Visualisation and modelling of renal capillaries from confocal images

A computer aided design was developed to support three-dimensional visualisation and modelling of vascular networks. Volume data comprised a series of images obtained using a Zeiss confocal laser scanning microscope. The profiles of vessels were automatically segmented using two-dimensional morphological filters. Segmented contours of the vessels were used to form a spatial model of the network. The centre points of segmented contours were used to derive a three-dimensional graph representing the vascular network. The proposed method was applied to renal capillary networks of normal rats, and showed well the lobular structure of glomeruli. The average length of renal capillary networks was 6.09 mm. Three-dimensional models based on confocal data require much less effort than reconstructions based on serial sections, and can be adapted for any vascular patterns.     

Three-dimensional analysis of increased vasculature around the glomerular vascular pole in diabetic nephropathy

In diabetic nephropathy, several small vessels are frequently observed around the glomerular vascular pole in addition to the usual afferent and efferent arterioles. To elucidate the morphology of these abnormal small vessels, a three-dimensional study was performed by using computer-aided reconstruction techniques. In the present study, the renal tissue samples of 21 biopsy and 73 autopsy cases of diabetic glomerulonephropathy were examined. In addition to ordinary light microscopic observations, three series of serial sections from one autopsy and two biopsy cases were analysed. Five glomeruli with increased numbers of vessels around the vascular pole were reconstructed three-dimensionally. The vasculature in and around the glomerulus was analysed in detail by rotating and viewing in different planes via computer-generated three-dimensional images. These vessels anastomose to the lobular structure of the intraglomerular capillary network, mainly to the afferent branches through the widened vascular hilus. The distal end of the vessels anastomoses to the peritubular capillary. The increased vasculature is interpreted as neoangiogenesis resulting from diabetes, which may have a functional role in facilitating efferent blood flow from the glomerulus.Dr. Wen Min is a visiting research fellow from Norman Bethune University of Medical Sciences, China     

Development of an Image-Based Network Model of Retinal Vasculature

The paper presents an image-based network model of retinal vasculature taking account of the 3D vascular distribution of the retina. Mouse retinas were prepared using flat-mount technique and vascular images were obtained using confocal microscopy. The vascular morphometric information obtained from confocal images was used for the model development. The network model developed directly represents the vascular geometry of all the large vessels of the arteriolar and venular trees and models the capillaries using uniformly distributed meshes. The vasculatures in different layers of the retina, namely the superficial, intermediate, and deep layer, were modeled separately in the network and were linked through connecting vessels. The branching data of the vasculatures was recorded using the method of connectivity matrix of network (the graph theory). Such an approach is able to take into account the detailed vasculature of individual retinas concerned. Using the network model developed, a circulation analysis based on Poiseuille’s equation was carried out. The investigations produced predictions of spatial distribution of the pressure, flow, and wall shear stress in the entire retinal vasculature. The method developed can be used as a tool for continuous monitoring of the retinal circulation for clinical assessments as well as experimental studies.     

Development of vascular networks during the morphogenesis of intestinal villi in the fetal mouse

The formation of villi was investigated in the proximal small intestine of fetal mice between 12 and 19 days of gestation in association with the development of vascular networks. Three-dimensional vascular networks were observed with confocal laser scanning microscopy after perfusing the fetuses with RITC-labelled gelatin. Some specimens were simultaneously stained for laminin by immunofluorescence to examine the epithelio-mesenchymal boundary. At 12 and 13 days of gestation, the epithelium of small intestine formed cylindrical tubes, and a loose plexus of large vessels formed in the mesenchyme. At 14 days of gestation, a dense plexus of smaller vessels was formed inside of the loose plexus and some capillary branches from this dense plexus developed beneath the epithelium. The epithelium began to make ingrowths in places where no capillaries were situated beneath the epithelium. The capillaries formed a loop in a concavity made between the ingrowths. As development proceeded, this concavity deepened and formed a villus. The top of the capillary loop always places at the top of the mesenchyme in the villus and the capillary loop then developed into a capillary network within the villus. These results suggest that the initial formation of intestinal villi in the fetal mouse occurs by an ingrowth of the epithelium and the developing vascular nets appear to play some role in villi formation. Throughout the villus formation, the top of the capillary loop in a villus would be anchored to the epithelial basement membrane or to the adjoining matrixes.     

