Lower-limb veins are thicker and vascular reactivity is decreased in a rat PCOS model: concomitant vitamin D3 treatment partially prevents these changes.

Polycystic ovary syndrome (PCOS) causes vascular damage to arteries; however, there are no data for its effect on veins. Our aim was to clarify the effects of dihydrotestosterone (DHT)-induced PCOS both on venous biomechanics and on pharmacological reactivity in a rat model and to test the possible modulatory role of vitamin D3 (vitD). PCOS was induced in female Wistar rats by DHT treatment (83 µg/day, subcutaneous pellet). After 10 wk, the venous biomechanics, norepinephrine (NE)-induced contractility, and acetylcholine-induced relaxation were tested in saphenous veins from control animals and from animals treated with DHT or DHT with vitD using pressure angiography. Additionally, the expression levels of endothelial nitric oxide synthase (eNOS) and cyclooxygenase (COX-2) were measured using immunohistochemistry. Increased diameter, wall thickness, and distensibility as well as decreased vasoconstriction were detected after the DHT treatment. Concomitant vitD treatment lowered the mechanical load on the veins, reduced distensibility, and resulted in vessels that were more relaxed. Although there was no difference in the endothelial dilation tested using acetylcholine (ACh), the blocking effect of N(G)-nitro-l-arginine methyl ester (l-NAME) was lower and was accompanied by lower COX-2 expression in the endothelium after the DHT treatment. Supplementation with vitD prevented these alterations. eNOS expression did not differ among the three groups. We conclude that the hyperandrogenic state resulted in thicker vein walls. These veins showed early remodeling and altered vasorelaxant mechanisms similar to those of varicose veins. Alterations caused by the chronic DHT treatment were prevented partially by concomitant vitD administration.

Heritability of venous biomechanics.

OBJECTIVE: Altered venous biomechanics may contribute to the pathogenesis of venous diseases, and their heritability is less known. METHODS AND RESULTS: Seventy-eight monozygotic twin pairs (aged 42.4 ± 16.8 years) and 24 same-sex dizygotic twin pairs (aged 50.5 ± 16.1 years) were examined. Anteroposterior and mediolateral diameters of the common femoral vein were measured by ultrasonography. Measurements were made both in supine and in standing body positions, with or without controlled forced expiration (Valsalva test). High correlation of diameter, capacity, and distensibility values was found between twin pairs. The univariate heritability (A), shared (C), and unshared (E) environmental effects model has shown 39.3% genetic component of the variance of low pressure, 37.9% of high-pressure venous capacity, and 36.4% of maximal capacity changes, even after elimination of sex, age, and body weight effects. Bivariate Cholesky analysis revealed substantial covariance of inherited body weight and venous capacity components (57.0%-81.4%). CONCLUSIONS: Femoral vein capacity and elasticity depend ≈30% to 40% on genetic factors, and this value in the standing body position can reach 50%. A relatively high genetic covariance was found between weight and femoral vein capacity and elasticity. Our work might yield some new insights into the inheritance of venous diseases that are associated with altered venous biomechanics and help elucidate the involved genes.

Pharmacological reactivity of resistance vessels in a rat PCOS model - vascular effects of parallel vitamin D3 treatment.

The aim of this study was to clarify the effects of dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS) on pharmacological reactivity of a resistance vessel in a rat model and the possible modulatory role of 1,25-(OH)2-cholecalciferol (vitamin D3). The PCOS model was induced in adolescent female Wistar rats by a 10-week DHT treatment. Norepinephrine induced contractility and acetylcholine relaxation were tested in arterioles by pressure arteriography in control as well as DHT- and DHT plus vitamin D3-treated (DHT+D3) animals. Decreased vasoconstriction and dilatation were detected after DHT treatment. Concomitant vitamin D3 treatment increased the contractile response and resulted in more relaxed vessels. Endothelial dilation tested with acetylcholine was lower after DHT treatment, this effect was not depend on vitamin D3 supplementation. In conclusion, hyperandrogenic state resulted in reduced endothelium- and smooth muscle-dependent vasorelaxation and constriction with a complete loss of nitric oxide (NO)-dependent relaxation compared with controls. These alterations caused by chronic DHT treatment were partially reversed by concomitant vitamin D3 administration.

Biomechanics of resistance artery wall remodeling in angiotensin-II hypertension and subsequent recovery.

