Fibrin glue eliminates the need for packing after complex liver injuries.

Hemostasis after traumatic liver injury can be extremely difficult to obtain, particularly in coagulopathic patients who have suffered extensive liver damage. We determined the ability of a fibrin glue preparation (FG) to terminate ongoing bleeding using a new, clinically relevant porcine model of complex hepatic injury. Anesthetized swine (n = 6, 18 to 19 kg) received an external blast to the right upper abdomen and were immediately anticoagulated with intravenous heparin (200 u/kg). Uncontrolled hemorrhage from blast continued from time of injury (t = 0 minutes) to t = 15 minutes. Lactated Ringer's solution was infused to keep mean arterial pressure (MAP) > 80 mm Hg until the end of experiment (t = 90 minutes). Animals underwent routine surgical techniques to control bleeding, and FG was employed in the event these measures failed. Estimated blood loss and fluid resuscitation volume were measured. Serial MAP, arterial base excess, and temperature were recorded. Animals were severely injured with significant blood loss prior to laparotomy (26 +/- 6 cc/kg) and during routine surgical efforts to arrest hemorrhage (11 +/- 2 cc/kg). Bleeding could not be controlled with standard techniques in any animal. FG rapidly controlled hemorrhage and eliminated the need for packing. Re-bleeding was noted in only one animal (portal vein injury). FG can control severe hepatic hemorrhage when surgical techniques fail. Further work in the clinical arena is warranted to determine the potential benefits of FG in arresting hemorrhage in hemodynamically unstable coagulopathic patients with complex hepatic injuries.     

Premedication with meloxicam exacerbates intracranial haemorrhage in an immature swine model of non-impact inertial head injury

Meloxicam is a cyclo-oxygenase-2 (COX-2) preferential non-steroidal anti-inflammatory drug with very effective analgesic and anti-inflammatory effects in swine. Previous reports in piglets have demonstrated that meloxicam also inhibits COX-1 and reduces production of thromboxane significantly. We use preinjury analgesia in our immature swine (3-5-day-old piglets) model of brain injury using rapid head rotations without impact. In 23 consecutive subjects we found that premedication with meloxicam (n = 6) produced a significantly higher mortality rate (5/6 or 83%) than buprenorphine (n = 17, 1/17 or 6%, P < 0.02). On gross neuropathological examination of the meloxicam-treated swine, we observed massive subdural and subarachnoid bleeding which were not present in buprenorphine-premedicated animals. To our knowledge there are no previous reports in swine of increased bleeding or platelet inhibition associated with meloxicam administration and further research is needed to define mechanisms of action in piglets. We caution the use of meloxicam in swine when inhibition of platelet aggregation might adversely affect refinement of experimental research protocols, such as in stroke, trauma and cardiac arrest models.     

藕节止血作用研究

本实验通过测定毛细管凝血时间(CT)、剪尾法出血时间(BT)、活化部分凝血酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)、优球蛋白溶解时间(ELT)等指标研究藕节醇提物及其不同极性段的体内止血作用.结果表明,藕节醇提物显著缩短小鼠CT、BT,显示很好的止血作用,可能是主要通过缩短APTT、PT,延长ELT...     

善宁联合垂体后叶素治疗肝硬化合并上消化道出血疗效观察

目的探讨醋酸奥曲肽(善宁)联合垂体后叶素治疗肝硬化合并上消化道出血的临床效果。方法选择2015年10月~2016年2月在我院消化内科接受治疗的肝硬化合并上消化道出血患者80例,随机分为对照组和观察组各40例,对照组给予去甲肾上腺素口服治疗,观察组在对照组的基础上应用善宁联合垂体后叶素静脉治疗,并对两组患者的止血效果进行比较。结果观察组的止血有效率为95%,对照组为60%(χ2=14.050,P=0.000),观察组止血效果显著高于对照组,差异具有统计学意义(P0.05)。结论善宁联合垂体后叶素治疗肝硬化合并上消化道出血的疗效显著,是治疗肝硬化合并上消化道出血的有效药物,值得临床推广应用。     

Iron protects porcine plasma coagulation kinetics from degradation by <Emphasis Type="Italic">Crotalus atrox</Emphasis> venom

