大蒜素对脓毒症大鼠肠黏膜屏障的保护作用

目的 研究大蒜素对脓毒症大鼠肠黏膜屏障功能的保护作用,并初步探讨其机制.方法 24只雄性SD大鼠随机(随机数字法)分为假手术组、脓毒症模型组、大蒜素治疗组,每组8只.脓毒症模型采用盲肠结扎穿孔(cecum ligation and punctue,CLP),6h及12 h后用大蒜素(30 mg/kg,ip)干预,假手术组及模型组于同时间点给予等量生理盐水.24 h后处死大鼠检测血清D-乳酸、二胺氧化酶(diamine oxidase,DAO)活性、荧光异硫氰酸盐葡聚糖(fluorescence isothiocyanate dextran,FITC-Dextran,FD-40)水平;检测肠组织肿瘤坏死因子[α(tumor necrosis factor alpha,TNF-α)、白介素-6(interleukin-6,IL-6)、丙二醛(malondialdehyde,MDA)水平及超氧化物歧化酶(superoxidedismutase,SOD)活性,并取部分小肠组织进行组织病理学分析.统计方法采用单因素方差分析.结果 与假手术组比较,CLP组大鼠血清D[乳酸、DAO活性及FD40水平明显升高[D-乳酸(nmol/mL):(599.4±101.1) vs.(149.2±20.63),t=11.84,P＜0.01;DAO (ng/mL):(302.1±64.56)vs.(76.57±14.76),t=9.433,P＜0.01;FD-40 (ng/mL):(6664.0±1437.0)vs.(1446.0±205.0),t=9.704,P＜0.01];病理切片示CLP组大鼠肠道形态学损伤明显;肠组织中TNF-α、IL-6、MDA水平明显升高[TNF-α (pg/mL):(186.35±20.43)vs.(58.76±8.94),t=17.23,P＜0.01;IL-6 (pg/mL):(763.25±85.23)vs.(125.36±14.37),t=22.54,P＜0.01;MDA(nmol/mg prot):(29.36±3.27)vs.(7.24 ±0.85),t=16.61,P＜0.01],SOD活性降低[SOD(U/mg prot):(35.75 ±6.53)vs.(73.26±8.35),t=10.57,P＜0.01].大蒜素显著减少脓毒症诱导的血清中D哥酸、DAO活性及FD-40的升高[D-乳酸(nmol/mL):(330.1±81.77)vs.(599.4±101.1),t=7.086,P＜0.01;DAO (ng/mL):(171.8±49.70)vs.(302.1±64.56),t=5.45,P＜0.01;FD-40(ng/mL):(3349.0±1 167.0)vs.(6664.0±1437.0),t=6.165,P＜0.01];病理切片显示大蒜素减轻肠道形态学损伤;大蒜素明显抑制肠组织中TNF-α、IL-6、MDA水平[TNF-α (pg/mL):(95.37±12.68)vs.(186.35±20.43),t=12.29,P＜0.01;IL-6 (pg/mL):(354.27±46.27)vs.(763.25 ±85.23),t=14.45,P＜0.01;MDA (nmol/mgprot):(16.27±3.14)vs.(29.36±3.27),t=9.831,P＜0.01],增加SOD活性[SOD (U/mg prot):(55.35±6.23)vs.(35.75±6.53),t=5.522,P＜0.01].结论 大蒜素对CLP诱导的肠黏膜屏障功能具有保护作用,其机制可能与抑制炎症和氧化应激有关.     

严重腹腔感染时小肠隐窝中Tcf-4表达的变化及意义

目的 探讨大鼠严重腹腔感染时T细胞因子4(Tcf-4)表达的变化规律及意义. 方法健康成年Wistar大鼠40只被随机分为对照组(仅行单纯剖腹手术)和腹腔感染[采用盲肠结扎穿孔术(CLP)制备严重腹腔感染模型]后12、24和48 h组,每组10只.应用免疫组化及逆转录-聚合酶链反应(RT-PCR)检测各组大鼠小肠隐窝Tcf-4阳性细胞及Tcf-4 mRNA表达的改变.结果 免疫组化结果表明,对照组大鼠肠黏膜隐窝底部细胞中Tcf-4仅有微弱表达,而CLP后12 h肠黏膜隐窝底部细胞中Tcf-4阳性细胞数明显增多,24 h达峰值,至48 h仍显著高于对照组(P均＜0.01).RT-PCR结果表明,与对照组(0.19±0.01)比较,CLP后12 h Tcf-4 mRNA明显上升(0.21±0.01,P＜0.01),至24 h达峰值(0.28±0.02,P＜0.01),随后逐渐下降,到48 h(0.20±0.01)仍显著高于对照组(P＜0.05).结论 严重腹腔感染早期可诱导Tcf-4表达,提示Tcf-4可能与肠上皮干细胞的增殖分化密切相关.     