BACKGROUND/AIMS: To identify the relationship between systemic and local hemodynamics, as well as segmental biomechanical properties in a musculocutaneous resistance artery during angiotensin-II hypertension and its recovery. METHODS: Rats were infused with angiotensin-II using implanted osmotic minipumps (ALZET 2ML4, 150 ng/kg/min) for 4 weeks. Measurements were made either immediately following infusion or after an additional 4-week recovery period. Parallel controls were created. Segmental geometry and blood flow were determined in vivo on microsurgically exposed segments of the saphenous arterial branch (350 mum). Pressure-radius plots of excised cylindrical segments were recorded by pressure arteriography. RESULTS: Eutrophic hypertensive wall remodeling developed, with reduced passive radius, increased wall thickness, elevated low-stress elastic modulus, reduced norepinephrine contraction, and reduced endothelium-mediated dilation. Relaxed wall geometry fully healed in 4 weeks of recovery, but an increased contractility and a reduced in vivo lumen persisted. Regional hemodynamic resistance correlated positively with systemic arterial pressure and wall thickness in vivo, and negatively with in vivo lumen size throughout these studies. CONCLUSION: A partial recovery of the biomechanical parameters was found. Healing of eutrophic hypertensive remodeling of the resistance artery wall is a complex biomechanical process, not a simple reversal of the original pathological sequel.

A novel videomicroscopic technique for studying rat ureteral peristalsis in vivo.

OBJECTIVES: Existing techniques do not allow analyzing the fine movements of the ureteral wall during in vivo peristaltic contractions. A videomicroscopic technique has been developed to study the active mechanical displacements of the rat ureter. METHODS: The middle portion of the ureter in a length of 16-18 mm was elevated from its base by microsurgical preparation, encased in a specific tissue chamber and continuously superfused with physiological saline. Contractions were recorded by videomicroscopy. A number of characteristic points on the surface were identified by the pattern of vasa vasorum. Their movements were analyzed in a coordinate system defined by the axial and radial directions of the segment. Identified surface points on the ureter moved along characteristic trajectory loops during contractile cycles. RESULTS: In addition to the synchronized longitudinal and circumferential contractions, typical axial displacement cycles could be identified. Our observations demonstrate that longitudinal contractions might be more important in transporting the urine as thought earlier because of the axial tether of the ureter. CONCLUSIONS: (1) A longitudinal contraction ring preceding the circumferential one axially distends the distal segments. (2) Initial phase of the longitudinal contraction ring promotes bolus volume rearrangement toward the passive diameter dilation. (3) Longitudinal contraction with the maximum circumferential contraction ring just behind it helps pushing the urine bolus downward. (4) Ureteral segments proximal to the longitudinal contraction ring will be passively axially stretched which also helps their filling.

[Master course in biomedical engineering]. / Egészségügyi mérnöki mesterképzés.

The Bologna Declaration aims at harmonizing the European higher education structure. In accordance with the Declaration, biomedical engineering will be offered as a master (MSc) course also in Hungary, from year 2009. Since 1995 biomedical engineering course has been held in cooperation of three universities: Semmelweis University, Budapest Veterinary University, and Budapest University of Technology and Economics. One of the latter's faculties, Faculty of Electrical Engineering and Informatics, has been responsible for the course. Students could start their biomedical engineering studies - usually in parallel with their first degree course - after they collected at least 180 ECTS credits. Consequently, the biomedical engineering course could have been considered as a master course even before the Bologna Declaration. Students had to collect 130 ECTS credits during the six-semester course. This is equivalent to four-semester full-time studies, because during the first three semesters the curriculum required to gain only one third of the usual ECTS credits. The paper gives a survey on the new biomedical engineering master course, briefly summing up also the subjects in the curriculum.

Gender, hyperandrogenism and vitamin D deficiency related functional and morphological alterations of rat cerebral arteries.

Hyperandrogenism is a risk factor of cerebrovascular diseases as androgens can alter markedly the regulation of cerebrovascular tone. We examined the combined impact of androgen excess and vitamin D deficiency (VDD), a common co-morbidity in hyperandrogenic disorders, on remodeling and testosterone-induced vascular responses of anterior cerebral arteries (ACA) in order to evaluate the interplay between androgens and VDD in the cerebral vasculature. Male and female Wistar rats were either fed with vitamin D deficient or vitamin D supplemented diet. Half of the female animals from both groups received transdermal testosterone treatment. After 8 weeks, vessel lumen, wall thickness and testosterone-induced vascular tone of isolated ACA were determined using pressure microangiometry and histological examination. Androgen receptor protein expression in the wall of cerebral arteries was examined using immunohistochemistry. In female rats only combined VDD and testosterone treatment decreased the lumen and increased the wall thickness of ACA. In males, however VDD by itself was able to decrease the lumen and increase the wall thickness. Vascular reactivity showed similar alterations: in females, testosterone constricted the ACA only after combined VDD and hyperandrogenism, whereas in males VDD resulted in increased testosterone-induced contractions in spite of decreased androgen receptor expression. In conclusion, a marked interplay between hyperandrogenism and VDD results in inward remodeling and enhanced testosterone-induced constrictions of cerebral arteries, which might compromise the cerebral circulation and thus, increase the risk of stroke in the long term. In addition, the early cerebrovascular manifestation of VDD appears to require androgen excess and thus, depends on gender.

