诊断超声联合微泡对肿瘤微循环的作用

目的 探讨高机械指数诊断超声联合微泡对大鼠Walker-256肿瘤微循环的作用。 方法 将29只皮下荷Walker-256肿瘤SD大鼠随机分为超声微泡组(n=15)、单纯超声组(n=7)和假照组(n=7):对超声微泡组采用声辐射力脉冲(ARFI)成像模式下诊断超声连续激励20次辐照肿瘤,同时经尾静脉推注微泡0.04 ml;对单纯超声组在行超声辐照的同时以等量生理盐水代替微泡;对假照组则采用假照方式,仅推注等量微泡溶液,但不发射超声能量。对所有动物于辐照前、辐照后即刻、10、20 min行CEUS检查。最后每组随机选取3只动物获取肿瘤组织标本,行病理学检查。 结果 辐照后即刻,超声微泡组辐照区几乎无造影剂充填,呈负性显影,肿瘤区平均造影峰值强度(PI)由25.17%减低到12.01%(P<0.01);单纯超声组及假照组辐照后即刻可见造影剂快速充填,灌注良好(P>0.05)。10 min后,超声微泡组造影可见血流逐渐恢复,但PI仍降低;20 min后肿瘤血流基本完全恢复,呈高灌注(P>0.05)。 结论 高机械指数诊断超声联合微泡能特异性地暂时降低大鼠Walker-256皮下移植瘤的微循环。     

新型聚内酯超声造影剂的微循环流变学研究

目的观察聚内酯微泡的微循环血流变学特征，评价微泡滞留的程度，滞留对局部血流动力学的影响。方法27只Wistar大鼠分为2组，分别经颈动脉和颈静脉注射不同剂量的荧光微泡，活体显微镜观察微泡在肠系膜微循环的流变学特点。结果动脉注射后微血管静止微泡的个数随剂量的增加而增多，600μl／kg剂量组静止微泡的发生率为0．85％（11／1300）。静脉注射聚内酯微泡后在所有30个光镜视野中未观察到微泡在微循环的阻塞。红细胞的流动速度与微泡的平均流速有很好的相关性（r=0．89，P〈0．05）。结论聚内酯超声造影剂微血管流变学特性与红细胞相似，有极少微泡会暂时粘附嵌陷在小动脉或毛细血管。     

自制高分子微泡超声造影机械指数与频率和峰值强度的相关性

目的 探讨兔体内自制高分子微泡CEUS机械指数和频率与峰值强度的相关性。方法 采用单乳化法制备包裹全氟戊烷的嵌段共聚物微泡造影剂,固定其他超声扫描条件,对6只新西兰大白兔于机械指数0.04、0.09、0.17、0.34和超声频率3.5、5.0、6.5、8.0 MHz下进行肾脏CEUS,分析机械指数和频率与峰值强度的相关性;静脉注射10倍造影剂量微泡,观察毒性反应。结果 自制微泡平均粒径为（3.92±1.43）μm。兔体内峰值强度与机械指数呈正相关（r=0.99,P<0.05）,与超声频率呈负相关（r=-0.93,P<0.05）;静脉注射大剂量微泡后,未见明显毒性反应。结论 采用自制高分子微泡作为造影剂对兔进行CEUS时,峰值强度与机械指数呈正相关,与超声频率呈负相关;微泡安全性良好。     

