Psychological Distress and Adolescents' Cyberbullying under Floods and the COVID-19 Pandemic: Parent-Child Relationships and Negotiable Fate as Moderators.

Since the outbreak of coronavirus disease 2019 (COVID-19), adolescents in 70 countries have suffered the COVID-19 pandemic and flood disasters simultaneously. Although antecedent cyberbullying variables have attracted significant research attention, the effects of psychological distress and the potential mechanisms of cyberbullying among adolescents under multiple disasters remains unclear. Based on social-ecological system theory, this study examines the moderating effects of parent-child relationships and the negotiable fate on the relationship between psychological distress and cyberbullying. A total of 1204 middle school students (52.4% boys) who suffered from floods and the COVID-19 pandemic from Zhengzhou City, China, are the participants. The results reveal that psychological distress was positively related to adolescent cyberbullying during a disaster. Parent-child relationships and negotiable fate significantly moderate the relationship between psychological distress and cyberbullying. Specifically, high parent-child relationships and a high negotiable fate could protect adolescents from the negative effects of psychological distress of cyberbullying. For adolescents with low or high parent-child relationships and low negotiable fate, the links between psychological distress and cyberbullying are stronger. These findings underline the significance of considering the interaction of psychological distress, parent-child relationships, and negotiable fate when examining adolescents' cyberbullying during disasters.

VE-1902-A direct thrombin inhibitor with reversible covalent mechanism of action shows efficacy with reduced bleeding in rodent models of thrombosis.

INTRODUCTION: High incidence of bleeding events remains a key risk for patients taking anticoagulants, especially those in need of long-term combination therapy with antiplatelet agents. As a consequence, patients may not receive clinically indicated combination antithrombotic therapy. Here, we report on VE-1902, a member of a novel class of precision oral anticoagulants (PROACs) that combines effective anticoagulation with reduced bleeding in preclinical testing. METHODS AND RESULTS: Acting through covalent, reversible active-site modification of thrombin similar to a previously described molecule [1], VE-1902 shows potency and selectivity for thrombin inhibition in human plasma comparable to clinically relevant direct thrombin inhibitors (DTI) such as argatroban and dabigatran (thrombin generation assay ETP EC50 = 1.3 µM compared to 0.36 µM and 0.31 µM for argatroban and dabigatran; >100-fold selectivity against related serine proteases). Unlike the current anticoagulants, VE-1902 does not significantly inhibit thrombin-mediated platelet activation in in vivo models of thrombosis. In the thrombin generation assay, the compound inhibits thrombin formation without significantly delaying the initiation phase of the clotting cascade. These features are possibly responsible for the observed reduced bleeding in tail bleeding and saphenous vein bleeding models. Consistent with this novel pharmacological profile, VE-1902 shows efficacious anticoagulation in several fibrin-driven animal models of thrombosis (arteriovenous shunt, venous stasis thrombosis, and thrombin-induced thromboembolism models), whereas it does not significantly prevent arterial occlusion in the platelet dependent FeCl3 model. CONCLUSIONS: By leaving platelet activation following vascular injury mostly unaffected, VE-1902, and the PROACs more generally, represent a new generation of precision anticoagulants with reduced bleeding risk.

Chronic effects of pulmonary artery stenosis on hemodynamic and structural development of the lungs.

Pulmonary artery (PA) stenosis is a difficult obstructive defect to manage since clinicians cannot know a priori which obstructions to treat and when. Prognosis of PA stenosis and its chronic effects on lung development are poorly understood. This study aimed to characterize the hemodynamic and structural effects of PA stenosis during development. Fourteen male Sprague-Dawley rats underwent left PA (LPA) banding at age 21 days, and 13 underwent sham operation. Hemodynamic and structural impacts were studied longitudinally at 20, 36, 52, 100, and 160 days. Chronic LPA banding resulted in a significant reduction in LPA flow (P < 0.0001) and size of both proximal LPA (P < 0.0001) and distal LPA (P < 0.01), as well as a significant increase in flow and size of the right PA (P < 0.05) throughout development. Flows and sizes adapted such that normal levels of wall shear were restored after banding. At 160 days, LPA banding resulted in a significant decrease in left lung volume and an increase in right lung volume but no significant differences in total lung volume. There was an elevation of proximal LPA pressure as well as right ventricular hypertrophy in the banded animals. The banded lung exhibited arterial disorganization, loss of vessels, and enlargement of its bronchial arteries, whereas the contralateral lung showed signs of vascular pathology. There are consequences on development of both lungs in the presence of an LPA stenosis at young age. These results suggest that early intervention may be necessary to optimize left lung growth and minimize right lung vascular pathology.

