Peritruncal Coronary Endothelial Cells Contribute to Proximal Coronary Artery Stems and Their Aortic Orifices in the Mouse Heart

Avian embryo experiments proved an ingrowth model for the coronary artery connections with the aorta. However, whether a similar mechanism applies to the mammalian heart still remains unclear. Here we analyzed how the main coronary arteries and their orifices form during murine heart development. Apelin (Apln) is expressed in coronary vascular endothelial cells including peritruncal endothelial cells. By immunostaining, however, we did not find Apln expression in endothelial cells of the aorta during the period of coronary vessel development (E10.5 to E15.5). As a result of this unique expression difference, Apln^CreERT2/+ genetically labels nascent coronary vessels forming on the heart, but not the aorta endothelium when pulse activated by tamoxifen injection at E10.5. This allowed us to define the temporal contribution of these distinct endothelial cell populations to formation of the murine coronary artery orifice. We found that the peritruncal endothelial cells were recruited to form the coronary artery orifices. These cells penetrate the wall of aorta and take up residence in the aortic sinus of valsalva. In conclusion, main coronary arteries and their orifices form through the recruitment and vascular remodeling of peritruncal endothelial cells in mammalian heart.     

An analysis of the age and size of 483 human embryos

The crown-rump length of 483 fixed human embryos of Carnegie stages 6-23 was analyzed and median and predicted mean lengths were calculated. The results were compared with those of other series and confirmed that early human growth rates are different from those of macaques with which human embryo growth has previously been compared. The study indicated that it is possible to predict: 1. the median size of an embryo of given Streeter horizon or Carnegie stage; 2. the age of a fresh embryo, or one of undisputed Streeter staging, by comparison with mean figures of other authorities; and 3. the corrected age of an embryo of known length or known Streeter staging or both in terms of postovulation age. Since teratogens may reduce the embryonic growth rate this information is relevant in the analysis of teratogenic factors in human development.     

Renin immunohistochemistry in the mesonephros and metanephros of the pig embryo

Summary The occurrence and distribution of renin was investigated in meso- and metanephric kidneys of pig embryos in various gestational stages. The immunohistochemical peroxidase-antiperoxidase-method (PAP) was used on paraffin sections after application of an antiserum against mouse renin which cross reacts with pig renin. Renin immunoreactivity was already found in the mesonephros of 21 day pig embryos (crown-rump(CR)-length 12 mm) with the strongest reaction in the media of the juxtaglomerular afferent arteriole. Efferent vessels, mesonephric arteries, and the aortic wall also contained scattered renin-positive cells. In the definitive kidney, renin was not detected prior to the 25 mm CR-length-stage. In 45 mm embryos, immunocytochemical staining was observed not only in the media of kidney arteries and arterioles, but also in proximal tubules after pinocytic absorption of filtered renin. TEM-studies revealed that the media of both the mesonephric and the developing metanephric arteries and arterioles contains epithelioid cells whose ultrastructure is very similar to that of renin-producing cells in the adult organ. The observed distribution of renin-producing cells along the entire renal arterial tree points to the possibility that the major function of the renin-angiotensin system in the fetal animal is to participate in the stabilization of renal perfusion pressure.     

Renin immunohistochemistry in the mesonephros and metanephros of the pig embryo

The occurrence and distribution of renin was investigated in meso- and metanephric kidneys of pig embryos in various gestational stages. The immunohistochemical peroxidase-antiperoxidase-method (PAP) was used on paraffin sections after application of an antiserum against mouse renin which cross reacts with pig renin. Renin immunoreactivity was already found in the mesonephros of 21 day pig embryos (crown-rump(CR)-length 12 mm) with the strongest reaction in the media of the juxtaglomerular afferent arteriole. Efferent vessels, mesonephric arteries, and the aortic wall also contained scattered renin-positive cells. In the definitive kidney, renin was not detected prior to the 25 mm CR-length-stage. In 45 mm embryos, immunocytochemical staining was observed not only in the media of kidney arteries and arterioles, but also in proximal tubules after pinocytic absorption of filtered renin. TEM-studies revealed that the media of both the mesonephric and the developing metanephric arteries and arterioles contains epithelioid cells whose ultrastructure is very similar to that of renin-producing cells in the adult organ. The observed distribution of renin-producing cells along the entire renal arterial tree points to the possibility that the major function of the renin-angiotensin system in the fetal animal is to participate in the stabilization of renal perfusion pressure.     