丙酮酸乙酯对严重腹腔感染时肠黏膜过氧化损伤的防治作用

目的探讨丙酮酸乙酯对严重腹腔感染大鼠肠黏膜过氧化损伤的防治作用。方法将36只SD大鼠按照随机数字表法分成对照组、腹腔感染组和治疗组，每组12只。腹腔感染组采用盲肠结扎加穿孔术（CLP）制作严重腹腔感染模型;治疗组行CLP后皮下注射丙酮酸乙酯40mg／kg，每8h1次；对照组仅行单纯剖腹手术。各组于术后24h和48h分别取小肠组织进行病理学观察，记录小肠黏膜病理损伤分级，同时检测小肠及血清丙二醛（MDA）含量、小肠髓过氧化物酶（MP0）活性。结果术后24h和48h腹腔感染组血清和小肠MDA水平均显著高于对照组（P均〈0．05）。且两者呈显著正相关（r=0．867，P〈0．05）；治疗组较腹腔感染组小肠黏膜病理性损害明显减轻，病理分级显著降低（P均〈0．05）；治疗组血清MDA和小肠MPO水平均明显低于腹腔感染组（P均〈0．05）。结论严重腹腔感染时过氧化损伤导致肠黏膜屏障功能障碍，丙酮酸乙酯具有减轻大鼠肠黏膜过氧化损伤的作用。     

Effect of linomide on gut immune cell distribution and on TNF-alpha in plasma and ascites: an experimental study in the septic rat

A significant reduction of the pan T lymphocytes as well as CD4+ and CD8 subsets of cells in the gut mucosa of the septic rats has previously been demonstrated. In contrast, the populations of major histocompatibility complex (MHC) class II-positive cells and macrophages increased. The aim of this study was to evaluate if the immunomodulator Linomide influenced the immune cell distribution in the small intestinal mucosa in sepsis and, furthermore, if these changes coincide with changes in the concentration of tumor necrosis factor-alpha (TNF-alpha) in plasma or ascites. Polymicrobial sepsis was induced in rats by cecal ligation and puncture (CLP). Three different experimental groups were used: CLP, Linomide p.o. + CLP, and Linomide i.p.+ CLP, with adequate controls. Specimens were taken from the small bowel for immunohistologic staining and grading of mucosal injury. The following monoclonal antibodies were used: W3/25, OX8, R73, OX6, and ED1. All slides were examined by one "blinded" examiner. Mucosal injury was graded from 0 to 5. The immunostained tissues were also analyzed by an automatic color-based image system. All controls had a normal appearance of the mucosa (grade 0-1), whereas the septic animals had a median grade of III (II-IV) mucosal injury. Linomide i.p. + CLP decreased mucosal damage to median I (0-IV, P < 0.05). Linomide had no effects on the immune cell distribution in controls. In CLP rats, a significant reduction in both CD4+ and CD8+ T lymphocytes as well as an increased number of macrophages and MHC class II-positive cells was seen in the villi as compared with sham-operated controls (P < 0.05). Linomide attenuated these changes for CD8+ and T lymphocytes and macrophages. Sepsis caused increased concentrations of TNF-alpha in portal blood and ascites 3 h from CLP induction. This increase was attenuated by Linomide.     

脓毒症大鼠肠黏膜免疫屏障功能的变化

目的 研究脓毒症对大鼠肠黏膜免疫屏障功能的影响.方法 60只SD大鼠随机(随机数字法)分为对照组(n=15)和脓毒症组(n=45),采用盲肠结扎穿孔术(CLP)建立脓毒症模型.模型建立后3 h、6 h和12 h留取回肠黏膜和全血标本.分别进行肠黏膜形态学观察、肠防御素5(RD-5)及肠三叶因子3(TFF_3)Mrna表达水平检测、肠黏膜淋巴细胞凋亡分析,以及外周血中肠源性细菌DNA定性检测.结果 CLP所致脓毒症导致大鼠回肠黏膜明显损害,主要表现为上皮脱落、固有层分离、毛细血管出血和溃疡形成;脓毒症组模型建立后3 h即出现RD-5和TFF_3 Mrna表达显著性减少(与正常组比较,P<0.05),且6 h和12 h组进行下降(与3 h组比较,P<0.05),肠黏膜淋巴细胞凋亡数亦显著增加(P<0.05);同时,脓毒症组全血肠源性细菌DNA扩增全部阳性.结论 脓毒症时大鼠肠黏膜免疫屏障功能显著减退,且随脓毒症的发展而进行性恶化.     

