Quantitative in-vivo studies on angiogenesis in a rat sponge model.

A method for quantitative in-vivo studies on angiogenesis is described in this article. It is based on subcutaneous implantation of sterile polyester sponges in the rat and subsequent measurement of blood flow in the implants as they become vascularized. The blood flow in an implant was measured in terms of per cent 133Xe-saline clearance 6 min after the radio-isotope was injected into the sponge via a cannula attached to it. Since originally the sponge contained no blood vessels, the development of blood flow would represent a neovascularization. Histological examination of implants removed at fixed time intervals confirmed that the sponges were initially encapsulated by granulation tissue and gradually infiltrated by host blood vessels. Under standard conditions, the 133Xe clearance from sponges 16 days post-implantation approached the clearance obtained in normal skin. The new blood vessels in the sponges were reactive to vasodilator prostaglandin-E2 and vasoconstrictor noradrenaline applied topically. Furthermore, we have shown that local administration of endothelial cell growth supplement accelerated angiogenesis while protamine delayed its onset. Thus the model offers a new means for objective, continuous and reproducible studies on the controlling mechanisms of angiogenesis.     

Angiopoietin-1 promotes tumor angiogenesis in a rat glioma model

Angiopoietins have been implicated in playing an important role in blood vessel formation, remodeling, maturation, and maintenance. However, the role of angiopoietins in tumor angiogenesis remains uncertain. In this study, expression of human angiopoietin-1 (hAng-1) and angiopoietin (hAng-2) was amplified in the rat glioma cell line GS9L by stable transfection using an inducible tet-off system. Transfected cells were implanted intracerebrally into syngenic Fischer 344 rats. We demonstrated by means of magnetic resonance imaging that increased hAng-1 expression promoted a significant in vivo growth of intracerebral gliomas in rats. Overexpression of hAng-1 resulted in more numerous, more highly branched vessels, which were covered by pericytes. On the other hand, tumors derived from hAng-2-overexpressing cells were smaller than empty-plasmid control tumors. The tumor vasculature in these tumors was composed of aberrant small vascular cords, which were associated with few mural cells. Our results indicate that in the presence of hAng-1, tumors induce a more functional vascular network, which led to better tumor perfusion and growth. On the other hand, overexpression of hAng-2 led to less intact tumor vessels, inhibited capillary sprouting, and impaired tumor growth.     

基于FRET荧光蛋白探针的活体定量分子影像方法探究

目的荧光共振能量转移(fluorescent resonance energy transfer,FRET)探针由于自身仍存在诸多局限,其在活体成像领域尚未得到广泛应用。本文旨在解决FRET探针在活体定量分子影像应用中所存在的瓶颈问题,即如何利用FRET探针精准计算目标物浓度,以及如何基于FRET探针实现三维成像。方法基于探针和目标物结合时的化学反应平衡关系,提出一种新型分析方法,以精确定量待测物[钙调素(calmodulin,Ca M)]的浓度。同时对于FRET探针的活体分子成像,结合荧光透射成像(3DFMT)方法,建立了可进行三维时空动态FRET检测的平台系统,对FRET探针的测量结果(即待测物浓度)进行实时定量的三维重建。结果准确地定量了待测物Ca M浓度,并得到了高质量的3D-FRET分布影像。结论提出FRET探针在活体成像应用领域两个关键问题的解决方案,为基因编码FRET探针向活体分子影像领域的推广奠定了重要的技术基础。     

小柴胡汤联合丹那唑抑制子宫内膜异位症大鼠血管生成的研究

目的:探讨小柴胡汤、丹那唑及二者联合用药对子宫内膜异位症(EM)模型大鼠血管生成因子和异位内膜微血管密度的影响。方法:EM模型大鼠随机分为不用药组(U)、小柴胡汤(XCH)、丹那唑组(D)和联合用药组(S),设假手术组(C)为对照。四周后观察各组异位子宫内膜体积和形态学的变化,计数腹腔液巨噬细胞,放射免疫法测定各组大鼠外周血、腹腔液及巨噬细胞培养液白细胞介素－8(IL－8)、肿瘤坏死因子(TNF-α)的含量,免疫组化法测定血管内皮生长因子(VEGF)在异位内膜中的表达,并通过CD34标记异位子宫内膜血管,测定其微血管密度(MVD)。结果:XCH组、D组和S组异位内膜呈现不同程度萎缩,腺体明显减少,腹腔液巨噬细胞计数减少,XCH组、D组及S组大鼠外周血、腹腔液及巨噬细胞培养液IL－8、TNF-α含量降低,异位内膜VEGF表达减弱,MVD明显减少,其中以S组最为明显。结论:小柴胡汤和丹那唑可以抑制EM模型大鼠异位内膜的血管生成,使异位内膜萎缩,当二者联合用药时,作用更强。     

