饲料硒和维生素E对大鼠机体抗氧化能力的影响

 用克山病病区粮配成基础低硒饲料,补充硒和/或维生素E组成四种不同水平的饲料,饲喂雄性断乳大鼠,观察其对机体抗氧化能力的影响。评价指标是用抗坏血酸诱发的红细胞溶血率、被O~-_2(超氧阴离子)氧化的血红蛋白量和组织中的TBA值。动物饲养13周后,自尾静脉取血,测定溶血率和血红蛋白被氧化的百分率,和全血SeGSHPx(含硒谷胱甘肽过氧化物酶)活力。15周后将动物断头杀死,立即取出心脏和肝脏测定SeGSHPx活力和TBA值。结果表明在克山病病区粮的饲料中补充硒或维生素E,或者二者同时补充均明显提高组织中的SeGSHPx活力和降低组织中的TBA值。不论在硒缺乏时或硒充足时,饲料中补充维生素E显著降低抗坏血酸诱发的红细胞溶血率,对O~-_2氧化血红蛋白无保护作用。在维生素E缺乏时,仅补充硒对溶血无作用。不论饲料中维生素E缺乏或者充足,补充硒对O~-_1氧化血红蛋白均有显著保护作用。     

妊娠晚期胎羊宫内外科手术的实验研究

目的　以妊娠晚期孕羊为研究对象 ,探讨宫内外科手术的可行性 ,为开展胎儿外科研究建立动物实验模型。方法　通过对妊娠 12 0d左右的 4只双胎孕羊进行宫腔手术 ,切除双胎中 1只胎羊的脚趾或唇部皮肤 ,观察妊娠结果。并取分娩后羔羊脑组织及手术切除部位皮肤作组织形态学观察和评价。结果　妊娠羊经阴道足月分娩后羔羊均成活 ,脚趾及唇部切除处未见疤痕形成 ,皮肤组织形态学观察对照组与实验组未见明显差异 ;脑组织形态学检查仅有 1例实验组羔羊有轻度病理学变化 ,与对照羔羊有差异 ,其余 3例实验羔羊与对照羔羊脑组织结构均正常 ,未见有明显的病理学改变。结论　妊娠羊经宫内胎羊外科手术后能够持续妊娠状态并经阴道自然分娩 ,产出羔羊的精神状态及病理组织学观察结果表明 ,利用妊娠晚期孕羊进行胎儿外科动物实验研究是可行的 ,为进一步开展人类先天性畸形胎儿的早期治疗奠定了动物实验基础     

蝮蛇抗栓酶防治脑血管疾病的机理及其对微循环作用的研究

通过脑膜微循环、脑神经细胞酶活性测量、脑组织病理及超微结构实验研究和１３４例缺血性脑病病人临床治疗证明,抗栓酶治疗缺血性脑病的效果是确切的;其机理除降低血粘度、解聚、溶纤外,还发现有增强微循环血流动力学、保护血管内皮细胞,改善微血管舒张功能的调控作用和对脑细胞酶活性及脑神经细胞的保护作用。     

啮齿类动物插管技术用于药动学采血的研究进展

近几十年来,人们持续关注实验动物的福利,并在全球范围的科学研究中整合、协调和监督实验动物的使用。"3R原则"是被广泛认可接受的标准和规范的核心要义和精髓所在。药代动力学研究中包含了必不可少的动物实验,如何保护实验动物福利是本专业发展到一定阶段和水平需要考虑的问题。国内外科研人员逐渐建立并优化在啮齿类动物(主要为大鼠和小鼠)上进行插管手术的技术,能让实验动物在清醒及自由活动状态下完成采血等实验。本文通过整理相关文献,对药动学研究中使用的插管技术进行梳理和总结,希望对国内该研究领域的实验动物福利保护有所助力。     

微波对豚鼠耳蜗微循环的影响

本文对７４只豚鼠,通过颈静脉注入大剂量的速尿,建立了豚鼠急性耳蜗微循环障碍动物模型。利用动态观测手段中的激光多普勒测试技术及静态观察方法的螺旋韧带血管纹红细胞计数技术,探讨了微波对成年豚鼠耳蜗微循环的保护作用。为保护和改善动物的听力水平提供更多的资料。     

