Intervascular Spaces Research Articles

Macular Telangiectasia Type 2: Case Report and Clinical Review

Background: Macular telangiectasia type 2 is a bilateral condition of unknown etiology, typically manifesting during the fourth to sixth decades of life. Its prevalence is likely underestimated as there is low disease awareness, both amongst clinicians and patients, and it is often misdiagnosed as age-related macular degeneration in later stages. It is important to understand the pathophysiology, natural history, prognosis, and potential complications to manage this condition properly and provide accurate patient education. Case Report: A 75-year-old white male presented with gradual worsening of vision at all distances for years. Fundus examination revealed bilateral foveal circular red spot with parafoveal pigment clumping in the left eye along with bilateral scattered refractile crystalline deposits. Optical coherence tomography of the macula revealed internal limiting membrane drape in both eyes with focal disruptions to the ellipsoid zone in the left eye. Optical coherence tomography angiography revealed telangiectatic vessels, evident in the superficial and deep plexus, increased intervascular spaces involving the superficial and deep plexus, as well as right-angled vessels and foveal avascular zone irregularities. There was no evidence of progression to proliferative disease and the patient’s vision had remained stable for six years. Conclusion: Macula telangiectasia type 2 is a neurodegenerative disease with the potential to cause vision loss. Newer imaging modalities, specifically optical coherence tomography and optical coherence tomography angiography, have become important diagnostic tools and useful in identifying this disease at earlier stages and differentiating it from other macular degenerative conditions. CE Notification: This article is available as a COPE accredited CE course. You may take this course for 1-hour credit. Read the article and take the qualifying test to earn your credit. Click here to Enroll (https://www.crojournal.com/macular-telangiectasia-type-2-case-report-and-clinical-review) Please check COPE course expiry date prior to enrollment. The COPE course test must be taken before the course expiry date.

DORSAL Plication Technique for the Treatment of Congenital Ventral Penile Curvature: Long-Term Outcomes of 72 Cases

A total of 78 patients aged 11 to 17 years were diagnosed with congenital ventral penile curvature and underwent surgery with the dorsal plication technique between 2005 and 2014. To investigate the long-term outcomes of 72 patients who underwent dorsal penile plication for the treatment of congenital ventral penile curvature without hypospadias. In all cases, the intervascular space between the deep dorsal vein and dorsal artery was dissected, and tunical plication was carried out with non-absorbable 3-0 polyamide sutures and the complication and satisfaction rates of the patients were determined in the postoperative seventh year. At the final postoperative follow-up, the patients' satisfaction with the operation was found to be 95.8%. Shortening of the penis (0.5-1 cm) in five cases, recurrence with less than a 20-degree curvature in two cases, palpable sutures in two cases was observed and no patients reported erectile dysfunction. The limitations of our study can be considered as the absence of pharmacological erection in the preoperative evaluation, failure to evaluate penile length at the last postoperative follow-up due to the continued development of the penis, inability to evaluate erectile function at the beginning, postoperative erectile capacity being assessed in only some of the operated cases, all operations being performed by a single surgeon in the same center, and the absence of standardized questionnaires for postoperative satisfaction or adverse events. According to the results of this study, dorsal plication is a relatively simple method with a low risk and high success rate for the treatment of congenital ventral penile curvatures.

Optical Coherent Tomography-Angiography in the Research of Myopia Complicated by Choroidal Neovascularization

Purpose: to determine the features of myopic choroidal neovascularization (mCNV) using optical coherent tomography-angiography (OCTA) in patients with complicated myopia during anti-VEGF therapy.Patients and methods. A prospective study of 25 people (40 eyes) with complicated myopia was carried out, which consisted in a comparative analysis of the qualitative and quantitative OCTA characteristics of 29 eyes with myopic CNV and 11 paired intact eyes. OCTA was used in the Angio Retina and Angio Analytics modes based on automatic software. The phenotypic features of CNV before treatment, during anti-VEGF therapy (Ranibizumab) and at the end of the observation period were taken into account.Results. The average follow-up period is 26.12 (12.36; 35.02) months. Two mCNV phenotypes were established: glomerular (44.83 %) and flat (55.17 %) as regularized (18.75 %) and tangled (81.25 %) vascular network patterns. Hypointensive peripheral halo during the entire period of the neovascular complex activity was visualized regardless of the phenotype. Significant differences in the flat phenotype were revealed: neuroepithelial detachment (87.50 %) was more common, the Select Area and Flow Area of the neovascular complex was significantly greater both before treatment and at the end of the observation period, the mCNV course of continued growth after injection of anti-VEGF did not occur, recurrence rate is much higher. The area and perimeter of the foveal avascular zone (FAZ) in the eyes with mCNV increased significantly by the end of the observation. The acirculatory index (AI) was determined to be significantly higher, and the Retinal capillary flow density in the fovea region 300 μm wide around the FAZ (FD) was lower in the eyes with mCNV compared to the control group. The nonactive neovascular complex were characterized by depletion of the vascular network with a predominance of intervascular space, vessels in the form of a residual skeleton, blindly end, dim, small capillaries and end loops are missing. Halo with nonactive CNV is not found.Conclusion. OCTA identifies the features of the course of myopic CNV depending on the phenotype, allows to evaluate the effectiveness of anti-VEGF therapy and the degree of progression of degenerative changes in the macular region.

