Carbon dioxide differentially affects the cytokine release of macrophage subpopulations exclusively via alteration of extracellular pH

BACKGROUND: The improved outcome after endoscopic surgery has been attributed to less surgical trauma. However, the underlying mechanisms are not fully understood, and direct effects of CO2 used for pneumoperitoneum, cellular acidification, and/or the lack of air contamination have been postulated to additionally modulate immune functions during endoscopic surgery. We investigated the effects of CO2 incubation, extracellular acidification, and air contamination on the inflammatory response of two distinct macrophage populations. METHODS: R2 and NR 8383 rat macrophage cell lines were used. Interleukin-6 (IL-6) and nitric oxide after lipopolysaccharide (LPS) stimulation were determined in these sets of experiments: incubation in 100% CO2, 5% CO2, and room air for 2h; incubation at pH 7.4, 6.5, and 5.5 for 2 h in 5% CO2; and incubation in 100% CO2, 5% CO2 and room air in fixed pH 6.3. The extracellular pH was monitored during incubation. We determined the alteration of intracellular pH in cells subjected to extracellular acidification by fluorescence microscopy. RESULTS: Extracellular pH decreased to 6.3 during 100% CO2 incubation. IL-6 release was reduced after CO2 incubation in NR 8383 cells and increased in R2 cells (p < 0.05). It was not altered by air incubation. Decreasing the extracellular pH to 6.5 mimicked the effects of CO2 and a decrease to 5.5 suppressed IL-6 release in both cell lines. In fixed pH at 6.3, CO2 and air incubation had no effect. CO2 and pH had no impact on nitric oxide release and vitality. Intracellular pH decreased with extracellular acidification without significant difference between the two cell lines. CONCLUSIONS: A decrease in extracellular pH during incubation in CO2 differentially affects IL-6 release in macrophage subpopulations. This may explain contradictory results in the literature. Moreover, we demonstrated that air contamination does not affect macrophage cytokine release. The decrease in extracellular pH is the primary underlying mechanism of the alteration of macrophage cytokine release after CO2 incubation, and it appears that the ability to maintain intracellular pH is not determined by the effects of CO2 or extracellular acidification.     

In vitro inhibition of superoxide anion production and superoxide dismutase activity by zinc in human spermatozoa

The in vitro effect of zinc on superoxide anion (O2-) generation and on SOD-like activity in spermatozoa of infertile men was investigated. The formation of superoxide anion was stimulated by NADPH and the level of superoxide anion was measured by the reduction of ferricytochrome c. Both Percoll-isolated (n = 14) and washed spermatozoa (n = 14) exposed to 1 mmol/L zinc (60 min, 37 degrees C), released less (p < 0.002 and p < 0.04, respectively) superoxide anions than did zinc-untreated spermatozoa. These results implicate a possible role for zinc as a scavenger of excessive superoxide anions produced by defective spermatozoa in semen after ejaculation. Additionally, zinc was found to dose-dependently inhibit superoxide dismutase (SOD)-like activity of spermatozoa in vitro. The inhibition of SOD-like activity by an equal concentration of zinc (1 mmol/L) was less pronounced in oligospermic (p < 0.002; n = 16) and asthenozoospermic (p < 0.0005; n = 20) than in normozoospermic samples (p < 0.0001; n = 20). This differential ability of zinc to inhibit SOD-like activity may be relevant to the physiological function of spermatozoa in fertilization. The evidence that zinc may elicit an inhibition of both superoxide anion production and SOD-like activity in human spermatozoa, indicate the existence of novel, zinc-related mechanism(s) involved in the oxidative events occurring after ejaculation, with a possible modulatory effect on germ cell function.     