自制纳米级E-选择素靶向超声微泡对缺血心肌分子成像

目的 采用自制的携E-选择素抗体的纳米级靶向超声造影剂对大鼠缺血再灌注损伤后的缺血心肌进行超声分子成像,评估其检测缺血心肌的可行性。方法 采用生物素-链亲和素方法制备纳米级靶向微泡,应用荧光显微镜及流式细胞仪检测微泡形态及抗体连接效率;将26只SD大鼠随机分为E-选择素靶向微泡组(MB_E组,n=10)、IGg抗体微泡组(MB_IGg组,n=10)、普通微泡组(MB_C组,n=6),制备心肌缺血再灌注损伤模型,将冠状动脉左前降支阻断15 min后恢复灌注,于再灌注后6 h经尾静脉分别注射3种微泡行CEUS。后行病理学检查。结果 MB_E组缺血区域声学强度(VI)显著高于非缺血区(P<0.05),也显著高于MB_C组、MB_IGg组缺血区域的VI(P<0.05);MB_C组、MB_IGg组缺血区域与非缺血区的VI差异无统计学意义(P>0.05)。结论 纳米级E-选择素靶向超声微泡可以早期检测到缺血心肌,有望实现缺血心肌的"记忆"成像。     

低频超声联合SonoVue微泡促进裸鼠基因体内转染的可行性

目的 探讨利用低频超声联合SonoVue微泡造影剂促进基因转染人前列腺癌裸鼠皮下移植瘤的可行性。方法 采用人前列腺癌PC-3细胞建立裸鼠皮下移植瘤模型,成瘤后随机分为空白组（仅注射生理盐水）、单纯质粒组（注射生理盐水和质粒混悬液）、低频超声组（注射生理盐水和质粒混悬液后超声辐照）和低频超声+微泡组（注射超声微泡造影剂和质粒混悬液后超声辐照）。超声辐照条件：功率3 W/cm²,占空比5：5,频率80 kHz,辐照10 min。基因转染3天后在激光共聚焦显微镜下观察各组绿色荧光蛋白表达情况,并分析其平均荧光强度;同时行常规病理检查,观察低频超声联合微泡辐照对组织的损伤程度。结果 4组中,仅低频超声+微泡组可见明显绿色荧光,与其他各组差异均有统计学意义（P均<0.05）;空白组、单纯质粒组和低频超声组均未见明显绿色荧光。各组均未见明显组织损伤。结论 SonoVue微泡造影剂在低频超声辐照下能够安全有效地促进pEGFP基因转染进入人前列腺癌裸鼠皮下移植瘤组织内。     

载睫状神经营养因子聚乙二醇-乳酸/羟基乙酸共聚物微泡联合超声对视网膜神经节细胞的保护作用

目的 制备载睫状神经营养因子(CNTF)的聚乙二醇(PEG)-乳酸/羟基乙酸共聚物(PLGA)超声微泡造影剂(CNTF-PEG-PLGA),观察其联合超声对视神经损伤大鼠视网膜神经节细胞(RGCs)的保护作用。 方法 采用双乳化法制备CNTF-PEG-PLGA,并检测其基本特性。随机选取SD大鼠145只(双眼兼用),将其分为7组,采用视神经钳夹法制作大鼠视神经损伤模型。治疗后应用荧光金(FG)逆行标记法比较各组大鼠RGCs存活数;视网膜组织病理切片观察视网膜的形态结构改变,评价载药微泡及联合超声的安全性;生长相关蛋白-43(GAP-43)免疫组化染色,观察大鼠视网膜GAP-43的表达情况。结果 CNTF-PEG-PLGA微泡平均粒径(312.5±57.35)nm,包封率62.35%,载药量0.298 μg/mg,体外释放28天时微泡释放率达93.60%。FG标记RGCs示,在每个观察时间点G组平均RGCs计数均显著高于其他各损伤组(P<0.05),但仍低于A组(P<0.05);G组GAP-43表达可持续到伤后4周,且明显高于其他各组(P<0.05);视网膜组织病理切片示玻璃体腔注射微泡后视网膜未见明显炎性细胞浸润现象。结论 载睫状神经营养因子PEG-PLGA微泡联合超声可增强药物对视神经损伤大鼠视网膜神经节细胞的保护作用,延长药物作用时间。     