[New results in the research of the biomechanics of the venous system]. / Ujabb eredmények a vénás rendszer biomechanikájának kutatásában.

The upright posture of man had been a major evolutional challenge. The mechanisms responsible for orthostatic tolerance mostly affect the venous system. In this paper, we discuss new results regarding the biomechanics of the venous system highlighting a rather neglected field, the biomechanical properties of the vein wall. These properties change according to localization of veins, age, gender and body mass. The anti-gravitational adaptation of veins is a complex process involving all three layers of the venous wall. Local myogenic and humoral mechanisms as well as systemic hormonal and nervous influences control the adaptive processes in the veins. Long term adaptation involves structural and functional remodeling of the venous wall. Disorders of the veins mostly cause pathological remodeling. Hemodynamic factors (pressure and flow) together with inflammatory processes may lead to pathological alterations, changing the biomechanical properties of the vein wall, which further contribute to the reservation and progression of venous dysfunction. Appropriate testing of venous function can reveal biomechanical disorders even in clinically asymptomatic patients. Thus, biomechanical investigation of veins not only helps to understand the underlying pathomechanism but it also can contribute to early diagnosis and follow-up of venous disorders. When recognized in time, pathological remodeling can be prevented or treated. In this way, the incidence of venous disorder could be cut back reducing both human suffering and material loss.

High pressure-low flow remodeling of the rat saphenous vein wall.

Objective To better understand factors that may play a role in the development of varicosities. Methods We induced combined flow-pressure disturbance in the saphenous system of the rat by performing chronic partial clipping of the main branch. Biomechanical and quantitative histological testing was undertaken. Results A rich microvenous network developed. Bloodflow decreased to 0.65 ± 0.18 µl/s (control side, 3.5 ± 1.4 µl/s) and pressure elevated to 6.8 ± 0.7 mmHg (control side, 2.3 ± 0.2 mmHg, p < 0.05). Involution of the wall and lumen was observed (16.5%, 28.7% and 35.5% reduction in outer diameter, wall thickness and wall mass respectively, p < 0.05). Elevated macrophage (CD68) and cell division (Ki67) activity was observed. Elastic tissue and smooth muscle actin became less concentrated in the inner medial layers. Conclusions Low-flow induced morphological shrinking of the lumen in veins may override pressure-induced morphological distension. Loosening of the force-bearing elements during flow-induced wall remodeling may be an important pathological component in varicosity.

Environmental stress and vestibular inputs modulate cardiovascular responses to orthostasis in hypertensive rats.

The frequent accompaniment of hypertension by orthostatic circulatory disorders prompted us to investigate the effect of repeated and sustained head-up and head-down tilt positions on cardiovascular responses in spontaneously hypertensive rats vs. Wistar rats using radiotelemetric implants. Repeated orthostasis caused a transient elevation in blood pressure (7.3±1.7 mmHg) and heart rate (39.7±10.5 BPM), while repeated antiorthostasis led only to reversible tachycardia (85.6±11.7-54.3±16.8 BPM) in spontaneously hypertensive rats. In contrast to the Wistar rats, sustained tilt failed to affect the blood pressure or heart rate in spontaneously hypertensive rats because the environmental stress of being placed in horizontal tilt cages prior to the sustained tilt test induced marked changes in cardiovascular parameters. Non-specific stress responses were eliminated both by the anxiolytic diazepam and a sub-anesthetic dose of chloralose. Unlike diazepam, chloralose amplified the orthostatic pressor responses in the Wistar rats. In contrast to diazepam preventing the pressor response and associated tachycardia in spontaneously hypertensive rats, chloralose elicited this effect during both sustained orthostasis (36.0±7.3 mmHg, 63.7±21.8 BPM) and antiorthostasis (42.9±10.9 mmHg, 82.8±25.4 BPM), with a reduced baroreflex sensitivity. However, during sustained orthostasis, removal of the vestibular input led to a depressor response with bradycardia (12.5±3.2 mmHg, 59.3±17.3 BPM), whereas antiorthostasis only reduced blood pressure (20.5±7.1 mmHg) in the spontaneously hypertensive rats. We conclude that repeated tilts induce a transient pressor response and/or tachycardia in spontaneously hypertensive rats. Cardiovascular parameters are suppressed by diazepam, whereas chloralose evokes both blood pressure and heart rate responses during sustained tilts, which are primarily elicited by baroreflex suppression in hypertension. Vestibular inputs support cardiovascular tolerance to sustained postural changes in a rat model of human 'essential' hypertension.