Development and arterial supply of the supraventricular crest during the human embryonic and fetal periods

Several works have concerned themselves with the anatomy of the supraventricular crest, for example, analyzing its role in the physiology of the right ventricle; nevertheless, its structure and arterial supply have been less studied. We have studied the morphogenesis of the architecture and the arterial supply of the supraventricular crest, in 25 embryos and human fetuses of 13-71 mm crown-rump length. The muscular organization of the crest (proper muscular bundles and parietal bundles of the right ventricle) and the development of the supraventricular crest's artery as well as its trajectory and its distribution during the fetal period were examined.     

Early prenatal attainment of adult metacarpal-phalangeal rankings and proportions.

As shown in 56 human embryos and fetuses between 15 and 104 mm in crown-rump length, "adult" metacarpal-phalangeal length rankings are attained by the seventh intrauterine week and near-adult bone-to-bone ratios or proportions by the theirteenth week. Micrometric measurements of optically-projected histological hand sections show relative elongation of the distals between the 15-29 mm and 30-44 mm crown-rump range, and relative reduction to radiogrammetrically-determined adult proportions by the 90-104 mm CRL.     

Thalamic arterial pattern: an endocast and scanning electron microscopic study in normotensive male rats

Rat cerebral vasculature serves as a model for study of the pathophysiology of stroke in humans. Human thalamic arteries show a high incidence of stroke. The objective is to describe the thalamic arterial vascular pattern in normotensive male rats as the initial step for quantitative histochemical studies of enzyme activities in the walls of these vessels. Intracardiac injections of methyl methacrylate monomer provide detailed vascular endocasts. The thalamic vascular bed defined by in situ dissection, serial reconstruction, and light and scanning electron microscopy of endocasts contained four groups of vessel: ventral medial thalamic arteries, thalamic branches from the posterior cerebral artery, and ventral lateral and ventral anterior thalamic arteries. Thalamic vessels are muscular arterioles that, after three to four bipinnate branches, feed into a continuous capillary bed (no loops). The parent vessels and their subsequent branches have been evaluated in terms of their mean internal diameters, mean interbranch intervals, and branch angles. The arterial patterns to rat and human thalami are very similar, with the exception of the anterior choroidal artery which is missing in the rat. The branches supplying the thalamus in both the rat and human are closely associated with the circle of Willis; however, the constituent parts of the circle in rat vary from the pattern in human brain. The rat thalamic arteries show morphological features similar to those seen in the stroke-prone ganglionic arteries in the human basal ganglia.     

The vasculature of the rat embryo: an electron microscope study

The ultrastructure of portions of the arterial and venous systems of the 11.5 day old Wistar rat embryos has been studied by scanning and transmission electron microscopy. The vessels at this stage of development are in the form of capillaries, and the arterial and venous types can be distinguished by the morphology of the endothelial cells by SEM. The endothelial cells of the arterial vessels gave prominent nuclear bulges and numerous microvilli apart from their spindle shape, whilst those of the veins appear flattened, are polygonal in shape, and have few microvilli. Transmission electron microscopy shows that the endothelial cells of the arteries and veins are identical in structure. The ultrastructure of these cells resembles that of endothelial cells at later stages of development including the adult type in that mature forms of cytoplasmic organelles are obtained. In studies on the intercellular junctions and fenestrations with lanthanum nitrate, the impression is formed that the vessels at this stage are impermeable to small molecular size particles, compared with adult capillaries. This suggests that cytoplasmic vesicles must play a major role in the transport of macromolecules in the 11.5 day embryonic vessels.     