Spatial Regulation of Thrombopoiesis in the Bone Marrow

Blood platelets play key roles in hemostasis and thrombosis and are the second most abundant cell type in the circulation. Due to their short life span of only a few days, anuclear platelets are continuously replenished and thus provide a classic system to study hematopoiesis. In mammals, platelets are produced by megakaryocytes (MKs) that are predominantly residing in the bone marrow (BM). MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Unfortunately, previous studies on megakaryopoiesis were often limited by 2D imaging and cutting artefacts when analyzing bone sections, potentially resulting in underestimation of MK-to-vessel contacts and MK volumes. We studied megakaryopoiesis by visualizing MKs in their 3D environment. To this end, murine bones were simultaneously stained for MKs and endothelial cells, fixed, chemically cleared and imaged by Light Sheet Fluorescence Microscopy (LSFM). Thus, we achieved 3D-reconstructions of the complete and intact bone with subcellular resolution. Through imaging of MKs in the intact BM, we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. We developed and compared different image processing pipelines and simulation scenarios for precise identification of MKs in 3D light-sheet fluorescence microscopy of uncut murine bones. By combining in vivo two-photon microscopy and in situ LSFM with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. To complement limited imaging approaches computational simulations represent an important, well-controllable tool. Typically, simulation studies use artificial meshes as templates to minimize the computational effort or due to the lack of experimental data. Unfortunately, such simplified artificial templates for MKs and the vasculature can bias simulations and lead to misinterpretations as we show here. Using the segmented cell and vessel objects of true 3D images can overcome those limitations providing a simulation framework that has the prerequisites to maximally reflect the physiological situation. Thus, imaging and simulations go hand in hand when the respective 3D cell and vessel objects perfectly serve as biological templates for advanced simulations. We demonstrate reliable whole-bone analysis in silico, and found that MKs influence neutrophil and HSC migration as biomechanical restrainers modulating cell mobility and extravasation. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche (1). Furthermore, we identify MKs as biomechanical restraints for bone marrow cell mobilization. As MKs themselves do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.(1) Stegner D, van Eeuwijk JMM, Angay O, Gorelashvili MG, Semeniak D, Pinnecker J, Schmithausen P, Meyer I, Friedrich M, Dütting S, Brede C, Beilhack A, Schulze H, Nieswandt B, Heinze KG. Thrombopoiesis is spatially regulated by the bone marrow vasculature, Nat Commun. 2017 8(1):127. DisclosuresNo relevant conflicts of interest to declare.

Choriocapillaris Imaging Using Multiple En Face Optical Coherence Tomography Angiography Image Averaging

