Creating Interactive Three-Dimensional Applications to Visualise Novel Stent Grafts That Aid in the Treatment of Aortic Aneurysms.

Three-Dimensional (3D) medical animations incorporated into applications are highly beneficial for clinical outreach and medical communication purposes that work towards educating the clinician and patient. Aortic aneurysms are a clinically important area to communicate with multiple audiences about various treatment options; both abdominal and thoracic aortic aneurysms were selected to create 3D animations and applications to educate medical professionals and patients regarding treatment options. Fenestrated endovascular aortic repair (FEVAR) and thoracic endovascular aortic repair (TEVAR) are both tried and tested minimally invasive surgical methods for treating thoracic aortic aneurysms respectively. The Terumo Aortic Custom Relay Proximal Scalloped stent graft and Fenestrated Anaconda stent graft were both designed specifically for these procedures; however, it can be difficult to visually communicate to clinicians and patients in a straightforward way how these devices work. Therefore, we have developed two interactive applications that use 3D visualisation techniques to demonstrate how these aortic devices function and are implemented. The objective of these applications is to engage both clinicians and patients, therefore demonstrating that the addition of anatomically accurate 3D visualisations within an interactive interface would have a positive impact on public engagement while also ensuring that clinicians will have the best possible understanding of the potential uses of both devices, enabling them to exploit their key features to effectively broaden the treatable patient population.Detailed anatomical modelling and animation was used to generate realistic and accurate rendered videos showcasing both products. These videos were integrated into an interactive application within a modern, professional graphic interface that allowed the user to explore all aspects of the stent device. The resulting applications were broken down into three modules: deployment, clinical performance and features. Following application development, these applications were evaluated by professionals in the field. Overall, positive feedback was received regarding the user-friendly nature of the applications and highly effective animations to showcase the products. The clinical applications and feature modules were particularly successful, while the deployment modules had a neutral response. Biomedical applications such as these show great potential for communicating the key features of medical devices and promoting discussion between clinicians and patients; further testing would need to be conducted on a larger group of participants in order to validate the learning effectiveness of the applications.

Using Confocal Microscopy to Generate an Accurate Vascular Model for Use in Patient Education Animation.

Hypertension is a condition requiring lifelong medication, where patients often feel well with or without treatment. Uncontrolled hypertension, however, can lead to permanent remodelling processes that occur to the vascular structure, which are seldom understood by the public. As a result, a significant burden is placed on healthcare systems globally as a result of the effects of hypertension and lack of adherence to prescribed treatment.Improving patient education through well-designed interactive applications and animation is a known strategy that can improve adherence rates to medication. In the context of hypertension, little attention has been given to helping patients understand the unseen damage that occurs to vessels exposed to high blood pressure. However, generating an accurate representation of a vessel and the changes that occur can be challenging. Using microscopy data is one way for creating an anatomically correct model, but this often needs careful consideration as data cannot be directly imported. Here we describe methods for creating an accurate 3D model of a small artery using confocal microscopy data. This model can then be animated to demonstrate the substructures and pathological changes that occur in hypertensive conditions to better inform patients about the dangers of uncontrolled blood pressure.

Interaction between α1B - and other α1 - and α2 -adrenoceptors in producing contractions of mouse spleen.

We have investigated the interaction of α1 - and α2 -adrenoceptor subtypes in producing isometric contractions to NA in mouse whole spleen. The α1 -adrenoceptor antagonist prazosin (10-8  M) or the α2 -adrenoceptor antagonist yohimbine (10-6  M) alone produced only small shifts in NA potency in wild type (WT) mice, but the combination produced a large shift in NA potency. In spleen from α1A/D -KO mice, the effects of prazosin and the combination of prazosin and yohimbine were similar to their effects in WT mice. Hence, in α1A/D -KO mice, in which the only α1 -adrenoceptor present is the α1B -adrenoceptor, prazosin still antagonized contractions to NA. The α1A -adrenoceptor antagonist RS100329 (3 × 10-9 M) produced significant shifts in the effects of higher concentrations of NA (EC50 and EC75 levels) and the α1D -adrenoceptor antagonist BMY7378 (3 × 10-8 M) produced significant shifts in the effects of lower concentrations of NA (EC25 and EC50 levels). The effects of BMY7378 and RS00329 demonstrate α1D -adrenoceptor and α1A -adrenoceptor components and suggest that the α1B -adrenoceptor interacts with an α1D -adrenoceptor, and to a lesser extent an α1A -adrenoceptor, at low, and an α1A -adrenoceptor at high, NA concentrations. This study demonstrates the complex interaction between α1 - and α2 -adrenoceptor subtypes in producing contractions of mouse spleen and may have general implications for α-adrenoceptor mediated control of smooth muscle.