含棉籽浓缩蛋白饲料添加酵母硒对草鱼生长性能和肌肉品质的影响

研究旨在探讨含棉籽浓缩蛋白(Cottonseed protein concentrate)饲料添加酵母硒(Yeast selenium, YS)对草鱼(Ctenopharyngodon idella)生长性能和肌肉品质的影响。设计了4种等氮等脂实验饲料,分别是在含棉籽浓缩蛋白饲料中添加0 (YS0)、0.3 (YS3)、0.6 (YS6)和0.9 mg/kg (YS9)酵母硒;实验选取初始体重为(309.74±0.36) g的草鱼240尾,随机分成4组,分别投喂4种实验饲料,在池塘网箱中开展为期54d的养殖实验。结果显示:(1)在生长性能方面,与YS0组相比, YS3和YS6组草鱼在生长性能上无显著性差异(P>0.05), YS9组草鱼的终末体重、增重率和特定生长率显著降低(P<0.05)。(2)在肌肉品质方面, YS3组草鱼肌肉的粗蛋白含量、硒含量、羟脯氨酸含量和硬度显著高于YS0组(P<0.05),肌肉纤维直径显著低于YS0组(P<0.05)。研究表明,在含棉籽浓缩蛋白的饲料中添加适量酵母硒(饲料硒含量为0.6 mg/kg),对草鱼的生长性能无影响,且可通过...     

大鼠心脏低温保存及高钾停跳保护作用的ESR研究

用ESR研究大鼠离体心脏低温长时间缺血后再灌注时产生的自由基,并观察心肌超微结构的变化;对临床心脏直视手术中常用的高钾停跳液的保护作用机理进行了分析。实验结果表明超氧自由基信号强度随缺血温度的降低而明显减弱。高钾停跳液保护的大鼠离体心脏于低温(4℃)缺血后再灌15s,4h内超氧自由基信号强度变化不明显。缺血4h后再灌注,实验组心脏全部于15s内自动复跳,超微结构的变化轻微。以上改变与对照组比较有明显的差异。本实验为低温保存心脏及高钾停跳液的临床应用提供了实验依据。     

ATP-MgCl_2对心肌缺血再灌注产生O~-_■的清除作用

McCord的研究结果表明,心肌缺血再灌注性损伤是活性氧自由基——主要是超氧阴离子自由基的毒性引起的。我们的研究证明,ATP-MgCl_2心脏冷停搏保护液在动物实验和临床心脏外科手术中,对缺血缺氧心肌有着十分满意的保护作用。 我们用电子自旋共振波谱仪(ESR)分别检测了以含氧灌注液、含氧灌注液加入SOD和含氧灌注液加入ATP—MgCl_2对离体兔心再灌注时氧自由基的变化,以阐明ATP-MgCl_2是否具有清除活性氧自由基的作用。     

低硒心肌损伤与钾离子通道蛋白的关系研究

目的:心肌上的离子通道蛋白与心肌损伤有很大的关系,本研究通过低硒喂养对C57BL/6小鼠心肌组织损伤的影响及其对钾通道蛋白的改变。方法:将实验小鼠分为4组:对照组,低硒30天组,低硒90天组和低硒180天组。采用低硒饲料(硒含量0.0045μg/g)喂养的方法建立低硒小鼠模型,对照组给予正常饲料(硒含量0.256μg/g),与低硒组同时喂养;硒含量的测定和HE染色方法观察心肌损伤情况,Western Blotting方法检测其钾通道蛋白的表达。结果:低硒饲料喂养小鼠的心脏硒含量与正常饲料喂养的硒含量相比明显降低(P0.01);并出现轻微的心肌损伤,钾通道蛋白的表达量在低硒30天组,低硒90天组和低硒180天组下调(P0.01)。结论:成功建立低硒小鼠模型,低硒能引起小鼠心肌损伤,这种改变可能有心脏的钾通道蛋白的表达水平有关。     