Imaging of the choriocapillaris in vivo is challenging with existing technology. Optical coherence tomography angiography (OCTA), if optimized, could make the imaging less challenging. To investigate multiple en face image averaging on OCTA images of the choriocapillaris. Observational, cross-sectional case series at a referral institutional practice in Los Angeles, California. From the original cohort of 21 healthy individuals, 17 normal eyes of 17 participants were included in the study. The study dates were August to September 2016. All participants underwent OCTA imaging of the macula covering a 3 × 3-mm area using OCTA software (Cirrus 5000 with AngioPlex; Carl Zeiss Meditec). One eye per participant was repeatedly imaged to obtain 9 OCTA cube scan sets. Registration was first performed using superficial capillary plexus images, and this transformation was then applied to the choriocapillaris images. The 9 registered choriocapillaris images were then averaged. Quantitative parameters were measured on binarized OCTA images and compared with the unaveraged OCTA images. Vessel caliber measurement. Seventeen eyes of 17 participants (mean [SD] age, 35.1 [6.0] years; 9 [53%] female; and 9 [53%] of white race/ethnicity) with sufficient image quality were included in this analysis. The single unaveraged images demonstrated a granular appearance, and the vascular pattern was difficult to discern. After averaging, en face choriocapillaris images showed a meshwork appearance. The mean (SD) diameter of the vessels was 22.8 (5.8) µm (range, 9.6-40.2 µm). Compared with the single unaveraged images, the averaged images showed more flow voids (1423 flow voids [95% CI, 967-1909] vs 1254 flow voids [95% CI, 825-1683], P < .001), smaller average size of the flow voids (911 [95% CI, 301-1521] µm2 vs 1364 [95% CI, 645-2083] µm2, P < .001), and greater vessel density (70.7% [95% CI, 61.9%-79.5%] vs 61.9% [95% CI, 56.0%-67.8%], P < .001). The distribution of the number vs sizes of the flow voids was skewed in both unaveraged and averaged images. A linear log-log plot of the distribution showed a more homogeneous distribution in the averaged images compared with the unaveraged images. Multiple en face averaging can improve visualization of the choriocapillaris on OCTA images, transforming the images from a granular appearance to a level where the intervascular spaces can be resolved in healthy volunteers.

Thrombopoiesis is spatially regulated by the bone marrow vasculature

In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.

Vascular anatomy in children with univentricular hearts regarding transcatheter bidirectional Glenn anastomosis

Transcatheter stent-secured Glenn anastomosis, aiming to reduce the invasiveness of palliation in patients with univentricular heart defects, has been reported in large experimental animals. The advent of biodegradable stents and tissue-engineered vascular grafts will make this procedure a reality in human patients. However, the relationship between the superior vena cava (SVC) and the right pulmonary artery (RPA) is different in humans. To characterise vascular anatomy in children with univentricular hearts, regarding technical aspects and device design for this procedure. Retrospective analysis of 35 thoracic computed tomography angiograms at a mean age of 18.1±22.4 months. Two types of arrangement between the SVC and the RPA were identified: anatomy convenient for immediate wire passage and stent deployment between the two vessels (60%); and pattern of early RPA branching, requiring the perforation wire to traverse the intervascular space to avoid entrance into the upper RPA branch (40%). In patients with the convenient vascular arrangement, the vessels were nearly perpendicular, having immediate contact, with the posterior SVC aspect partially "wrapping" the adjacent RPA in most patients. In patients with early RPA branching, the mean shortest SVC-to-central RPA distance was 4.3±2.7mm. For the total population, the mean length of proximal SVC that allowed stent deployment without covering the brachiocephalic vein was 15.6±5.1mm. A trumpet-shaped covered stent in a craniocaudal orientation reaching from the SVC into the prebranching RPA seems most suitable for achieving bidirectional Glenn anastomosis percutaneously in humans. However, the short length of the proximal SVC and the presence of early RPA branching pose challenges for optimal design of the dedicated device.

Characteristics of fundus image in macular telangiectasia type 2

Objective To observe the fundus image characteristics of macular telangiectasia type 2 (MacTel type 2) patients. Methods A total of 8 patients (16 eyes) diagnosed of MacTel type 2 were included in this study. There were 4 males and 4 females, age ranged from 44 to 69 years old with a median age of (59.88±7.85) years. All patients received examination of best-corrected visual acuity (BCVA), slit lamp microscope, indirect ophthalmoscopy, fundus color photography, fundus autofluorescence (AF), fundus fluorescein angiography (FFA), spectral domain optical coherence tomography (OCT) and macular pigment optical density (MPOD). Four eyes of 2 patients received OCT angiography examination at the same time. Classification was made according to the Gass and Blodi′s criteria. The follow-up time was from 1 to 19 months with the average time of (11.00±8.91) months. The clinical characteristics were observed and analyzed. Results The BCVA was 0.07 - 0.8. There were 1 eye in stage 1, 1 eye in stage 2, 6 eyes in stage 3, 8 eyes in stage 4. The disease showed a bilateral appearance with a low progression. Fundus features included loss of retinal transparency (14 eyes, 87.5%), blunted retinal venule (15 eyes, 93.75%), pseudo-lamellar hole (5 eyes, 31.25%), pigment proliferation (9 eyes, 56.25%). FFA findings were telangiectatic capillaries predominantly temporal to the foveola in the early phase and a diffuse hyperfluorescence in the late phase. Spectral domain OCT features included depletion of the retinal inner, outer structures, cavity (7 eyes, 43.75%), and atrophy of the neurosensory retina (9 eyes, 56.25%). On AF, reduced foveal masking due to loss of macular pigment can be observed. The loss of macular pigment could also be seen on MPOD. OCTA showed the increased intervascular spaces, broken regular network of foveal avascular zone (FAZ), right-angled vessel dipping, dilatations, traction of superficial and deep capillary layers in both the superficial and deep layers, especially in the deep layer. Only one eye of one patient proceeded from stage 4 to stage 5. Conclusions Characteristic features of MacTel type 2 included cavities and outer retina atrophy on OCT; loss of MP on MPOD; broken regular network of FAZ, morphological and structural abnormalities of superficial and deep layers of perifoveal capillary network on OCTA. Key words: Retinal Diseases/diagnosis; Macula lutea/injuries; Disease attributes