严重烫伤后腹腔巨噬细胞游离钙及抗原提呈功能的实验研究

我们对严重烫伤小鼠腹腔巨噬细胞内游离钙PM(?)[Ca~(2 )]和抗原提呈(AP)功能进行了动态观察,结果显示:体外未受刺激的PM(?)[Ca~(2 )]在烫伤后12、24、72h均显著升高。这证明烫伤后早期PM(?)在体内就已被激活。加A23187刺激的PM(?)[Ca~(2 )]在伤后虽无显著升高,但也提示PM(?)处于高度活化,甚至过度活化的状态。严重烫伤后可使PM(?)的AP能力明显低下,其不仅发生早,而且持续时间较久,可能已成为伤后特异性免疫功能受抑制的主导因素。M(?)体内活性的改变则可能是烧伤后M(?)免疫功能异常的基础。     

严重烫伤后腹腔巨噬细胞游离钙及抗原提呈功能的实验研究

我们对严重烫伤小鼠腹腔巨噬细胞内游离钙ＰＭ［Ｃａ２＋］ｉ和抗原提呈（ＡＰ）功能进行了动态观察，结果显示：体外未受刺激的ＰＭ［Ｃａ２＋］ｉ在烫伤后１２、２４、７２ｈ均显著升高。这证明烫伤后早期ＰＭ在体内就已被激活。加Ａ２３１８７刺激的ＰＭ［Ｃａ２＋］ｉ在伤后虽无显著升高，但也提示ＰＭ处于高度活化，甚至过度活化的状态。严重烫伤后可使ＰＭ的ＡＰ能力明显低下，其不仅发生早，而且持续时间较久，可能已成为伤后特异性免疫功能受抑制的主导因素。Ｍ体内活性的改变则可能是烧伤后Ｍ免疫功能异常的基础。     

Carbon dioxide directly suppresses spontaneous migration, chemotaxis, and free radical production of human neutrophils

BACKGROUND: Carbon dioxide (CO(2)) insufflation during laparoscopy has been shown to dampen the systemic stress response to surgery. This is related to a suppression of peritoneal macrophage functions. In vivo data suggest that CO(2) can also affect neutrophils (polymorphonuclear cells, PMNs), the most abundant cell type in the inflamed peritoneal cavity. Nonetheless, the direct effects of CO(2) on PMNs have not yet been investigated. METHOD: PMNs were isolated from peripheral blood of healthy volunteers and incubated with (1) CO(2) (100% CO(2), pH 6.2), (2) hypoxic control (95% helium/5% CO(2), pH 7.4), and (3) control (95% air/5% CO(2), pH 7.4). Spontaneous and IL-8-induced migrations (chemokinesis and chemotaxis) during 2 h of exposure to different gases were measured with a transwell chamber system. The release of reactive oxygen species (ROS, luminometry) was determined after 15-min and 2-h exposures. In other sets of experiments, PMNs were exposed for 2 h or 4 h and kept under normal conditions for 18 h with lipopolysaccharide (LPS) stimulation thereafter. Final viability and apoptosis were assessed with fluorometry. RESULTS: Exposure to 100% CO(2) completely blocked spontaneous and IL-8 induced migration of PMNs (p < 0.001 vs. controls). Neutrophil migration was slightly diminished in the hypoxic control group. PMA-stimulated ROS production was reduced even after short exposure to 100% CO(2)(p < 0.05). We observed a slight increase of caspase-3/7 activity after exposure to 100% CO(2) and/or hypoxia; however, total viability was not affected. CONCLUSIONS: CO(2) incubation directly and temporarily suppresses the proinflammatory functions of PMNs; this is caused only partially by the concomitant hypoxia. This effect will contribute to the dampened inflammatory response to laparoscopic surgery. Further studies are needed to investigate whether the temporary suppression of neutrophil functions could affect the clearance of bacterial contaminations.     

The effect of preoperative corticosteroids on peritoneal macrophage function after laparoscopic and open abdominal surgery in a rat model

Background

Peritoneal macrophages play an important role in the immune response after abdominal operations. The stress response after these operations has been associated with impaired phagocytosis by peritoneal macrophages. This study examined the influence of minimally invasive techniques and preoperative corticosteroid administration on postoperative peritoneal macrophage phagocytic activity.

Methods

After IACUC approval, 66 Sprague Dawley rats were randomly divided into 7 groups: baseline animals (B), anesthesia controls (AC), open cecectomy (OC), and laparoscopic cecectomy (LC). Within the AC, OC, and LC groups, half received intraperitoneal (IP) dexamethasone (10 mg/kg) 1 hour before surgery (+S), and the other half received an equal volume of normal saline IP (−S). Animals were observed postoperatively for 24 hours and were then euthanized. Peritoneal macrophages were harvested via intraperitoneal lavage. A phagocytosis assay was performed to calculate the net phagocytosis and percent response to the effector agent. Statistical analysis was performed using analysis of variance and a Student t test between groups. A P value of <.05 was considered significant.