LHRHa靶向微泡造影剂的制备及体外寻靶实验

目的 制备人卵巢癌靶向超声造影剂,观察其体外寻靶能力。方法 采用生物素-链霉亲和素法制备促黄体生成素释放激素类似物(LHRHa)靶向脂质微泡,以免疫荧光染色验证LHRHa与脂质微泡的结合情况,并以普通脂质微泡作为对照,在倒置显微镜下观察LHRHa靶向脂质微泡对人卵巢癌A2780/DDP的体外寻靶能力。结果 LHRHa靶向脂质微泡免疫荧光阳性;体外寻靶实验显示LHRHa靶向脂质微泡能够与表面存在LHRH受体的A2780/DDP细胞特异性结合,而普通脂质微泡则不能结合。结论 利用生物素-链霉亲和素方法可成功制备LHRHa靶向脂质微泡造影剂,该靶向造影剂具有体外寻靶能力。     

超声激励荧光微泡对兔乳腺癌转移淋巴结的释放作用

目的 观察超声激励荧光微泡空化对兔乳腺癌转移淋巴结的荧光释放作用。 方法 使用二甲基亚砜(DMSO)溶解绿色细胞膜荧光分子探针(DiO),抽取少量溶解液与脂质微泡混和,通过高速机械振荡制成荧光微泡。选取16只荷VX2乳腺癌的新西兰大白兔随机分成荧光微泡联合超声空化组和单纯荧光微泡组。荧光微泡联合超声空化组经瘤周皮下注射1 ml荧光微泡并按摩,待瘤周引流淋巴结超声显影后,采用脉冲式治疗超声间歇辐照淋巴结5次,共30 min;单纯荧光微泡组同样经瘤周皮下注射荧光微泡并按摩,但给予治疗超声假照。剖取淋巴结标本行冰冻组织切片,于激光共聚焦显微镜下观察淋巴结内的荧光分布情况,并定量分析荧光面积、累积光密度(IOD)及平均光密度(AOD)。 结果 荧光微泡联合超声空化组淋巴结的荧光面积、IOD和AOD均高于单纯荧光微泡组(P均<0.05)。 结论 荧光脂质微泡经瘤周注射不仅可进入兔乳腺癌模型的淋巴管使淋巴结显影,且可在治疗超声的激励作用下实现荧光物质在局部淋巴结高浓度释放。     

微泡增强的超声空化阻断肿瘤微循环的病理观察

目的 探讨微泡增强的超声空化阻断肿瘤微循环的病理机制。方法 建立兔肝VX2转移瘤模型18只,随机分为超声微泡组(n=6)、单纯超声组(n=6)及假照组(n=6)。对超声微泡组经耳缘静脉注射微泡,并进行超声辐照;对单纯超声组以生理盐水代替微泡,并进行超声辐照;对假照组注射生理盐水,并进行超声假照。观察辐照后各组病理变化。结果 大体解剖观察,超声微泡组辐照面可见较多出血点;单纯超声组及假照组辐照后肿瘤组织均未见明显改变。光镜下观察,超声微泡组肿瘤组织可见大片出血,血管内皮细胞损伤、连续性中断,红细胞外溢;单纯超声组可见局灶性小片状出血,血管内皮尚完整;假照组肿瘤组织未见明显出血,血管内皮完整。电镜下观察,超声微泡组肿瘤血管内皮破损严重,可见大量红细胞渗出,线粒体肿胀及吞饮小泡;单纯超声组及假照组仅见内质网略水肿。结论 微泡增强的超声空化阻断兔VX2肝脏肿瘤微循环的机制可能为血管机械性损伤。     