内脏器官活动的神经调节

内脏器官活动调节的研究,是生理学最重要的问题之一。在人和动物的身体内经常进行着各种非常复杂的、彼此间十分协调的生理过程,这些过程的进行是由有机体的全部器官和生理系统来实现的。例如,肌肉活动的时候,可以观察到心脏、肺、肝脏、脾脏和其他器官的活动都有显著的变化。由于心脏收缩的加强,使增加了通过正在活动着的肌肉的血流速度,     

Effects of erythrocyte flexibility on microvascular perfusion and oxygenation during acute anemia

Responses to exchange transfusion using red blood cells (RBCs) with normal and reduced flexibility were studied in the hamster window chamber model during acute moderate isovolemic hemodilution to determine the role of RBC membrane stiffness in microvascular perfusion and tissue oxygenation. Erythrocyte stiffness was increased by 30-min incubation in 0.02% glutaraldehyde solution, and unreacted glutaraldehyde was completely removed. Filtration pressure through 5-microm pore size filters was used to quantify stiffness of the RBCs. Anemic conditions were induced by two isovolemic hemodilution steps using 6% 70-kDa dextran to a hematocrit (Hct) of 18% (moderate hemodilution). The protocol continued with an exchange transfusion to reduce native RBCs to 75% of baseline (11% Hct) with either fresh RBCs (RBC group) or reduced-flexibility RBCs (GRBC group) suspended in 5% albumin at 18% Hct; a plasma expander (6% 70-kDa dextran; Dex70 group) was used as control. Systemic parameters, microvascular perfusion, capillary perfusion [functional capillary density (FCD)], and oxygen levels across the microvascular network were measured by noninvasive methods. RBC deformability for GRBCs was significantly decreased compared with RBCs and moderate hemodilution conditions. The GRBC group had a greater mean arterial blood pressure (MAP) than the RBC and Dex70 groups. FCD was substantially higher for RBC (0.81 +/- 0.07 of baseline) vs. GRBC (0.32 +/- 0.10 of baseline) and Dex70 (0.38 +/- 0.10 of baseline) groups. Microvascular tissue Po(2) was significantly lower for Dex70 and GRBC vs. RBC groups and the moderate hemodilution condition. Results were attributed to decreased oxygen uploading in the lungs and obstruction of tissue capillaries by rigidified RBCs, indicating that the effects impairing RBC flexibility are magnified at the microvascular level, where perfusion and oxygenation may define transfusion outcome.     

Red blood cells prevent inhibition of hypoxic pulmonary vasoconstriction by nitrite in isolated, perfused rat lungs

Nitrite reduction to nitric oxide (NO) may be potentiated by a nitrite reductase activity of deoxyHb and contribute to systemic hypoxic vasodilation. The effect of nitrite on the pulmonary circulation has not been well characterized. We explored the effect of nitrite on hypoxic pulmonary vasoconstriction (HPV) and the role of the red blood cell (RBC) in nitrite reduction and nitrite-mediated vasodilation. As to method, isolated rat lungs were perfused with buffer, or buffer with RBCs, and subjected to repeated hypoxic challenges, with or without nitrite. As a result, in buffer-perfused lungs, HPV was reduced at nitrite concentrations of 7 muM and above. Nitrite inhibition of HPV was prevented by excess free Hb and RBCs, suggesting that vasodilation was mediated by free NO. Nitrite-inhibition of HPV was not potentiated by mild acidosis (pH = 7.2) or xanthine oxidase activity. RBCs at 15% but not 1% hematocrit prevented inhibition of HPV by nitrite (maximum nitrite concentration of approximately 35 muM) independent of perfusate Po(2). Degradation of nitrite was accelerated by hypoxia in the presence of RBCs but not during buffer perfusion. In conclusion, low micromolar concentrations of nitrite inhibit HPV in buffer-perfused lungs and when RBC concentration is subphysiological. This effect is lost when RBC concentration approaches physiological levels, despite enhanced nitrite degradation in the presence of RBCs. These data suggest that, although deoxyHb may generate NO from nitrite, insufficient NO escapes the RBC to cause vasodilation in the pulmonary circulation under the dynamic conditions of blood flow through the lungs and that RBCs are net scavengers of NO.     

