 105 minutes, and thenshowed fluctuant decreasing. Its blood flow perfusion volume had beengradually progressive decreased since 195 minutes in kidney anddecreasing since 270 minutes in liver. The time during which started tocontinuously decrease of kidney blood flow perfusion volume wassignificantly earlier than that of liver (P<0.01). In lymph plasma group,high level of blood flow perfusion volume of kidney maintained up to 75minutes and had been closed to control group since 75 minutes (P>0.05).The blood flow perfusion volume of liver was approached to the level ofpre-experiment and control group, except several times higher than that ofcontrol group by chance (P>0.05). Since 270 minutes, the kidney and livermicro-local blood flow perfusion volume was significantly higher than thatof model group (P<0.05, P<0.01).In model group, mesenteric arteriole diameter remained contractilestate throughout the experiment and it was significantly narrowedcompared with pre-experiment and control group (P<0.01, P<0.05), but inlymph plasma group, arteriole diameter recovered to the level ofpre-experiment and control group after infusion with lymph plasma(P<0.05), and contracted starting from 330 minutes. The mesenteric venulediameter progressive contracted since 150 minutes in model group, whichwas significantly lower than that of control group and lymph plasma groups(P<0.05, P<0.01), but the venular diameter maintained normal levelconstantly in lymph plasma group. Meanwhile, blood flow condition in model group slowed down from line flow to granular-line flow, to granularflow even to slow granular flow which resulted in multiple score valueincreased significantly compred with control and lymph plasma groups(P<0.01, P<0.05), and there appeared erythrocyte aggregation and bleeding.But in lymph plasma group, there was no change of blood flow conditionuntil 300 minutes, and since then the multiple score value was higher thanthat of control group, but lower than that of model group. The changes ofarteriole blood flow condition were later than that of the venule in modelgroup and lymph plasma groups, and blood flow condition of lymphplasma group was significantly better than that of the model group (P<0.01,P<0.05). After infusion with LPS, there were progressively increasingleukocytes adherent to mesenteric venules in model group, and numbers ofadherent leukocytes were significantly increased compared with controland lymph plasma groups (P<0.01, P<0.05). The adherent numbers oflymph plasma group maintained a low level the same as those beforeinfusing with lymph plasma, although it had been increasing slowly since270 minutes, but still significantly less than that of model group (P<0.01).Since 30 minutes after LPS challenge in model and lymph plasma groups,the decreased contractile parameters and maximum contractile diameter ofmesenteric micro-lymphatic had recoverd to normal levels ofpre-experiment and control group (P>0.05), although the changes ofmicro-lymphatic diameter were displayed. The normal level of spontaneouscontractile parameters maintained up to 180 minutes, except that LD-Indexincreased occasionally in model group (P<0.05). The spontaneouscontractile frequency of micro-lymphatic had no significant change in threegroups within 180 minutes.After LPS challenge for 180 minutes, the concentration of P-selectinwas significantly increased in plasma and homogenate of lung and liver inmodel group (P<0.05, P<0.01). In lymph plasma group, the concentrationof plasma P-selectin was higher than that of control group (P<0.05), but P-selectin of plasma and pulmonary homogenate was significantly lowerthan that of model group (P<0.01). There was no significant difference ofP-selectin in renal homogenate between the model and lymph plasmagroups (P>0.05). At the same time, the level of ICAM-1 in pulmonary andhepatic homogenate in model group was significantly higher than that oflymph plasma and control groups (P<0.01, P<0.05). After challenge LPSfor 360 minutes, the level of ICAM-1 in plasma and homogenate of lung,liver and kidney was significantly higher than that of control group (P<0.01,P<0.05), while the level of ICAM-1 in homogenates of lymph plasmagroup were all closed to control group after 180 minutes. Although theICAM-1 of plasma and homogenates of lung and kidney was significantlyhigher than that of control group (P<0.01, P<0.05), the level of ICAM-1was significantly lower than that of model group (P<0.05).After LPS challenge for 180 and 360 minutes, the MPO activity inhomogenate of lung, liver and kidney in model group was significantlyhigher than that of control group (P<0.01, P<0.05), and the Na+-K+-ATPaseactivity was obviously lower than that of control group (P<0.01, P<0.05).The MPO activity in renal homogenate of lymph plasma group showed nostatistically significant difference with control group, and pulmonary andhepatic homogenates was higher than that of control group, but thepulmonary was lower than that of model group and MPO of liver waslower than that of model group in 180 minutes(P<0.01, P<0.05). Exceptthe activity showed no significant difference between lymph plasma andcontrol groups at 180 minutes, every one of Na+-K+-ATPase activity inhomogenates of lymph plasma group was significantly lower than that ofcontrol group, but those were obviously higher than model group.The results of detecting biochemical indexes of kidney and liverfunction in plasma showed that the concentration of BUN, Cre, AST, andALT significantly increased in model and lymph plasma groups afterinfusion with LPS for 180 minutes and 360 minutes, which compared with control group (P<0.01). In lymph plasma group, the level of BUN and Cresignificantly decreased compared with model group at 360 minutes(P<0.05, P<0.01), the level of AST and ALT were all lower than that ofmodel group at 180 minutes and 360 minutes (P<0.05, P<0.01).Pathomorphologic study showed that the tissue structures were foundserious damage in lung, kidney, liver of model group, and only mild lesionscould be found in organs of lymph plasma group. The W/D ratio ofabove-mentioned organs in both groups was higher than that of controlgroup (P<0.01, P<0.05), which in accordance with the tissue morphologicdamage. The W/D ratio of lung in lymph plasma group was significantlylower than that of model group (P<0.05). The rats of control group werelong-term life. The survival time of lymph plasma was (11.80±2.67) hours,it was significantly than that of model group(P<0.01), which was(7.21±1.33) hours.The results above suggests that normal lymph plasma plays a positiveinterference effect on endotoxic shock induced by LPS, including impro-ving hypotension and tissue hypoperfusion volume, depressing organsdysfunction and morphologic damage and prolonging survival time. Theinterference mechanisms of lymph plasma on ES may be concerned withthe improvement of microcirculatory dysfunction, alleviatingion of theproduction of cell adhesion molecules (such as P-selectin and ICAM-1) andreducing leukocytes adherent to microvascular wall and detained in organs.Thus protecting micro blood flow condition and recovering tissue bloodflow perfusion, improving cellular metabolism and membrane pump, all ofthese help to reverse shock. The tactics of normal lymph plasma to interfereendotoxic shock may present a new way to clinical treatment.