An ultrastructural study of vascular proliferation and vascular alkaline phosphatase activity in the developing cerebral cortex of the rat

Alkaline phosphatase cytochemistry was employed to study the distribution of this enzyme in blood vessels during vascular differentiation and maturation during the postnatal development of the rat cerebral cortex. Enzyme reaction product was present in early vascular sprouts, and also throughout the subsequent maturation and differentiation of capillaries and arterial vessels. Cerebral capillaries appeared to be patent soon after the fusion of a sprout tip with another vessel; no evidence for delayed or synchronous opening was obtained. The distribution of alkaline phosphatase reaction product in vessel walls changed during vascular maturation. In vascular sprouts, reaction product was found mainly in the narrow lumen. As vessels became patent, reaction product appeared also on abluminal surfaces, at first chiefly in the narrow spaces between overlapping vascular cells. As vessels matured, reaction product became more generally distributed around the abluminal surface. In relatively mature capillaries and arterial vessels, it was restricted largely to endothelial cell surfaces and the spaces between smooth muscle cells. The significance of this distribution is unknown. Some possible explanations, including the possibility of artifact, are discussed. No alkaline phosphatase reaction product was found in differentiated veins.     

An electron microscope study of the differentiating capillaries of the mouse neurohypophysis

Summary The developing capillaries of the mouse neurohypophysis were studied in the electron microscope to elucidate the fine structural differentiation of the vascular component of the neuro-hemal contact zones in the external median eminence and the neural lobe.In the embryo the growth of the superficial net of the primary plexus of the hypophysial portal system is largely manifested by the presence of proliferation areas located within the capillary plexus covering the surface of the median eminence. Presumptive shallow capillary loops diverge from these areas in the first postnatal week. Differentiation of the capillary wall follows the pattern outlined for continuous capillaries. A few fenestrae appear in the endothelium of immature, superficial vessels at the 17th gestational day, increase in frequency during the following embryonic days, and occur regularly in the postnatal animal.In the neural lobe the internal capillaries proliferate by vascular sprouts emanating from the vessels on the surface of the gland. At the end of embryonic time an extensive net has developed, composed of capillaries with immature characteristics. Proliferation is largely finished by the end of the third postnatal week, when mature capillaries dominate the picture. Formation of attenuated, porous areas is a postnatal process, apart from single fenestrae appearing in the walls of a few immature capillaries in late fetal life.The structural possibilities for an onset of neurohypophysial function in the mouse is discussed.     

Development of the intrarenal vascular system of the puppy kidney

This study investigates the development of the vascular system of the puppy kidney (1-21 days after birth) after preparing casts of the renal vessels. At two days, the intrarenal vascular system distal to the afferent arteriole is strikingly different than that of the adult. The glomeruli of the outer cortex consist of a single dilated vessel while those of the mid and inner cortex possess an increasingly larger number of capillary loops. The efferent arterioles vary greatly in appearance from outer to inner cortex. Those in the nephrogenic zone are characteristically short and narrow and join a larger venous vessel termined a sinusoidal capillary. An efferent system somewhat similar to that of the adult is seen in the mid and inner cortex. One of the most obvious differences noted between the puppy and adult kidney is the relative lack of peritubular capillary networks throughout the cortex of the puppy kidney. The puppy possesses large, irregular vessels termed sinusoidal capillaries. The most rudimentary sinusoids are found in the outer cortex with more mature vessels in the inner cortex. The vascular arrangement of the efferent arteriole and sinusoidal capillary appears as a post-glomerular shunt. Functionally, the shunt would direct blood flow away from the proximal tubule and thus could result in a low extraction ratio and Tm for secreted solutes.     

Development of the intrarenal vascular system of the puppy kidney.