血必净对脓毒症大鼠回肠黏膜形态学的影响

目的 观察不同给药途径及不同给药时间情况下,血必净注射液对脓毒症大鼠回肠黏膜形态学的影响.方法 91只健康Sprague-Dawley大鼠由中山大学实验动物中心提供,随机分为正常对照组(n=7)、脓毒症组(n=21)、灌胃给药+脓毒症组(预处理组)(n=21)、静脉给药+脓毒症组(预处理组)(n=21)和脓毒症+静脉给药组(治疗组)(n=21).除正常对照组外,其余各组按手术后3 h,6 h,12 h再分3组,每组7只.采用盲肠结扎穿孔法制作脓毒症模型.正常对照组不予任何处理,灌胃给药+脓毒症组于术前2 h灌胃给药(5 mL/kg血必净);静脉给药+脓毒症组于术前2 h尾静脉给药(5 mL/kg血必净);脓毒症+静脉给药组于术后2 h尾静脉给药(5 mL/kg血必净).正常对照组不作任何处理,直接留取组织标本;其余各组于术后3 h,6 h,12 h分别活杀大鼠留取回肠标本,用以进行形态学观察及回肠上皮损伤指数测定.用SPSS 13.0统计软件进行统计学分析;组间比较用方差分析,P<0.05为差异具有统计学意义.结果 预处理组及治疗组大鼠回肠上皮损伤程度均较脓毒症组减轻(P<0.01);预处理组中,静脉给药较灌胃给药的保护效果更好(P<0.05).结论 血必净注射液能减轻脓毒症大鼠回肠黏膜的损伤程度,预先静脉给药效果更佳.     

Endotoxemia inhibits intestinal adaptation in a rat model of short bowel syndrome

Sepsis is commonly associated with or complicates short bowel syndrome (SBS). The purpose of the present study was to investigate the effects of endotoxemia on intestinal adaptation in a rat model of SBS. Male Sprague-Dawley rats were divided into three experimental groups: Sham rats underwent bowel transection and re-anastomosis, SBS rats underwent 75% small bowel resection, and SBS-LPS rats underwent bowel resection and were given lipopolysaccharide. Bowel weight, organ weights, and parameters of intestinal adaptation (bowel and mucosal weights, mucosal DNA and protein, villus height, and crypt depth) were determined on day 15 following operation. The results of this study demonstrate that SBS rats showed a significant increase (vs. Sham) in jejunal and ileal bowel and mucosal weight, mucosal DNA and protein, villus height, and crypt depth. SBS-LPS animals demonstrated lower (vs. SBS rats) final body weight (215 +/- 7 vs. 287 +/- 10 g, P < 0.05), overall weight in duodenum (98+/- 2 vs. 119 +/-5 mg/cm, P < 0.05) and jejunum (144 +/- 9 vs. 189 +/- 16 mg/cm, P < 0.05), mucosal weight in jejunum (54 +/- 5 vs. 69 +/- 5 mg/cm, P < 0.05) and ileum (31 +/- 2 vs. 37 +/- 3 mg/cm, P < 0.05), mucosal DNA in jejunum (89 +/- 11 vs. 120 +/- 11 microg/cm, P < 0.05) and ileum (46 +/- 6 vs. 61 +/- 4 microg/cm, P < 0.05), jejunal crypt depth (152 +/- 19 vs. 189 +/- 12 microm, P < 0.05), and ileal villus height (405 +/- 63 vs. 515 +/- 30 pm, P < 0.05). In addition, the SBS group had no late (second week) mortality, whereas the SBS-LPS group had an 17% late mortality rate. In conclusion, in a rat model of SBS-LPS, endotoxemia appears to inhibit structural intestinal adaptation and increase mortality.     

丙酮酸乙酯对脓毒症大鼠肠黏膜屏障功能保护作用的研究

目的 观察丙酮酸乙酯(ethyl pyruvate ,EP)干预治疗对脓毒症大鼠生存率和肠黏膜屏障的影响.方法 ①EP对脓毒症大鼠生存率的影响:无特定病原雄性SD大鼠100只随机分为假手术组(A组)、脓毒症组(B组)、EP早期治疗组(C组)及EP延迟治疗组(D组),每组25只,利用盲肠结扎穿孔法(cecal ligation and puncture,CLP)制作大鼠脓毒症模型,各组均于术后6、12、18、24、36、48、60、72 h腹腔内注射给药3 mL,C、D组分别于术后6、12 h开始予EP(40 mg/kg),A、B两组同法予等量林格乳酸钠溶液(ringer lactate solution ,RLS),每隔12 h记录死亡情况,分析比较5 d生存率;②EP对脓毒症大鼠肠黏膜屏障的影响:80只无特定病原雄性SD大鼠随机分为四组,每组20只,分组及给药方法与方法一相同,术后24、48 h各处死10只.测定各时间点血浆D-乳酸、DAO的变化,同时用透射电镜观察术后48 h肠黏膜上皮细胞超微结构的变化.采用Kaplan- Meier生存分析法进行生存分析,多组均数间比较采用单因素方差分析的方法, 多组均数间两两比较采用SNK-q检验,P＜0.05为差异有统计学意义.结果 A、B、C、D四组大鼠5 d生存率分别为100%、24%、68%、56%,与B组相比,C、D组大鼠5 d生存率明显提高(P<0.05),C、D组间差异无统计学意义(P＞0.05);与B组相比,C、D组术后24 h和48 h血浆D-乳酸含量明显下降(P<0.01);与B组相比,C组、D术后24 h和48 h血浆DAO活性明显下降(P<0.01),C、D组术后24、48 h血浆D-乳酸含量、DAO活性差异无统计学意义(P＞0.05);电镜下C、D组肠黏膜上皮细胞损伤较B组明显减轻,细胞间紧密连接较清楚.结论 脓毒症时肠黏膜损伤严重,EP早期与延迟干预治疗能有效保护肠黏膜屏障,提高5 d生存率,具有抗脓毒症作用 .     