While the administration of antivenom to treat hemotoxic snake bite injury remains the gold standard of therapy, we have demonstrated that modifying human fibrinogen with iron and carbon monoxide renders it resistant to fibrinogenolytic snake venom enzymes. In order to translate these findings into a possible biometal-based therapy complementary to antivenom administration, a preclinical model that possesses fibrinogen that closely mimics the human molecule in response to iron and carbon monoxide needed to be identified. The goal of this investigation was to determine if a swine model could serve in this capacity by assessing the thrombelastographic response of porcine plasma to iron and carbon monoxide exposure, without or with further exposure to the fibrinogenolytic venom of the viper Crotalus atrox. Using plasma obtained from eight swine, it was determined that their plasma responded to iron and carbon monoxide in a manner similar to that of human plasma by displaying enhanced coagulation kinetics. However, in sharp contrast to the response seen with human plasma, only iron significantly protected porcine plasma coagulation kinetics from C. atrox venom degradation. Therefore the pig is an animal beyond humans that could derive benefit from the biometal-focused therapy of iron infusion to protect against venom mediated compromise of coagulation. Thus, future investigation to assess the effects of iron administration to attenuate the effects of fibrinogenolytic envenomation with a pig model is justified.     

Variability in response to cryoprecipitate treatment for hemostatic defects in uremia

Cryoprecipitate is frequently administered as treatment for hemostatic defects in patients with uremia. The only published data supporting this approach however, involves seven patients described by Janson and colleagues in whom bleeding times were shortened and bleeding complications reduced after cryoprecipitate infusion. We retrospectively reviewed our institution's experience with cryoprecipitate in this setting. Five patients had sufficiently complete data for evaluation of the efficacy of therapy with cryoprecipitate, including pretreatment bleeding time greater than 15 minutes, normal coagulation studies, and platelet count greater than 100,000/microliters. Two patients had normalization of their bleeding time and a favorable clinical outcome after cryoprecipitate infusion. Three patients failed to shorten their bleeding time after cryoprecipitate infusion or, in one case, multiple infusions. One of these latter patients had correction of his abnormal bleeding time after subsequent administration of deamino-8-D-arginine vasopressin (DDAVP). We conclude that the hemostatic response to cryoprecipitate therapy is variable, and that cryoprecipitate therapy does not achieve restoration of normal hemostasis in some patients with uremic bleeding.     

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.     

Nonclinical aspects of venous thrombosis in pregnancy

Pregnancy is a hypercoagulable state which carries an excess risk of maternal venous thrombosis. Endothelial injury, alterations in blood flow and activation of the coagulation pathway are proposed to contribute to the hypercoagulability. The risk for thrombosis may be accentuated by certain drugs and device implants that directly or indirectly affect the coagulation pathway. To help ensure that these interventions do not result in adverse maternal or fetal outcomes during pregnancy, gravid experimental animals can be exposed to such treatments at various stages of gestation and over a dosage range that would identify hazards and inform risk assessment. Circulating soluble biomarkers can also be evaluated for enhancing the assessment of any increased risk of venous thrombosis during pregnancy. In addition to traditional in vivo animal testing, efforts are under way to incorporate reliable non‐animal methods in the assessment of embryofetal toxicity and thrombogenic effects. This review summarizes hemostatic balance during pregnancy in animal species, embryofetal development, biomarkers of venous thrombosis, and alterations caused by drug‐induced venous thrombosis. Birth Defects Research (Part C) 105:190–200, 2015. © 2015 Wiley Periodicals, Inc.     

胶原/纤维蛋白止血效果观察

目的研究胶原/纤维蛋白对新西兰兔的止血作用,并与胶原蛋白海绵止血效果作比较。方法选用胶原/纤维蛋白止血贴,对新西兰兔耳部动、静脉出血、耳表创面、股动脉割伤、肝损伤、体表创面进行止血试验,同时与胶原蛋白海绵止血效果作比较,观察其止血时间、失血量、敷料与创面的粘合等情况,并定期观察创面愈合、体内吸收和抗炎情况。结果胶原/纤维蛋白复合止血贴组、胶原蛋白海绵组新西兰兔耳动、静脉、耳表创面、股动脉割伤、标准肝创伤的止血时间与对照组比较,差异显著（P〈0.05）。胶原/纤维蛋白复合止血贴组新西兰兔耳动、静脉、耳表创面的止血时间与胶原蛋白海绵组,差异显著（P〈0.05）。胶原/纤维蛋白复合止血贴组、胶原蛋白海绵组动物的耳表创面、标准肝创伤失血量与对照组比较,差异显著（P〈0.05）。体内标准肝创伤、体表创伤后期观察,胶原/纤维蛋白复合止血贴与胶原蛋白海绵均能在21d内完全吸收,未见炎症反应。结论胶原/纤维蛋白复合止血贴对新西兰兔耳动脉割伤、耳静脉割伤、耳标创伤、股动脉割伤和标准肝损伤模型都具有明显的止血作用,体表创面伤口恢复良好,体内吸收速度快,具有一定的抗炎作用,而且在新西兰兔耳动脉、耳静脉割伤和耳表创伤的止血效果明显优于胶原蛋白海绵。胶原/纤维蛋白复合止血贴是一种较安全有效的局部止血生物材料。     