Examining Vascular Structure and Function Using Confocal Microscopy and 3D Imaging Techniques.

The structure of the blood vessel wall has historically been studied using thin cut sections using standard histological stains. In the mid-80s laser scanning confocal microscopes became available and offered investigators the chance to examine the 3D structure of thicker sections (i.e. ~60 µm depth penetration for a typical vascular wall). Unfortunately, desktop computers lagged far behind in their capacity to process and display large 3D (confocal) data sets. Even extremely highly priced graphics workstations of the early to mid-90s offered little in the way of flexible 3D viewing. Today's gaming PCs provide the kind of processing power that 3D confocal users have been waiting for. Coupled with high end animation software, virtual reality and game design software, we now have the capacity to exploit the huge data sets that modern microscopes can produce. In this chapter, the vascular wall will be used as an example of a biological tissue that can benefit from these developments in imaging hardware and software.

ß2-Adrenoceptor signaling in airway epithelial cells promotes eosinophilic inflammation, mucous metaplasia, and airway contractility.

The mostly widely used bronchodilators in asthma therapy are ß2-adrenoreceptor (ß2AR) agonists, but their chronic use causes paradoxical adverse effects. We have previously determined that ß2AR activation is required for expression of the asthma phenotype in mice, but the cell types involved are unknown. We now demonstrate that ß2AR signaling in the airway epithelium is sufficient to mediate key features of the asthmatic responses to IL-13 in murine models. Our data show that inhibition of ß2AR signaling with an aerosolized antagonist attenuates airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus-production responses to IL-13, whereas treatment with an aerosolized agonist worsens these phenotypes, suggesting that ß2AR signaling on resident lung cells modulates the asthma phenotype. Labeling with a fluorescent ß2AR ligand shows the receptors are highly expressed in airway epithelium. In ß2AR-/- mice, transgenic expression of ß2ARs only in airway epithelium is sufficient to rescue IL-13-induced AHR, inflammation, and mucus production, and transgenic overexpression in WT mice exacerbates these phenotypes. Knockout of ß-arrestin-2 (ßarr-2-/-) attenuates the asthma phenotype as in ß2AR-/- mice. In contrast to eosinophilic inflammation, neutrophilic inflammation was not promoted by ß2AR signaling. Together, these results suggest ß2ARs on airway epithelial cells promote the asthma phenotype and that the proinflammatory pathway downstream of the ß2AR involves ßarr-2. These results identify ß2AR signaling in the airway epithelium as capable of controlling integrated responses to IL-13 and affecting the function of other cell types such as airway smooth muscle cells.

Sensory innervation of perivascular adipose tissue: a crucial role in artery vasodilatation and leptin release.

AIMS: Electrical field stimulation (EFS) elicits robust sensory neurogenic relaxation responses in the rat isolated mesenteric arterial bed but these responses are absent or difficult to demonstrate in isolated arteries. We believe that this mismatch is due to the absence of perivascular adipose tissue (PVAT) as it is conventionally removed in studies on isolated vessels. We aimed to determine whether sensory nerves are expressed in PVAT, their physiological roles and their possible interactions with PVAT-derived adipokines. METHODS AND RESULTS: Using confocal imaging, enzyme immunoassay (EIA), myography, vascular perfusion, and multiplex analysis of rat mesenteric arteries, we show that PVAT is crucial for the roles of sensory nerves in control of vasomotor tone and adipokine release. Immunofluorescence double staining showed co-expression of calcitonin gene-related peptide (CGRP; sensory neurotransmitter) and PGP9.5 (neuronal marker) in PVAT of mesenteric arteries. CGRP release from dissected PVAT, measured using EIA, was increased by capsaicin which activates sensory nerves. EFS in both mesenteric arteries and perfused mesenteric arterial beds, with and without PVAT, demonstrated neurogenic relaxation in the presence of PVAT, which was greatly attenuated in preparations without PVAT. Neurogenic relaxation due to EFS was associated with release of leptin in PVAT-intact mesenteric arterial beds, which was abolished in preparations without PVAT. Exposure to low oxygen was associated with an attenuated leptin and adiponectin release, but an increase in IL-6 release, from mesenteric arterial beds. Exogenous leptin augmented relaxation to CGRP in mesenteric arteries. CONCLUSION: These data show, for the first time, expression of sensory nerves within PVAT and that PVAT is crucial for sensory neurogenic vasorelaxation and crosstalk with adipocytes leading to leptin release, which may augment CGRP-mediated relaxation; leptin release is abolished after exposure to conditions of reduced oxygenation.