Inhibition of experimental abdominal aortic aneurysm in a rat model by way of tanshinone IIA.

BACKGROUND: The purpose of the present study was to investigate whether tanshinone IIA (Tan IIA), one of the major lipophilic components of Salvia miltiorrhiza Bunge, could inhibit the development of elastase-induced experimental abdominal aortic aneurysms (AAAs). METHODS: Male Sprague-Dawley rats (n = 12/group) were randomly distributed into three groups: Tan IIA, control, and sham. The rats from the Tan IIA and control groups underwent intra-aortic elastase perfusion to induce AAAs, and the rats in the sham group were perfused with saline. Only the Tan IIA group received Tan IIA (2 mg/rat/d). The maximum luminal diameter of the abdominal aorta was measured before and 5, 12, 18, and 24 d after perfusion. The systolic blood pressure was measured twice using the tail cuff technique before administration and death. Aortic tissue samples were harvested at 24 d and evaluated using reverse transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and Miller's elastin-Van Gieson staining. RESULTS: The rats in the control group had significantly increased aortic sizes compared with the sham group after 24 days (P < 0.05), and the Tan IIA group had a significant reduction in aortic size (Tan IIA versus control, P < 0.05) without affecting blood pressure (P > 0.05). The overexpression of matrix metalloproteinase-2, metalloproteinase-9, monocyte chemotactic protein-1, and inducible nitric oxide synthase and the depletion of elastic fibers and vascular smooth muscle cells induced by elastase perfusion were significantly decreased by Tan IIA treatment (P < 0.05). CONCLUSIONS: Tan IIA inhibited the development of elastase-induced experimental AAAs by suppressing proteolysis, inflammation, and oxidative stress and preserving vascular smooth muscle cells. It could be a new pharmacologic therapy for AAAs.

Localized control of exsanguinating arterial hemorrhage: an experimental model.

UNLABELLED: To develop an arterial injury model for testing hemostatic devices at well-defined high and low bleeding rates. MATERIAL AND METHOD: A side-hole arterial injury was created in the carotid artery of sheep. Shed blood was collected in a jugular venous reservoir and bleeding rate at the site of arterial injury was controlled by regulating outflow resistance from the venous reservoir. Two models were studied: uncontrolled exsanguinating hemorrhage and bleeding at controlled rates with blood return to maintain hemodynamic stability. Transcutaneous Duplex ultrasound was used to characterize ultrasound signatures at various bleeding rates. RESULTS: A 2.5 mm arterial side-hole resulted in exsanguinating hemorrhage with an initial bleeding rate of 400 ml/min which, without resuscitation, decreased to below 100 ml/min in 5 minutes. After 17 minutes, bleeding from the injury site stopped and the animal had lost 60% of total blood volume. Reinfusion of shed blood maintained normal hemodynamics and both high and low bleeding rates could be maintained without hemorrhagic shock. Bleeding rate at the arterial injury site was held at 395±78 ml/min for 8 minutes, 110±11 ml/min for 15 minutes, and 12±1 ml/min for 12 minutes. Doppler flow signatures at the site of injury were characterized by high peak and end-diastolic flow velocities at the bleeding site which varied with the rate of hemorrhage. CONCLUSION: We have developed a hemodynamically stable model of acute arterial injury which can be used to evaluate diagnostic and treatment methods focused on control of the arterial injury site.

The three-dimensional micro- and nanostructure of the aortic medial lamellar unit measured using 3D confocal and electron microscopy imaging.

Changes in arterial wall composition and function underlie all forms of vascular disease. The fundamental structural and functional unit of the aortic wall is the medial lamellar unit (MLU). While the basic composition and organization of the MLU is known, three-dimensional (3D) microstructural details are tenuous, due (in part) to lack of three-dimensional data at micro- and nano-scales. We applied novel electron and confocal microscopy techniques to obtain 3D volumetric information of aortic medial microstructure at micro- and nano-scales with all constituents present. For the rat abdominal aorta, we show that medial elastin has three primary forms: with approximately 71% of total elastin as thick, continuous lamellar sheets, 27% as thin, protruding interlamellar elastin fibers (IEFs), and 2% as thick radial struts. Elastin pores are not simply holes in lamellar sheets, but are indented and gusseted openings in lamellae. Smooth muscle cells (SMCs) weave throughout the interlamellar elastin framework, with cytoplasmic extensions abutting IEFs, resulting in approximately 20 degrees radial tilt (relative to the lumen surface) of elliptical SMC nuclei. Collagen fibers are organized as large, parallel bundles tightly enveloping SMC nuclei. Quantification of the orientation of collagen bundles, SMC nuclei, and IEFs reveal that all three primary medial constituents have predominantly circumferential orientation, correlating with reported circumferentially dominant values of physiological stress, collagen fiber recruitment, and tissue stiffness. This high resolution three-dimensional view of the aortic media reveals MLU microstructure details that suggest a highly complex and integrated mural organization that correlates with aortic mechanical properties.