电针足三里穴对腹腔脓毒症大鼠肠缺血及氧自由基损伤的作用研究

目的 探讨电针足三里穴对脓毒症大鼠肠缺血和氧自由基损伤的保护作用.方法 采用盲肠结扎穿孔术(CLP)制备大鼠脓毒症模型.雄性Wistar大鼠32只,随机分为CLP+电针足三里(CLP/EA)组、CLP+假电针(CLP/SEA)组、迷走神经切断+CLP+SEA(VA/CLP/SEA)组、VA+CLP+EA(VA/CLP/EA)组,每组8只.EA组持续针刺双侧足三里穴30 min,刺激强度为2～3 mA,2～100 Hz;SEA组采用相同频率和强度刺激非经非穴(足三里外侧旁开0.5 cm)30 min;VA组于CLP前切断迷走神经.各组大鼠于CLP后6 h测定空肠黏膜血流量(JMBF),处死动物取空肠组织,测定丙二醛(MDA)含量、黄嘌呤氧化酶(XOD)和二胺氧化酶(DAO)活性及肠组织含水量.结果 与CLP/SEA组比较,CLP/EA组JMBF和DAO活性显著增加,XOD和MDA及肠组织含水量显著降低(P均<0.05);VA/CLP/SEA组和VA/CLP/EA组JMBF和DAO活性显著降低,XOD和MDA水平显著升高,且组织含水量明显高于CLP/EA组(P均<0.05).VA/CLP/EA组与VA/CLP/SEA组各指标均无明显差异(P均＞0.05).结论 电针足三里可显著增加CLP大鼠JMBF和DAO活性,减轻肠组织水肿和脂质过氧化损伤;切断腹腔迷走神经能减轻或消除电针的作用,增强小肠组织过氧化反应,加重肠组织水肿和黏膜细胞损害.电针足三里穴对肠缺血和氧自由基损伤的保护机制可能与兴奋胆碱能通路有关.     

多器官功能障碍综合征时肠淋巴细胞再循环的变化

目的观察大鼠肠缺血--再灌注致多器官功能障碍综合征(MODS)后肠淋巴细胞归巢的改变,从肠黏膜免疫角度探讨肠淋巴细胞归巢在MODS中的作用.方法采用随机分组方法,用夹闭肠系膜动脉根部45 min、再灌注6 h制备大鼠MODS模型.MODS 1组大鼠(n=10)于再灌注第5 h从肠系膜淋巴管插管引流肠淋巴液1 h,检测淋巴细胞数及T、B细胞比例,同时取肠、肝、肺、肾组织进行病理组织学观察;对照1组(n=6)大鼠仅单纯施行肠淋巴液引流.MODS 2组大鼠(n=6)于再灌注第3 h从肠系膜淋巴管插管引流肠淋巴液2 h,肠淋巴细胞在体外经51Cr标记后,于第6 h回输入大鼠的体内,1 h后取上述各组织或器官以检测51Cr-淋巴细胞在体内的分布; 对照2组单纯施行肠淋巴液引流及淋巴细胞标记后回输.结果肠缺血-再灌注致MODS时,MODS组大鼠由肠黏膜迁移至血循环肠淋巴细胞总数[(0.28±0.15)×107/h]较对照组[(2.69±0 .61)×107/h]显著降低;而归巢至肠黏膜的肠淋巴细胞增加,派伊尔(Peyer)淋巴结及小肠内所分布的 51Cr-淋巴细胞量分别占总51Cr细胞量为(5.04±1.23)%和(3. 23±1.69)%,显著高于对照组(2.69±2.19)% 和(1.11±0.75)%,P均<0.05,并伴随肠淋巴内毒素含量及肿瘤坏死因子-α(TNF-α)浓度显著增加及重要器官或组织功能损害.结论肠淋巴细胞归巢增加是MODS发病机制的一个重要方面.     