This study investigates the development of the vascular system of the puppy kidney (1-21 days after birth) after preparing casts of the renal vessels. At two days, the intrarenal vascular system distal to the afferent arteriole is strikingly different than that of the adult. The glomeruli of the outer cortex consist of a single dilated vessel while those of the mid and inner cortex posses an increasingly larger number of capillary loops. The efferent arterioles vary greatly in appearance from outer to inner cortex. Those in the nephrogenic zone are characteristically short and narrow and join a larger venous vessel termined a sinusoidal capillary. An efferent system somewhat similar to that of the adult is seen in the mid and inner cortex. One of the most obvious differences noted between the puppy and adult kidney is the relative lack of peritubular capillary networks throughout the cortex of the puppy kidney. The puppy possesses large, irregular vessels termed sinusoidal capillaries. The most rudimentary sinusoids are found in the outer cortex with more mature vessels in the inner cortex. The vascular arrangement of the efferent arteriole and sinusoidal capillary appears as a post-glomerular shunt. Functionally, the shunt would direct blood flow away from the proximal tubule and thus could result in a low extraction ratio and Tm for secreted solutes.     

人大脑内部微血管构筑——组织化学显示法研究

本文应用碱性磷酸酶组织化学对血管内皮的染色方法,光镜观察了3例人大脑内部的微血管构型。结果,皮质短动脉进入皮质内的分枝去向有:1.皮质返枝;2.水平枝;3.下降支。皮质长动脉末端的分枝类型分为:1.血管栅栏样分枝型;2.烛台样或小锚样分枝型;3.树根样分枝型。看到了从微动脉、毛细血管到微静脉的连续性通路。论述了皮质动脉与静脉之间在形态学方面的差别及皮质内血管吻合的几种形式,为更好地理解大脑皮质内微循环类型提供了形态学依据。     

Mechanisms of neovascularization. Vascular sprouting can occur without proliferation of endothelial cells

The kinetics of endothelial cells during microvascular growth were studied using a model of inflammation-induced neovascularization of the rat cornea. Inflammation was produced by central silver nitrate cauterization, cellular proliferation was assessed by tritiated-thymidine autoradiography and nuclear counts on plastic sections, and formation of new vessels was studied on whole-mount preparations after vascular perfusion with colloidal carbon. The 3H-thymidine-labeling index of endothelial cells was significantly higher than normal at 1 day following cauterization, although neither mitotic figures nor vascular sprouts were present. The labeling index reached a peak at 2 days, when cell division was evidenced by mitotic figures and doubling of the number of nuclei per section. Actual vascular sprouting also began during the 1- to 2-day interval. To determine whether vascular sprouting was dependent upon endothelial cell division, proliferation was suppressed by X-irradiation (2000 or 8000 rads) prior to cauterization. In irradiated corneas displaying no cellular proliferation, vascular sprouting at 2 days was similar to that in contralateral shielded corneas. Vascular growth continued in irradiated corneas between 2 and 4 days, but at 4 days the length of the vascular ingrowth was reduced to 66.7 and 53.4% of control after 2000 and 8000 rads, respectively. Vascular ingrowth did not progress between 4 and 7 days. This study demonstrates that initial vascular sprouting does not require proliferation of endothelial cells, although under ordinary circumstances DNA synthesis has been stimulated and is in progress at the time of sprouting. After initial sprouting without proliferation, limited vascular growth can continue for about 2 more days but subsequently ceases. Ultrastructural evaluation suggested that migration and redistribution of existing endothelial cells from the limbal vessels enable vascular sprouting and elongation without cellular proliferation.     

Regulated angiogenesis and vascular regression in mice overexpressing vascular endothelial growth factor in airways

Angiogenesis and vascular remodeling occurs in many inflammatory diseases, including asthma. In this study, we determined the time course and reversibility of the angiogenesis and vascular remodeling produced by vascular endothelial growth factor (VEGF) in a tet-on inducible transgenic system driven by the CC10 promoter in airway epithelium. One day after switching on VEGF expression, endothelial sprouts arose from venules, grew toward the epithelium, and were abundant by 3 to 5 days. Vessel density reached twice baseline by 7 days. Many new vessels were significantly larger than normal, were fenestrated, and penetrated the epithelium. Despite their mature appearance at 7 days suggested by their pericyte coat and basement membrane, the new vessels started to regress within 3 days when VEGF was switched off, showing stasis and luminal occlusion, influx of inflammatory cells, and retraction and apoptosis of endothelial cells and pericytes. Vessel density returned to normal within 28 days after VEGF withdrawal. Our study showed the dynamic nature of airway angiogenesis and regression. Blood vessels can respond to VEGF by sprouting angiogenesis within a few days, but regress more slowly after VEGF withdrawal, and leave a historical record of their previous extent in the form of empty basement membrane sleeves.     