Glutathione protects chemokine-scavenging and antioxidative defense functions in human RBCs

Oxidant stress, in vivo or in vitro, isknown to induce oxidative changes in human red blood cells (RBCs). Ourobjective was to examine the effect of augmenting RBC glutathione(GSH) synthesis on 1) degenerative protein loss and2) RBC chemokine- and free radical-scavenging functions inthe oxidatively stressed human RBCs by using banked RBCs as a model.Packed RBCs were stored up to 84 days at 1-6°C in Adsol or inthe experimental additive solution (Adsol fortified with glutamine,glycine, and N-acetyl-L-cysteine). Supplementingthe conventional additive with GSH precursor amino acids improved RBCGSH synthesis and maintenance. The rise in RBC -glutamylcysteineligase activity was directly proportional to the GSH content andinversely proportional to extracellular homocysteine concentration,methemoglobin formation, and losses of the RBC proteins band 3, band4.1, band 4.2, glyceraldehyde-3-phosphate dehydrogenase, and Duffyantigen (P < 0.01). Reduced loss of Duffy antigencorrelated well with a decrease in chemokine RANTES (regulated uponactivation, normal T-cell expressed, and secreted) concentration. Weconclude that the concomitant loss of GSH and proteins in oxidatively stressed RBCs can compromise RBC scavenging function. Upregulating GSHsynthesis can protect RBC scavenging (free radical and chemokine) function. These results have implications not only in a transfusion setting but also in conditions like diabetes and sickle cell anemia, inwhich RBCs are subjected to chronic/acute oxidant stresses.

     

Glutathione peroxidase activities during selenium depletion of adult female rats and during selenium repletion of their offspring

The main purpose of the present investigation was to produce young rats with severe selenium deficiency, but with no clinical signs of this deficiency, and to examine their liver and red blood cell (RBC) glutathione peroxidase activities during selenium repletion. To achieve this goal, female breeders were fed a selenium-deficient diet beginning 2 weeks before mating. The liver glutathione peroxidase activity of the dams was significantly lower than the activity of comparable nonpregnant females after 5 and 10 weeks of selenium depletion. This difference arose exclusively during the period of pregnancy. In contrast, the RBC glutathione peroxidase activity was significantly increased during this period. Only traces of liver enzyme activity were found in the offspring, and the RBC enzyme activity was only 2% of that of the selenium-repleted controls. Body weight was retarded in the male offspring. However, no severe signs of clinical selenium deficiency were observed. The glutathione peroxidase activity in the liver and RBCs of the offspring was determined after 0, 2, 4, 7, 14, and approximately 40 days of selenium repletion. The liver enzyme activity increased faster in females than in males, while the opposite was found for the RBCs. After 14 days of selenium repletion, the glutathione peroxidase activity of the liver was essentially restored, and the RBC enzyme activity was about half that of the control values. This type of rat may prove useful in studies in which young selenium-deficient rats are preferable, as well as in studies of selenium functions that might not be directly related to the role of selenium in glutathione peroxidase.     

Inborn defects in the antioxidant systems of human red blood cells

Red blood cells (RBCs) contain large amounts of iron and operate in highly oxygenated tissues. As a result, these cells encounter a continuous oxidative stress. Protective mechanisms against oxidation include prevention of formation of reactive oxygen species (ROS), scavenging of various forms of ROS, and repair of oxidized cellular contents. In general, a partial defect in any of these systems can harm RBCs and promote senescence, but is without chronic hemolytic complaints. In this review we summarize the often rare inborn defects that interfere with the various protective mechanisms present in RBCs. NADPH is the main source of reduction equivalents in RBCs, used by most of the protective systems. When NADPH becomes limiting, red cells are prone to being damaged. In many of the severe RBC enzyme deficiencies, a lack of protective enzyme activity is frustrating erythropoiesis or is not restricted to RBCs. Common hereditary RBC disorders, such as thalassemia, sickle-cell trait, and unstable hemoglobins, give rise to increased oxidative stress caused by free heme and iron generated from hemoglobin. The beneficial effect of thalassemia minor, sickle-cell trait, and glucose-6-phosphate dehydrogenase deficiency on survival of malaria infection may well be due to the shared feature of enhanced oxidative stress. This may inhibit parasite growth, enhance uptake of infected RBCs by spleen macrophages, and/or cause less cytoadherence of the infected cells to capillary endothelium.     