靶向微泡造影剂促进兔心肌梗死骨髓干细胞移植后血管新生及心肌力学改变

目的 应用斑点追踪技术评价靶向微泡造影剂注射后兔心肌梗死区域骨髓干细胞移植疗效。 方法 制备靶向微泡超声造影剂(携CD34单克隆抗体)。将36只新西兰大白兔随机分为对照组(单纯移植组)、普通造影剂组(移植+C组)及靶向造影剂组(移植+T组),分别于急性心肌梗死(AMI)前、AMI后3天、干细胞移植术后4周行心肌超声造影(MCE),采用彩色编码参数量化(PQ)技术对比各组干细胞移植前后梗死区域心肌灌注参数,同时对3组动物心肌梗死干细胞移植区域心肌的径向应变率(SrR)、圆周应变率(SrC)、旋转率(RotR)、收缩期S峰值及心肌扭转角度(Rot)进行斑点追踪分析,并于移植4周后检测微血管密度。 结果 干细胞移植后各心肌节段的A、β和A×β值均较本组内移植前改善,移植+T组改善最为明显(P均<0.05)。各组左心室前壁的SrR、SrC、RotR及Rot均较本组内移植前增高(P均<0.01),并与左心室射血分数相关。 结论 靶向微泡造影剂对兔骨髓干细胞移植后微血管新生具有一定作用。     

Microvascular rheology of Definity microbubbles after intra-arterial and intravenous administration.

The microvascular rheology and extent of pulmonary retention of second-generation microbubble ultrasound contrast agents has not previously been well characterized. We assessed the microvascular behavior of Definity, a lipid-shelled microbubble agent containing perfluoropropane gas, using intravital microscopy of either rat spinotrapezius muscle or mouse cremaster muscle. Immediately after intra-arterial injection, which was performed to model pulmonary retention, larger microbubbles (> 5 microm) were entrapped within small arterioles and capillaries. The retention fraction of microbubbles was low (1.2% +/- 0.1%) and entrapment was transient (85% dislodged by 10 minutes), resulting in no adverse hemodynamic effects. Leukocyte or platelet adhesion at the site of entrapment was not seen. After intravenous injection, no microbubble entrapment was observed and the velocities of microbubbles in arterioles, venules, and capillaries correlated well with those of red blood cells. We conclude that after intravenous injection and pulmonary passage, the microvascular rheology of Definity microbubbles is similar to that of red blood cells. Microbubble entrapment within the pulmonary microcirculation after venous injection should be negligible and transient. These findings are important for establishing the safety of this agent.     

THE STATE OF THE VESSELS OF THE MESENTERY IN SHOCK PRODUCED BY CONSTRICTING THE LIMBS AND THE BEHAVIOR OF THE VESSELS FOLLOWING HEMORRHAGE

1. Direct observations of the arteries, arterioles, capillaries, veins, and lymphatics in the mesentery of anesthetized cats put into shock by incomplete occlusion of the circulation of the limbs showed that: (a) Marked constriction of the arteries and arterioles, produced by muscular contraction, occurred usually within an hour after incomplete occlusion of the limbs, lasted several hours, and finally gave way in most instances to relaxation an hour or more before death. The constriction reduced the blood supply to the mesentery and intestine and the venous return from them. It did not, however, interrupt the blood flow. No pooling or stagnation of blood was seen even as a terminal phenomenon. (b) The veins of the mesentery also became constricted but showed less tendency to dilate as death approached. The lymphatics likewise became somewhat narrowed. Even during the terminal stage the leukocytes moved along without change in shape or sticking to the walls of the capillaries or venules. (c) Hematocrit determinations showed progressive hemoconcentration of moderate degree. (d) Autopsy usually showed the presence of small hemorrhages in many parts of the body, especially the heart, liver, spleen, and lungs. (e) Bilateral nephrectomy, suprarenalectomy, and pancreatectomy did not significantly alter the morphological picture elicited by shock induced by restriction of the circulation to the limbs. 2. Removal of large amounts of blood was always followed within a short time by constriction of arteries, arterioles, veins, and lymphatics of the mesentery. 3. Fall in arterial pressure produced by pithing was not accompanied by change in diameter of the arteries, arterioles, veins, or lymphatics, or by blanching of the mesentery or gut.     