Topography and structure of vasculature in developing cortex of rat kidney

Summary Young Wistar and SIV rats fixed by retrograde vascular perfusion were used for scanning and transmission electron microscopic, as well as light microscopic observations of the developing renal cortex and its vasculature. The latter was additionally observed by scanning electron microscopy of vascular casts effectuated by injection of methacrylate into the renal blood vessels.Disappearance of the initial nephron anlages — the vesicles — took place in the observed animals 2–3 days after birth, and another 2–3 days later the S-shaped bodies with the developing glomeruli differentiated into more advanced nephrons. The vascular system in the developing renal cortex consists of arterial supplying vessels, the sinusoid-like capillaries, and, continuous with the latter, large vessels which terminate in the arcuate vein. The arterial vessels assume their adult pattern already in the differentiating kidney, except their terminal — peripheral branching which is represented by arterial capillaries continuous with the drainage system. When nephronogenesis terminates the arterial capillaries are transformed into the young afferent vessels and the peripheral plexus of sinusoid-like capillaries receives its blood supply from the young efferent vessels. The sinusoid-like capillaries and large vessels give origin to the peritubular network of capillaries in the adult kidney, although their differentiation continues once nephronogenesis has been accomplished. They show some ultrastructural characteristics similar to those observed in sinusoids of other organs and they also differentiate in the same manner, by a conformation to the growing tubules.This work is dedicated to Professor Otto Bucher, M.D. (Lausanne), in honour of his 65th birthdayThe author wishes to thank Mrs. Madeleine Wirth and Miss Solange Gros for their excellent technical assistance     

小鼠大脑皮质发育过程中神经细胞、胶质细胞及血管之间的相互关系

目的探讨小鼠大脑皮质发育过程中神经细胞、胶质细胞及血管发生之间的关系。方法应用免疫荧光、5-溴脱氧尿嘧啶核苷(Brd U)和墨汁血管灌注等技术,对胚胎受孕15d(E15)至生后90d(P90)小鼠大脑内神经干细胞、神经前体细胞、神经胶质细胞和血管进行形态学观察。结果 E15左右大脑皮质内开始出现血管网,随着年龄的增长血管分支逐渐增多,血管体密度呈现增长趋势;成年后(P60)血管密度基本稳定。大脑皮质部位小血管走行方向与放射状胶质细胞长突起延伸方向一致,呈平行分布;大量放射状胶质细胞伸出膨大的足板参与血脑屏障的构筑;增殖的神经干细胞主要沿着血管走行方向及放射状胶质细胞长突起延伸方向进行迁移。结论在大脑皮质发育过程中,神经干细胞不断增殖,并沿着血管走行方向及放射状胶质细胞长突起延伸方向进行迁移。神经细胞、神经胶质细胞参与血管壁的构成,三者之间相互诱导,共同维持血脑屏障的完整与功能调节。     

Observations of vascularization in the spinal cord of mouse embryos, with special reference to development of boundary membranes and perivascular spaces