Evolution of Primary Hemostasis in Early Vertebrates

Hemostasis is a defense mechanism which protects the organism in the event of injury to stop bleeding. Recently, we established that all the known major mammalian hemostatic factors are conserved in early vertebrates. However, since their highly vascularized gills experience high blood pressure and are exposed to the environment, even very small injuries could be fatal to fish. Since trypsins are forerunners for coagulation proteases and are expressed by many extrapancreatic cells such as endothelial cells and epithelial cells, we hypothesized that trypsin or trypsin-like proteases from gill epithelial cells may protect these animals from gill bleeding following injuries. In this paper we identified the release of three different trypsins from fish gills into water under stress or injury, which have tenfold greater serine protease activity compared to bovine trypsin. We found that these trypsins activate the thrombocytes and protect the fish from gill bleeding. We found 27 protease-activated receptors (PARs) by analyzing zebrafish genome and classified them into five groups, based on tethering peptides, and two families, PAR1 and PAR2, based on homologies. We also found a canonical member of PAR2 family, PAR2-21A which is activated more readily by trypsin, and PAR2-21A tethering peptide stops gill bleeding just as trypsin. This finding provides evidence that trypsin cleaves a PAR2 member on thrombocyte surface. In conclusion, we believe that the gills are evolutionarily selected to produce trypsin to activate PAR2 on thrombocyte surface and protect the gills from bleeding. We also speculate that trypsin may also protect the fish from bleeding from other body injuries due to quick contact with the thrombocytes. Thus, this finding provides evidence for the role of trypsins in primary hemostasis in early vertebrates.     

尖吻蝮蛇凝血酶对手术切口止血有效性及安全性的临床研究

目的:评价注射用尖吻蝮蛇凝血酶单次注射对外科手术切口止血的临床有效性及安全性。方法:采用随机、双盲单模拟、安慰剂平行对照的研究方法。将入选研究的82例随机分为研究组(61例,术前30分钟静注尖吻蝮蛇凝血酶2u)和对照组(21例,术前30分钟静注模拟尖吻蝮蛇凝血酶2u),并对手术切口、止血时间、出血量、单位面积出血量,以及机体凝血功能和安全性指标等进行观察。结果:研究组平均止血时间[(128.49±71.04)s]、切口平均出血量[(2.70±1.94)g]及平均单位面积出血量[(0.19±0.17)g.cm-2]较对照组[(177.19±77.31)s、(4.35±2.10)g、(0.35±0.17)g.cm-2]明显减少(P<0.05)。在凝血功能及安全性指标等方面,两组结果相似(P>0.05),整个试验过程无不良事件。结论:尖吻蝮蛇凝血酶对腹部切口有较好的止血作用和安全性。     

Porcine Model of Hemophilia A

Hemophilia A is a common X chromosome-linked genetic bleeding disorder caused by abnormalities in the coagulation factor VIII gene (F8). Hemophilia A patients suffer from a bleeding diathesis, such as life-threatening bleeding in the brain and harmful bleeding in joints and muscles. Because it could potentially be cured by gene therapy, subhuman animal models have been sought. Current mouse hemophilia A models generated by gene targeting of the F8 have difficulties to extrapolate human disease due to differences in the coagulation and immune systems between mice and humans. Here, we generated a porcine model of hemophilia A by nuclear transfer cloning from F8-targeted fibroblasts. The hemophilia A pigs showed a severe bleeding tendency upon birth, similar to human severe hemophiliacs, but in contrast to hemophilia A mice which rarely bleed under standard breed conditions. Infusion of human factor VIII was effective in stopping bleeding and reducing the bleeding frequency of a hemophilia A piglet but was blocked by the inhibitor against human factor VIII. These data suggest that the hemophilia A pig is a severe hemophilia A animal model for studying not only hemophilia A gene therapy but also the next generation recombinant coagulation factors, such as recombinant factor VIII variants with a slower clearance rate.     