Results

Significant differences were observed between groups. The B group had a response rate of 94.2% ± 56.7%, which was not different from the AC groups (−S, P = .28; +S, P = .16) or the LC-S group (P = .9). The lowest phagocytic activity rate was in the OC-S group with a response rate of 33.8% ± 28.5%. The highest phagocytic response rates occurred in the AC +S (145.2% ± 60.2%) and LC +S (198.1% ± 103.5%). These were not significantly different from each other (P = .3). The LC +S group had a significantly higher percent response than all of the other groups. The phagocytic response rate of the OC +S group was not different from either the AC-S group (P = .07) or the LC-S group (P = .8); however, it was less than the AC +S group (P = .02) and the LC +S group (P = .003).

Conclusion

Open cecectomy resulted in greater impairment of the phagocytic activity of peritoneal macrophages than laparoscopic cecectomy. The addition of preoperative corticosteroids improved phagocytic activity back to baseline function. The combination of minimally invasive surgical technique and preoperative corticosteroid administration resulted in the greatest postoperative phagocytic function of peritoneal macrophages in a rat model.     

氯胺酮对嗜中性粒细胞粘附分子表达和超氧自由基产生的影响

目的 观察氯胺酮对人嗜中性粒细胞（PMN）粘附分子表达和超氧自由基（O2^-)产生的影响，以探讨其抗炎特征。方法 （1）将氯胺酮加入已离全血细胞，分别经甲硫氨酰-亮氨酰-苯丙氨酸（FMLP）或豆蔻酰佛波醇乙酯（PMA）刺激，孵化和单抗标定后，通过用分析了解β2-结合素（β2-integrin)和L-选择素（L-selectin)在PMN表面的表达情况；（2）分离并孵化PMN，利用细胞色素C还原色谱的改变来了解氯胺酮对FMLP或PMA诱导产生O2^-的抑制效果。结果 氯胺酮可显著抑制PMN受刺激引起PMN的β2-结合素表达，L-选择素蜕落和O2^-产生，氯胺酮抑制β2-结合素表达与其立体构型无关。结论 氯胺酮抑制PMN粘附和自由基生成，此作用不是通过与特异受体的反应实现的，对全身性炎症反应综合征（SIRS）和缺血再灌注损伤病人的麻醉镇静时宜选用该药。     

Effects of homocysteine and ginsenoside Rb1 on endothelial proliferation and superoxide anion production

BACKGROUND: Homocysteine (Hcy) is an independent risk factor for cardiovascular disease by its multiple effects on vascular cells and throbmosis factors, which may be involved in oxidative stress mechansims. Ginsenoside Rb1, a constituent of ginseng, bears various beneficial effects on the cardiovascular system. In the present study, we investigated the effect of Hcy on endothelial proliferation and a protective effect of ginsenoside Rb1 on the action of Hcy. METHODS: We initially incubated a mouse lymph node endothelial cell line (SVEC4-10) with increasing concentrations of Hcy or for different time periods and then assessed cell proliferation by using [(3)H]-thymidine incorporation. We then incubated SVEC4-10 cells with Hcy (50 microM) for 24 h with or without Rb1 (10 microM) to examine its inhibitory effect on the proliferation. These experiments were repeated in human umbilical vein endothelial cells (HUVECs). To explore the underlying molecular mechanisms, we measured superoxide anion, a reactive oxygen species (ROS), by using dihydroethidium (DHE) staining. RESULTS: SVEC4-10 cells treated with Hcy (50, 100, and 200 microM) for 24 h significantly reduced cell proliferation by 43%, 42%, and 40%, respectively, as compared with control cells (P < 0.01). SVEC4-10 cells treated with Hcy (50 microM) for 12 and 24 h showed a significant reduction of cell proliferation (P < 0.05). In HUVECs, Hcy (50 microM) significantly reduced cell proliferation by 55% as compared with control cells (P < 0.05). In the presence of Rb1, Hcy-induced inhibition of cell proliferation was effectively blocked in both SVEC4-10 and HUVECs. Furthermore, Hcy (50 microM) significantly increased superoxide anion production by 23% in SVEC4-10 as compared with control cells (P < 0.05). However, in the presence of Rb1, Hcy increased superoxide anion production by only 8%, showing that RB1 almost completely blocked the effect of Hcy. CONCLUSION: Hcy significantly inhibits endothelial proliferation with increased production of superoxide anion, which is effectively blocked by ginsenoside Rb1. This study provides some new aspects of Hcy-induced endothelial dysfunction, and suggests a potential role of Rb1 to block Hcy action, which may have clinical applications.     