The nature and extent of histopathologic injury in human avulsed arteries and veins and in experimentally avulsed monkey arteries

To determine the end point of histopathologic damage in avulsed arteries, the forearm arteries of five monkeys being sacrificed were avulsed longitudinally and samples of proximal and distal arteries prepared for light microscopy and transmission and scanning electron microscopy. A severe and consistent circumferential skip lesion was found on the luminal surface involving the intima and media. In 30 percent of vessels, histopathologic damage extended more than 3.0 cm from the rupture point. Similar circumferential tears occurred on the luminal surface of resected human avulsed arteries collected at the time of replantation surgery. No consistent lesions were noted in resected veins from human avulsed amputations. It is possible that in the human artery (as in the monkey) circumferential lesions frequently extend many centimeters from the rupture point and therefore beyond resection distances. Lesions present in the vessel after resection and microsurgical repair might be the site of thrombosis and subsequent occlusion.     

Growth allometry of the myocardium in human embryos (from stages 15 to 23).

The relative growth of the myocardium was studied in 27 staged human embryos (Carnegie stages). The volume of the myocardium was determined for each embryo according to Cavalieri's principle (by using point-counting planimetry to determine the area of the profiles of the myocardium). The volume of the myocardium (variable Y) was correlated to embryonic crown-rump length (variable X in millimeters) and age (in days). The bivariate allometric equation was used as Y = aXb. The scatterplot was discontinuous, presenting two trends during the postsomitic period. The first part was composed of embryos staged from stages 15 to 20, and the second part by embryos staged from stages 21 to 23. The breakpoint between these different trends was found at the level of stage 20 (embryo of 22 mm in crown-rump length and age nearly of 52 days). From stages 15 to 20, the growth rate of the myocardium was allometrically negative. On the other hand, from stages 21 to 23 this growth rate was moderately allometrically positive. These differences in growth of the myocardium were analyzed and, at least partially, might be due to the functional circulatory increase in the peripheral vascular bed in correlation to the cardiac hemodynamic demand required at the end of the embryonic period proper.     

Arterial repair after microvascular anastomosis. Scanning and transmission electron microscopy study

In order to study the morphological aspects of endothelial regeneration and vascular wall reaction after microvascular anastomosis, rat femoral arteries were sectioned and successively sutured (end-to-end anastomosis) with microsurgical techniques. Control arteries and anastomosed vessels (recovered after 1, 4, 7, 14, 21, 30, 60, 120, 180 and 360 days) were studied by means of scanning (SEM) and transmission electron microscopy (TEM). The reendothelialization phenomena started after 7 days and were mainly evident at 21 days. Areas of subendothelial connective tissue with fibrin deposition remained exposed to the blood stream up to 21-30 days. Thrombus formations or post-anastomotic stenosis have been occasionally observed. Regenerating endothelium showed evident morphological differences from the control. These changes mainly consisted of shortened cell length, absence of pinocytotic vesicles, presence of cytoplasmic prolongations, and microvillous proliferations. The arterial wall showed subintimal thickening. The anastomotic site appeared completely covered by new endothelium after 30-60 days. Subintimal vascular wall changes (thickening of the media) as well as slight alterations of endothelial cells (shortened length, reduced number of pinocytotic vesicles) were evident in 60-day vessels. Lumen reduction, due to the protruding of endothelial-covered sutures, was occasionally observed in 60- to 120-day arteries. Endothelial cell morphology normalized after 60-120 days. However, thickening of the media and occasional lumen reduction were observed also after 180-360 days. Although the endothelial regeneration phenomena were clearly evident after 2 weeks, nevertheless the reestablishment of arterial wall took longer time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fourth ventricular floor in human embryos: scanning electron microscopic observations

The ultrastructural surface features of the normal fourth ventricular floor of seven human embryos ranging from Carnegie stage 14 to stage 19 (crown-rump length: 7.6-16.2 mm) were examined by using scanning electron microscopy (SEM). Low-power SEM views showed the median sulcus, sulcus limitans, and neuromeres, transient structures characteristic of the earlier embryonic period. High-power SEM observation revealed supraependymal cells (SE cells) and supraependymal fibers (SE fibers) which exhibited a characteristic localization, as well as generalized surface-membrane modifications such as microvilli and cilia. SE cells could be classified into two major groups. The type 1 SE cells seem to possess neuronal functions, as deduced from morphological similarities to their counterparts in adults and the specialized distribution closely related to neuromeres. The type 2 SE cell morphologically resembled the phagocytic SE cell described in related literature. SE fibers ran a course either rostrocaudally in the median sulcus or mediolaterally on the neuromeres, most frequently near the interneuromeric cleft; they made contact with type 1 SE cells and ependymal surface modifications and then penetrated the ependymal layer.     