Autonomic nerves and perivascular fat: interactive mechanisms.

The evidence describing the autonomic innervation of body fat is reviewed with a particular focus on the role of the sympathetic neurotransmitters. In compiling the evidence, a strong case emerges for the interaction between autonomic nerves and perivascular adipose tissue (PVAT). Adipocytes have been shown to express receptors for neurotransmitters released from nearby sympathetic varicosities such as adrenoceptors (ARs), purinoceptors and receptors for neuropeptide Y (NPY). Noradrenaline can modulate both lipolysis (via α2- and ß3-ARs) and lipogenesis (via α1- and ß3-ARs). ATP can inhibit lipolysis (via P1 purinoceptors) or stimulate lipolysis (via P2y purinoceptors). NPY, which can be produced by adipocytes and sympathetic nerves, inhibits lipolysis. Thus the sympathetic triad of transmitters can influence adipocyte free fatty acid (FFA) content. Substance P (SP) released from sensory nerves has also been shown to promote lipolysis. Therefore, we propose a mechanism whereby sympathetic neurotransmission can simultaneously activate smooth muscle cells in the tunica media to cause vasoconstriction and alter FFA content and release from adjacent adipocytes in PVAT. The released FFA can influence endothelial function. Adipocytes also release a range of vasoactive substances, both relaxing and contractile factors, including adiponectin and reactive oxygen species. The action of adipokines (such as adiponectin) and reactive oxygen species (ROS) on cells of the vascular adventitia and nerves has yet to be fully elucidated. We hypothesise a strong link between PVAT and autonomic fibres and suggest that this poorly understood relationship is extremely important for normal vascular function and warrants a detailed study.

Visualization and analysis of vascular receptors using confocal laser scanning microscopy and fluorescent ligands.

The use of fluorescent ligands to analyze receptor distribution is increasing in popularity. This is due to the ever growing number of fluorescent ligands and the increased sensitivity of microscope-based technologies. Image-analysis methods have advanced to a stage where quantification of fluorescent signals is relatively simple (if used appropriately). In this chapter we describe a method of analyzing the 2D and 3D distribution of fluorescent ligands in segments of blood vessels. In addition, we introduce the issues surrounding the accurate analysis of colocalization of two different fluorescent ligands.

Themed section: Imaging in pharmacology.

This themed section on Imaging in Pharmacology comprising reviews and original articles arose from two symposia held at the Summer Meeting of the British Pharmacological Society in Edinburgh 8-10 July 2009 on Developments in Receptor Imaging and Imaging and Targeting Inflammation in Stroke and Atherosclerosis. The reports cover a broad spectrum of pharmacological studies, from whole animal imaging to gene expression and emphasize the importance of imaging techniques in pharmacology. The development of each new imaging methodology brings pharmacology closer to the ambitious goal of being able to image (simultaneously) each component part of the G-protein-coupled receptor signalling process.

Confocal myography for the study of hypertensive vascular remodelling.

Hypertension is associated with vascular structural alterations known as "vascular remodelling", which initially are adaptive but in the long run, lead to vascular damage and loss of function. Despite decades of study, there is still modest information on the 3-dimensional (3D) arrangement of vascular cells and extracellular matrix (ECM) and how they change under pathological situations. To address this problem we developed a technique which combines fluorescence confocal microscopy, pressure myography and image analysis, "confocal myography", which permits the study of intact resistance-sized vessels at cellular level and at physiological pressure. With the aid of this method, we have identified, in arteries from hypertensive rats, abnormal orientation of endothelial, smooth muscle cells (SMC) and elastic fibres; elongation and denudation of endothelial cells, and adventitial hypercellularity. Confocal myography offers a new approach to the study of vascular remodelling in intact small arteries from a 3D point of view.

Imaging the vascular wall using confocal microscopy.

