Optimizing quantitative in vivo fluorescence imaging with near‐infrared quantum dots

Quantum dots (QDs) are fluorescent nanoparticles with broad excitation and narrow, wavelength‐tunable emission spectra. They are used extensively for in vitro fluorescence imaging studies and more recently for in vivo small animal and pre‐clinical studies. To date there has been little concern about the selection of QD size (and thus emission wavelength peak) and excitation wavelengths, as they have little relevance to the results of in vitro studies. In vivo imaging, however, poses additional constraints, such as the scattering and absorption by tissue, which may influence the signal intensity at the body surface. Here, we demonstrate that longer‐wavelength excitation and emission yield less quantization error in measured relative fluorescence intensity, using three near‐infrared QDs (QD655, QD705 and QD800) applied to in vivo lymphatic imaging, and a range of excitation wavelengths from the blue to the red. Statistically significant differences in quantization error were observed between nearly all pairs of excitation wavelengths (445–490, 503–555, 575–605, 615–665 and 671–705 nm). Similarly, quantization error decreased with longer emission wavelengths (655, 705 and 800 nm). Light absorbance and scattering were demonstrated to be more potent factors than absorbance efficiency of QDs in producing quantization error in the measured fluorescence intensity. As a result, while wavelengths can be adjusted for qualitative experiments, the longest possible wavelengths should be used if quantification is desired during QD imaging experiments. Copyright © 2010 John Wiley & Sons, Ltd.     

Interactions between ingested kaolinite and the intestinal mucosa in rat: proteomic and cellular evidences

Although some of the effects of clay ingestion by humans and animals, such as gastrointestinal wellness and the increase in food efficiency are well known, the underlying mechanisms are not yet fully understood. Therefore, the interactions between the intestinal mucosa and kaolinite particles and their effects on mucosal morphology were observed using light microscopy (LM), transmission electron microscopy (TEM), conventional (CSEM) and environmental (ESEM) scanning electron microscopy combined with an EDX micro-analysis system. Kaolinite consumption, given with free access to rats, varied considerably from one animal to the other but was regular through time for each individual. Some kaolinite particles appeared chemically dissociated in the lumen and within the mucus barrier. Aluminium (Al) originating from ingested clay and present in the mucus layer could directly cross the intestinal mucosa. A significant increase in the thickness of the villi with large vacuoles at the base of the mucosal cells and a decrease in the length of enterocyte microvilli characterized complemented animals. The proteomic analyses of the intestinal mucosa of complemented rats also revealed several modifications in the expression level of cytoskeleton proteins. In summary, kaolinite particles ingested as food complement interact with the intestinal mucosa and modify nutrient absorption. However, these data, together with the potential neurotoxicity of Al, need further investigation.     

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.     

Fibrinogen and von Willebrand factor-independent platelet aggregation in vitro and in vivo

BACKGROUND: Fibrinogen (Fg) has been considered essential for platelet aggregation. However, we recently demonstrated formation of occlusive thrombi in Fg-deficient mice and in mice doubly deficient for Fg and von Willebrand factor (Fg/VWF(-/-)). METHODS AND RESULTS: Here we studied Fg/VWF-independent platelet aggregation in vitro and found no aggregation in citrated platelet-rich plasma of Fg/VWF(-/-) mice. Surprisingly, in Fg/VWF(-/-) plasma without anticoagulant, adenosine diphosphate induced robust aggregation of Fg/VWF(-/-) platelets but not of beta(3)-integrin-deficient (beta(3) (-/-)) platelets. In addition, beta(3) (-/-) platelets did not significantly incorporate into thrombi in Fg/VWF(-/-) mice. This Fg/VWF-independent aggregation was blocked by thrombin inhibitors (heparin, hirudin, PPACK), and thrombin or thrombin receptor activation peptide (AYPGKF-NH(2)) induced aggregation of gel-filtered Fg/VWF(-/-) platelets in 1 mm Ca(2+) PIPES buffer. Notably, aggregation in PIPES buffer was only 50-60% of that observed in Fg/VWF(-/-) plasma. Consistent with the requirement for thrombin in vitro, hirudin completely inhibited thrombus formation in Fg/VWF(-/-) mice. These data define a novel pathway of platelet aggregation independent of both Fg and VWF. Although this pathway was not detected in the presence of anticoagulants, it was observed under physiological conditions in vivo and in the presence of Ca(2+)in vitro. CONCLUSIONS: beta(3) integrin, thrombin, and Ca(2+) play critical roles in this Fg/VWF-independent aggregation, and both plasma and platelet granule proteins contribute to this process.     