基于Micro-CT的家兔腹盆腔器官血管数字化三维模型的构建

目的 通过乳胶-氧化铋对比剂与显微计算机断层扫描(Micro-CT)技术相结合的方法,完成家兔腹盆腔器官内血管系统的三维数字化图像重建。方法 6只4月龄雌性健康家兔作为研究对象。试验动物均进行腹腔麻醉。麻醉成功后纵行切开家兔腹壁,暴露盆腹腔血管,完成腹主动脉插管(用于子宫、膀胱、小肠的对比剂在体灌注)及肾动脉插管(用于肾脏的对比剂在体灌注)。选择乳胶-氧化铋混合溶液(氧化铋颗粒/乳胶溶液=1 g/mL)作为血管灌注对比剂。血管灌注成功后选取家兔的子宫、膀胱、小肠和肾脏作为研究器官。利用SkyScan 1076型Micro-CT对目标器官进行扫描,使用NRecon软件对扫描获得的原始图像进行处理和转化。采用Mimics 17.0软件对目标器官内的血管图像进行三维重建,获得家兔不同器官内的血管树三维图像,分析家兔不同器官内不同血管树的分级和分支特点。结果 目标器官的血管树数字化三维重建模型均构建成功。家兔膀胱血管树的最小可测量血管分级为二级,直径为(0.41±0.08)mm;子宫血管树的最小可测量血管分级为三级,直径为(0.39±0.08)mm;小肠和肾脏血管树的最小可测量血管分级均为四级,直径分别为(0.27±0.04)mm和(0.19±0.03)mm。结论 采用乳胶-氧化铋混合对比剂进行动物血管灌注Micro-CT成像,通过相关软件可成功重建动物器官内血管树的三维图像,可显示家兔器官血管树的走行并测量血管直径,对于小动物腹盆腔器官血管系统尤其是微小型血管网数字化三维模型的建立具有较大优势,为小动物腹盆腔器官疾病模型中血管解剖结构的研究提供了新的思路和方法。     

Microvascular architecture of the human urinary bladder wall: a corrosion casting study

The vascular system of the urinary bladder wall effectively performs its function in spite of considerable spatial changes due to the filling/voiding cycle. However, only a few studies have dealt with the microvascular architecture of the bladder wall and only two, using old-fashioned techniques, were devoted to the human bladder. This study presents the microvasculature of the human bladder wall visualized by scanning electron microscopy of vascular corrosion casts. Postoperative bladder specimens obtained from patients with advanced bladder tumors were filled with small amount (80 ml) of saline and perfused via at least four largest arteries with anticoagulant-containing saline followed by paraformaldehyde/glutaraldehyde fixative and Mercox resin. After polymerization of the resin, the vascular casts were macerated with potassium hydroxide, cleaned with formic acid and water and freeze dried. Only regions of the bladder wall distant to the tumor were examined in light and scanning electron microscopes. The almost empty state of the bladder was manifested by extensive folding of the mucosa and tortuosity of almost all vessels other than capillaries. The branches of main arteries and veins formed an adventitial/serosal plexus which directly supplied/drained the capillary network of the muscularis and sent long perpendicular vessels to the mucosal plexus. These vessels had straight or coiled course depending on whether they terminated at the top or at the base of the mucosal folds. The rich mucosal plexus followed the folds parallel to their surface and gave off short, straight, mostly perpendicular twigs communicating with the subepithelial capillary network. Apart from very few vascular interconnections between the mucosal plexus and the muscularis, the submucosa was generally avascular. The subepithelial capillary network showed extreme density and uneven contours of the capillaries, only in less folded areas of trigone and urethral orifice the network was looser and capillaries thinner. The capillary system of the muscularis was poorly developed. Due to its architecture, tortuosity, and coiling/uncoiling capabilities, the microvasculature of the human urinary bladder wall seems to efficiently accommodate changes associated with cyclic contraction and stretching. Disturbances in blood flow induced by overdistension of the bladder reported in several studies may be due to pressure of the urine affecting the patency of the vessels rather than to the spatial insufficiency of the vascular system.     