The early stages of vascularizations of the spinal cord of the mouse were studied by graphic reconstruction techniques and electron microscopy. Vascular sprouts arise from the perineural vascular plexus (PNVP) to invade the cord of 10-day embryos. These enter the cord most frequently via the lateral surface between the dorsal root and the ventral root; less frequently, they enter via the ventral and/or dorsal surfaces and anastomose with sprouts that have entered via the lateral surface. During the development of intramedullary blood vessels there are essential changes both in the basal laminae covering the neural parenchyma of the cord and in the relationship between the neural tissue and vascular walls. The basal laminae of the developing spinal cord were classified into three categories. The first is the perineural, external, or primary neural, basal lamina (PNBL), which is the earliest of the three in formation and covers the entire external surface of the cord. The second one is the internal, or secondary neural, basal lamina (INBL), which invests the internal surface of the neural tissue facing the walls of invading blood vessels. The third type is the perivascular basal lamina (PVBL), which surrounds the vascular wall. Blood vessels enter the spinal cord by penetrating the PNBL. Since the PVBL and INBL are absent or incomplete in early stages of vascularization, the neural tissue is in direct contact with intramedullary blood vessels. However, following their development, boundary membranes are formed, separating the neural tissue from neighboring vessels, a situation characteristic of capillaries in the mature CNS. Perivascular spaces are seen along the course of developing vessels and secondarily become continuous with the extramedullary connective tissue space. They are neither artifact nor intramedullary extensions of extramedullary connective tissue space along invading sprouts. The boundary membranes are formed by connection of membrane plaques or by fusion of the INBL and PVBL.     

Differentiation of thymus and thymocytes: A study in foetal guinea-pigs using alkaline phosphatase as a label of thymocytes

The differentiation of the guinea-pig thymus and thymocytes was studied using alkaline phosphatase (AP) as a marker of the thymocytes. The stem cell was assumed to be AP-negative. Cortical thymocytes contained a high activity of AP, which was lost or greatly diminished during the differentiation into medullary thymocytes and into T cells. Only approximately 1 per cent of the peripheral lymphocytes contained AP-activity. The role of these cells is obscure.

In the morphological study three developmental stages were observed. At the beginning the AP-activity was concentrated in the periphery of the thymus anlage, and was first observed in a 23-day-old foetus. At about 28 days the foetal thymus contained homogeneously distributed AP-positive and AP-negative cells. The cortex and medulla began to differentiate at the age of 31–33 days and the definite differentiation formed the third developmental stage. Medulla achieved its maximal proportion at birth. The results are in agreement with earlier studies concerning the development of the guinea-pig thymus.

     

Vascular tumors in the region of the breast

Summary Three vascular tumors in the breast region with different degrees of differentiation are presented. The first neoplasm is a haemangiosarcoma (of the vascular neoplasms, these tumors are the type which occur most frequently in the breast). Haemangiosarcomas show an infiltrative growth of atypical blood capillaries, frequently with formation of highly cellular and solid capillary sprouts. Ultrastructurally, the tumor cells are characterized as endothelial, also in the region of the capillary sprouts. The second tumor (an angiosarcoma in Stewart-Treves-syndrome, STS) is characterized by an intensive endothelial proliferation. Solid spindle-celled regions are also found in which the tumor cells correspond to undifferentiated mesenchymal cells, but other cells possessing properties of smooth muscle cells and pericytes may be found.The third tumor corresponds light and electron microscopically to a haemangiopericytoma of the soft tissue. The pericytic character of the tumor cells is most clearly seen in the immediate vicinity of the vessels. With increasing distance from the capillaries, the tumor cells take on the characteristics of fibroblasts. The tumors reflect the diversity of the angioplastic differentiation potential of the mesenchyme.     

Tumor vessel development and maturation impose limits on the effectiveness of anti-vascular therapy

The effect of anti-vascular agents on the growth of experimental tumors is well studied. Their impact on tumor vasculature, the primary therapeutic target of these agents, is not as well characterized, even though this primarily determines treatment outcome. Hypothesizing that the response of vessels to therapy is influenced by their stage of maturation, we studied vascular development and the vascular effects of therapy in several transplanted murine tumor models. Based on size, perfusion, endothelial cell (EC) proliferation, and the presence of pericytes, tumor vessels segregated into three categories. Least mature were highly proliferative, nonperfused EC sprouts emanating from functional vessels. Intermediate were small, perfused vessels which, like the angiogenic sprouts, were not covered by pericytes. Most mature were larger vessels, which were predominantly pericyte-covered with quiescent ECs and few associated sprouts. Thus, a developmental order, similar to that described during physiological neovascularization, was evident among vessels in growing tumors. This order markedly influenced tumor vessel response to anti-vascular therapy with recombinant interleukin-12. Therapy reduced tumor vessel density, which was attributable to a decrease in angiogenic sprouts and induction of EC apoptosis in pericyte-negative vessels. Although the great majority of vessels in growing tumors lacked pericyte coverage, selective loss of less mature vessels with therapy significantly increased the fraction of pericyte-positive vessels after therapy. These data indicate that the therapeutic susceptibility of tumor vasculature to recombinant murine IL-12 and, potentially, other anti-vascular agents is limited by its level of maturation. An implication is that tumor susceptibility is similarly limited, making pericyte coverage of tumor vasculature a potential indicator of tumor responsiveness.     