断指再植术后患者血清丙二醛含量及超氧化物歧化酶活性变化

目的 探讨断指再植患者术后血清丙二醛(malondialdehyde,MDA)含量及超氧化物歧化酶(superoxide dismutase,SOD)活性变化特点.方法 对40例患者分别于手术后1、24、48、72 h,第7、14 d检测血清MDA含量和SOD活性,另外取30例健康成年人检测血清MDA含量和SOD活性作为对照组进行对比分析.结果 断指再植患者术后1h至7d血清MDA含量逐渐升高,血清SOD活性明显降低,与对照组比较差异有统计学意义(P＜0.05),术后14 d血清中MDA及SOD基本达到对照组水平(P＞0.05).结论 术后检测MDA含量及SOD活性的变化可以指导临床用药,有效地防治血管危象的发生.     

Effect of free radical scavengers on myocardial function and Na+, K+-ATPase activity in stunned rabbit myocardium

Objectives. The role of reactive oxygen species (ROS) in the mechanism of myocardial stunning was investigated. Material and methods. Isolated Langendorff-perfused rabbit hearts were subjected to 15?min normothermic ischemia followed by 10?min reperfusion with Krebs-Henseleit solution±mannitol or histidine. Results. In hearts reperfused without free radical scavenger the left ventricular developed pressure as well as its maximal positive and negative first derivatives (+dP/dt, ?dP/dt) was significantly depressed, whereas end diastolic pressure (LVEDP) increased when compared to preischemic values. Treatment with mannitol had little protective effects, whereas singlet oxygen scavenger histidine significantly improved the recovery of LVEDP and ?dP/dt. Sarcolemmal Na⁺, K⁺-ATPase activity (control, 400±41 nmol P_i.min^?1.mg^?1) was depressed in untreated stunned hearts (260±27 nmol P_i.min^?1.mg^?1), but was almost completely recovered in hearts pretreated with histidine (364±27 nmol P_i.min^?1.mg^?1). The inhibition of Na⁺, K⁺-ATPase was only slightly prevented by mannitol (302±29 nmol P_i.min^?1.mg^?1l). Conclusions. The results suggest that ROS-induced inhibition of Na⁺, K⁺-ATPase activity is involved in the mechanism of postischemic contractile dysfunction and support the view that singlet oxygen may be one of the major causes of oxidative injury during ischemia and reperfusion.     

Detrusor mitochondrial lipid peroxidation and superoxide dismutase activity in partial bladder outlet obstruction of rabbits

OBJECTIVES: Bladder outlet obstruction has shown to damage detrusor mitochondria with impaired detrusor contractility. One likely cause for mitochondrial injury is reactive oxygen species (ROS)-induced damages, including lipid peroxidation injury. We designed this study to examine this hypothesis. MATERIALS AND METHODS: Placing a silicon ring around the bladder neck of male New Zealand rabbits induced bladder outlet obstruction. The bladders were removed 3 (N = 6), 7 (N = 6), and 14 days (N = 8) later. Sham operated animals (N = 6 for each time period) served as the controls. Contractile function of the bladder was assessed by the response of the detrusor strips to bethanechol and field stimulation. Detrusor mitochondrial superoxide dismutase (SOD) activity and mitochondrial content of malondialdehyde (MDA) were determined. Detrusor contents of phosphocreatine and adenine triphosphate (ATP) were assayed. RESULTS: (1) Outlet obstruction induced an increase in the bladder weight and a decrease in the contractile function; (2) mitochondrial SOD activity significantly elevated in every time period of the obstruction, indicating a persistently increased ROS generation; (3) detrusor MDA level increased in 3-day obstruction animals. It returned to the control level in 7- and 14-day groups; (4) phosphocreatine content was significantly reduced in every time period of the obstruction; (5) ATP content was significantly decreased in 3- and 7-day groups; while 14-day obstruction group contained similar level as the sham-operated group. CONCLUSIONS: This study shows that bladder outlet obstruction increases generation of ROS and enhances lipid peroxidation of detrusor mitochondria. The resulted mitochondrial damages might sustain, leading to persistently depressed energy production and impaired detrusor contractility.     