间充质干细胞体内迁移研究进展

间充质干细胞（MSC）具有低免疫源性、多向分化、强大的支持造血和免疫调节功能,同时具有来源广泛,易于体外分离和扩增的特点,目前广泛应用于临床疾病的治疗。研究资料表明,MSC输注后广泛分布于受者各组织器官,然而MSC体内靶向迁移是达到临床治疗的高效性和安全性的关键所在,本文对静脉输注后MSC的体内分布特点和增强MSC体内靶向迁移策略研究的新进展作一综述。     

E‐selectin targeting to visualize tumorsin vivo

Generally angiogenic factors induce the expression of E‐selectin in vascular endothelial cells in the tumors. In this study, we employed an anti‐E‐selectin monoclonal antibody to target tumors in vivo and evaluated an optical imaging reagent to visualize tumor regions. The anti‐E‐selectin antibody was conjugated on the surface of liposomes, which encapsulated the near‐infrared fluorescent substances Cy3 or Cy5.5. The liposomes efficiently recognized human umbilical vein endothelial cells only when E‐selectin was induced by angiogenic factors such as TNF‐α in vitro. Cy5.5 encapsulated into liposomes that were conjugated with an anti‐E‐selectin antibody successfully visualized Ehrlich ascites tumor cells when transplanted into mice. Thus, E‐selectin targeting with liposomes containing a near‐infrared fluorescent dye was found effective in visualizing tumors in vivo. This strategy should be extremely useful as a method to identify sentinel lymphatic nodes and angiogenic tumors as well as use for drug delivery to tumor cells. Copyright © 2010 John Wiley & Sons, Ltd.     

血管性帕金森综合征

随影像学发展和尸解病例增多 ,血管性帕金森综合征已渐为人们所认识。本文对血管性帕金森综合征的病因、发病机制、病理改变、临床表现、血液流变学、经颅多普勒、头颅CT、MRI改变 ,诊断与鉴别诊断以及治疗和康复等研究进展进行综述     

结直肠癌前哨淋巴结体内外定位方法的比较及其隐匿转移检测研究

目的比较结直肠癌患者体内外前哨淋巴结（SLN）定位法各自的检出率、准确性和特异性并分析淋巴结隐匿转移检测的临床应用价值。方法86例结直肠癌行前哨淋巴结定位检测,体外和体内法分别为40例和46例．所有获得的前哨淋巴结及非前哨淋巴结均给予连续切片及以CK20为标记物的免疫组织化学分析．计算两组患者的淋巴结检出率、特异性、准确性、假阴性率及病理分期提高率。结果体外组和体内组在肿瘤大小、位置、分期及分化程度上差异无统计学意义（x2分别=0．59、0．97、0．67、0．76,P均〉0．05）。体外组和体内组前哨淋巴结平均检出数分别为2．62±1．95和2．33±0．91．检出率分别为87．50％和89．13％,准确性分别为90．00％和85．71％。假阴性率分别为10．00％和14．29％,两组差异均无统计学意义（t=0．50,X2分别=1．13、1．82、0．63,P均〉0．05）。两组患者病理分期提高率分别为15．38％和20．69％,差异无统计学意义（x2=0．92,P〉0．05）,全组病例总体分期提高率为18．18％。结论体外法和体内法具有相似的的栓出率和准确性,但体外法简单易行,可减少手术时间;对前哨淋巴结进行免疫组织化学检测可发现隐匿转移从而提高部分患者的病理分期,最终使患者从综合治疗中获益。     

In vivo perfusion estimation using subharmonic contrast microbubble signals.