Effects of colchicine administration on the endothelial cells of the developing semilunar heart valves of the chick embryo

Summary Recent ultrastructural studies have revealed that differences exist in endothelial cell shape and cytoskeletal architecture between the arterial and ventricular faces of developing semilunar valves. In the present work we analyzed the morphologic response of the valvular endothelial cells of chick embryos to colchicine by light microscopy, scanning electron microscopy and transmission electron microscopy. The results show that colchicine administration during the stages of valve morphogenesis causes a very conspicuous disruption of the endothelial layer of the arterial face of the valves. The cells appear rounded and show massive surface blebbing. These alterations were not present in the endothelial cells on the ventricular face of the valves at the same stages. On the basis of these results we suggest that a difference in the degree of cell differentiation exists between the endothelial cells of the arterial and ventricular faces of the cusps and that this difference may have morphogenetic significance.     

Macroscopic distribution of coronary atherosclerotic lesions in cholesterol-fed rabbits.

In the present study we macroscopically examined a change in the distribution of coronary atherosclerosis in cholesterol-fed rabbits. Rabbits were fed a cholesterol-enriched diet for 15 weeks, then replaced by a normal diet, and were sacrificed at 15, 24, 32 and 42 weeks after the start of the experiment. The coronary atherosclerosis in the cholesterol-fed rabbits was distributed more densely in the proximal portion than in the middle and distal portions, and the lesions were severe at 24 and 32 weeks after the start of the experiment. comparison of lesions in the three portions at these time points showed that the percentages of lesion areas in the proximal portion, the middle portion and the distal portion were approximately 51%, 21 to 25% and 0.2 to 3.7%, respectively. Macroscopic observation of the coronary atherosclerotic lesions showed that the lesions formed over the vessel lumen in the proximal portion within the range of approximately 5 mm from the orifice of the left coronary artery. In the middle portion, the lesions formed predominantly around the orifices of branches as small patchy lesions from 1 to 3 mm in diameter. These findings support previous histopathological reports that suggested that the incidence of stenosis in the proximal portion was high, and the incidence of lesion occurrence in the middle and the distal portions varied. The method, macroscopical investigation of the coronary artery, is useful for analyzing coronary atherosclerosis in the rabbit.     

Taste bud papillae on the retromolar mucosa of the rat, mouse and golden hamster

The retromolar mucosa of the rat, mouse and golden hamster was observed by light and scanning electron microscopy. Numerous taste bud papillae, each of which formed a low round eminence containing one to several taste buds, were present in the posterior region of the retromolar mucosa, and were especially concentrated in the vicinity of the orifices of the molar glands. This topographical coincidence suggests that the retromolar mucosa of these animals has a functional role as a taste organ. Microridges, arranged in various patterns and small pits, were observed on the surface of the keratinized epithelium of the rat and mouse retromolar mucosa. It appears that the development of numerous microridges is adapted for varied stimuli in the oral cavity.     

Blood vasculature of the lymph node in the dog: anatomical evidence for participation of extrahilar arterial vessels in the blood supply of the cortex.

The organization of the arterial vessels of dog lymph nodes (LN) was studied using methods of visualization of the vasculature by systemic injection of different tracers (colloidal carbon, Micropaque resin and methylmethacrylate) followed by observation of the samples by light microscopy (after clearing of the thick sections of LN) or scanning electron microscopy (corrosion casts). LN from all of the three groups of nodes studied (tracheobronchial, paratracheal and popliteal) showed an extensive network of arterial vessels encircling the capsule of the organ. We found that branches of these capsular arteries penetrated deeply into the cortical domain of LN. The capsule-originating vessels appeared to have a significant participation in the blood supply of the LN parenchyma at the cortical domains of the organs. Our findings are in contrast with current views on the angiology of the LN that consider that virtually all of the arterial capillaries of the LN parenchyma come from hilar arteries. We propose, therefore, that important segments of the LN cortex receive their blood supply from capsular arteries rather than from hilar vessels.     

Ontogeny of human fetal lymph nodes.