A scanning electron microscope study of vascular development in the dental papilla of prenatal rat molars

Summary Prenatal development of the vascular supply to the dental papilla was studied in the maxillary first molar teeth of rats from 18.5 to 22.0 days gestation, using the vascular casting/scanning electron microscope method. Five pulp horns developed in order, central, distal, mesial, disto-lingual and mesio-lingual with the development of the dental papilla. The first vessels that invaded each pulp horn were slightly depressed and formed an irregular network. The newly developed blood vessels were found to grow by sprouting and loop formation. After the invasion, blood vessels at the top of the horn became wider and then diminished in size to form a dense vascular network. The growth of the blood vessels in the latter stages is thought to take place mainly at the tops of the horns, and it is suggested that narrower capillaries arise from wide vessels. A dense and flattened vascular network consisting of thin blood vessels was formed when mesenchymal cells were beginning to differentiate into odontoblasts. The increase in density is thought to correlate with the differentiation of odontoblasts.     

Corneal neovascularization as studied by scanning electron microscopy of vascular casts

Rat corneas were cauterized chemically, and the induced neovascularization was studied by scanning electron microscopy of vascular casts. Casts were prepared by filling the pericorneal and intracorneal vessels with an acrylic monomer by an intracardiac injection. The initial response to injury was a vasodilation of pericorneal vessels and the appearance of impressions in the walls of veins and venules consistent with those of marginating leukocytes. The first new vascular buds emerged from the pericorneal venules and capillaries at 27 hours after injury. These sprouts lengthened and multiplied by 72 hours to produce a rich anastomosing plexus. New vessels were not seen arising from arteries and arterioles. By 7 days, numerous channels reached the cautery site and, by 13 days, many of the redundant intracorneal vessels had regressed leaving large looping channels connecting either with a pericorneal artery or a vein. The casts of those continuous with the artery had surface features suggesting arterial or arteriolar differentiation, whereas the smooth surfaces of the remaining channels were consistent with those of veins or capillaries. Transmission electron microscopy confirmed this differentiation by documenting intracorneal vessels with the smooth muscle and prominent endothelial cell nuclei which characterize arteries and arterioles. Vascular casts have several advantages in the study of neovascularization. They depict the three-dimensional characteristics of new vessel formation and reflect the vascular and cellular events in the accompanying acute inflammatory response; define more readily than histologic sections the time that the first new buds appear; emphasize capillaries and venules as the predominant source of new vessels; and suggest that certain new intracorneal vessels assume morphologic features of arteries or arterioles, whereas others retain capillary or venous characteristics.     

Demonstration of the intralobular lymphatics in the guinea pig pancreas by an enzyme-histochemical method

Intralobular lymphatics in the guinea pig pancreas were demonstrated enzyme-histochemically showing the extent, distribution and fine structure by combined light and transmission electron microscopy. 5'-nucleotidase(5'-Nase)-positive lymphatic vessels were present throughout the pancreas. Intralobular lymphatics among the acini were comparatively rare and generally independent of the blood capillaries, pancreatic ducts and acini. These lymphatics revealed the usual structural features, such as typical intercellular junctions and very tenuous vascular walls without continuous basal laminae. Fine precipitates of the cerium-based reaction product for 5'-Nase activity were found to be associated with cell membranes of the lymphatic endothelium and pinocytotic vesicles. Lymphatics were not closely related to the endocrine islets, although alkaline phosphatase(ALPase)-positive blood capillaries were well developed. Collecting lymphatic vessels with valves with weaker 5'-Nase activity were also detected in the interlobular connective tissue. ALPase activity, absent in the lymphatics, was positive in the blood capillaries, suggesting that it is also a useful way of demonstrating, histochemically, the blood capillaries in the guinea pig pancreas.     