Pulmonary oxidant stress in murine sepsis is due to inflammatory cell nitric oxide

OBJECTIVE: Pulmonary oxidant stress is an important pathophysiologic feature of acute lung injury. It is unclear whether nitric oxide contributes to this oxidant stress. Thus, we examined the role of inducible nitric oxide synthase (iNOS) in pulmonary oxidant stress in murine sepsis and the differential contribution of different cellular sources of iNOS. DESIGN: Randomized, controlled animal study. SETTING: Research laboratory of an academic institution. SUBJECTS: Male iNOS+/+, iNOS-/- C57Bl/6 mice, and bone-marrow transplanted iNOS chimeric mice: +to- (wild-type iNOS+/+ donor bone-marrow transplanted into iNOS-/- recipient mice) and the reciprocal -to+ chimeras. INTERVENTIONS: Animals were randomized to sepsis (n = 264), induced by cecal ligation and perforation, vs. naive groups (n = 138). MEASUREMENTS AND MAIN RESULTS: In septic iNOS-/- vs. wild-type iNOS+/+ mice, sepsis-induced pulmonary oxidant stress (33 +/- 11 [mean +/- sem] vs. 365 +/- 48 pg 8-isoprostane/mg protein, p < .01) and nitrosative stress (0.0 +/- 0.0 vs. 0.9 +/- 0.4 micromol 3-nitrotyrosine/mmol para-tyrosine, p < .05) were abolished, despite similar septic increases in pulmonary myeloperoxidase activity in both (86 +/- 20 vs. 83 +/- 12 mU/mg protein, p = .78). In +to- iNOS chimeric mice (iNOS localized only to donor bone-marrow-derived inflammatory cells), cecal ligation and perforation resulted in significant pulmonary oxidant stress (368 +/- 81 pg 8-isoprostane/mg protein) and nitrosative stress (0.6 +/- 0.2 micromol 3-nitrotyrosine/mmol para-tyrosine), similar in degree to septic wild-type mice. In contrast, pulmonary oxidant and nitrosative stresses were absent in septic -to+ iNOS chimeras (iNOS localized only to recipient parenchymal cells), similar to iNOS-/- mice. CONCLUSIONS: In murine sepsis-induced acute lung injury, pulmonary oxidant stress is completely iNOS dependent and is associated with tyrosine nitration. Moreover, pulmonary oxidant stress and nitrosative stress were uniquely dependent on the presence of iNOS in inflammatory cells (e.g., macrophages and neutrophils), with no apparent contribution of iNOS in pulmonary parenchymal cells. iNOS inhibition targeted specifically to inflammatory cells may be an effective therapeutic approach in sepsis and acute lung injury.     

Changes in gut mucosal nitric oxide synthase (NOS) activity after thermal injury and its relation with barrier failure

This study was designed to investigate changes in mucosal NOS activity after burns and its relation to barrier failure. In Experiment 1, female specific pathogen free (SPF) Sprague-Dawley rats underwent 35% total body surface area (TBSA) burn. One to six days after burn, intestinal permeability was determined from the plasma leakage of fluorescein isothiocyanate (FITC)-dextran 4400, intestinal mucosal cNOS and iNOS activity were assayed using Griess' reagent, and the cellular localization of iNOS was examined using immunostaining. In Experiment 2, S-methylisothiourea (SMT) was given (5 mg/kg, i.p. every 12 h) for 2 days to suppress inducible NOS (iNOS) activity after thermal injury. On postburn Day 2, the effect of SMT on gut mucosal NOS activity, intestinal permeability, and barrier function were evaluated. The activity of iNOS increased 24 h after the injury and up to a maximum of twofold on postburn Day 2, and decreased thereafter. The increase in iNOS activity in gut mucosa correlated well with the increase in intestinal permeability, an index for barrier failure (r = .776, p = .0002). Results from iNOS immunostaining showed that changes in mucosal iNOS activity after the burn occurred mainly in the enterocytes rather than in the macrophages. Administration of SMT decreased mucosal iNOS activity, intestinal permeability, and bacterial translocation incidence to mesenteric lymph node concurrently. In conclusion, thermal injury induces intestinal mucosal iNOS, which is principally in the enterocytes. The increased intestinal iNOS activity was closely related to barrier failure. SMT inhibited intestinal mucosal iNOS activity and prevented barrier failure as demonstrated by a decrease in BT occurrence and intestinal permeability.     