Endothelial cell injury in venule and capillary induced by contrast ultrasonography

The aim of the present study was to test the hypothesis that microvascular endothelial cells (EC) are subject to the bioeffects induced by contrast ultrasound (US) because of their proximity to the circulating microbubbles. We examined EC injury in each microvessel section (arteriole, capillary or venule) in rat mesenteries among the following five groups: three controls (sham operation, microbubble injection alone, US exposure with saline injection), and two contrast-US groups (US exposure at a 1-Hz or 30-Hz frame rate with microbubble injection). Propidium iodide (PI), a fluorescent indicator of cell injury, was employed to visualize impaired EC. PI-positive nuclei were equally few among the three controls. Contrast-US increased PI-positive cells in capillaries (1-Hz frame rate, 2.4 +/- 2.2 cells per 0.1-mm vessel length, p = 0.09; 30-Hz frame rate, 4.3 +/- 1.8 cells, p < 0.01) and in venules (1-Hz frame rate, 4.1 +/- 2.5 cells, p < 0.05; 30-Hz frame rate, 13.8 +/- 3.6 cells, p < 0.01) compared with sham operation (0.10 +/- 0.22 cells). The finding indicates that diagnostic contrast US potentially causes EC injury, particularly in venules and capillaries.     

Subpleural microvascular flow velocities and shear rates in normal and septic mechanically ventilated rats

Changes in pulmonary microhemodynamics are important variables in a large variety of pathological processes. We used in vivo fluorescent videomicroscopy of the subpleural microvasculature in mechanically ventilated rats to directly monitor microvascular flow velocity (FV) and shear rate in pulmonary arterioles, capillaries, and venules in healthy rats and in septic rats 20 h after cecal ligation and puncture (CLP). Observations were made through a small thoracotomy after injection of fluorescent microspheres (D = 1 microm) into the systemic circulation. The FVs were calculated off-line by frame-by-frame measurements of the distance covered by individual microspheres per unit of time. In healthy rats, inspiratory FV were 1322 +/- 142 microm/s in subpleural arterioles and 599 +/- 25 microm/s in capillaries. The highest FV was found in venules (1552 +/- 132 microm/s). The calculated shear rates were 547 +/- 62/s in arterioles and 619 +/- 19/s in capillaries. The highest shear rates were detected in venules (677 +/- 59/s). No significant changes in FV and shear rates were observed throughout the 1-h observation period in any of the microvascular compartments. Pulmonary microvascular FV and shear rates found in sham-operated rats in the CLP experiments were not significantly different from values of healthy rats. The CLP caused a significant increase in leukocyte sequestration in the lungs and a mean of 27% to 34% decrease in FV in all sections of the pulmonary microvasculature (P < 0.001 in capillaries and P < 0.05 in venules). Also, CLP caused a 23% decrease in capillary shear rate that reached only borderline statistical significance (P < 0.06) and a significant 35% decrease in mean shear rate in venules (P < 0.05). Fluorescent videomicroscopy is offered as a stable and reproducible method for in vivo determinations of pulmonary microhemodynamics in clinically relevant models of sepsis.     

Microvascular dysfunction and skeletal muscle oxygenation assessed by phase-modulation near-infrared spectroscopy in patients with septic shock

Objective Sepsis is now considered a disease of the microcirculation. Little is known about the various sepsis-induced changes responsible for microvascular dysfunction. We investigated human microvascular function, regulation, oxygenation, and cellular metabolism during subacute septic shock.Design and setting Prospective case-control study in a nine-bed polyvalent surgical ICU of a university hospital.Patients and participants A prospectively enrolled group of 26 patients (13 with septic shock, 13 nonseptic postsurgical patients) and 15 healthy volunteer controls.Measurements and results The absolute tissue hemoglobin concentrations (oxygenated hemoglobin and deoxyhemoglobin) were measured noninvasively in arterioles, capillaries, and venules by phase-modulation near-infrared spectroscopy in the human brachioradial muscle during a series of venous occlusions and an arterial occlusion (ischemia) induced by applying a pneumatic cuff. These measurements were used to calculate tissue blood volume, postischemic hemoglobin resaturation time, microvascular compliance, and O₂ consumption. Patients with sepsis had significantly higher tissue blood volume values and lower compliance than healthy controls. They also had longer postischemic hemoglobin resaturation times than the other two groups and blunted resaturation curves. O₂ consumption was lower in patients with sepsis than in healthy controls. In patients with septic shock cuff-induced ischemia left O₂ consumption unchanged, whereas in healthy volunteers it reduced O₂ consumption to values almost matching those of patients with septic shock.Conclusions These findings show that septic shock alters microvascular muscle function and regulation. Diminished local VO₂ presumably reflects maldistribution and faulty autoregulation of local blood flow.     