OBJECTIVE: The purpose of this study was to quantify perfusion in vivo using contrast-enhanced subharmonic imaging (SHI). METHODS: A modified LOGIQ 9 scanner (GE Healthcare, Milwaukee, WI) operating in gray scale SHI mode was used to measure SHI time-intensity curves in vivo. Four dogs received intravenous contrast bolus injections (dose, 0.1 mL/kg), and renal SHI was performed. After 3 contrast agent injections, a microvascular staining technique based on stable (nonradioactive) isotope-labeled microspheres (BioPhysics Assay Laboratory Inc, Worcester, MA) was used to quantify the degree of perfusion in 8 sections of each kidney. Low perfusion states were induced by ligating surgically exposed segmental renal arteries followed by contrast agent injections and microvascular staining. Digital clips were transferred to a personal computer, and SHI time-intensity curves were acquired in each section using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Subharmonic fractional blood volumes were calculated, and the perfusion was estimated from the initial slope of the fractional blood volume uptake averaged over 3 injections. Subharmonic perfusion data were compared with the gold standard (ie, the microspheres) using linear regression analysis. RESULTS: In vivo gray scale SHI clearly showed flow and, thus, perfusion in the kidneys with almost complete suppression of tissue signals. In total, 270 SHI time-intensity curves were acquired, which reduced to 94 perfusion estimates after averaging. Subharmonic perfusion estimates correlated significantly with microsphere results (r = 0.57; P < .0001). The best SHI perfusion estimates occurred for high perfusion states in the anterior of the kidneys (r = 0.73; P = .0001). The corresponding root mean square error was 2.4%. CONCLUSIONS: Subharmonic perfusion estimates have been obtained in vivo. The perfusion estimates were in reasonable to good agreement with a microvascular staining technique.     

In vivo quantification of magnetically labelled cells by MRI relaxometry

Cellular MRI, which visualizes magnetically labelled cells (cells*), is an active research field for in vivo cell therapy and tracking. The simultaneous relaxation rate measurements (R₂*, R₂, R₁) are the basis of a quantitative cellular MRI method proposed here. U937 cells were labelled with Molday ION Rhodamine B, a bi‐functional superparamagnetic and fluorescent nanoparticle (U937*). U937* viability and proliferation were not affected in vitro. In vitro relaxometry was performed in a cell concentration range of [2.5 × 10⁴–10⁸] cells/mL. These measurements show the existence of complementary cell concentration intervals where these rates vary linearly. The juxtaposition of these intervals delineates a wide cell concentration range over which one of the relaxation rates in a voxel of an in vivo image can be converted into an absolute cell concentration. The linear regime was found at high concentrations for R₁ in the range of [10⁶– 2 × 10⁸] cells/mL, at intermediate concentrations for R₂ in [2.5 × 10⁵– 5 × 10⁷] cells/mL and at low concentrations for R₂* in [8 × 10⁴– 5 × 10⁶] cells/mL. In vivo relaxometry was performed in a longitudinal study, with labelled U937 cells injected into a U87 glioma mouse model. Using in vitro data, maps of in vivo U937* concentrations were obtained by converting one of the in vivo relaxation rates to cell concentration maps. MRI results were compared with the corresponding optical images of the same brains, showing the usefulness of our method to accurately follow therapeutic cell biodistribution in a longitudinal study. Results also demonstrate that the method quantifies a large range of magnetically labelled cells*. Copyright © 2016 John Wiley & Sons, Ltd.     

In vivo analysis of vascularization and biocompatibility of electrospun polycaprolactone fibre mats in the rat femur chamber

In orthopaedic medicine, connective tissues are often affected by traumatic or degenerative injuries, and surgical intervention is required. Rotator cuff tears are a common cause of shoulder pain and disability among adults. The development of graft materials for bridging the gap between tendon and bone after chronic rotator cuff tears is essentially required. The limiting factor for the clinical success of a tissue engineering construct is a fast and complete vascularization of the construct. Otherwise, immigrating cells are not able to survive for a longer period of time, resulting in the failure of the graft material. The femur chamber allows the observation of microhaemodynamic parameters inside implants located in close vicinity to the femur in repeated measurements in vivo. We compared a porous polymer patch (a commercially available porous polyurethane‐based scaffold from Biomerix?) with electrospun polycaprolactone (PCL) fibre mats and chitosan (CS)‐graft‐PCL modified electrospun PCL (CS‐g‐PCL) fibre mats in vivo. By means of intravital fluorescence microscopy, microhaemodynamic parameters were analysed repetitively over 20 days at intervals of 3 to 4 days. CS‐g‐PCL modified fibre mats showed a significantly increased vascularization at Day 10 compared with Day 6 and at Day 14 compared with the porous polymer patch and the unmodified PCL fibre mats at the same day. These results could be verified by histology. In conclusion, a clear improvement in terms of vascularization and biocompatibility is achieved by graft‐copolymer modification compared with the unmodified material.     