Blood vessels are capable of structural changes in a dynamic process called 'vascular remodelling', which involves cell growth, death, phenotypic change and migration, as well as extracellular matrix synthesis and degradation. An integrated view of the interrelationships of the different elements of the arterial wall is made possible by fluorescence confocal microscopy which enables collection of serial optical sections of relatively thick specimens without the need to cut them as with conventional histology. With the aid of image analysis software, these serial sections can be further reconstructed to obtain 3-D images, where the structures of interest are localized and quantified. Confocal microscopy can be combined with pressure myography to obtain, simultaneously, information on vascular function and 3-D structure at near-to-physiological conditions. There are a vast number of fluorescent compounds useful for imaging vessel structure and function. Nuclear dyes allow the identification of the different types of vascular cells and the quantification of their number, shape and orientation. The speed of confocal image acquisition and processing makes it possible to scan entire intact arteries stained with fluorescent kits or antibodies to locate infrequent events such as cell apoptosis, proliferation or migration. Confocal microscopy is not only useful for imaging vascular wall structure, but also to visualize and quantify, by the intensity of fluorescence, the generation of vascular cell factors such as nitric oxide or superoxide anion. In conclusion, confocal microscopy and image analysis software provide insight into vascular wall structure and function and the active process of vascular remodelling in physiological and pathological situations.

Alterations in rabbit aorta induced by types I and II pyrethroids.

Since pyrethroids are involved in reactive oxygen species production and no investigations have yet been performed on smooth muscle cell integrity, we studied the influence of permethrin- and cypermethrin-treatment on rabbit aorta using confocal laser scanning fluorescence microscopy, which allows cell viability to be assessed within the wall of living rabbit aorta. The data obtained show that the pyrethroid-treatment (10-100µM) impairs the smooth muscle cell viability. A double-labeling protocol allowed us to distinguish cytotoxic effects of permethrin- and cypermethrin-treatment in aortic rings. In conclusion, permethrin seems to induce more oxidative stress on the aorta wall than that cypermethrin does.

Postnatal alterations in elastic fiber organization precede resistance artery narrowing in SHR.

Resistance artery narrowing and stiffening are key elements in the pathogenesis of essential hypertension, but their origin is not completely understood. In mesenteric resistance arteries (MRA) from spontaneously hypertensive rats (SHR), we have shown that inward remodeling is associated with abnormal elastic fiber organization, leading to smaller fenestrae in the internal elastic lamina. Our current aim is to determine whether this alteration is an early event that precedes vessel narrowing, or if elastic fiber reorganization in SHR arteries occurs because of the remodeling process itself. Using MRA from 10-day-old, 30-day-old, and 6-mo-old SHR and normotensive Wistar Kyoto rats, we investigated the time course of the development of structural and mechanical alterations (pressure myography), elastic fiber organization (confocal microscopy), and amount of elastin (radioimmunoassay for desmosine) and collagen (picrosirius red). SHR MRA had an impairment of fenestrae enlargement during the first month of life. In 30-day-old SHR, smaller fenestrae and more packed elastic fibers in the internal elastic lamina were paralleled by increased wall stiffness. Collagen and elastin levels were unaltered at this age. MRA from 6-mo-old SHR also had smaller fenestrae and a denser network of adventitial elastic fibers, accompanied by increased collagen content and vessel narrowing. At this age, elastase digestion was less effective in SHR MRA, suggesting a lower susceptibility of elastic fibers to enzymatic degradation. These data suggest that abnormal elastic fiber deposition in SHR increases resistance artery stiffness at an early age, which might participate in vessel narrowing later in life.

Evidence for involvement of alpha1D-adrenoceptors in contraction of femoral resistance arteries using knockout mice.

The role of alpha(1D)-adrenoceptors in vasoconstrictor responses to noradrenaline in mouse femoral resistance arteries was investigated using wire myography in alpha(1D)-adrenoceptor knockout (alpha(1D)-KO) and wild-type (WT) mice of the same genetic background.alpha(1D)-KO mice were 2.5-fold less sensitive than WTs to exogenous noradrenaline and BMY 7378 was significantly less potent against noradrenaline in alpha(1D)-KO mice than in WTs, showing a minor contribution of alpha(1D)-adrenoceptors in response to noradrenaline. Prazosin and 5-methyl-urapidil were equally effective against noradrenaline in alpha(1D)-KO and WT mice. Chloroethylclonidine produced a significantly greater attenuation of the response to noradrenaline in alpha(1D)-KO mice than in WTs. Responses to electrical field stimulation (EFS), at 2-20 Hz for 10 s and 0.09 ms pulse width were significantly smaller overall in alpha(1D)-KOs than in WTs although no significant differences were seen at the different frequencies.BMY 7378 produced significantly greater inhibition of responses at 2 and 5 Hz than at higher frequencies in WTs. In alpha(1D)-KOs, this greater sensitivity to BMY 7378 at lower frequencies was not apparent, confirming that the effect of BMY 7378 was due to blockade of alpha(1D)-adrenoceptors. Prazosin and 5-methyl-urapidil had similar inhibitory effects on responses to EFS in alpha(1D)-KO and WT mice. Chloroethylclonidine inhibited responses to EFS to a significantly greater extent in alpha(1D)-KO mice. The present study with alpha(1D)-KO mice shows that alpha(1D)-adrenoceptors contribute to vasoconstrictor responses to exogenous and neurally released noradrenaline in femoral resistance arteries.

