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.     

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.     

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     

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。     

Mouse model of angiogenesis.

Neovascularization of ischemic muscle may be sufficient to preserve tissue integrity and/or function and may thus be considered to be therapeutic. The regulatory role of vascular endothelial growth factor (VEGF) in therapeutic angiogenesis was suggested by experiments in which exogenously administered VEGF was shown to augment collateral blood flow in animals and patients with experimentally induced hindlimb or myocardial ischemia. To address the possible contribution of postnatal endogenous VEGF expression to collateral vessel development in ischemia tissues, we developed a mouse model of hindlimb ischemia. The femoral artery of one hindlimb was ligated and excised. Laser Doppler perfusion imaging (LDPI) was employed to document the consequent reduction in hindlimb blood flow, which typically persisted for up to 7 days. Serial in vivo examinations by LDPI disclosed that hindlimb blood flow was progressively augmented over the course of 14 days, ultimately reaching a plateau between 21 and 28 days. Morphometric analysis of capillary density performed at the same time points selected for in vivo analysis of blood flow by LDPI confirmed that the histological sequence of neovascularization corresponded temporally to blood flow recovery detected in vivo. Endothelial cell proliferation was documented by immunostaining for bromodeoxyuridine injected 24 hours before each of these time points, providing additional evidence that angiogenesis constitutes the basis for improved collateral-dependent flow in this animal model. Neovascularization was shown to develop in association with augmented expression of VEGF mRNA and protein from skeletal myocytes as well as endothelial cells in the ischemic hindlimb; that such reparative angiogenesis is indeed dependent upon VEGF up-regulation was confirmed by impaired neovascularization after administration of a neutralizing VEGF antibody. Sequential characterization of the in vivo, histological, and molecular findings in this novel animal model thus document the role of VEGF as endogenous regulator of angiogenesis in the setting of tissue ischemia. Moreover, this murine model represents a potential means for studying the effects of gene targeting on nutrient angiogenesis in vivo.     

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.     

Protamine and mast-cell-mediated angiogenesis in the rat.

Different doses of protamine sulphate (PS) given s.c. (at 12-h intervals) were tested for signs of non-specific toxicity measured as effect on body weight and small-gut proliferation as well as on mast-cell secretion and mast-cell-mediated mitogenesis in the mesenteric windows following i.p. injection of Compound 48/80, a potent mast cell secretagogue, in normal rats. In a non-toxic dose range, the effect of PS on mast-cell-mediated angiogenesis, effected by 48/80, was quantified as the number of vessels per mm of mesenteric window in histological sections at x 400. No intelligible dose-effect relationship was discernible between the dose of PS given and the effect on angiogenesis. Only in a tight interval, at 40 mg PS/kg but not at 20 or 60 mg PS/kg, was the angiogenesis statistically significantly suppressed. Hence, it was concluded that PS can be angiostatic but does not exert a more general angiostatic effect in the autogenous systems used.     

Quantitative studies of the effect of progesterone on endometrial morphology of the spayed rat.

The effects of various dosages and of various time periods of treatment with progesterone have been studied in the spayed, mature rat. Test objects were the cells of the luminal epithelium and of the endometrial stroma which were examined by qualitative and quantitative electron microscopy. No significant response was observed in epithelial or stromal cells until after 12 hrs of progesterone treatment. The nuclei of both cell types were then more circular than earlier with reduced long diameters. The functional significance of this change in configuration is unclear since only in the stromal cells was it followed by nuclear growth. Further, after 12 hrs of treatment the relative amounts of mitochondria and granular endoplasmic reticulum of stromal cells were reduced while the volume of the stromal cell cytoplasm appeared enlarged. This is taken as evidence that progesterone causes an intracellular oedema probably by decreasing cell membrane permeability. This response is probably not specific for the stroma but also includes the luminal epithelium, although the volume of the epithelial cell cytoplasm could not be determined here. Nucleolar enlargement did occur in stromal cells and was observed after 12 hrs of treatment but was not significant until after 24 hrs. At this point of time the net amount of granular endoplasmic reticulum in stromal and epithelial cells was increased indicating an increased protein synthesis in both cell types. However, only in the stromal cells was this associated with nucleolar enlargement, which supports the idea that progesterone stimulates protein synthesis through different mechanisms in the two cell types. Testing various dosages of progesterone showed that 0.5 mg had an effect similar to 5 mg of progesterone. When 0.05 mg progesterone was injected the only effect observed was an increase in the amount of apical vesicles of the luminal epithelium, showing that the epithelium is more sensitive to progesterone than the stroma.     