Developing lymph nodes from 30 human embryos and fetuses with crown-rump lengths (CRL) of 18 mm (5.6 wk) to 245 mm (26 wk) were examined by light microscopy. The nodes were embedded in araldite, and the sections examined were approximately 1 mu in thickness. The development of nodes was divided into three stages: 1. the lymphatic plexus and connective tissue invagination (30 mm to 67 mm CRL); 2. the early fetal lymph node (43 mm to ,5 mm CRL); and 3. the late fetal lymph node (CRL greater than 75 mm). The lymphatic plexus was formed by connective tissue invaginations and bridges which divided a lymph sac into a meshwork of channels and spaces. Connective tissue invaginations were endothelially-lined and were surrounded by lymphatic space. Reticular cells, macrophages, and blood vessels were found in these invaginations. Early fetal lymph nodes were formed from invaginations when the cellular density and lymphocyte content increased. The lymphatic space surrounding the early node was the developing subcapsular sinus. With further development the early node became packed with lymphocytes, increasing the cellular density and size of the node. The connective tissue surrounding the subcapsular sinus condensed to form the capsule. Afferent lymphatic vessels pierced the capsule. Capillaries, veins, postcapillary venules, and occasional arteries were found in early and late nodes.     

Development of the salivary glands in embryos of Ixodes ricinus (Acari: Ixodidae)

We examined Ixodes ricinus embryos between 18 and 28 days of development with light, scanning and transmission electron microscopy. The differences in inner structure attested to establish three successive developmental stages: days 18-20, day 23, and days 26-28. Between 18 and 20 days the embryos are at early stages of organogenesis. Salivary glands cannot be identified at that stage. In 23-day-old embryos salivary glands are already outlined but the structure of alveoles is still different from that in larvae in which the embryonic development has been completed. Gland cells start to form alveoles and become active between 26 and 28 days of the development.     

Gradual Thawing Improves the Preservation of Cryopreserved Arteries

This study was designed to test a slow, controlled, automated process for the thawing of cryopreserved arteries, whereby specimen warming is synchronized with the warming of its environment. Segments of minipig iliac artery, 4-5 cm in length, were subjected to controlled, automated cryopreservation in a biological freezer at a cooling rate of 1 degrees C/min to -120 degrees C, followed by storage in liquid nitrogen at -196 degrees C for 30 days. Following storage, the arterial segments were subjected to rapid (warming rate of approximately 100 degrees C/min) or gradual (1 degrees C/min) thawing. Thawed specimens were processed for light microscopy and for scanning and transmission electron microscopy, Cell death was determined by the TUNEL method. Metalloproteinase (MMP) expression was estimated by immunohistochemical analysis. Most of the cryopreserved vessels subjected to rapid thawing showed spontaneous fractures, mainly microfractures, whereas these were absent in slowly thawed specimens. In rapidly thawed vessels, the proportion of damaged cells was double that observed in those thawed more gradually. Increased intensity and extent of MMP-2 expression was shown by rapidly thawed specimens. The slow-thawing protocol tested avoids the formation of spontaneous fractures and microfractures and the accumulation of fluid within the arterial wall tissue. This results in improved tissue preservation.     

Histological and histochemical characteristics of the bovine notochord.

In 20 bovine embryos and fetuses 6-65 mm long (crown-rump length) and 23 to 60-70 days old, the structure and localization of acid and neutral mucopolysaccharides and glycogen in their notochord were investigated. Also, the localization in the notochord was examined of the activity of alkaline and acid phosphatases, alpha-glycerophosphate-, glucose-6-phosphate-, isocitrate-, glutamate-, lactate- and succinic- dehydrogenases, and nicotinamide-adenine-dinucleotide- and nicotinamide-adenine-dinucleotide-phosphate- diaphorases. It was found that the bovine notochord begins decomposing at the end of embryonal and the beginning of fetal development (45-50 days old) and that in the fetus aged 55-65 days it no longer represents an unbroken cord of notochordal cells. Secretory activity of notochordal cells which produce the notochordal sheath starts very early (in 10 mm-long embryos), and interruptedly increases up to the end of the embryonal developmental period when regression appears at the beginning of the fetal period. These findings agree with findings in the human embryo where, however, they relate to earlier developmental periods.