Direct demonstration of beta1- and evidence against beta2- and beta3-adrenoceptors, in smooth muscle cells of rat small mesenteric arteries.

1 Recent evidence supports additional subtypes of vasodilator beta-adrenoceptor (beta-AR) besides the 'classical' beta(2). The aim of this study was to investigate the distribution of beta-ARs in the wall of rat mesenteric resistance artery (MRA), to establish the relative roles of beta-ARs in smooth muscle and other cell types in mediating vasodilatation and to analyse this in relation to the functional pharmacology. 2 We first examined the vasodilator beta-AR subtype using 'subtype-selective' agonists against the, commonly employed, phenylephrine-induced tone. Concentration-related relaxation was produced by isoprenaline (pEC(50): 7.70+/-0.1) (beta(1) and beta(2)). Salbutamol (beta(2)), BRL 37344 (beta(3)) and CGP 12177 (atypical beta) caused relaxation but were 144, 100 and 263 times less potent than isoprenaline; the 'beta(3)-adrenoceptor agonist' CL 316243 was ineffective. 3 In arteries precontracted with 5-HT or U 46619, isoprenaline produced concentration-related relaxation but salbutamol, BRL 37344, CGP 12177 and CL 316243 did not. SR 59230A, CGP 12177 and BRL 37344 caused a parallel rightward shift in the concentration-response curve to phenylephrine indicating competitive alpha(1)-AR antagonism, explaining the false-positive 'vasodilator' action against phenylephrine-induced tone. Endothelial denudation but not L-NAME slightly attenuated isoprenaline-mediated vasodilatation in phenylephrine and U 46619 precontracted MRA. 4 The beta-AR fluorescent ligand BODIPY TMR-CGP 12177 behaved as an irreversible beta(1)-AR antagonist in MRA and bound to the surface and inside vascular smooth muscle cells in intact vascular wall. Beta-ARs in smooth muscle cells were observed in a perinuclear location, consistent with the location of Golgi and endoplasmic reticulum. 5 Binding of BODIPY TMR-CGP 12177 was inhibited by BAAM (1 microM) in all three vascular tunics, confirming the presence of beta-ARs in adventitia, media and intima. Binding in adventitia was observed in both neuronal and non-neuronal cell types. Lack of co-localisation with a fluorescent ligand for alpha-ARs confirms the selectivity of BODIPY TMR-CGP 12177 for beta-ARs over alpha-ARs. 6 Our results support the presence of functional vasodilator beta(1)-ARs and show that they are mainly located in smooth muscle cells. Furthermore, we have demonstrated, for the first time, the usefulness of BODIPY TMR-CGP 12177 for identifying beta-AR distribution in the 'living' vascular wall.

3-d image analysis of fluorescent drug binding.

Fluorescent ligands provide the means of studying receptors in whole tissues using confocal laser scanning microscopy and have advantages over antibody- or non-fluorescence-based method. Confocal microscopy provides large volumes of images to be measured. Histogram analysis of 3-D image volumes is proposed as a method of graphically displaying large amounts of volumetric image data to be quickly analyzed and compared. The fluorescent ligand BODIFY FL-prazosin (QAPB) was used in mouse aorta. Histogram analysis reports the amount of ligand-receptor binding under different conditions and the technique is sensitive enough to detect changes in receptor availability after antagonist incubation or generic manipulations. QAPB binding was concentration dependent, causing concentration-related rightward shifts in histogram. In the presence of 10 microM phenoxybenzamine (blocking agent), the QAPB (50 nM) histogram overlaps the autofluorescence curve. The histogram obtained for the 1D knockout aorta lay to the left of that control and 1B knockout aorta, indicating a reduction in 1D receptors. We have shown, for the first time, that it is possible to graphically display binding of a fluorescent drug to a biological tissue. Although our application is specific to adrenergic receptors, the general method could be applied to any volumetric, fluorescence-image-based assay.