Reduced erythrocyte deformability alters pulmonary hemodynamics

Isolated rat lungs were perfused with suspensions containing normal and stiffened erythrocytes (RBCs) to assess the effect of altered RBC deformability on pulmonary hemodynamics. RBC suspensions were prepared using cells previously incubated in isosmolar phosphate-buffered saline with or without 0.0125 or 0.01875% glutaraldehyde. Washed RBCs were resuspended in isosmolar 4% albumin saline solution. Isolated rat lungs were perfused with control and stiffened cells by the use of a perfusion system that allowed rapid switching between suspensions. Pressure-flow (P/Q) curves were constructed by measuring pulmonary arterial pressure (Ppa) over a range of flow rates. In a second set of experiments, P/Q curves were generated for perfusion with control and stiffened cells (0.0125% glutaraldehyde) before and after vasoconstriction with a synthetic prostaglandin analogue (U 46619). RBC deformability was quantified in all experiments by determination of filtration time of a dilute cell suspension through a 4.7 microns Nuclepore filter. Incubation with 0.0125 or 0.01875% glutaraldehyde produced a 6 or 21% decrease in RBC deformability, respectively. These decreases in deformability were associated with significant increases in Ppa at each flow rate. The increases in Ppa correlated significantly with the degree of RBC stiffening. With 0.0125% glutaraldehyde, the P/Q curve was shifted upward without a change in slope, whereas incubation with 0.01875% glutaraldehyde resulted in a significant increase in slope. Vasoconstriction and perfusion with stiffened RBCs had additive effects on Ppa. These findings suggest that decreases in RBC deformability cause physiologically significant elevations in hemodynamic resistance in the pulmonary circuit independent of vasoactivity.     

Oxidative insult in sheep red blood cells induced by T-butyl hydroperoxide: the roles of glutathione and glutathione peroxidase

Three different types of red blood cells (RBC) were used: (i) RBC from sheep having genetically high GSH (ii) RBC from sheep with genetically low GSH and (iii) RBC from high-GSH sheep treated with CDNB to deplete GSH. Incubation of these RBC with t-butyl hydroperoxide (tBHP, 3 mM) for 10 min caused the formation of TBARS, oxidation of haemoglobin and degradation and aggregation of membrane proteins in RBC from low-GSH sheep and GSH-depleted RBC. By contrast, RBC from high-GSH sheep (normal RBC) did not show the degradation and aggregation of membrane proteins within the first 10 min. Dithiothreitol (DTT) was highly effective in preventing the tBHP-mediated oxidation of haemoglobin, the formation of TBARS and the degradation and aggregation of membrane proteins in both normal RBC and low-GSH RBC. However, DTT did not provide protection in GSH-depleted RBC or normal RBCs in the presence of 1.5 mM mercaptosuccinate (MCS), a potent inhibitor of GSH peroxidase (GSHPx). The ability of GSH to prevent the oxidation of haemoglobin and the degradation and aggregation of membrane proteins was abolished in the presence of MCS. These results indicate that the protective function of DTT involves a GSH-dependent mechanism. Both GSH and GSHPx play key roles in this enzymatic system. In the light of the complete protection of RBC against oxidation induced by tBHP in the presence of DTT or GSH, the GSH/GSHPx system appears to act directly as a tBHP scavenger. The activities of four well-known antioxidants, Butylated hydroxytoluene, ascorbate, alpha-tocopherol and desferrioxamine were also tested in this study to cast further light on the role of free radical scavenging in protection from tBHP mediated free radical insult.     