Vascular network in papillae of dog oral mucosa using corrosive resin casts with scanning electron microscopy

The purpose of this study was to undertake a three-dimensional analysis of the vascular network of the lamina propria in the gingiva, alveolar mucosa, buccal mucosa, palate, and lingual mucosa of the dog. Using the corrosive resin casting technique, casts of the vascular network were prepared and examined by scanning electron microscopy. In the oral mucosa, larger arteries in the submucosa divide into smaller branches that enter the lamina propria. These branches form one or more layers of vessels at the base of the papillae of the lamina propria, the so-called subpapillary vascular network. Here the vessels divide again and enter the papillae to form a subepithelial capillary network. The configuration of the capillary loops within each papilla of the lamina propria is determined by the shape of the papilla. The characteristic shape of the loops resembles a hairpin. The capillary loops in the lingual papillae are larger and more complex than capillary loops found elsewhere in the oral mucosa. The mucosa of the posterior portion of the hard palate, the soft palate, and the tongue contain many venous valves.     

Characterization of solid tumor microvasculature: a three-dimensional analysis using the polymer casting technique

Using a polymer casting technique in conjunction with scanning electron microscopy, the three-dimensional characterization of tumor microvasculature as a function of age of renal adenocarcinoma in the rat kidney is undertaken. The microvasculature of the rat tumor model is compared with VX2 carcinoma in the rabbit leg muscle. Light microscopy and transmission electron microscopy on the rat tumor model are performed to correlate the features seen under scanning electron microscopy of vascular casts. The casts show marked differences between tumor and normal microvasculature. The tumor vascular architecture appears disarrayed with prevalent atypical features such as coils, ribbons, sheets, dense capillary networks, saccular dilatations, leaky and otherwise highly irregular vessels. Sprouts of new growth capillaries are seen throughout the tumor casts. Compressed vessels are present and become more pronounced in older tumors. These features are not observed in normal controls treated under identical conditions. The application of this high resolution three-dimensional casting technique to tumor studies is promising for research in basic tumor mechanics as well as in the effects of tumor vasculature on mediating radiation and chemotherapy and the fundamental mechanisms of metastasis.     

同时显示肾微血管和细胞构筑的实验研究

目的：在媒染微血管的同时显示肾内细胞构筑，以了解肾微血管与肾小管之间的相互关系。方法：用单宁酸一氯化铁法(TA—be)媒染肾微血管，再以苏木精、H—E法复染来观察肾小管细胞结构。结果：经复染的2组切片各级血管和细胞均染色良好，血管壁平滑肌纤维、内皮细胞界限清晰，细胞核蓝染，H—E染色的细胞质呈深浅不同的红色。因切片较厚，保证了微血管的立体感，细胞的染色效果却不及薄切片的鲜艳。结论：TA—Fe法配合苏木精或HE复染，可同时显示肾微血管和细胞构筑。     

Angioarchitecture of rabbit iris

We made a scanning electron-microscopic study of the angioarchitecture of the rabbit iris using vascular resin casts, and compared the vascular structure in miosis to that in mydriasis. There were three vascular layers in the iris: the anterior capillary layer, arteriolo-venular layer and posterior capillary layer. The anterior capillary layer was a network which covered the anterior surface of the iris. The posterior capillary layer was a peculiar network composed of many capillary folds, which were arranged radially. The arteriolo-venular layer was sandwiched between the two capillary layers. In this layer, arterioles and venules ran radially toward the pupil. The peripupillary region lacked the posterior capillary layer. In miosis, the vessels of the peripheral iris were straightened radially, while those in the peripupillary region were folded. In mydriasis, the vessels were very tortuous in the peripheral region, while those in the peripupillary region were stretched laterally. The change in the angioarchitecture of the iris was suited to pupillomotoric activity.     