Decrease of red blood cell filterability seen in intensive care. II. Red blood cell crenelation “in vivo” as morphological evidence of increased red blood cell viscosity in low flow states

We have reported the finding of numerous spiculed erythrocytes in blood stored for 15–21 days with acid citrate dextrose, drawing attention to the possible decrease of stored red cell deformability because of the decrease of the surface to volume ratio. The presence of anisotropy with crenated spheres, equivalent to the internal crystallization of hemoglobin, is closely related to these altered red cell parameters. The increase of erythrocyte filtration time in blood stored with acid citrate dextrose correlated well with the duration of storage up to the 21st day, r = 0.74 > 3 Sr, y = 1.104x + 1.853, n = 47, as well as with the increase of the proportion of crenated red cells.

Using higher magnification (×700–1000) of the microcirculation in the great omentum of shocked dogs, crenated spheres could be seen within the arterioles, venules and capillaries; during refractory shock, nearly all the red cells became crenated spheres. The observation of echinocytes “in vivo” is a morphological proof of the damage to deformability of normal red blood cells in low flow states.     

Role of CD11/CD18 in shear rate-dependent leukocyte-endothelial cell interactions in cat mesenteric venules.

In vivo microscopy was used to assess the relationships among shear rate (and shear stress), leukocyte rolling velocity, and leukocyte adherence in a cat mesentery preparation. Shear rate in individual venules and arterioles of 25-35 microns diameter were varied over a wide range by graded occlusion of an arterial loop. There was a linear decline in leukocyte rolling velocity (Vwbc) as red cell velocity (Vrbc) was reduced. The ratio Vwbc/Vrbc remained constant despite variations in shear stress from 5-25 dyn/cm2. A reduction in shear stress was associated with an increased leukocyte adherence, particularly when Vwbc was reduced below 50 microns/s. Reduction in wall shear rate below 500 s-1 in arterioles allowed 1-3 leukocytes to adhere per 100 microns length of vessel, while venules exposed to the same shear rates had 5-16 adherent leukocytes. In arterioles, leukocyte rolling was only observed at low shear rates. At shear rates less than 250 s-1 leukocyte rolling velocity was faster in arterioles than venules, and the ratio Vwbc/Vrbc for arterioles was 0.08 +/- 0.02, which was fourfold higher than the ratio obtained in venules at similar shear rates. Pretreatment with the CD18-specific antibody (mAb) IB4 increased leukocyte rolling velocity in venules by approximately 20 microns/s at red cell velocities below 2,000 microns/s. mAb IB4 largely prevented the leukocyte adherence to arterioles and venules, and increased the ratio Vwbc/Vrbc observed in venules at low shear elicit a CD18-dependent adhesive interaction between leukocytes and microvascular endothelium, and that differences in shear rates cannot explain the greater propensity for leukocyte rolling and adhesion in venules than arterioles.     

Label free measurement of retinal blood cell flux,velocity, hematocrit and capillary width in the living mouse eye

Measuring blood cell dynamics within the capillaries of the living eye provides crucial information regarding the health of the microvascular network. To date, the study of single blood cell movement in this network has been obscured by optical aberrations, hindered by weak optical contrast, and often required injection of exogenous fluorescent dyes to perform measurements. Here we present a new strategy to non-invasively image single blood cells in the living mouse eye without contrast agents. Eye aberrations were corrected with an adaptive optics camera coupled with a fast, 15 kHz scanned beam orthogonal to a capillary of interest. Blood cells were imaged as they flowed past a near infrared imaging beam to which the eye is relatively insensitive. Optical contrast of cells was optimized using differential scatter of blood cells in the split-detector imaging configuration. Combined, these strategies provide label-free, non-invasive imaging of blood cells in the retina as they travel in single file in capillaries, enabling determination of cell flux, morphology, class, velocity, and rheology at the single cell level.OCIS codes: (170.4460) Ophthalmic optics and devices, (330.4300) Vision system - noninvasive assessment, (110.1080) Active or adaptive optics, (330.7324) Visual optics, comparative animal models     