The effects of pentoxifylline on renal function and free radical production in unilateral ureteral obstruction

There is an ongoing discussion about ureteral obstruction-related renal dysfunction. In this study, we aimed to test the effect of pentoxifylline (PTX) on both kidneys in unilateral ureteral obstruction (UUO), and to determine its interaction of with prostaglandin E₂ (PGE₂), and diclofenac sodium (DIS). A sham operation was performed in group 1. Placebo, PTX, DIS, and PTX+DIS were administrated to groups 2, 3, 4 and 5, respectively. The left ureter was ligated in all groups except group 1. At 24 h, technetium 99m diethylenetriamine penta-acetic acid scintigraphy was performed to determine renal function. Additionally, the tissue levels of thiobarbituric acid reactive substances (TBARS) and PGE₂ in both kidneys were measured to determine cytotoxic and cytoprotective mechanisms. When the ipsilateral kidneys were evaluated: (1) UUO significantly reduced DTPA uptake and none of the medications used prevented the reduction, (2) UUO significantly increased TBARS production, and only PTX prevented the increase, (3) UUO caused a significant increase in PGE₂ production, and only DIS significantly decreased this. When the contralateral kidneys were evaluated: (1) UUO significantly increased DTPA uptake but DIS and PTX+DIS prevented this, (2) UUO significantly elevated TBARS levels and DIS and PTX+DIS caused an additional elevation, (3) UUO significantly increased PGE₂ production, and only DIS prevented this. In conclusion, UUO caused ipsilateral renal hypofunction and contralateral hyperfunction, which are related to increased TBARS and PGE₂ levels. PTX markedly decreased free radical activity in the ipsilateral kidney. While PTX showed a placebo effect, DIS prevented the compensatory contralateral renal response through increased TBARS and decreased PGE₂ levels. The beneficial effect of PTX on the ipsilateral kidney, and the hazardous effect of DIS on the contralateral kidney may be explained by more complex interactions among TBARS, PGE₂, PTX, DIS and UUO-related renal dysfunction.     

Carbon monoxide production from desflurane and six types of carbon dioxide absorbents in a patient model

BACKGROUND: Desflurane is known to produce high concentrations of carbon monoxide (CO) in desiccated sodalime or Baralyme (Allied Healthcare Products, St. Louis, MO). Desiccated absorbents without strong bases like potassium hydroxide or sodium hydroxide are reported to produce less or no CO at all. The purpose of this study is to compare the concentration of CO in an anesthesia circuit for desflurane with six different types of completely desiccated CO(2) absorbents with less strong bases than sodalime. METHODS: A patient model was simulated using a circle anesthesia system connected to an artificial lung. Completely desiccated CO(2) absorbent (950 g) was used in this system. A low flow anesthesia (500 ml min(-1)) was maintained using desflurane. For immediate quantification of CO production a portable gas chromatograph was used. RESULTS: Peak concentrations of CO were very high in Medisorb (Datex-Ohmeda, Hoevelaken, The Netherlands) and Spherasorb (Intersurgical, Uden, The Netherlands) (13317 and 9045 p.p.m., respectively). It was lower with Loflosorb (Intersurgical, Uden, The Netherlands) and Superia (Datex-Ohmeda, Hoevelaken, The Netherlands) (524 and 31 p.p.m., respectively). Amsorb (Armstrong, Coleraine, N. Ireland) and lithium hydroxide produced no CO at all. CONCLUSION: Medisorb and Spherasorb are capable of producing large concentrations of CO when desiccated. Loflosorb and Superia produce far less CO under the same conditions. Amsorb and lithium hydroxide should be considered safe when desiccated.