Vitamin D deficiency causes inward hypertrophic remodeling and alters vascular reactivity of rat cerebral arterioles.

BACKGROUND AND PURPOSE: Vitamin D deficiency (VDD) is a global health problem, which can lead to several pathophysiological consequences including cardiovascular diseases. Its impact on the cerebrovascular system is not well understood. The goal of the present work was to examine the effects of VDD on the morphological, biomechanical and functional properties of cerebral arterioles. METHODS: Four-week-old male Wistar rats (n = 11 per group) were either fed with vitamin D deficient diet or received conventional rat chow with per os vitamin D supplementation. Cardiovascular parameters and hormone levels (testosterone, androstenedione, progesterone and 25-hydroxyvitamin D) were measured during the study. After 8 weeks of treatment anterior cerebral artery segments were prepared and their morphological, biomechanical and functional properties were examined using pressure microangiometry. Resorcin-fuchsin and smooth muscle actin staining were used to detect elastic fiber density and smooth muscle cell counts in the vessel wall, respectively. Sections were immunostained for eNOS and COX-2 as well. RESULTS: VDD markedly increased the wall thickness, the wall-to-lumen ratio and the wall cross-sectional area of arterioles as well as the number of smooth muscle cells in the tunica media. As a consequence, tangential wall stress was significantly lower in the VDD group. In addition, VDD increased the myogenic as well as the uridine 5'-triphosphate-induced tone and impaired bradykinin-induced relaxation. Decreased eNOS and increased COX-2 expression were also observed in the endothelium of VDD animals. CONCLUSIONS: VDD causes inward hypertrophic remodeling due to vascular smooth muscle cell proliferation and enhances the vessel tone probably because of increased vasoconstrictor prostanoid levels in young adult rats. In addition, the decreased eNOS expression results in endothelial dysfunction. These morphological and functional alterations can potentially compromise the cerebral circulation and lead to cerebrovascular disorders in VDD.

Gender differences in biomechanical properties of intramural coronary resistance arteries of rats, an in vitro microarteriographic study.

The prevalence of ischemic heart disease is lower in premenopausal females than in males of corresponding age. This should be related to gender differences in coronary functions. We tested whether biomechanical differences exist between intramural coronary resistance arteries of male and female rats. Intramural branches of the left anterior descending coronary artery (uniformly approximately 200microm in diameter) were isolated, cannulated and studied by microarteriography. Intraluminal pressure was increased from 2 to 90mmHg in steps and steady-state diameters were measured. Measurements were repeated in the presence of vasoconstrictor U46619 (10(-6)M) and the endothelial coronary vasodilator bradykinin (BK) (10(-6)M). Finally, passive diameters were recorded in calcium-free saline. A similar inner radius and a higher wall thickness (41.5+/-2.9microm vs. 31.4+/-2.7microm at 50mmHg in the passive condition, p<0.05) resulted in lower tangential wall stresses in male rats (18.9+/-1.9kPa vs. 24.9+/-2.5kPa at 50mmHg, p<0.05). Isobaric elastic modulus of vessels from male animals was significantly smaller at higher pressures. Vasoconstrictor response was significantly stronger in male than in female animals. Endothelial relaxations induced by BK were not different. This is the first demonstration that biomechanical characteristics of intramural coronary resistance arteries of a mammalian species are different in the male and female sexes. Higher wall thickness and higher vascular contractility in males are associated with similar endothelial function and larger high-pressure elasticity compared to females. These gender differences in biomechanics of coronary resistance arteries of rats may contribute to our better understanding the characteristic physiological and pathological differences in humans.

Generalized changes in venous distensibility in postthrombotic patients.