Characteristics and quantification of vascular changes in macular telangiectasia type 2 on optical coherence tomography angiography

BackgroundMacular telangiectasia type 2 (MacTel 2) is a bilateral idiopathic, rare neurodegenerative disease with alterations in the macular capillary network leading to vision loss and is the most common of...

Venous Nicking Without Arteriovenous Contact

Arteriovenous nickings (AVNs) in the retina are the cause of retinal vein occlusions and are also surrogates of cerebrovascular aging. The prevalent mechanistic model of AVNs stating that arteries crush veins remains somewhat unchallenged despite the lack of evidence other than fundus photographs. Here, we observed that venous nicking may be observed in the absence of physical contact with an arteriole. This observational study, conducted from January 2013 to September 2014, included 7 patients showing remodeling of a venous segment close to a retinal arteriole without arteriovenous overlap were imaged by adaptive optics imaging. Affected venous segments showed a variable association of nicking, narrowing, deviation, and opacification. Venous segments were deviated toward the arterioles in 6 of the 7 cases. The degree of venous narrowing ranged from 40% to 77%, while at these sites, the width of the intervascular space ranged from 16 µm to 42 µm. Similar features were identified in typical AVNs. Arteriovenous nickings do not necessarily involve an arteriovenous compression. Instead, the topology of venous changes suggests a retractile process originating in the intervascular space. These findings have important implications for the understanding of retinal vein occlusions and of cerebrovascular aging.

Review of the genus Eutetrarhynchus Pintner, 1913 (Trypanorhyncha: Eutetrarhynchidae), with the description of Eutetrarhynchus beveridgei n. sp.

The genus Eutetrarhynchus Pintner, 1913 is revised. Eutetrarhynchus beveridgei n. sp. is described from the spiral intestine of the dwarf whipray, Himantura walga (Müller & Henle) (Myliobatiformes: Dasyatidae), from the South China Sea off the Malaysian part of Borneo. The new species is characterised by a slender, elongate scolex, two oval bothria, muscular bulbs, retractor muscles inserting at the base of the bulbs, and the presence of gland-cells and prebulbar organs. The tentacular armature is typical heteroacanthous with heteromorphous hooks. Eutetrarhynchus beveridgei n. sp. is allocated to the genus due to its distinct segment morphology featuring two internal seminal vesicles and scattered testes occupying the complete intervascular space. It differs from congeners in its relatively small size, much smaller scolex regions and in the presence of a basal armature with a distinct basal swelling. Eutetrarhynchus cortezensis Friggens & Duszynski, 2005 is transferred to Dollfusiella Campbell & Beveridge, 1994, as D. cortezensis n. comb., on the basis of its segment morphology, with testes in a linear arrangement and the absence of internal seminal vesicles. A new generic diagnosis and a key for the identification of species of Eutetrarhynchus is provided.