A new abdominal cavity chamber to study the impact of increased intra-abdominal pressure on microcirculation of gut mucosa by using video microscopy in rats

OBJECTIVE: In experimental studies of capillary blood flow that use intravital video microscopy, organs are exposed in observation chambers implanted into the animal. In this article we describe an abdominal cavity chamber for intravital video microscopy of gut mucosa microcirculation during increased intra-abdominal pressure. DESIGN: Prospective, experimental animal study. SETTING: Research laboratory at a university hospital. SUBJECTS: Male Wistar rats. INTERVENTIONS: The abdominal cavity chamber was designed for implantation into the abdominal wall of rats after laparotomy, thus creating an expanded hermetic, abdominal cavity volume. Animals were assigned to three levels of intra-abdominal pressure: controls (group 1), 10 mm Hg (group 2), and 15 mm Hg (group 3). Intra-abdominal pressure was increased by intra-abdominal insufflation of gas. By using a fluorescent marker, we quantitatively assessed mucosa perfusion index, functional capillary density, red blood cell velocity, capillary diameters, and flow motion during increased intra-abdominal pressure by intravital video microscopy. Results were expressed as mean +/- SEM. Significance of differences was determined by analysis of variance and multiple comparison of means with post hoc test (*p <.05 groups vs. control;p <.05 group 3 vs. group 2). MEASUREMENTS AND MAIN RESULTS: When compared with controls, animals subjected to an intra-abdominal pressure of 10 and 15 mm Hg showed a significant stepwise decrease in mucosa perfusion index (88%, 71%*, 22%*), functional capillary density (665.4 +/- 71.7, 461.6 +/- 71.9*, 375.1 +/- 2.0*cm(-1)), and red blood cell velocity (0.50 +/- 0.04, 0.33 +/- 0.03*, 0.04 +/- 0.06*mm/sec), indicating a stepwise impairment of mucosal microcirculation. Capillary diameters and flow motion did not change with respect to intra-abdominal pressure. CONCLUSIONS: This novel animal model of intravital intestinal video microscopy that uses an abdominal cavity chamber is a feasible and sensitive experimental tool to study intestinal microcirculation during increased intra-abdominal pressure. Intra-abdominal pressure likely results in a severe impairment of mucosal microcirculation.     

Arginine vasopressin compromises gut mucosal microcirculation in septic rats

OBJECTIVE: Arginine vasopressin (AVP) is increasingly used in the therapy of septic patients with hypotension. However, its effects on the microvascular networks have not been studied in detail. This study was designed to determine the effects of AVP infusion on the villus microcirculation of the septic rat ileum. DESIGN: Prospective, placebo-controlled, randomized, single-blinded trial. SETTING: University research laboratory. SUBJECTS: Fifteen male Sprague-Dawley rats. INTERVENTIONS: Twenty-four hours after cecal ligation and perforation to create sepsis (M1), rats (n = 8) received a continuous AVP infusion to increase mean arterial pressure by 20 mm Hg (M2) and 40 mm Hg (M3) from M1. In the control group (n = 7), an equivalent volume of normal saline was infused. MEASUREMENTS AND MAIN RESULTS: Videomicroscopy was performed on 6-10 villi of ileum mucosa at M1 and was repeated at M2 and M3. Blood was drawn to determine plasma levels of AVP and interleukin-6. At M1, both study groups were hypotensive compared with preseptic data (mean arterial pressure, -25%). The increase in mean arterial pressure was linked to supraphysiologic AVP plasma levels and was accompanied by a decrease in mean mucosal blood flow by 76% at M2 and 81% at M3 (p <.001 vs. control). Red blood cell velocity fell by 45% and 47%, respectively (p <.05 vs. control). Whereas periods of arrested villus blood flow increased from 8.1 +/- 2.6 secs/min to 43.8 +/- 5.2 and 47 +/- 6.2 secs/min at M2 and M3 (p <.001), the diameter of terminal arterioles remained unchanged. In addition, AVP infusion further augmented the sepsis-associated increase in interleukin-6 levels (AVP, 905 +/- 160 vs. control, 638 +/- 55 pg/mL; p =.022). CONCLUSIONS: This study provides evidence for severe abnormalities in gut mucosal blood flow after AVP infusion in septic rats, accompanied by an augmented inflammatory response to the septic injury. The effects of AVP on microvascular blood flow in this model may be related to AVP activities on larger arterioles (>40 microm), a concomitant reduction in cardiac output, or even both.     

A 4-amino analogue of tetrahydrobiopterin attenuates endotoxin-induced hemodynamic alterations and organ injury in rats