P19 progenitor cells progress to organized contracting myocytes after chemical and electrical stimulation: implications for vascular tissue engineering.

PURPOSE: To test the hypothesis that a level of chemical and electrical stimulation exists that allows differentiation of progenitor cells into organized contracting myocytes. METHODS: A custom-made bioreactor with the capability of delivering electrical pulses of varying field strengths, widths, and frequencies was constructed. Individual chambers of the bioreactor allowed continuous electrical stimulation of cultured cells under microscopic observation. On day 0, 1% dimethylsulfoxide (DMSO), known to differentiate cells into myocytes, was added to P19 progenitor cells. Additionally, for the next 22 days, electrical pulses of varying field strengths (0-3 V/cm), widths (2-40 ms), and frequencies (10-25 Hz) were continuously applied. On day 5, the medium containing DMSO was exchanged with regular medium, and the electrical stimulation was continued. From days 6-22, the cells were visually assessed for signs of viability, contractility, and organization. RESULTS: P19 cells remained viable with pulsed electrical fields <3 V/cm, pulse widths <40 ms, and pulse frequencies from 10 to 25 Hz. On day 12, the first spontaneous contractions were observed. For individual colonies, local synchronization and organization occurred; multiple colonies were synchronized with externally applied electrical fields. CONCLUSION: P19 progenitor cells progress to organized contracting myocytes after chemical and electrical stimulation. Incorporation of such cells into existing methods of producing endothelial cells, fibroblasts, and scaffolds may allow production of improved tissue-engineered vascular grafts.

A novel culture system shows that stem cells can be grown in 3D and under physiologic pulsatile conditions for tissue engineering of vascular grafts.

BACKGROUND: Currently available vascular grafts have been limited by variable patency rates, material availability, and immunological rejection. The creation of a tissue-engineered vascular graft (TEVG) from autologous stem cells would potentially overcome these limitations. As a first step in creating a completely autologous TEVG, our objective was to develop a novel system for culturing undifferentiated mouse embryonic stem cells (mESC) in a three-dimensional (3D) configuration and under physiological pulsatile flow and pressure conditions. MATERIALS AND METHODS: A bioreactor was created to provide pulsatile conditions to a specially modified four-well Labtek Chamber-Slide culture system. Undifferentiated mESC were either suspended in a 3D Matrigel matrix or suspended only in cell-culture media within the culture system. Pulsatile conditions were applied to the suspended cells and visualized by video microscopy. RESULTS: Undifferentiated mESC were successfully embedded in a 3D Matrigel matrix and could withstand physiological pulsatile conditions. Video microscopy demonstrated that the mESC in the 3D matrix were constrained to the wells of the culture system, moved in unison with the applied flows, and were not washed downstream; this was in contrast to the mESC suspended in media alone. CONCLUSIONS: Undifferentiated mESC can be grown in 3D and under pulsatile conditions. We will use these results to study the effects of long-term pulsatile conditions on the differentiation of mESC into endothelial cells, smooth muscle cells, and fibroblast cells with the long-term goal of creating a completely autologous TEVG.

Arterial enlargement, tortuosity, and intimal thickening in response to sequential exposure to high and low wall shear stress.