Angiomyofibroma of the Orbit

The aim of this study was to describe a novel primary orbital vascular tumor combining elements of a vascular leiomyoma (angioleiomyoma) and a cavernous hemangioma. A critical review of clinical records, diagnostic tests, and radiographic studies combined with histopathologic evaluation with standard and special histochemical staining and immunohistochemical investigations was conducted. A 44-year-old man slowly developed 5 mm of well-tolerated relative right proptosis with minimal motility disturbance and no visual decline. Computed tomography and magnetic resonance imaging demonstrated a medial and intraconal rounded mass that perfused slowly and whose anterior surface was well circumscribed. At surgery, the tumor was solid and pink with intersecting white bands and densely attached to surrounding normal tissues. The most adherent apical portion of the mass was left behind after subtotal excision. Histopathologically, only a partial pseudocapsule was discovered. The tumor was composed of cavernous channels, capillary zones, compressed lumens with linear strands of endothelium, and collections of muscular veins devoid of an elastica. Striking smooth muscle actin positivity was identified in disorganized masses of smooth muscle cells in the intervascular spaces and around the cavernous vascular units; these myocytes were intermixed with bundles of interstitial keloidal collagen. The endothelium was CD31 and CD34 positive for vascular endothelium and D2-40 negative for lymphatic endothelium. The authors have classified this hybrid tumor an angiomyofibroma with low neoplastic potential and features of a malformation. It is a composite variant of cavernous hemangioma associated with a conspicuous proliferation of anomalous disorganized smooth muscle cells (leiomyoma). Most of the lesion lacked a pseudocapsule, which impeded surgical delivery. Incomplete excision is recommended in such cases as preferable to the complications that could ensue from overly aggressive efforts at complete removal, particularly at the orbital apex. Supporting this position is the observation that incompletely excised cavernous hemangioma generally does not recur.

Expression of contractile proteins α-actin and myosin of smooth muscle cells and of type IV collagen in human placenta at placental insufficiency in III trimester of pregnancy

Changes of expression of contractile proteins (smooth muscle cell α-actin and myosin) and of type IV collagen in villous stroma of human placenta were studied at the diagnosed placental insufficiency (PI) in III trimester of pregnancy. The study revealed pronounced disturbances of expression of contractile proteins and type IV collagen at PI. It is shown that in perivascular sheaths of vessels of stem and intermediate villi there is present a much greater amount of cells expressing smooth muscle actin and myosin. These cells are arranged by the denser concentric layers and more compactly than in norm and fill the intervascular space inside the villi. The width of perivascular sheaths of vessels is higher, while vascular lumens are lower than in norm. In terminal villi the capillary walls are thickened and the number of pericytes immunopositive against the smooth muscle cell α-actin and myosin as well as type IV collagen is increased. The change of synthesis of the cytoskeletal contractile proteins and type IV collagen is shown to lead to structural disturbances of villi of different types and of perivascular areas and vessels, which doubtlessly indicates their participation in pathogenesis of placental dysfunction and of disturbance of placental hemodynamics.

Lycat and cloche at the Switch Between Blood Vessel Growth and Differentiation?

See related article, pages 1057–1064 The formation of the cardiovascular system starts in the mouse embryo at approximately embryonic day (E)7.0 to E7.5. The first blood vessels in the extraembryonic membranes, the major intraembryonic vessels, and the heart form by vasculogenesis, the in situ differentiation of mesodermal cells that give rise to “blood-islands.” The latter are composed of hemangioblasts, the common precursors of endothelial and blood cells. Hemangioblasts situated in the lumen of the blood islands will further differentiate into hematocytoblasts, the precursors of all 3 lineages of blood cells. In contrast, hemangioblasts lining the walls of the blood islands will give rise to angioblasts that form endothelial cells.1 Migrating angioblasts from the proximal lateral mesoderm assemble symmetrically at the lateral sides of the embryo to establish 2 preendocardial tubes. They fuse to give rise to the primordial heart.2 While vasculogenesis is still proceeding, the uniform blood islands begin to remodel to a network of large and small vessels by the process of angiogenesis, preferentially intussusceptive microvascular growth.3,4 Gene expression and targeting studies have identified vascular endothelial growth factor and its 2 receptors, KDR/flk-1 and flt-1, as critical for the formation and early remodeling of the blood islands. Vascular endothelial growth factor is produced by endodermal and mesodermal cells at the onset of hemangioblast formation, whereas its receptors are expressed in the future endothelial cells lining the blood islands.5 Flk-1−/− embryos lack blood islands throughout the embryo and yolk sac.6 In flt-1−/− embryos, blood islands do not properly remodel but form large blood channels.7 Inactivation of a single vascular endothelial growth factor allele caused multiple embryonic malformations including the heart, rudimentary dorsal aortae, and a reduced number of blood cells.8,9 All deletions were lethal between days E8.5 and E11 to …