大鼠肾标本连续切片的三维重建及形态学测量

目的 综合运用目前较成熟的技术,建立一个易施行的、可对大组织标本连续切片整体图像进行三维重建及形态学测量的方法。 方法 对正常大鼠肾的石蜡包埋标本进行全序列连续切片,HE染色后对切片图像进行数字化采集,经过拼接、配准步骤,建立切片整体图像的数字化数据集,对其内部的血管系统及肾盂进行分割及三维重建,并对重建结果进行形态学测量。 结果 切片的整体图像清晰,数字化图像数据集配准准确,三维重建结果再现了各种结构的立体特征及其在原组织块内的位置,在三维空间内,可进行无阻挡的自由观察,并且测量出了脉管系统的多项三维形态学指标,包括组织体积构成百分比、动脉长度及分支角度等。 结论 采用这种方法取得了较为满意的重建结果,可为其他组织连续切片的三维重建研究提供借鉴。     

Automatic multiscale enhancement and segmentation of pulmonary vessels in CT pulmonary angiography images for CAD applications

The authors are developing a computerized pulmonary vessel segmentation method for a computer-aided pulmonary embolism (PE) detection system on computed tomographic pulmonary angiography (CTPA) images. Because PE only occurs inside pulmonary arteries, an automatic and accurate segmentation of the pulmonary vessels in 3D CTPA images is an essential step for the PE CAD system. To segment the pulmonary vessels within the lung, the lung regions are first extracted using expectation-maximization (EM) analysis and morphological operations. The authors developed a 3D multiscale filtering technique to enhance the pulmonary vascular structures based on the analysis of eigenvalues of the Hessian matrix at multiple scales. A new response function of the filter was designed to enhance all vascular structures including the vessel bifurcations and suppress nonvessel structures such as the lymphoid tissues surrounding the vessels. An EM estimation is then used to segment the vascular structures by extracting the high response voxels at each scale. The vessel tree is finally reconstructed by integrating the segmented vessels at all scales based on a "connected component" analysis. Two CTPA cases containing PEs were used to evaluate the performance of the system. One of these two cases also contained pleural effusion disease. Two experienced thoracic radiologists provided the gold standard of pulmonary vessels including both arteries and veins by manually tracking the arterial tree and marking the center of the vessels using a computer graphical user interface. The accuracy of vessel tree segmentation was evaluated by the percentage of the "gold standard" vessel center points overlapping with the segmented vessels. The results show that 96.2% (2398/2494) and 96.3% (1910/1984) of the manually marked center points in the arteries overlapped with segmented vessels for the case without and with other lung diseases. For the manually marked center points in all vessels including arteries and veins, the segmentation accuracy are 97.0% (4546/4689) and 93.8% (4439/4732) for the cases without and with other lung diseases, respectively. Because of the lack of ground truth for the vessels, in addition to quantitative evaluation of the vessel segmentation performance, visual inspection was conducted to evaluate the segmentation. The results demonstrate that vessel segmentation using our method can extract the pulmonary vessels accurately and is not degraded by PE occlusion to the vessels in these test cases.     