INTRODUCTION: In situ biomechanical properties of peripheral large veins were compared between asymptomatic young patients who had previously unilateral femoro-popliteal deep venous thrombosis (DVT) and age-matched, healthy controls; the aim of this study was to assess local or generalized alterations of venous wall biomechanics in postthrombotic patients. PATIENTS AND METHODS: Inner diameters of both common femoral veins, right axillary vein, and right internal jugular veins were measured in two directions by ultrasonography. Venous pressure was altered by posture changes (standing and lying) and by application of graded and controlled Valsalva. Ten postthrombotic young patients without any symptoms and 11 age-matched control subjects were included. RESULTS: In postthrombotic patients, both the affected and unaffected common femoral vein diameters and capacities were larger at low transmural pressures than those for the control group, but they demonstrated significantly less distensibility when higher pressures were applied. Similarly, in the internal jugular vein, capacity without Valsalva was significantly higher in postthrombotic patients and distensibility was reduced (statistically significant in the erect position). Pressure-induced changes in axillary vein diameter were negligible. CONCLUSIONS: In situ diameter and capacity changes, and in situ distensibility of the femoral veins on both sides (i.e., the side of previous thrombosis as well as the disease-free side) and of the jugular veins are reduced in the young DVT patients compared to veins of the age-matched, healthy controls. The pathophysiological mechanism of generalized venous wall changes in these young DVT patients remains unknown.

[Biomechanics of intracranial aneurysms]. / Az aneurysmák biomechanikai tulajdonságai.

INTRODUCTION: Viscoelastic parameters of circumferential and meridional strips of ruptured and silent aneurysms were investigated (considered clinical and histological data either) in order to advance the prediction of risk of aneurysm rupture. METHOD: In our clinical practice, aneurysms managed by microsurgery aneurysm clipping were removed. Meridional and circumferential strips were cut. Strips were investigated by an uniaxial biomechanical instrument: distending force was recorded as the length of the strips was increased in steps. Normal stress-relaxation patterns were detected. The shape of strain curves well overlapped with the Standard Linear Solid Model curve, as had been expected. The viscosity, serial and parallel elastic moduli of the model were then computed. RESULTS: Linear correlation was demonstrated amongst peek distending force detected and aneurysm strip thickness. Steric inhomogeneity was detected at the meridional and circumferential strips. Strain-stress behaviour of ruptured and silent aneurysm specimen showed significant difference. Values of strips originated from patients suffered from hypertension as well as strips originated from aneurysms had been histologically found inflamed were higher. DISCUSSION: Results of these observations are going to be used to set three dimensional computer model in cooperation with IT team of Budapest University of Technology and Economics to advance rupture risk prediction.

Estrogen therapy may counterbalance eutrophic remodeling of coronary arteries and increase bradykinin relaxation in a rat model of menopausal hypertension.

OBJECTIVE: Hypertension causes adverse remodeling and vasomotor alterations in coronaries. Hormones such as estrogen may help counterbalance some of these effects. The aim of this study was to analyze the effects of ovariectomy and estrogen therapy in a rat model of menopausal hypertension induced by angiotensin II (AII). METHODS: We investigated diameter, tone, and mechanics of intramural coronaries taken from ovariectomized female rats (n = 11) that received chronic AII treatment to induce hypertension, and compared the results with those found in female rats that were also given estrogen therapy (n = 11). The "hypertensive control" group (n = 11) underwent an abdominal sham operation, and received AII. After 4 weeks of AII treatment, side branches of left anterior descendent coronary (approximately 200 µm in diameter) were isolated, cannulated with plastic microcannulas at both ends, and studied in vitro in a vessel chamber. The inner and outer diameter of the arteries were measured by microangiometry, and spontenuous tone, wall thickness, wall cross-sectional area, tangential stress, incremental distensibility, circumferential incremental elastic modulus, thromboxane agonist-induced tone, and bradykinin-induced dilation were calculated. RESULTS: In hypertension, intramural small coronaries show inward eutrophic remodeling after ovariectomy comparing with hypertensive controls. Estrogen therapy had an opposite effect on vessel diameter. Hormone therapy led to an increase in spontaneous tone, allowing for greater dilatative capacity. CONCLUSIONS: Estrogen may therefore be considered to counterbalance some of the adverse changes seen in the wall of intramural coronaries in the early stages of chronic hypertension.

Estrogen replacement therapy reverses changes in intramural coronary resistance arteries caused by female sex hormone depletion.