Most recently we have shown that 4-aminotetrahydrobiopterin (4-ABH4), an analogue of tetrahydrobiopterin (cofactor of NO synthase), even administered 2 h after endotoxin challenge, improves survival rate in rats. The following experiment was performed to examine the effects of 4-ABH4 with respect to endotoxin-induced hemodynamic alterations and organ failure. At 2 h after endotoxic challenge (10 mg kg(-1) body weight) animals received 4-ABH4 at a dose of 1, 10, or 100 mg kg(-1) body weight. The controls were treated similarly but received saline at the same volume. Eight hours after endotoxin challenge cardiac index and stroke volume were significantly increased in animals treated with 10 mg 4-ABH4 compared to controls (0.23 +/- 0.06 vs. 0.16 +/- 0.04 mL min(-1) kg(-1) and 0.29 +/- 0.05 vs. 0.22 +/- 0.03 mL beat(-1)) while mean arterial pressure and peripheral vascular resistance index did not significantly differ among the groups. Plasma alanine aminotransferase (ALT) and creatinine levels were significantly increased in endotoxin controls compared with laboratory controls (ALT: 1643 +/- 1436 vs. 74 +/- 17 U L(-1); Creatinine: 91 +/- 29 vs. 42 +/- 3 micromol L(-1)) which was attenuated in animals treated with 10 mg kg(-1) 4-ABH4 (ALT: 417 +/- 318 U L(-1); Creatinine: 78 +/- 26 micromol L(-1)). Moreover, endotoxin-induced lung edema and intestinal necrosis were significantly reduced by 4-ABH4. Our study provides information that tetrahydrobiopterin analogue, 4-ABH4, improves LPS induced hemodynamic conditions and organ injury. This may, at least in part, account for the previously observed protection of rats by 4-ABH4 against endotoxin-induced mortality in the same endotoxic shock model.     

胆碱酯酶抑制剂中毒大鼠肠屏障功能和形态结构变化及宾赛克嗪治疗作用的研究

目的 观察胆碱酯酶抑制剂类神经性毒剂中毒后大鼠肠屏障功能和组织形态结构的变化以及宾赛克嚷的治疗作用.方法 40只雄性Wistar大鼠按随机数字表法均分为对照组、维埃克斯染毒模型组及宾赛克嗪1、3、9 mg/kg治疗组.皮下注射维埃克斯13μg/kg染毒;宾赛克嗪组在染毒后5 min腹腔注射相应剂量药物.各组于染毒后3 h取血,检测血浆D-乳酸浓度及二胺氧化酶(DAO)活性;同时取小肠组织,观察肠黏膜形态和超微结构变化.结果 与对照组比较.模型组血浆D-乳酸浓度和DAO活性均明显升高[D-乳酸:(87.752±22.906)mg/L比(29.072±6.546)mg/L,DAO:(6.72±0.93)U/L比(2.99±0.43)U/L,均P<0.01];宾赛克嗪1、3、9 mg/kg组呈现剂量依赖性降低血浆D-乳酸浓度和DAO活性,其中宾赛克嗪9 mg/kg组可逆转染毒后血浆D-乳酸浓度[(45.290±11.141)mg/L]和DAO活性C(3.17±0.68)U/L]增高(均P<0.01).光镜下观察模型组大鼠空肠和回肠肠壁变薄,皱壁变短,结构紊乱,固有层毛细血管扩张充血,黏膜间质水肿等病理变化;电镜下观察模型组大鼠回肠上皮细胞发生坏死,细胞器损伤,紧密连接破坏等变化.宾赛克嗪1、3、9 mg/kg组呈现剂量依赖性抑制小肠的病理变化.结论 胆碱酯酶抑制剂中毒时出现肠黏膜上皮细胞损伤,肠黏膜屏障功能破坏,通透性增加;宾赛克嗪能抑制中毒肠黏膜屏障结构和功能的损伤.     

Alterations in Hepatic Gluconeogenesis, Prostanoid, and Intracellular Calcium during Sepsis

OBJECTIVE: The metabolic alterations observed during sepsis may be associated with changes in local concentrations of intracellular calcium (Ca2+) and prostanoid synthesis in the liver. The authors studied hepatocyte intracellular Ca2+ and the release of glucose and prostanoid in an in-vivo murine liver perfusion model. METHODS: Sepsis was induced in anesthetized, fasted rats by cecal ligation and puncture (CLP, n = 42). Hepatic glucose release was studied in control (n = 10) and CLP (n = 10) groups using a non-recirculating liver perfusion model with and without lactate as gluconeogenic substrate. Hepatocyte intracellular Ca2+ (n = 11) was measured using the selective indicator Fura-2 under basal and epinephrine (10(-5) M) stimulated conditions. 6-Keto-prostaglandin F1alpha (6-Keto) and thromboxane B2 (TxB2) were determined from liver perfusate by radioimmunassay (n = 11). Data were analyzed using t-tests and repeated-measures ANOVA. RESULTS: Plasma glucose was significantly lower in CLP groups compared with controls (74.9+/-6.6 vs 115.7+/-4.6 mg/dL, p < 0.05). Plasma lactate was significantly higher in CLP vs controls (3.7+/-0.4 vs 1.4+/-0.1 mM, p < 0.05). Glucose release in isolated perfused livers was significantly lower in CLP vs controls (8.5 vs 16+/-1.2 microM/g/hr, p < 0.001). With the addition of lactate + pyruvate to the perfusate, glucose output in CLP livers was significantly lower following 5 (9.9+/-0.7 vs 17.7+/-1.1 microM/g/hr, p < 0.05) and 10 (11.9+/-1.2 vs 20.6+/-1.3 microM/g/hr, p < 0.001) minutes of perfusion. The basal level of intracellular calcium ([Ca2+]i) in CLP rats (460.1+/-91.6 nM) was significantly higher than in control rats (196.3+/-35.5 nM) (p < 0.05). A significant increase (p < 0.05) in [Ca2+]i occurred after the addition of epinephrine in hepatocytes in control (196.3+/-35.5 vs 331.8+/-41.4 nM) but not CLP (460.1+/-91.6 vs 489.4+/-105 nM) rats. 6-Keto was significantly lower in CLP compared with controls at 30 minutes (25.7+/-3.9 vs 33.4+/-5.5 pg/mL, p < 0.05), whereas TxB2 was not significantly altered (52.1+/-34.7 vs 87.5+/-43.2 pg/mL). CONCLUSION: These results demonstrate that CLP sepsis is associated with an increase in hepatocyte intracellular free Ca2+ concentration along with attenuation of hormone-mediated Ca2+ mobilization and hepatic gluconeogenesis.     