In-vitro and in-vivo studies of a cytotoxin from Campylobacter jejuni

Studies were performed on a cytotoxin (CT) from human strains of Campylobacter jejuni isolated in Malaysia. CT was detected by cytopathic effect (CPE) on HeLa cells at titres from 8 to 32, in culture filtrates from 14 (48%) of 29 human isolates. The CPE correlated well with a quantitative 51Cr-release assay where a specific release of 54-68% was noted. CT production was lost after 5-7 subcultures. CT activity was also detected in 5 (26%) of 19 faecal filtrates from which CT-producing isolates were subsequently obtained. The mol. wt of CT was estimated by Sephadex G-50 chromatography to be greater than 30,000. In a suckling-mouse assay, CT consistently failed to demonstrate fluid accumulation after intragastric inoculation of culture filtrate. The Removable Intestinal Tie Adult Rabbit Diarrhoea (RITARD) assay was also used. Rabbits given CT-producing strains of C. jejuni developed bacteraemia and severe watery mucus-containing diarrhoea for the duration of the experiment with death of some animals. Rabbits given CT non-producing strains had less severe disease and none died. Rabbits given partially-purified CT had diarrhoea for 3 days but none died.     

Quantitative regression analysis of the cutaneous vascular territories in a rat model

Background  

The rat skin flap model has been widely used in experimental flap survival studies; however, most of these have been qualitative studies. The purpose of this study was to investigate the quantitative relationship between the diameter of a cutaneous artery and the area of skin that it supplies, and also to explore the factors that influence this relationship.     

Ginsenoside-Rg1 enhances angiogenesis and ameliorates ventricular remodeling in a rat model of myocardial infarction

Ginsenoside-Rg1 (Rg1) has been used in the traditional Chinese medicine for over 2,000?years. The present study was performed to test our hypothesis that Rg1 provides pro-angiogenic and anti-fibrotic benefits in the ischemic myocardium in a rat model of myocardial infarction. The expression of vascular endothelial growth factor (VEGF) and phosphorylation/activation of PI3K, Akt, and p38 MAPK signaling pathways were examined in human umbilical vein endothelial cells and in the myocardial samples of rats. In addition, the expression levels of TNF-?? and collagen I level, the number of newly formed blood vessels, the extent of myocardial fibrosis, and left ventricular function were measured in vivo. Our results demonstrated that administration of Rg1 increased VEGF expression levels, activated PI3K/Akt, and inhibited p38 MAPK in vitro and in vivo. Furthermore, Rg1 increased the density of newly formed vessels, decreased TNF-?? and collagen I expression levels and area of myocardial fibrosis, and improved left ventricle function in vivo. PI3K inhibitor LY294002 significantly attenuated Rg1-enhanced VEGF expression and capillary density. As well, inhibition of p38 MAPK slightly increased VEGF expression in vitro and in vivo, increased capillary density, and decreased TNF-?? and collagen I expression levels and area of myocardial fibrosis in vivo. Rg1-induced activation of PI3K/Akt also contributed to the downregulation of p38 MAPK. Thus, Rg1 is effective in promoting angiogenesis and attenuating myocardial fibrosis, resulting in ameliorated left ventricular function. The possible mechanisms may involve activation of PI3K/Akt, inhibition of p38 MAPK, and cross talk between the two signaling pathways.     

Quantitative studies of mitoses: hippocampus of neonatal rat

In continuation of our quantitative studies of mitoses in 15-day fetal rat brain, we have now studied mitoses in hippocampus of neonatal rat. In contrast to cortex in which neuroblast mitoses terminate prenatally, hippocampus in rodents has been known to exhibit mitoses well past birth. Our present study suggests that at birth (rat) 61% of these mitoses are situated in the medioventral tip of the germinal (ventricular and sub-ventricular) zone of lateral ventricle. However, the remaining 39% of mitoses may not be neural but represent mitoses of brain capillaries, similar to "deep mitoses" which we reported in 15-day fetuses. Like these, some of the neonatal mitoses are situated not in the germinal layer of the ventricle, but 44 +/- 21 microns deeper, near Ammon's horn or dentate gyrus; also, as in these previous studies, the spindles of all deep mitoses are not parallel to ventricular or hippocampal structures but perpendicular, presumably to promote their radial expansion known for capillaries at that age. Possibilities are considered that capillary cells are produced in excess and eventually undergo natural "capillary death", to follow natural "neuronal death".     