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.     

Evaluation of the porcine ameroid constrictor model of myocardial ischemia for therapeutic angiogenesis studies.

The porcine ameroid model of chronic myocardial ischemia has been widely used for the evaluation of coronary collateralization development. The impact of target vessel occlusion on the presence of myocardial ischemia, and the relationship between morphological, functional, and hemodynamic measurements in the context of therapeutic angiogenesis studies, however, has not been studied thus far. The authors therefore performed a systematic analysis of 94 animals undergoing ameroid constrictor placement around the left circumflex coronary artery (LCX) and, furthermore, a comprehensive evaluation including echocardiography and coronary angiography 26 +/- 1 (mean +/- SEM) days after ameroid placement. Complete LCX occlusion was observed in 34/94 animals (36%) and identified those with myocardial ischemia of the lateral wall, both at rest and under pharmacological stress. By applying a set of angiographic criteria (TIMI or= 1), another 27/94 animals with myocardial ischemia under conditions of pharmacological stress conditions could be identified. Interestingly, echocardiographic parameters of regional and global myocardial function were not correlated with myocardial blood flow or the degree of ischemia. There was no relationship between the extent of coronary collateralization, as assessed by angiography, echocardiographic parameters, or myocardial blood flow. The authors therefore conclude that complete occlusion of the ameroid instrumented coronary artery is not a prerequisite for successfully establishing the pathophysiology of myocardial ischemia. Defined angiographic criteria are important in identifying ischemic animals, thus reducing total animal numbers. Angiographic assessment of the degree of coronary collateralization, however, is not associated with myocardial blood flow or function and should not be used as a primary outcome measure of therapeutic angiogenesis studies in this model.     

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.     

Transplant arteriosclerosis in a rat aortic model.

Transplant arteriosclerosis (TA) has emerged as an obstacle to the long-term survival of transplanted organs, especially cardiac transplants. The animal models that have been used to study TA have not been fully characterized with regard to features such as the time course of cell proliferation and the sequence of cell types arriving in the developing intimal lesion. We present a model of TA based on a transplanted segment of abdominal aorta that helps address these questions. Two strains of rats (PVG x DA) underwent orthotopic aortic transplantation without immunosuppression and were killed at 14, 20, 40, and 60 days after transplantation. The within-strain control group displayed minimal evidence of cellular rejection with minimal to absent intimal lesions. In contrast, the allograft group showed a linearly increasing intimal lesion, up through 60 days after transplantation. The mechanism of intimal thickening was by an increase in cell number at the earlier time points with the later deposition of extracellular matrix. The early intimal lesion consisted mostly of mononuclear inflammatory cells (45%) with gradually increasing presence of smooth muscle cells (SMC) in the intima between 20 and 60 days. Conversely, the media showed gradual infiltration by macrophage-type cells with virtual loss of all SMC from the media by 40 days. The proliferative index showed a peak of 6% and 8% at 20 days in both the intima and media, respectively, and was preceded by the presence of macrophages. In fact, most of the proliferating cells at the earlier time points were either monocytes/macrophages, or were immediately adjacent to monocyte-/macrophage-rich regions. This straight artery segment model of transplant arteriosclerosis provides an easily quantifiable system in which the effects of different interventions (e.g., immunosuppressive regimens) can be tested.     

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.     

The need for modification of the polyvinyl sponge model of connective tissue growth. Histoligic and biochemical studies in the rabbit.

Quantitative histopathologic and biochemical comparisons were made between polyvinyl sponge capsular and sponge tissue in the rabbit on different days after subcutaneous implantation. Up to 9 days the predominant cell type in the capsular tissue is the fibroblast and in the sponge it is the neutrophil. During this time period the sponge tissue shows lower rates of (14C) proline and (14C) cytidine incorporation and lower rates of total (14C) collagen synthesis than the surrounding capsule. Different gel electrophoretic patterns of isolated radioactive proteins are found in sponge and capsule at 6 days. These biochemical differences appear to be related to the small number of fibroblasts, relative to granulocytes present in sponges during the first 9 days after implantation. It is suggested that future biochemical investigations of the early phase of connective tissue reactions (first 9 days) in this model utilize sponge capsular tissue within 1 cm of the sponge edge instead of the sponge and its contents.