高剂量生长抑素、奥美拉唑联合止血芳酸治疗急性上消化道出血合并凝血功能障碍患者的临床效果

目的:探讨高剂量生长抑素、奥美拉唑联合止血芳酸治疗急性上消化道出血合并凝血功能障碍患者的临床效果及安全性。方法:选择我院2014年1月~2017年12月收治的92例急性上消化出血合并凝血功能障碍的患者,并按随机数表法将其分为对照组和研究组。对照组予以常规剂量生长抑素、奥美拉唑联合止血芳酸治疗,研究组予以高剂量生长抑素治疗,其余奥美拉唑及止血芳酸用法同对照组。治疗后,比较两组的临床疗效、止血情况、住院时间,治疗前后血常规指标、凝血功能的变化及并发症的发生情况。结果:治疗后,研究组总有效率明显高于对照组[91.30%vs.74.42%](P0.05),而平均止血时间、再止血率及住院时间均明显短于对照组(P0.05);两组白细胞计数(WBC)、部分活化凝血酶原时间(APTT)及凝血酶原时间(PT)均较治疗前明显下降,血红蛋白(Hb)、红细胞计数(RBC)、红细胞压积(Hct)及血小板计数(PLT)均较治疗前明显上升,且研究组以上指标变化较对照组更明显(P0.05)。两组并发症的发生率比较差异均无统计学意义(P0.05)。结论:高剂量生长抑素、奥美拉唑联合止血芳酸治疗急性上消化道出血合并凝血功能障碍的效果明显优于常规剂量生长抑素、奥美拉唑联合止血芳酸治疗,其能够更有效缩短止血时间,避免再出血,且未增加药物不良反应,安全性高。     

Changes on hemostatic parameters induced by 17beta-estradiol,ethinylestradiol, and the 17beta-aminoestrogen pentolame in the male Wistar rat

Oral contraceptives containing estrogens increases the incidence of thromboembolic events. In contrast, administration of 17beta-aminoestrogens prolonged blood clotting time (BCT) in rodents. We studied the effect of estradiol (E(2)), ethinylestradiol (EE) and pentolame on some screening hemostatic tests. BCT was evaluated 24, 48, 72 and 96 h post-treatment. Rats received subcutaneously (s.c.) for five consecutive days E(2) (0.1-1000 microg), EE (1-1000 microg), pentolame (0.1-1000 microg), or vehicle (propyleneglycol 0.3 ml). At 48 h post-treatment E(2) (1000 microg) diminished BCT (32%, P<0.01), in contrast pentolame (1000 microg) augmented BCT by 41% (P<0.01). After 72 h, E(2) showed procoagulant effects with 10, 100 and 1000 microg doses (-45, -30, and -21%, respectively). Significant effects on BCT of EE were observed 72 h after with 1000 microg (-12%, P<0.05). Animals were treated s.c. for two consecutive days with E(2) (3mg/100g), pentolame (4 mg), or vehicle (0.1 ml). BCT, bleeding time (BT), platelet aggregation (PA), prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen concentration were determined. E(2) produced a significant diminution on BCT (-20%) after 72 h whereas pentolame increased BCT from 24 to 96 h (62%, maximal response at 48 h). APTT and PT coagulation times of the groups treated with E(2) and pentolame were lengthened (33 and 29%; 16 and 24%, respectively; P<0.05). Fibrinogen concentration increased (115%, P<0.01) only in the pentolame-treated group. Pentolame and E(2) produced any effects on BT and PA compared with control groups, indicating that platelet function was not modified. Our results indicate that E(2), EE and pentolame affects the plasmatic phase of the hemostatic mechanism.     

Implantable device for intravenous drug delivery in the rat.

The experimental design of studies using small laboratory animals may require repeated venous access for the application of drugs or diagnostics. An appropriate device should provide quick intravenous access without causing severe handling stress to the animals or the necessity of anaesthesia. In addition, the system should be suitable for repeated injections over several weeks. A silicone catheter is connected to an intravenous indwelling cannula with fixation wings. A closure stopper with injection port completes this system. The device is fixed subcutaneously on the back of the animal and the catheter is inserted into the jugular vein. The device is easily and quickly assembled and is more economic than commercial systems. It is easily implanted and allows repeated intravenous injections for several weeks without anaesthesia or restraint of the animals. The method allows repeated intravenous drug delivery over a couple of weeks in small animals.     