Ileal mucosal oxygen consumption is decreased in endotoxemic rats but is restored toward normal by treatment with aminoguanidine

OBJECTIVE: We sought to test the hypothesis that ileal mucosal oxygen consumption is impaired in endotoxemic rats. METHODS: Male Sprague-Dawley rats were injected intravenously with either Escherichia coli lipopolysaccharide (5 mg/kg) or a similar volume of vehicle. A segment of ileum was excised 8 hrs later, and the serosal and muscular layers of the bowel were stripped away from the mucosa. A strip of mucosa was mounted in a polarographic chamber containing air-saturated Krebs-Henseleit buffer plus 20 mM glucose, PO2 being monitored during a 10-min period. Some rats were injected intraperitoneally with the inducible nitric oxide synthase inhibitor, aminoguanidine (30 mg/kg per dose), or a similar volume of vehicle, at 1, 3 and 6 hrs after injection of lipopolysaccharide. RESULTS: In an initial experiment, the rate of oxygen consumption was significantly lower for mucosal samples from endotoxemic rats as compared with control rats (0.76+/-0.11 ng-atoms vs. 1.42+/-0.22 ng-atoms of 0/min per microg dry weight, respectively; n = 8 per group; p<.05). The rate of mucosal oxygen consumption was higher in aminoguanidine-treated as compared with vehicle-treated endotoxemic rats (1.25+/-0.11 ng-atoms and 0.73+/-0.07 ng-atoms of 0/min per microg, respectively; n = 7 and n = 6, respectively; p<.05). CONCLUSION: Endotoxemia is associated with diminished intestinal mucosal oxygen utilization due to an intrinsic acquired derangement in cellular respiration that is caused, at least in part, by an aminoguanidine-inhibitable mechanism.     

卡巴胆碱对脓毒症大鼠内脏组织缺血引起脂质过氧化损伤的作用研究

目的 探讨卡巴胆碱(CAR)对脓毒症大鼠脏器组织灌流和脂质过氧化损伤的影响.方法 雄性SD大鼠64只,采用盲肠结扎穿孔术(CLP)制备大鼠脓毒症模型.随机分为CAR处理组和CLP组,每组32只,术后即刻两组分别静脉注射CAR 10 μg/kg或等量生理盐水.每组16只用于观察12 h和24 h死亡率;其余16只于CLP后18 h测定平均动脉压(MAP)和肝、肾、空肠组织血流量;取血测定血浆丙氨酸转氨酶(ALT)和肌酐(Cr)水平;后处死动物,测定空肠组织二胺氧化酶(DAO)活性及肝、肾、空肠组织黄嘌呤氧化酶(XOD)活性、丙二醛(MDA)含量和组织含水量.结果 CAR组12 h和24 h死亡率分别为25.0%(4/16)和50.0%(8/16),显著低于CLP组[37.5%(6/16),75.0%(12/16),P均<0.05].CLP后18 h,两组MAP差异无统计学意义(P>0.05);CAR组肝、肾、空肠组织血流量均明显大于CLP组(P均<0.05),肾和空肠组织XOD活性、MDA含量及组织含水量显著低于CLP组(P均<0.05);脏器功能指标[ALT;(64.3±8.3)U/L,Cr:(96.4±7.0)μmol/L,DAO;(0.20±0.04)U/L]的改善程度也均显著优于CLP组EALT:(81.5±7.9)U/L,Cr:(117.1±6.7)μmol/L,DAO;(0.12±0.03)U/L,P均<0.053.结论 CAR能改善脓毒症大鼠内脏灌流、抑制氧自由基生成,减轻组织水肿和脏器功能损害.