REVIEW ARTICLEThrombus formation: direct real-time observation and digital analysis of thrombus assembly in a living mouse by confocal and widefield intravital microscopy

Summary.  We have developed novel instrumentation using confocal and widefield microscopy to image and analyze thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and a brightfield channel, and supports laser-induced injury through the microscope optics. Although this imaging facility requires interface of multiple hardware components, the primary challenge in vascular imaging is careful experimental design and interpretation. This system has been used to localize tissue factor during thrombus formation, to observe defects in thrombus assembly in genetically altered mice, to study the kinetics of platelet activation and P-selectin expression following vascular injury, to analyze leukocyte rolling on arterial thrombi, to generate three-dimensional models of thrombi, and to analyze the effect of antithrombotic agents in vivo .     

国产阿奇霉素体内外抗菌作用的研究

本研究的目的在于评价国产阿奇霉素的体内，外抗菌活性并与进口阿奇霉素及同类药罗红霉素与红霉素进行比较。结果表明，用琼脂二倍稀释法，阿奇霉素对临床分离的７０７株致病菌的体外抗菌谱与抗菌作用国产品与进口品相仿，阿奇霉素对呼吸道常见致病菌中的革兰氏阳性球菌具有强抗菌活性，对金葡菌，溶血性链球菌，肺炎链球菌，草绿色链球菌，奈瑟要球菌，嗜血流感力等ＭＩＣ≤０．０６ｍｇ／Ｌ，与罗红霉素，红霉素相似或稍强。革兰氏     

血管性帕金森综合征临床特点的分析

目的：探讨血管性帕金森综合征（VP）的临床特点及治疗。方法：对35例VP患者（VP组）和42例帕金森病（PD）患者（PD组）的临床资料进行比较分析。结果：VP组平均发病年龄晚于PD组,多合并有脑血管病危险因素,临床表现以步态障碍最为突出,头颅MRI主要表现以基底核区及皮质下白质腔隙性脑梗死为主,左旋多巴等药物对帕金森样症状的治疗有一定的改善作用。结论：VP的诊断应结合病史、临床表现和影像学特征进行综合分析。     

颈椎旋转（提）手法的在体力学测量

目的：研究颈椎旋转手法操作过程中力学参数的特征及其相互关系。方法：运用生物力学实测方法测量同一操作者对15例患者施行旋转手法过程中的力学参数（作用力、作用时间和加速度）,再通过数学方法,计算出旋转手法扳动过程所产生的位移及冲量。结果：①颈椎旋转手法的力学参数平均值：预加载力15.15±5.11kg,最大作用力27.24±8.81kg,扳动力14.29±5.15kg,扳动时间114.33±16.98ms,最大加速度为-0.35±0.11g,扳动位移为9.34±2.67mm,扳动冲量为22.49±7.11Ns;②左手操作与右手操作的旋转手法力学参数相比较,除最大加速度、扳动时间和扳动冲量以外,余项P值均〉0.05,差异无显著性意义;③预加载力和扳动力相比较,两者间差异无显著性意义（P〉0.05）;④经Pearson相关分析,预加载力、最大作用力和扳动力呈显著的正相关性（R〉0.8,P〈0.001）。结论：左右手施行旋转手法时作用力特征（包括预加载力、最大作用力、扳动力）是相近的;旋转手法从缓慢上牵开始到扳动操作结束整个过程具有一定的规律性：扳动力的大小取决于预加载力的大小。     

双光子成像在细胞移植与治疗中的应用

双光子成像技术具有活体三维深层成像的能力,是重要的活体成像工具,在针对生物组织相关的活体、原位研究中应用广泛。通过移植细胞建立研究模型,可以在真实的细胞微环境中进行过程与机理研究。结合双光子成像技术,可以对移植细胞进行在体形态学鉴定与功能评价,避免体外培养模型带来的差异。双光子成像技术促进了疾病模型的研究,为疾病与治疗等病理生理过程的研究提供了重要的研究手段。细胞移植已经用于中枢神经系统、心肌、骨髓以及抗肿瘤药物等研究模型建立中,本文对双光子成像技术的技术原理、应用领域进行综述,并探讨了该技术的发展前景。双光子成像技术以其较大的成像深度、较高的成像质量等特点,满足了在体成像的需求,在以动物为研究对象的研究中发挥了重要的作用,使我们对活细胞生理、病理和药理领域的认识得到极大的发展。      