We investigated the effects of sequential and prolonged exposure to high and low wall shear stress on arterial remodeling using a rabbit arteriovenous fistula (AVF) model. Blood flow was increased by approximately 17-fold to 20-fold when the AVF was open, and returned to normal when the AVF was occluded. Repeated opening and closing of the AVF resulted in sequential exposure of the artery to high and low wall shear stress. High flow and high wall shear stress induced arterial dilatation, elongation, and tortuosity, without intimal thickening. The common carotid artery was elongated 37% after 4 weeks of high flow, and was shortened 10% after 6 weeks of normal flow. Subsequent cycles of high flow induced less elongation, with less shortening after return to normal flow. Enlargement of the distal segment was more dramatic than in the proximal segment, despite exposure to the same volume of flow and the same initial high wall shear stress after creation of the AVF. The distal carotid segment enlarged more than did the proximal segment during each exposure to high flow. In segments of carotid artery exposed to low wall shear stress (<5 dynes/cm(2)) intimal thickening developed. These changes were maximal in the distal carotid segment, just before the AVF. Each cycle of low wall shear stress induced intimal thickening accompanied by medial hyperplasia. Intimal thickening was inhibited during periods of high flow when wall shear stress was high. Three cycles of flow alteration induced three layers of intimal thickening in the distal arterial segment, two layers of intimal thickening in the middle segment, and one layer of intimal thickening in the proximal segment. Long-term exposure to low wall shear stress induced severe intimal thickening and medial hyperplasia in different segments. Thus the response of the carotid artery afferent to an AVF varies along the length of the artery, with maximum enlargement, elongation, and tortuosity in the distal segment, just proximal to the AVF. Similarly, intimal thickening in response to low wall shear stress is maximal in the distal carotid artery. It appears that intimal thickening is related to local levels of low wall shear stress, and occurs when wall shear stress chronically falls to less than 5 dynes/cm(2).

High flow drives vascular endothelial cell proliferation during flow-induced arterial remodeling associated with the expression of vascular endothelial growth factor.

Endothelial cell activation and proliferation are the essential steps in flow-induced arterial remodeling. We investigated endothelial cell turnover in the early stages of high-flow in the rabbit common carotid arteries using an arteriovenous fistula (AVF) model by kinetic investigation of cell proliferation and cell molecular analysis. BrdU was administrated to label endothelial cells (ECs) in DNA synthetic phase (S-phase) of the cell mitotic cycle. Pulse labeling revealed that ECs entered S-phase at 1.5 days of AVF (0.93 +/- 0.19%). Endothelial cell labeling index (EC-LI) peaked at 2 days of AVF (8.90 +/- 0.87%) with a high index of endothelial cell mitosis (EC-MI, 1.67 +/- 0.47%). Endothelial cell density increased remarkably at 3 days of AVF with a significant decrease in EC-LI (54%) and EC-MI (60%). Study of kinetics of EC proliferation revealed that endothelial cells took 16-24 h to finish one cycle of cell mitosis. Tracking investigation of pulse BrdU-labeled endothelial cells at 1.5 days showed that more than 66% of endothelial cells were BrdU-labeled 1.5 days after labeling. VEGF, integrin alphanubeta3, PECAM-1, and VE-cadherin were upregulated significantly preceding endothelial cell proliferation and kept at high levels during endothelial cell proliferation. These data suggest that endothelial cell proliferation is the initial step in flow-induced arterial remodeling. Hemodynamic forces may drive endothelial cell downstream migration. Expression of VEGF and cell junction molecules contribute to flow-induced arterial remodeling.

Ultrastructure of endothelial cells under flow alteration.

Endothelial cells are stable and quiet in normal animals. They arrange regularly and have a smooth lumen surface and thin endothelial wall. According to Thoma's principle (1893) and Kamiya and Togawa's principle (1980) on the relationship of the vascular diameter to flow alteration, blood flow is in equilibrium to the diameter and in a physiological state. That is to say, there is no fast flow or slow flow. To understand the nature of the endothelial cells, we should investigate endothelial cells under flow alteration to break the equilibrium state. Endothelial cells under increased flow were studied in arteries with an arteriovenous fistula or in the capillaries of myocardium with volume-overloaded hearts or of the skeletal muscle by electrical stimulation. Those under decreased flow were studied by the closure of the fistula or by ceasing the stimulation. Endothelial cells in the coarctation of the arteries were also observed. Endothelial cells were activated by increased flow in the arteries and capillaries, while they were inactivated by decreased flow. Endothelial activation is characterized as lumen protrusions, increase of cytoplasmic organelles, abluminal protrusions, basement membrane degradation, internal elastic lamina degradation in the arteries, and sproutings in the capillaries. These are ultrastructurally comparable to angiogenesis. Endothelial inactivation is characterized by the decrease of endothelial cell number with apoptosis, which is ultrastructurally comparable to angioregression. We assume that endothelial cells respond to increased flow by angiogenesis and to decreased flow by angioregression.

Biphasic response of tropoelastin at the poststenotic dilation segment of the rabbit aorta.