The effect of injected RGD modified alginate on angiogenesis and left ventricular function in a chronic rat infarct model

Congestive heart failure (CHF) is a chronic disease with a high mortality rate. Managing CHF patients has been one of the most severe health care problems for years. Scaffold materials have been predominantly investigated in acute myocardial infarction (MI) studies and have shown promising improvement in LV function. In this study we examined whether surface modification of a biomaterial can influence the myocardial microenvironment and improve myocardial function in a rodent model of ischemic cardiomyopathy. In vitro cell culture and in vivo rat studies were performed. RGD peptides conjugated to alginate improved human umbilical vein endothelial cell (HUVEC) proliferation and adhesion when compared to a non-modified alginate group. Injection of the alginate hydrogel into the infarct area of rats 5 weeks post-MI demonstrated that both modified and non-modified alginate improve heart function, while LV function in the control group deteriorated. Both the RGD modified alginate and non-modified alginate increased the arteriole density compared to control, with the RGD modified alginate having the greatest angiogenic response. These results suggest that in situ use of modified polymers may influence the tissue microenvironment and serve as a potential therapeutic agent for patients with chronic heart failure.     

Quantitative studies on the supraoptic nucleus in the rat. I. Synaptic organization

Four types of synapses: axo-axonic, axo-dendritic, axon-spine, and axo-somatic were distinguished in the supraoptic nucleus in the rat. The density of synaptic terminals (boutons) in this nucleus is 35.17-10(6)/mm3 hence there are over 5 million boutons on each side, and on the average 596 per neuron. Only about one third of the axon terminals in this nucleus originate outside the nucleus and its immediate neighbourhood. Two thirds appear to be of intranuclear or otherwise of local origin. This could be explained by assuming either numerous intranuclear axon collaterals or interneurons having richly arborizing axons, or possibly both.     

Magnesium ions prevent phencyclidine-induced cerebrovasospasms and rupture of cerebral microvessels: direct in-vivo microcirculatory studies on the rat brain

Phencyclidine (PCP) abuse is reaching alarming proportions. PCP has recently been shown to induce hypertensive encephalopathies, microvascular cerebrovasospasm and acute intracerebral hemorrhage. Since we have shown in vitro that cerebral vasospasms induced by PCP could be completely reversed, or prevented, by use of organic calcium antagonists, we utilized a television microscope recording system to determine whether magnesium ions (Mg2+) could inhibit the ability of PCP to induce contraction of pial arterioles and its sequelae of microvascular damage. Administration of either MgCl2 or Mg aspartate HCl, i.a. or i.v. (1, 10, and 20 mumol/min), before or after administration of PCP produced dose-dependent inhibition (30-80%) of PCP-induced arteriolar spasms and the subsequent vascular damage. A variety of pharmacologic receptor antagonists and cyclooxygenase inhibitors failed to influence PCP-induced cerebrovasospasms. These data suggest that a naturally-occurring Ca2+ antagonist, viz. Mg2+, may be useful in the treatment of PCP intoxication and its cerebral vascular consequences.     

A reproducible in-vivo model of lymphatic malformation in rats

The aim of this study was to develop a reproducible rat model of lymphatic malformation. Different types of adjuvant, with and without vascular endothelial growth factor (VEGF)-C, was injected into the neck and floor of the mouth of rats. The rats were killed 2 months after the injection. Injected rats developed cystic lesions in the neck and floor of the mouth. Immunohistochemical examination revealed that the cysts were lined by endothelium, which expressed the lymphatic endothelial markers LYVE-1 and VEGF receptor-3. Raman spectra of the liquid contents of the cysts were similar in all injected rats. Transmission electron microscopy revealed that the endothelial cells had no basement membrane or surrounding pericytes. The cystic lesions were consistent with human lymphatic malformation. This animal model could be used to investigate pathogenesis of lymphatic malformation and its responses to candidate therapies.     

Modulation of angiogenesis in a model of chronic inflammation

Inflammation Research -     

Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model

Tissue-engineering (TE) applications include the isolation, culture, and seeding of cells into a suitable matrix or scaffold before in vivo transplantation. After transplantation, vascularization of the scaffold is a principal limiting factor for cell viability for the first 6-8 days posttransplantation. A model for systematic analysis of this process has been developed. Fertilized White Leghorn eggs were incubated (at 37.8 degrees C in 60% relative humidity) and opened on day 3 of incubation. Preadipocyte-seeded fibrin constructs were implanted in a specially designed plastic cylinder and placed through the opening on the surface of the chorioallantoic membrane (CAM) on day 8 of incubation. Vascularization of the constructs by chorioallantoic blood vessels was assessed for up to 8 days posttransplantation. The survival rate for embryos receiving transplanted constructs was about 90%. Histology confirmed transplant cell viability at day 4 posttransplantation and vascularization of the constructs by avian endothelial cells began at this time. A new in vivo model to study the effect of angiogenesis in TE constructs, including assessments of viability, proliferation, and differentiation of transplanted cells and biomaterial properties, is presented. Advantages include easy access to the vascular network of the CAM, lack of immunocompetence, low costs, and avoidance of animal experiments.     