The polysaccharide fucoidan inhibits microvascular thrombus formation independently from P- and L-selectin function in vivo

BACKGROUND: Adhesion molecules of the selectin family (mainly P- and L-selectin) have been suggested to mediate interactions between platelets, leukocytes and endothelial cells in thrombus formation. The polysaccharide fucoidan has anticoagulative properties, but is also able to bind and block the function of the selectins. Here, we investigated in vivo (i) if fucoidan can prevent microvascular thrombus formation, and (ii) whether this is potentially mediated by the inhibition of P-and/or L-selectin. MATERIALS AND METHODS: For this purpose, we used intravital microscopy in the mouse cremaster microcirculation in which thrombosis was induced photochemically by light exposure to individual arterioles and venules after intravenous (i.v.) injection of FITC-dextran. RESULTS: We found that intravenous administration of fucoidan significantly prolonged the time required for complete occlusion in arterioles and venules by almost seven- and nine-fold, respectively. In contrast, treatment with monoclonal antibodies against P- and L-selectin had no effect on the development of microvascular thrombosis. Fucoidan and also the anti-P-selectin antibody completely inhibited baseline venular leukocyte rolling in the cremaster muscle, indicating that these treatment regimes abolished P-selectin function. Importantly, fucoidan and the anti-P-selectin antibody had no effect on systemic platelet and leukocyte counts. On the other hand, we found that fucoidan treatment significantly altered coagulation parameters, including prothrombin time (Quick percentage), activated partial thromboplastin time (APTT) and thrombin clotting time (TCT), which may explain the potent in vivo anticoagulative effect of fucoidan observed here. CONCLUSIONS: Taken together, our novel findings suggest that fucoidan effectively prevents microvascular thrombus formation induced by endothelial damage in arterioles and venules in vivo. This protective effect of fucoidan is not attributable to inhibition of P- and L-selectin function but may instead be related to the anticoagulative capacity of fucoidan.     

EVIDENCE FOR A BLOOD-THYMUS BARRIER USING ELECTRON-OPAQUE TRACERS

In order to verify the existence of a blood-thymus barrier to circulating macromolecules, the permeability of the vessels of the thymus was analyzed in young adult mice using electron opaque tracers of different molecular dimensions (horseradish peroxidase, cytochrome c, catalase, ferritin, colloidal lanthanum). Results show that although blood-borne macromolecules do penetrate the thymus, their parenchyma] distribution is limited to the medulla of the lobe by several factors: (a) the differential permeability of the various segments of the vascular tree; (b) the spatial segregation of these segments within the lobe; (c) the strategic location of parenchymal macrophages along the vessels. The cortex is exclusively supplied by capillaries, which have impermeable endothelial junctions. Although a small amount of tracer is transported by plasmalemmal vesicles through the capillary endothelium, this tracer is promptly sequestrated by macrophages stretched out in a continuous row along the cortical capillaries and it does not reach the intercellular clefts between cortical lymphocytes and reticular cells. The medulla contains all the leaky vessels, namely postcapillary venules and arterioles. Across the walls of the venules, large quantities of all injected tracers escape through the clefts between migrating lymphocytes and endothelial cells; also the arterioles have a small number of endothelial junctions which are permeable to peroxidase, but do not allow passage of tracers of higher molecular weight. The tracers released by the leaky vessels penetrate the intercellular clefts of the medulla, but they never reach the cortical parenchyma, even at long time intervals after the injection. Therefore, a blood-thymus barrier to circulating macromolecules does exist, but is limited to the cortex. Medullary lymphocytes are freely exposed to blood-borne substances.