高压氧中毒小鼠脑微血管图像分析定量研究

本文采用计算机图像定量测量方法，对高压氧中毒后小鼠软脑膜微血管和大脑皮质毛细胞血管内皮细胞碱性磷酶活性进行了定量检测。结果表明，ＨＢＯＩ后小鼠软脑膜毛细血管开放数目减少，毛细血管密度降低。     

Prolonged exercise induces angiogenesis and increases cerebral blood volume in primary motor cortex of the rat

Plastic changes in motor cortex capillary structure and function were examined in three separate experiments in adult rats following prolonged exercise. The first two experiments employed T-two-star (T(2)*)-weighted and flow-alternating inversion recovery (FAIR) functional magnetic resonance imaging to assess chronic changes in blood volume and flow as a result of exercise. The third experiment used an antibody against the CD61 integrin expressed on developing capillaries to determine if motor cortex capillaries undergo structural modifications. In experiment 1, T(2)*-weighted images of forelimb regions of motor cortex were obtained following 30 days of either repetitive activity on a running wheel or relative inactivity. The proton signal intensity was markedly reduced in the motor cortex of exercised animals compared with that of controls. This reduction was not attributable to alterations of vascular iron levels. These results are therefore most consistent with increased capillary perfusion or blood volume of forelimb regions of motor cortex. FAIR images acquired during experiment 2 under normocapnic and hypercapnic conditions indicated that resting cerebral blood flow was not altered under normal conditions but was elevated in response to high levels of CO(2), suggesting that prolonged exercise increases the size of a capillary reserve. Finally, the immunohistological data indicated that exercise induces robust growth of capillaries (angiogenesis) within 30 days from the onset of the exercise regimen. Analysis of other regions failed to find any changes in perfusion or capillary structure suggesting that this motor activity-induced plasticity may be specific to motor cortex.These data indicate that capillary growth occurs in motor areas of the cerebral cortex as a robust adaptation to prolonged motor activity. In addition to capillary growth, the vascular system also experiences heightened flow under conditions of activation. These changes are chronic and observable even in the anesthetized animal and are measurable using noninvasive techniques.     

Myocardial capillaries: Increase in number by splitting of existing vessels

Summary 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.     

Enzymatic differentiation of arterial and venous segments of the capillary bed during the development of free muscle grafts in the rat

The revascularization of freely grafted muscles in the rat was studied by histochemical reactions that on frozen sections stain the arterial part of the capillary bed blue (alkaline phosphatase [AP] reaction) and the venous part of the capillary bed red (dipeptidyl peptidase IV [DPP IV] reaction). In 112 rats the extensor digitorum longus or soleus muscles were freely grafted and removed at various times up to 93 days following the surgery. In cross section, the capillaries of a normal muscle show a mosaic pattern of staining for the activity of the two enzymes. After grafting, DPP IV activity of capillaries is lost throughout the entire graft within a day or two; but within ischemic muscle, weak and diffuse AP staining persists in capillary remnants for up to 6 days. In the very periphery of the graft AP staining is also preserved in partially damaged capillaries. By 4 days, new AP-positive capillaries can be identified at the periphery of the graft, and in succeeding days AP-positive capillaries are found progressively nearer the center of the graft. At 7 days, the capillary/muscle-fiber ratios are 66% of normal in the periphery of the graft and 44% in the intermediate zone. DPP IV-stained capillaries are not seen until 7 days after grafting. By 60 days, when the grafts have become stabilized, the mosaic pattern of capillary staining has become reestablished. In mature grafts, the number of capillaries per unit area was slightly higher than that in control muscles, but the capillary/muscle-fiber ratio was slightly lower, owing to the smaller than normal cross-sectional areas of the regenerated muscle fibers.