OBJECTIVE: The purpose of this study was to assess the time course of tropoelastin gene expression in the poststenotic dilatation segment of rabbit aorta with experimental coarctation. METHODS: Midthoracic aortic coarctation was created in rabbits to produce a PSD. The time points of the study after coarctation were 1, 3, and 7 days and 2, 4, and 8 weeks (n = 3 each). Additional animals (n = 6) were subjected to hypercholesterolemia for analysis of tropoelastin expression in intimal lesions. Northern and Western blot analyses were used to quantitate tropoelastin messenger RNA (mRNA) and protein, and immunohistochemistry was used to analyze tropoelastin distribution. RESULTS: Thoracic aortic coarctation produced a moderate stenosis, which resulted in PSD. mRNA levels in the PSD segment decreased at days 1 and 3, followed by an increase at 2 and 4 weeks (P <.05 versus controls). This biphasic change in tropoelastin mRNA was associated with increase in tropoelastin protein levels at 2 and 4 weeks (P <.05 versus controls). PSD diameter reached a maximum at 4 weeks and did not increase significantly thereafter. The number of medial elastic laminae in PSD was reduced slightly, but media thickness was unchanged. Intimal lesions were much smaller in the PSD segment than in the proximal segment in animals with hypertension superimposed with hypercholesterolemia. Moreover, tropoelastin protein distributed not only in the intima but also in the media of the PSD. CONCLUSION: Tropoelastin gene expression is regulated in a biphasic pattern and precedes PSD formation. The differential distribution of tropoelastin in the media suggests a role for tropoelastin in the poststenotic adaptation response, which may provide increased elasticity to the PSD wall.

Arterial enlargement in response to high flow requires early expression of matrix metalloproteinases to degrade extracellular matrix.

This study investigated the effects of high flow and shear stress on the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-2 (TIMP-2) during flow-induced arterial enlargement using a model of arteriovenous fistula (AVF) creation on the carotid artery with the corresponding jugular vein in Japanese white male rabbits. Flow increased 8-fold 7 days after AVF. Endothelial cells (EC) and smooth muscle cells (SMC) proliferated with internal elastic lamina (IEL) degradation in response to high flow and shear stress. Expression of MMP-2 mRNA peaked at 2 days (1700-fold) and maintained high level expression. MMP-9 mRNA gave a 10.8-fold increase within 2 days and decreased later. Their proteins were detected in EC and SMC. Membrane type-1-MMP (MT1-MMP) mRNA increased 121-fold at 3 days and maintained high expression. TGF-beta1 was increased after AVF. Two-peak up-regulation of Egr-1 mRNA was recognized at 1 and 5 days of AVF. These results suggest that high flow and shear stress can mediate EC and SMC to express MMP-2 and MMP-9, which degrade cell basement membranes and IEL to induce arterial enlargement. The disproportional increase in MT1-MMP and TIMP-2 might contribute to MMP-2 activation. Egr-1 and TGF-beta1 might play important roles in this process.

Expression of TGF-beta1 and beta3 but not apoptosis factors relates to flow-induced aortic enlargement.

BACKGROUND: Cell proliferation and apoptosis are both involved in arterial wall remodeling. Increase in blood flow induces arterial enlargement. The molecular basis of flow-induced remodeling in large elastic arteries is largely unknown. METHODS: An aortocaval fistula (ACF) model in rats was used to induce enlargement in the abdominal aorta. Aortic gene expression of transforming growth factors beta (TGF-beta) and apoptosis-related factors was assessed at 1 and 3 days and 1, 2, 4, and 8 weeks. Expression levels were determined using a ribonuclease protection assay and western blotting. Cell proliferation and apoptosis were analyzed using BrdU incorporation and TUNEL techniques. RESULTS: Blood flow increased 5-fold immediately after ACF (P<0.05). Lumen diameter of the aorta was 30% and 75% larger at 2 and 8 weeks respectively than those of controls (P<0.05). mRNA levels of TGF-beta1 and TGF-beta3 increased after ACF, peaked at 3 days (P<0.05) and returned to normal level at 1 week and thereafter. Western blotting showed enhanced expression of TGF-beta1 at 3 days and TGF-beta3 at 1 and 3 days and 1 week (P<0.05). mRNA levels of Bcl-xS initially decreased at 1 day, 3 days and 1 week, followed a return to baseline level at 2 weeks. Cell proliferation was observed at all time points after ACF (P<0.001 vs. controls) with proliferation in endothelial cells more significant than smooth muscle cells. Apoptosis was not significant. CONCLUSIONS: Gene expression of TGF-beta1 and beta3 precedes arterial enlargement. Expression of apoptosis related factors is little regulated in the early stage of the flow-induced arterial remodeling.