Alterations in the choroid in hypercholesterolemic rabbits: Reversibility after normalization of cholesterol levels

Endothelial damage in atherosclerosis is characterized by abnormal vascular functionality. Hyperlipidemic patients show alterations in ocular vascularization. However, it is not known whether these alterations are reversible after the lipid profile returns to normal. This study evaluates a rabbit model of hypercholesterolemia, examining the ultrastructural changes in the choroid, and the changes in it after a period of normal blood-cholesterol values induced by a standard diet. Rabbits were divided into three groups: G0, fed a standard diet; G1A, fed a 0.5% cholesterol-enriched diet for 8 months; and G1B, fed a 0.5% cholesterol-enriched diet for 8 months followed by a standard diet for a further 6 months. Eyes were processed for transmission electron microscopy. G1A had a buildup of lipids at the suprachoroidea that compressed the vascular layers, and hypertrophy of endothelial and vascular smooth muscle cells. In G1B there was less lipid accumulation than in G1A, but this was not followed by reversal of the choroidal damage. The suprachoroidea thickness of G1B was still greater than in G0 due to abundant collagen fibers. The intervascular spaces of the choroid had fewer lipids than G1A but more collagen fibers than G0. The large- and medium-sized vessel layers and choriocapillaris were less compressed than in G1A but exhibited basal membrane and endothelial changes similar to those in G1A. Normalization of serum cholesterol levels is not enough to reverse cholesterol-induced vascular damage to the choroid. These choroidal changes could be compatible with a chronic ischemia that could produce retinal degeneration.

Effect of apoptosis and response of extracellular matrix proteins after chemotherapy application on human breast cancer cell spheroids

Multicellular tumor spheroid (MTS) represents a three-dimensional structural form of tumors in laboratory conditions, and it has the characteristics of avascular micrometastases or intervascular spaces of big tumors. Recent studies indicate that extracellular matrix (ECM) proteins play a critical role in tumor metastasis, therefore normal and cancer cells require an ECM for survival, proliferation and differentiation. Doxorubicin and Docetaxel are widely used in the therapy of breast cancer, as well as in in vivo and in vitro studies. In this study, we examined the effect of apoptosis and proliferation of cells on the human breast cancer cell line, MCF-7, by using p53, bcl-2 and Ki67 gene expression, and the tendency to metastasis with extracellular matrix proteins, laminin and type IV collagen after chemotherapy in the spheroid model. The apoptotic cell death in situ was detected by TUNEL method. TUNEL-positive cells and positive immunoreactivities of laminin, type IV collagen, p53 and, bcl-2 were detected in the control group. There was no laminin and type IV collagen immunoreactivities in spheroids of drug groups. While TUNEL-positive cells and p53 immunoreactivity were detected in Docetaxel, Doxorubicin and Docetaxel/Doxorubicin groups, p53 immunoreactivity was not observed in the Docetaxel group. There was no bcl-2 immunoreactivity in either drug group. In addition, we did not detect Ki67 immunoreactivity in both control and drug treatment groups. However, the absence of Ki67 protein in MCF-7 breast multicellular tumor spheroids is possibly related to the cells in G0 or S phase. These chemotherapeutic agents may affect the presence of ECM proteins in this in vitro model of micrometastasis of spheroids. These findings suggest that the possible mechanism of cell death in Doxorubicin and Docetaxel/Doxorubicin treatment groups is related to apoptosis through the p53 pathway. However, we considered the possibility that there is another control mechanism for the Docetaxel group.

Vasculogenesis and angiogenesis in the subcardinal venous plexus of quail mesonephros: spatial and temporal morphological analysis.

Vasculogenesis and angiogenesis are involved in a coordinated program for the development of the mesonephric subcardinal venous plexus of quail embryo. Vasculogenesis occurs between days 3 and 4 of incubation, while angiogenesis takes place from day 5 to day 7. Examination of vascular corrosion casts and whole mounts, and tissue sections labelled with specific markers to hemangioblast lineage (QH1, LEP100 and AcPase activity), allowed us to distinguish six phases in the formation of subcardinal plexus. (1) Appearance of isolated angioblast-like cells where the subcardinal plexus will form. (2) Alignment of angioblast-like cells into cellular strands. (3) Formation of compact vascular cords by association of angioblast-like strands. (4) Polygonal interconnection of vascular cords to constitute the primary subcardinal plexus. In this stage, isolated angioblast-like cells were present inside inter-vascular spaces. (5) The splitting of primary inter-vascular spaces by angiogenic sprouts to form secondary subcardinal plexus (outward angiogenesis). Isolated angioblast-like cells were not present in this stage. (6) Expansion of the secondary subcardinal plexus by insertion of slender transcapillary tissue pillars (inward angiogenesis) and angiogenic sprouts. We also describe three morphogenetic gradients during the development of the subcardinal plexus: ventral-to-dorsal, cranial-to-caudal and lateral-to-medial.