New aspects of vascular remodelling: the involvement of all vascular cell types.

Conventionally, the architecture of arteries is based around the close-packed smooth muscle cells and extracellular matrix. However, the adventitia and endothelium are now viewed as key players in vascular growth and repair. A new dynamic picture has emerged of blood vessels in a constant state of self-maintenance. Recent work raises fundamental questions about the cellular heterogeneity of arteries and the time course and triggering of normal and pathological remodelling. A common denominator emerging in hypertensive remodelling is an early increase in adventitial cell density suggesting that adventitial cells drive remodelling and may initiate subsequent changes such as re-arrangement of smooth muscle cells and extracellular matrix. The organization of vascular smooth muscle cells follows regular arrangements that can be modelled mathematically. In hypertension, new patterns can be quantified in these terms and give insights to how structure affects function. As with smooth muscle, little is known about the organization of the vascular endothelium, or its role in vascular remodelling. Current observations suggest that there may be a close relationship between the helical organization of smooth muscle cells and the underlying pattern of endothelial cells. The function of myoendothelial connections is a topic of great current interest and may relate to the structure of the internal elastic lamina through which the connections must pass. In hypertensive remodelling this must present an organizational challenge. The objective of this paper is to show how the functions of blood vessels depend on their architecture and a continuous interaction of different cell types and extracellular proteins.

Visualisation of morphological changes in living intact human microvessels using confocal microscopy.

Conventional techniques to visualise microvascular structure often involve fixed tissue slices that provide two-dimensional images. A previous study using diffusive labelling of fresh, dissected tissue samples with fluorescently-tagged endothelial markers demonstrated the possibility of examining the three-dimensional architecture of the microvasculature using confocal microscopy. The present study extends the use of this quick and simple method of diffusive labelling to examine the possibility of repeatedly measuring changes in the morphology of intact microvessel in response to pharmacological stimuli. Initially, three-dimensional surface-rendered images of the same microvessel derived from the placenta and subcutaneous biopsies demonstrated morphological and topological changes in response to temperature and increasing potassium changes of physiological salt solutions, respectively. Furthermore, a dose-response study was performed with subcutaneous microvessels using the potent vasodilator, adrenomedullin. Analysis of a series of z-stack, superimposed to form a single maximum brightness image, demonstrated an inverse dose-response relationship, with responses to increasing adrenomedullin concentrations (10(-12) to 10(-8) M). In vessels that had constricted in response to noradrenaline (diameters: 22.4 to 58.0 microm), physiological concentrations of 10(-12) M increased vessel diameter by 108% above baseline conditions. Control treatment using physiological salt solution did not demonstrate any changes. The technique described suggest that diffusive labelling with vascular endothelial markers such as ulex europeaus agglutinin I in live tissue samples may be used in conjunction with confocal microscopy to demonstrate heterogeneous morphological and topological changes in intact segments of the microvasculature.

Influence of elastin on rat small artery mechanical properties.

We have previously developed a method for estimating elastin content and organization in resistance arteries, where it is a minor component. The aim of the present study was to validate the method against a quantitative assay and to determine the relative importance of elastin content and organization for intrinsic elasticity of small arteries. Mesenteric third order branches (from 10-day-old, 1- and 6-month-old rats) and middle cerebral arteries (from 6-month-old rats) were pressurized. beta-Values were calculated from stress-strain relationships and used as indicators of intrinsic stiffness. The same pressure-fixed arteries were used to estimate elastin content and organization in the internal elastic lamina with confocal microscopy. Collagen and elastin contents were determined by Picrosirius Red staining and radioimmunoassay for desmosine, respectively. Confocal and desmosine assays gave similar results: no difference in elastin content of mesenteric vessels from 1- and 6-month-old rats, and a significant reduction in cerebral compared to mesenteric arteries. For all parameters (elastin and collagen content, fenestrae area and internal elastic lamina thickness) the best correlation was found between beta-values and fenestrae size. These data suggest that in small arteries: (1) confocal microscopy can be used as a method for the simultaneous study of changes in elastin content and organization; and (2) elastin organization might be a key determinant of intrinsic elastic properties.