Microvasculature in the mouse ovarian follicle demonstrated by a lectin angiography method

A lectin angiography method was applied to identify ovarian vasculature in 32-34 days old mice treated with gonadotropins. Blood was washed out by perfusion until the inferior vena cava became translucent. Horseradish-peroxidase (HRP)-conjugated Concanavalin A (Con A) solution (10-15 ml) was perfused into the systemic circulation via the left ventricle at the rate of 2-3 ml/min. The animals were left for a 30 min reaction interval. The lumina of the blood vessels were flushed with 5-10 ml of phosphate buffered saline (pH 7.4). The ovaries were excised and fixed by immersion in 4% paraformaldehyde and sectioned serially at thickness of 100, 200 or 400 microm using a Microslicer. The binding of HRP-conjugated-Con A to endothelial cells was visualized by using 3,3'-diaminobenzidine-4 HCl (DAB-4 HCl) reaction. By examining various sections the three-dimensional architecture of the vascular networks of the preovulatory Graafian follicles and corpora lutea can be established. Capillary networks of preovulatory Graafian follicles were identified in sections of ovaries removed 11 h after hCG injection. Then, capillary networks of the Graafian follicles increased due to the hypertrophic growth of the theca interna and extension of capillary branches into the follicles. Well-developed capillary networks of corpora lutea were found in ovaries removed 24 h after hCG injection. For these observations the 200 microm-sections were the most useful. The present modified lectin angiography is a useful method for visualizing the microvasculature of mouse ovaries.     

婴儿和足月胎儿下颌下腺的微血管构筑

用血管铸型扫描电镜法，观察了婴儿和足月胎儿下颌下腺的微血管构筑，腺泡微动脉自小叶内部行至腺泡周围分成毛细胞管网，或穿出小叶至小叶的基底面，沿小叶表面延伸并分成毛细血管网，腺泡毛细血管网排列稀疏。足月胎儿的纹状管周围为毛细血管后微静脉和微静脉交织吻合成的网状微静脉丛。婴儿的纹状管周围为排列密集的毛细血管网。足月胎儿的腺泡毛细血管汇入纹状管周围微静脉丛，婴儿的与纹状管周围毛细血管网相连。小叶间导管周围     

Thymic vasculature: organizer of the medullary epithelial compartment?

The epithelial component of the thymic environment is organized into discrete cortical and medullary compartments that mediate different aspects of thymocyte differentiation. The processes controlling the growth and organization of these epithelial compartments are poorly defined. In this study we have used a novel approach to define the three-dimensional organization of thymic epithelial (TE) compartments to demonstrate that the organization of the medullary TE compartment is very complex. A spatial relationship of medullary thymic epithelium with vascular elements of the thymus was demonstrated by simultaneous immunohistochemical labeling of vascular elements and medullary TE. Medullary TE was often arranged as perivascular cuffs surrounding intermediate-sized vessels, but was not associated with either the capillary network or large centrally located vessels. Similar analyses of RAG-2(-/-) thymi revealed a striking physical association of medullary TE with vascular elements. Ultrastructural analysis of the RAG-2(-/-) thymus indicated a preferential association of focal accumulations of medullary TE with post-capillary venules. These data suggest that discrete segments of the thymic vasculature provide cues that act in concert with thymocyte-derived stimuli to effect normal development of the thymic environment.     

Vascular architecture of human urinary bladder carcinoma: a SEM study of corrosion casts

The vascular architecture of five advanced invasive papillary tumours of the urinary bladder was investigated using corrosion casting and scanning electron microscopy. The superficial vasculature was composed predominantly of capillary systems of two types: dense flat networks with numerous interconnections and tightly packed tortuous loops, forming multiple irregular folds that reflected the papillary morphology of the tumours. The capillaries were supplied and drained by numerous straight nonanastomosing arterioles and venules, which arose by way of multiple branching of larger vessels originating from the mucosal plexus of the bladder. Differences between the tumours in the spatial arrangement of these vessels probably reflect different growth dynamics. The intramural parts of the tumours contained a chaotic network of straight, uniform capillaries with numerous sprouts, which was very different from the superficial capillary system. It is postulated that different angiogenesis-targeted growth factors may be expressed in the phases of exophytic growth and muscularis invasion of the tumour, leading to the formation of different microvascular patterns.