Transverse Vein Differentiation Associated with Gas Space Formation—Fate of the Middle Cell Layer in Leaf Sheath Development of Rice

In monocotyledons, the leaf vascular network consists of a hierarchical sequence of vertical vascular bundles and numerous transverse veins that interconnect adjacent vertical veins. In the leaf sheath of these species, especially grasses, lysigenous gas cavities (gas spaces) are developed into intervascular spaces and provide a gas conducting system to non-aerial parts under flooded conditions. The spatial relationship between gas space formation and transverse vein differentiation was investigated using the leaf sheath of rice (Oryza sativa L.). Histochemical observation showed that patterns of differentiation of the transverse vein are distinct from those of vertical vascular bundles. On the other hand, gas spaces are formed through the processes of cell death (collapse). Both events are initiated at a specific cell position in the middle layers of the leaf sheath, from which the vascular system of the leaf is derived; this indicates that differentiation of transverse veins is associated with gas space formation. The cell-to-cell movement of fluorescein isothiocyanate-conjugated dextran injected into middle layer cells coincided with the area where cell collapse occurred, indicating a close relationship between the middle and adaxial cell layers, but not abaxial cell layers. A uniform cell number between each transverse vein in the leaf sheath suggested the involvement of spatial regulation in transverse vein formation regardless of clonal history at the later stage of leaf vein canalization.

Fluorescence histochemical demonstration of adrenergic terminations in the human choroid.

With the existing literature on the aspects of anatomic and physiological ocular choroid membrane variables in mind, the authors verify the possibility of applying the methods of Axelsson et al and of Lindvall and Bjorklund, as simplified by Furness and Costa for fluorescent microscopy, to the study of human choroid nerve topographic distribution. The material for the study was obtained from three human eyeballs, two enucleated because of malignant melanoma of the choroid and one because of neoplasia of the ciliary body. A binocular dissecting microscope was used. A fluorescent microscope was used to observe the histological specimens and photographs were taken. The following results are presented and discussed: 1) the fluorescent paravascular nervous fibres and periarterial nervous plexuses divided into preterminal and terminal fibres; 2) in the suprachoroidal layer, there were small groups of polygonal nervous branches with or without this connection; 3) the most intervasal plexus development was in the posterior part of the vascular layer with preterminal and terminal fibres, 4) in the intervascular space, melanocytes and free adrenergic nervous terminations were observed; 5) adrenergic fibres were not observed in the choriocapillaris of the choroid membrane; 6) there were adrenergic neurons in the vascular layer.

Myocardial capillaries: Increase in number by splitting of existing vessels

To study myocardial vascular development, stereological parameters were estimated in 24 Wistar rat hearts of six different age groups, from newborn to adult. The vascular surface density showed a sharp increase in the first 2 weeks, a peak around the age of 2 weeks, and then a steady decrease until it flattened in adulthood. In contrast, the vascular volume percentage, when plotted against age, decreased continuously with the greatest change in the first week, after which the curve flattened. These findings are compatible with an increase in the number of capillaries with a concomitant decrease of their diameters. Qualitative scrutiny of the histology did indeed support the idea that vessels become thinner. Reconstructions of the histological sections showed the same change three dimensionally. The reconstructions also demonstrated very small holes that seemed to go through the capillaries in the younger stages. Corrosion casts of the blood vessels were made using a casting resin. This was injected into the umbilical artery of rat embryos from 15 days gestation to birth. In postnatal rats of six age groups methacrylate was injected directly into the left ventricle. These casts supported the stereological data by showing an increase in number and decrease in diameter of capillaries, while during pre- and postnatal development, the intervascular spaces lengthened from small, irregular spaces to long, rectangular ones. Small holes, the probable precursors of such spaces, were clearly visible in the wider vessels of the youngest stages. All data point to an interesting mode of capillary growth, i.e. growth by division of existing vessels.