Gene expression of tropoelastin is enhanced in the aorta proximal to the coarctation in rabbits.

To assess elastin biosynthesis in the aortic wall in response to acute elevation of blood pressure, we studied the aortic gene expression of tropoelastin in a rabbit midthoracic aortic coarctation model. The time points of the study were 1, 3, and 7 days and 2, 4, and 8 weeks after coarctation. Additional animals were subjected to hypercholesterolemia for analysis of tropoelastin expression in the intimal lesion. mRNA for tropoelastin was quantitated by Northern blot analysis and its distribution was revealed by in situ hybridization. The 65-kDa tropoelastin was analyzed by Western blotting and immunohistochemistry. Tropoelastin mRNA proximal to the coarctation was increased at 2 weeks and returned to baseline by 8 weeks (P < 0.05 versus control). Changes in 65-kDa tropoelastin corresponded to those of mRNA. Tropoelastin gene was expressed mainly in the intima and in the outer media at the proximal region to the stenoses, which was particularly remarkable in the intimal lesion. The results indicate that tropoelastin gene expression was enhanced in the early remodeling response to elevated blood pressure. The distribution of newly synthesized tropoelastin in the outer media suggests a reenforcement role of tropoelastin, which preserves mechanical resiliency in response to changes in tensile stress.

Subnormal shear stress-induced intimal thickening requires medial smooth muscle cell proliferation and migration.

Arterial intimal thickening is consisted of predominately smooth muscle cells (SMC). The source of these SMCs and mechanisms response for their changes have not been well cleared. Using a model of rabbit common carotid artery (CCA) shear induced intimal thickening, we sought to identify and describe the source of SMCs in intima. The enlarged CCA 28 days after arteriovenous fistula (AVF) creation was subjected to subnormal wall shear stress (WSS) for 1, 3, and 7 days by closure of the AVF. To determine SMC proliferation, BrdU pulse labeling of SMCs was performed. BrdU-labeled SMCs were tracked over time to further confirm SMC migration. In response to subnormal WSS intimal thickening developed progressively. BrdU-labeled SMCs localized in the subendothelial area. When the BrdU-labeled medial SMCs were tracked 1 day after AVF closure, progenies of these BrdU-incorporated SMCs increased by 4.8-fold with 75% of them in the intima. They were 12-fold increased with 83% in the intima 7 days after. En face examination showed an accumulation of SMCs in internal elastic lamina gap after AVF closure, which later migrated into subendothelial area. In situ hybridization revealed increased TGF-beta1 mRNA expression in intimal SMCs. This study demonstrates that the medial SMCs are the predominant cells in subnormal WSS-induced intimal thickening. Early expression of TGF-beta1 may play an important role in the process of intimal thickening.

Physiological angiogenesis in electrically stimulated skeletal muscle in rabbits: characterization of capillary sprouting by ultrastructural 3-D reconstruction study.

Physiological angiogenesis occurs in electrically stimulated skeletal muscles. It is known to start as capillary sproutings, but has not yet been well characterized as ordinary angiogenesis. To characterize the sprouting process during physiological angiogenesis, we carried out an ultrastructural 3-D reconstruction study for the extensor digitorum longus of three adult rabbits under electrical stimulation for 7 days. In addition, hemodynamic and morphological studies were carried out after stimulation for 3, 7, and 14 days. The electrical stimulation induced a twofold increase in femoral artery blood flow and tissue blood flow. This was associated with an increase in capillary density of the muscle by more than twofold at 7 and 14 days. Sproutings frequently appeared at 7 days (4.3 +/- 1.4 x 10(3) sproutings per mm3, 13.3 +/- 6.9 microm in length). All sprouting tips consisted of endothelial cytoplasmic protrusions (ECP). Besides sproutings, there were communicating networks consisting segmentally of ECP (11.6 +/- 5.6 x 10(3) networks per mm3). Endothelial cytoplasmic protrusions began to appear at 3 days, were frequent at 7 days, and disappeared at 14 days, which corresponded well to the changes in blood flow and capillary density. We consider that in physiological angiogenesis, sproutings start as ECP, which contact other capillaries to form networks and become hollowed to form new capillary networks.