OBJECTIVE: We tested the hypothesis that female sex hormone depletion and estradiol replacement therapy significantly influences the biomechanical properties of intramural coronary resistance arteries. DESIGN: Female rats (n=30) were divided into three groups. In group O, rats were subjected to bilateral ovariectomy. Group HRT was subjected to bilateral ovariectomy and estradiol replacement therapy. Rats in group C served as controls. One month after ovariectomy, intramural coronary arteries (approximately 200 microm in diameter) branching from the left anterior descending coronary were isolated, cannulated and studied by microarteriography. Intraluminal pressure was increased in steps between 0 and 90 mm Hg. The steady state diameter at each step was measured. These measurements were repeated in the presence of U46619, a thromboxane (TX) A2 receptor agonist (at a concentration of 10(-6) M), and bradykinin (BK; at 10(-6) M). Finally, Ca2+-free Krebs-induced passive diameter (PD) was measured in each group. RESULTS: Ovariectomy increased spontaneous myogenic tone of coronary arteries (p<0.05), which was normalized by estrogen replacement. Ovariectomy decreased distensibility observed at low pressure, although passive diameter was not changed. Estrogen replacement decreased wall stress and elastic modulus (p<0.05). The thromboxane A2 agonist induced the largest contraction in the ovariectomized group, whereas bradykinin-induced relaxation was the largest in the estrogen replacement group (p<0.05). CONCLUSION: Estradiol hormone replacement therapy (HRT) may exert a beneficial effect on myocardial perfusion in menopause by opposing the deterioration of biomechanical properties of intramural coronary resistance vessels induced by female sex hormone depletion.

Sex hormone replacement therapy reverses altered venous contractility in rats after pharmacological ovariectomy.

OBJECTIVE: Female sex hormones have several important effects on the venous system. We earlier found that hormone replacement has a significant effect on venous distensibility, but effects of menopause and hormone replacement on venous contractility have never been studied. Therefore, and because the changes we found earlier in distensibility were most likely caused by alterations of contractility, we examined the changes in contractility of saphenous vein caused by depletion and replacement of sex hormones in female rats. DESIGN: Twenty Sprague-Dawley rats were pharmacologically ovariectomized by triptorelin. Ten of these rats received combined sex hormone replacement (HRT) with estradiol propionate and medroxyprogesterone acetate. The rest were given vehicle. Ten animals without ovariectomy served as controls. After 3 months of treatment, segments of the saphenous vein were dissected. Pressure-diameter curves were recorded in relaxed, contracted, and control states. RESULTS: Venous diameter, adjusted for body weight, was significantly decreased after pharmacological ovariectomy. HRT increased the diameter. The presence of sex hormones augmented norepinephrine contraction measured at physiological pressures (control: 19.2 +/- 2.3%; pharmacological ovariectomy: 15.2 +/- 1.4%, p < 0.05 and 17.8 +/- 2.2% following HRT). Myogenic (spontaneous) tone of the saphenous vein did not change after ovariectomy, but it was lowered by hormone replacement (control: 8 +/- 1.1%; ovariectomy: 6.9 +/- 2.5%; ovariectomy + HRT: 2.7 +/- 1.1%, p < 0.05). CONCLUSIONS: Sex hormone depletion induces significant alterations in contractility of the saphenous vein, which could perturb venous capacitance function and distensibility. This effect has a potential role in the development of hypertension and venous varicosity, and these changes could possibly be prevented by HRT.

Vacuole formation in the endothelium of rat extremity vessels depends on fixation techniques and vessel type.

Applying immersion fixation for electron microscopy, huge clear endothelial membrane-bound vacuoles of 0.1-3 microm diameter were noted in the extremity veins of Sprague-Dawley rats. Histological and electron microscopic histochemical methods were applied to determine whether they were the product of programmed cell death or any other kind of cell damage. Image analyzer was used to measure the total area of the vacuoles in the endothelium cells. Neither lipid content nor acidic phosphatase activity could be identified in the vacuoles. In saphenous and brachial veins, the vacuoles occupied 20.6 +/- 2.21% and 18 +/- 2.45% of the endothelium, respectively. Venous endothelium of two different strains of rat also contained the vacuoles. No such structures appeared in extremity arteries. Long-term tilting did not influence vacuolization. Using in vivo whole body fixation, only pinocytotic and dense microvesicles, but no vacuoles were noted. In conclusion, the clear vacuolar structures represent neither lipid inclusions nor secondary lysosomes. The method of tissue fixation is critical when venous endothelial vesicles are investigated. It is presumed that the vacuoles originated from intra- or intercellular microstructures, and that in case of the collapsible vein segments, their size is increased under the pathological-hypoxic and low-pressure-conditions of in vitro fixation.