Imaging modalities for the in vivo surveillance of mesenchymal stromal cells

Bone marrow stromal cells exist as mesenchymal stromal cells (MSCs) and have the capacity to differentiate into multiple tissue types when subjected to appropriate culture conditions. This property of MSCs creates therapeutic opportunities in regenerative medicine for the treatment of damage to neural, cardiac and musculoskeletal tissues or acute kidney injury. The prerequisite for successful cell therapy is delivery of cells to the target tissue. Assessment of therapeutic outcomes utilize traditional methods to examine cell function of MSC populations involving routine biochemical or histological analysis for cell proliferation, protein synthesis and gene expression. However, these methods do not provide sufficient spatial and temporal information. In vivo surveillance of MSC migration to the site of interest can be performed through a variety of imaging modalities such as the use of radiolabelling, fluc protein expression bioluminescence imaging and paramagnetic nanoparticle magnetic resonance imaging. This review will outline the current methods of in vivo surveillance of exogenously administered MSCs in regenerative medicine while addressing potential technological developments. Furthermore, nanoparticles and microparticles for cellular labelling have shown that migration of MSCs can be spatially and temporally monitored. In vivo surveillance therefore permits time‐stratified assessment in animal models without disruption of the target organ. In vivo tracking of MSCs is non‐invasive, repeatable and non‐toxic. Despite the excitement that nanoparticles for tracking MSCs offer, delivery methods are difficult because of the challenges with imaging three‐dimensional systems. The current advances and growth in MSC research, is likely to provide a wealth of evidence overcoming these issues. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of dietary supplementation with creatine on homocysteinemia and systemic microvascular endothelial function in individuals adhering to vegan diets

The incidence of cardiovascular diseases in vegetarian individuals is lower than that in the general population. Nevertheless, individuals who adhere to vegan diets have a higher prevalence of hyperhomocysteinemia with eventual adverse effects on vascular reactivity. Creatine supplementation (CrS) reduces plasma homocysteine levels and enhances vascular reactivity in the microcirculation. Thus, we investigated the effects of CrS on systemic microcirculation and homocysteine blood levels in strict vegan subjects. Forty‐nine strict vegan subjects were allocated to the oral CrS (5 g micronized creatine monohydrate daily for three weeks; n = 31) and placebo (n = 18) groups. Laser speckle contrast imaging coupled with acetylcholine skin iontophoresis was used to evaluate cutaneous microvascular reactivity, and intravital video‐microscopy was used to evaluate skin capillary density and reactivity before and after CrS. We demonstrated that CrS reduces the plasma levels of homocysteine and increases those of folic acid. After the CrS period, the homocysteine levels of all of the vegan subjects normalized. CrS also induced increases in baseline skin functional capillary density and endothelium‐dependent capillary recruitment in both normo‐ (N‐Hcy) and hyperhomocysteinemic (H‐Hcy) individuals. CrS increased endothelium‐dependent skin microvascular vasodilation in the H‐Hcy vegan subjects but not in the N‐Hcy vegan subjects. In conclusion, three weeks of oral CrS was sufficient to increase skin capillary density and recruitment and endothelium‐dependent microvascular reactivity. CrS also resulted in plasma increases in folic acid levels and reductions in homocysteine levels among only the H‐Hcy individuals.     

DNA三角双锥荧光分子探针的制备及初步生物学评价

目的  构建基于DNA三角双锥纳米结构（DBN）的荧光分子探针,于活体动物水平探索探针的生物学性质。方法  采用一步退火法,制备携带侧臂链的DBN;与DyLight 755偶联的寡聚核苷酸单链A20（DyLight 755-A20）混匀,制备荧光分子探针DyLight 755-DBN;将DyLight 755-DBN通过尾静脉注射实验鼠体内,于不同时间点处死,取感兴趣脏器测定荧光计数,探索分子探针的体内分布情况;将DyLight 755-DBN通过尾静脉注射体内,于不同时间点行小动物活体成像研究。结果  成功制备携带侧臂链的DBN,利用聚丙烯酰胺凝胶电泳表征;通过与DyLight 755-A20等摩尔量杂交,成功制备荧光分子探针DyLight 755-DBN。体内分布实验显示DyLight 755-DBN进入体内后,荧光信号主要集中在肾脏、肝脏、脾脏、胃;注射分子探针5~15 min,在实验鼠的肺部有一定的荧光信号,但15 min后,荧光信号几乎没有。活体成像结果显示DyLight 755-DBN进入实验鼠体内主要集中在肝脏和胃,膀胱中维持较强的荧光信号。结论  荧光分子探针DyLight 755-DBN是一种性质优良的分子影像探针。