Mechanical behavior of the erythrocyte in microvessel stenosis

The passage of red blood cells (RBCs) through capillaries is essential for human blood microcirculation. This study used a moving mesh technology that incorporated leader-follower pairs to simulate the fluid-structure and structure-structure interactions between the RBC and a microvessel stenosis. The numerical model consisted of plasma, cytoplasm, the erythrocyte membrane, and the microvessel stenosis. Computational results showed that the rheology of the RBC is affected by the Reynolds number of the plasma flow as well as the surface-to-volume ratio of the erythrocyte. At a constant inlet flow rate, an increased plasma viscosity will improve the transit of the RBC through the microvessel stenosis. For the above reasons, we consider that the decreased hemorheology in microvessels in a pathological state may primarily be attributed to an increase in the number of white blood cells. This leads to the aggregation of RBCs and a change in the blood flow structure. The present fundamental study of hemorheology aimed at providing theoretical guidelines for clinical hemorheology.     

Hemoglobin-mediated selenium export from red blood cells

On the basis of the fact that selenium from selenite binds to hemoglobin (Hb), we investigated the missing process in the selenium export from red blood cells (RBCs), i.e., the transfer of selenium bound to Hb to RBC membrane proteins. To elucidate the molecular events of the Hb-associated selenium export from RBC, a Hb–Se complex was synthesized from thiol-exchange of Cys-β93 in Hb with penicillamine-substituted glutathione selenotrisulfide, as a model of major metabolic intermediates, and then interactions between the Hb–Se complex and RBC inside-out vesicles (IOVs) were examined. Selenium bound to Hb was transferred to the IOV membrane on the basis of the intrinsic interactions between Hb and the cytoplasmic domains of band 3 protein (CDB3). The observed selenium transfer was inhibited by the pretreatments of IOVs with iodoacetamide and the α-chymotrypsin digestion, indicating that the Hb mediates the selenium transfer to the thiol groups of CDB3. In addition, it was found that deoxygenated Hb, with a high binding affinity for CDB3, more favorably transferred selenium to the IOV membranes than oxygenated Hb, with a low affinity. When selenium export from RBC to the plasma was examined by continuously introducing nitrogen gas, the selenium export rate was promoted with an increase in the rate of deoxygenated Hb. Overall, these data suggested that Hb could possibly play a role in the selenium export from RBC treated with selenite in an oxygen-linked fashion. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Red blood cell washing,nitrite therapy,and antiheme therapies prevent stored red blood cell toxicity after trauma–hemorrhage

Transfusion of stored red blood cells (RBCs) is associated with increased morbidity and mortality in trauma patients. Pro-oxidant, pro-inflammatory, and nitric oxide (NO) scavenging properties of stored RBCs are thought to underlie this association. In this study we determined the effects of RBC washing and nitrite and antiheme therapy on stored RBC-dependent toxicity in the setting of trauma-induced hemorrhage. A murine (C57BL/6) model of trauma–hemorrhage and resuscitation with 1 or 3 units of RBCs stored for 0–10 days was used. Tested variables included washing RBCs to remove lower MW components that scavenge NO, NO-repletion therapy using nitrite, or mitigation of free heme toxicity by heme scavenging or preventing TLR4 activation. Stored RBC toxicity was determined by assessment of acute lung injury indices (airway edema and inflammation) and survival. Transfusion with 5 day RBCs increased acute lung injury indexed by BAL protein and neutrophil accumulation. Washing 5 day RBCs prior to transfusion did not decrease this injury, whereas nitrite therapy did. Transfusion with 10 day RBCs elicited a more severe injury resulting in ~90% lethality, compared to <15% with 5 day RBCs. Both washing and nitrite therapy significantly protected against 10 day RBC-induced lethality, suggesting that washing may be protective when the injury stimulus is more severe. Finally, a spectral deconvolution assay was developed to simultaneously measure free heme and hemoglobin in stored RBC supernatants, which demonstrated significant increases of both in stored human and mouse RBCs. Transfusion with free heme partially recapitulated the toxicity mediated by stored RBCs. Furthermore, inhibition of TLR4 signaling, which is stimulated by heme, using TAK-242, or hemopexin-dependent sequestration of free heme significantly protected against both 5 day and 10 day mouse RBC-dependent toxicity. These data suggest that RBC washing, nitrite therapy, and/or antiheme and TLR4 strategies may prevent stored RBC toxicities.