雏鸡源致病性粪肠球菌的分离鉴定及其耐药性分析

【背景】粪肠球菌(Enterococcus faecalis)是人和动物肠道正常菌群之一,也是一种条件性致病菌。近年来,粪肠球菌引起人和动物感染的报道越来越多。【目的】探明引起某养鸡场雏鸡发病死亡的病原及其致病性和有效治疗药物。【方法】结合临床症状和病理剖检,开展病原菌分离、生理生化特性检测和16S rRNA基因序列分析、致病性试验、耐药分析和对患病鸡群的药物治疗。【结果】患病鸡有昏睡、瘫痪或共济失调等临床症状;肝、脾肿大,肝脏发黄、少量出血点、质脆易碎,肠道粘膜增厚、出血,脑轻微水肿;从肝脏组织分离得到一株革兰氏阳性球菌,经纯化培养后命名为CJ517;依据该菌株形态特征、生理生化特性和16S rRNA基因序列分析鉴定为粪肠球菌;致病性试验显示CJ517菌株能致死小鼠,致死率为66.67%;该菌对头孢噻肟、磷霉素、丁胺卡那等药物敏感,对多西环素、卡那霉素、新霉素、氟苯尼考等药物耐药;经用敏感药物和提高免疫力结合治疗后,鸡群病情得到控制。【结论】研究结果可为临床诊断和治疗动物粪肠球菌感染提供参考。     

去甲肾上腺素冰生理盐水联合垂体后叶素治疗上消化道出血的疗效观察

目的探讨去甲肾上腺素冰生理盐水口服联合静滴垂体后叶素治疗上消化道出血的临床效果。方法选择2015年10月~2016年11月我院消化内科住院治疗的上消化道出血患者100例,随机分为对照组和观察组各50例,对照组应用去甲肾上腺素生理盐水口服治疗,观察组应用去甲肾上腺素冰生理盐水口服联合垂体后叶素静脉用药治疗,并对两组患者的临床止血效果进行比较。结果观察组的止血有效率为96%,对照组为60%(χ~2=18.881,P=0.000),两组止血效果比较差异具有统计学意义(P0.05)。结论去甲肾上腺素冰生理盐水口服联合静滴垂体后叶素治疗上消化道出血的效果显著,明显优于去甲肾上腺素生理盐水,值得临床应用。     

Inflammatory, hemostatic, and clinical changes in a baboon experimental model for heatstroke.

The mortality and neurological morbidity in heatstroke have been attributed to the host's inflammatory and hemostatic responses to heat stress, suggesting that immunomodulation may improve outcome. We postulated that an experimental baboon model of heatstroke will reproduce human responses and clinical outcome to allow testing of new therapeutic strategies. Eight anesthetized juvenile baboons (Papio hamadryas) were subjected to heat stress in an incubator maintained at 44-47 degrees C until rectal temperature attained 42.5 degrees C (moderate heatstroke; n = 4) or systolic arterial pressure fell to <90 mmHg (severe heatstroke; n = 4) and were allowed to recover at room temperature. Four sham-heated animals served as a control group. Rectal temperature at the end of heat stress was 42.5 +/- 0.0 and 43.3 +/- 0.1 degrees C, respectively. All heat-stressed animals had systemic inflammation and activated coagulation, indicated by increased plasma IL-6, prothrombin time, activated partial thromboplastin time, and D-dimer levels, and decreased platelet count. Biochemical markers and/or histology evidenced cellular injury/dysfunction: plasma levels of thrombomodulin, creatinine, creatine kinase, lactic dehydrogenase, and alanine aminotransferase were increased, and varying degrees of tissue damage were present in liver, brain, and gut. No baboon with severe heatstroke survived. Neurological morbidity but no mortality was observed in baboons with moderate heatstroke. Nonsurvivors displayed significantly greater coagulopathy, inflammatory activity, and tissue injury than survivors. Sham-heated animals had an uneventful course. Heat stress elicited distinct patterns of inflammatory and hemostatic responses associated with outcome. The baboon model of heatstroke appears suitable for testing whether immunomodulation of the host's responses can improve outcome.     

Inflammatory cell participation in coagulation

Vascular cells, and leukocytes in particular, have evolved a formidable machinery to initiate and amplify coagulation. Through multiple, receptor-mediated recognitions this process provides a cellular microenvironment of limited proteolytic activation that contributes to the maintenance of the hemostatic balancein vivo. However, the ability of leukocytes to generate thrombin is also a fundamental aspect of inflammatory responses, and has far-reaching implications in the pathophysiology of vascular diseases. The scope of this article is to revisit the most recent contributions to the cellular and molecular mechanisms of leukocyte-mediated coagulation, and to highlight their role in the pathogenesis of vascular injury.     

Congenital Hypofibrinogenemia in Five Members of a Family

The bleeding tendency in five members of one family with fibrinogen levels ranging from 58 mg. % to 158 mg. % was mild and chiefly related to dental extractions. Abruptio placentae in one patient produced severe bleeding. Reports of menstrual bleeding patterns in patients with defects of hemostatic mechanisms suggest that normal platelets, vascular function and extrinsic and possibly intrinsic coagulation systems, except for fibrinogen, control menstrual blood loss. An autosomal dominant gene with variable penetrance may determine fibrinogen levels.