Quantitative studies on the supraoptic nucleus in the rat. II. Afferent fiber connections

Summary A quantitative electron microscopic study of synaptic terminal degeneration was performed in the supraoptic nucleus (NSO) after a variety of major transections or ablations, destroying or interrupting in different combinations the afferent pathways known from earlier and own light microscopic degeneration studies. Solutions of a set of equations, expressing the percentage degenerations in synaptic profiles after different combinations in which the several pathways are interrupted by the various interferences, enabled the authors to give the following percentage numbers for afferent synapses from different sources.32.7% of supraoptic afferents originate from the brain stem probably representing the monoaminergic innervation of this nucleus. The medial basal hypothalamus (21.0%), amygdala (13.5%), septum (13.5%), hippocampus (8.5%) and olfactory tubercle and further rostral cortical region (17.0%) are the other main sites of origin of supraoptic nucleus afferents. There are no supraoptic afferents from the optic nerve, superior cervical ganglion or fimbria hippocampi.Abbreviations A nucleus accumbens - AB nucleus amygdaloideus basalis - AC nucleus amygdaloideus centralis - AL nucleus amygdaloideus lateralis - AM nucleus amygdaloideus medialis - ATV area tegmenti ventralis (Tsai) - C caudate-putamen - CA commissura anterior - CC corpus callosum - CFV commissura fornicvis ventralis - CO chiasma opticum - CP commissura posterior - D nucleus tractus diagnolis - DM nucleus dorsomedialis - DS decussationes supraoptica - F columna fornicis - FH fimbria hippocampi - FLM fasciculus longitudinalis medialis - FP fornix praecommissuralis - FS fornix superior - G globus pallidus - GD gyrus dentatus - HI hippocampus - IC capsula interna - IP nucleus interpeduncularis - LM lemniscus medialis - M medial forebrain bundle (MFB) - MM nucleus medialis thalami, pars medialis - NA nucleus arcuatus - R nucleus rhomboideus - RE nucleus reuniens - RV nucleus ruber - S stria medullaris thalami - SD nucleus dorsalis septi - SF nucleus fimbrialis septi - SG substantia grisea centralis - SL nucleus lateralis septi - SM nucleus medialis septi - SN substantia nigra - ST nucleus interstitialis striae terminalis - T tractus olfactorius lateralis - TD tractus diagonalis (Broca) - TO tractus opticus - TSTH tractus striohypothalamicus - TU tuberculum olfactorium - VM nucleus ventromedialis     

Effect of bioactive borate glass microstructure on bone regeneration,angiogenesis, and hydroxyapatite conversion in a rat calvarial defect model

Borate bioactive glasses are biocompatible and enhance new bone formation, but the effect of their microstructure on bone regeneration has received little attention. In this study scaffolds of borate bioactive glass (1393B3) with three different microstructures (trabecular, fibrous, and oriented) were compared for their capacity to regenerate bone in a rat calvarial defect model. 12 weeks post-implantation the amount of new bone, mineralization, and blood vessel area in the scaffolds were evaluated using histomorphometric analysis and scanning electron microscopy. The amount of new bone formed was 33%, 23%, and 15%, respectively, of the total defect area for the trabecular, oriented, and fibrous microstructures. In comparison, the percent new bone formed in implants composed of silicate 45S5 bioactive glass particles (250–300 μm) was 19%. Doping the borate glass with copper (0.4 wt.% CuO) had little effect on bone regeneration in the trabecular and oriented scaffolds, but significantly enhanced bone regeneration in the fibrous scaffolds (from 15 to 33%). The scaffolds were completely converted to hydroxyapatite within the 12 week implantation. The amount of hydroxyapatite formed, 22%, 35%, and 48%, respectively, for the trabecular, oriented, and fibrous scaffolds, increased with increasing volume fraction of glass in the as-fabricated scaffold. Blood vessels infiltrated into all the scaffolds, but the trabecular scaffolds had a higher average blood vessel area compared with the oriented and fibrous scaffolds. While all three scaffold microstructures were effective in supporting bone regeneration, the trabecular scaffolds supported more bone formation and may be more promising in bone repair.