Evaluation of nerves in Mohs micrographic surgery: histologic mimickers of perineural invasion and nervous tissue on frozen section.

BACKGROUND: Perineural invasion (PNI) is an important histologic finding and may be a negative prognostic factor for squamous cell carcinoma (SCC). It may be associated with more-aggressive tumor behavior. Mohs surgeons encounter microscopic PNI regularly and must be able to diagnose it accurately to guide care decisions. OBJECTIVE: To describe benign histologic mimickers of PNI and neural structures in SCC commonly encountered on frozen, hematoxylin and eosin-stained sections and to review how to differentiate them from PNI. METHODS AND MATERIALS: Review of the literature regarding histologic mimickers of PNI and additional contributions to frozen section PNI and nerve tissue mimickers. RESULTS: We describe benign findings, including arrector pili muscles, eccrine muscles, vessels, granulomatous inflammation, and eddies of SCC, that may each be mistaken for nerves or PNI. We discuss the ways in which they may be distinguished on frozen sections and review other commonly encountered entities that resemble PNI. CONCLUSION: Perineural inflammation and peritumoral fibrosis are common mimickers of PNI on frozen section, although other mimickers exist on permanent sections. Normal structures may appear "neural" by way of frozen tissue orientation, processing, or inflammation and thus must be differentiated from nerve tissue and PNI during Mohs surgery.

Comparison of vascular smooth muscle cells in canine great vessels.

BACKGROUND: Elucidating the histological characteristics of normal vascular smooth muscle cells (VSMCs) is important for understanding mechanisms of development, disease etiology and the remodeling and/or regeneration process of the vessel. However, knowledge regarding VSMCs is focused primarily on the artery. Although the characteristics of each great vessel are documented, few studies have examined VSMCs in parallel within each great vessel. The present study focused on comparing characteristics of canine VSMCs within the aorta (Ao), branch pulmonary artery (bPA), main pulmonary artery (mPA) and inferior vena cava (IVC), simultaneously. RESULTS: Western blot and immunohistochemistry were used to determine VSMC protein content for alpha smooth muscle actin (ASMA), calponin, myosin heavy chain (MHC) and its isozyme SM2, and non-muscle myosin heavy chain B (SMemb). Thickness and ratio of the VSMC layer were also measured. Expression levels of ASMA, calponin and SM2 significantly differed between vessels, except between mPA and either bPA, Ao and IVC vessels. Expression levels of MHC were significantly different in all vessels, whilst expression of SMemb was significantly different in the Ao compared with either bPA and mPA vessels. All vessels were significantly different with respect to total wall and VSMC layer thickness. The ratio between VSMC layer and total wall thickness was significantly different for each vessel, except between bPA and mPA vessels. Histological analysis of the IVC revealed that the VSMC layer does not line evenly and continuously through the long axis or transverse sections. With respect to the pulmonary artery, calponin was expressed to a greater extent in the mPA compared with the bPA (P < 0.01*). In contrast, MHC and SM2 were expressed to a greater extent in the bPA compared with the mPA (P < 0.01*). Differences in VSMC distribution indicate structural differences in the proximal and distal pulmonary artery bifurcation. CONCLUSION: Our results show that the VSMC expression pattern in each great vessel is unique and suggestive of the developmental differences between great vessels. We believe this study provides basic data for the pathology, etiology and regenerative capability of the vessels.

Myoendothelial contacts, gap junctions, and microdomains: anatomical links to function?

In several species and in many vascular beds, ultrastructural studies describe close contact sites between the endothelium and smooth muscle of <∼20nm. Such sites are thought to facilitate the local action of signaling molecules and/or the passage of current, as metabolic and electrical coupling conduits between the arterial endothelium and smooth muscle. These sites have the potential for bidirectional communication between the endothelium and smooth muscle, as a key pathway for coordinating vascular function. The aim of this brief review is to summarize the literature on the ultrastructural anatomy and distribution of key components of MECC sites in arteries. In addition to their traditional role of facilitating electrical coupling between the two cell layers, data on the role of MECC sites in arteries, as signaling microdomains involving a spatial localization of channels, receptors and calcium stores are highlighted. Diversity in the density and specific characteristics of MECC sites as signaling microdomains suggests considerable potential for functional diversity within and between arteries in health and disease.

Prospective self-gating for swallowing motion: a feasibility study in carotid artery wall MRI using three-dimensional variable-flip-angle turbo spin-echo.

Three-dimensional black-blood MRI is a promising noninvasive imaging technique for the assessment of atherosclerotic carotid artery disease. However, this technique is inherently susceptible to motion. In particular, swallowing can result in considerable wall motion at the carotid bifurcations, which may induce drastic image degradation or substantial overestimation of wall thickness. Self-gating techniques have previously been shown to be capable of resolving and compensating for cardiac or respiratory motion during MRI. This work presents a self-gating-based prospective motion gating scheme that is combined with a three-dimensional variable-flip-angle turbo spin-echo sequence (SPACE) for detecting swallowing motion. Self-gating signal readouts along the superior-inferior direction during each repetition time period are used to derive the projection profiles of the imaging volume. Based on cross-correlation analysis between the projection profiles and the corresponding reference profiles, swallowing motion can be detected and the motion-contaminated data will subsequently be discarded and reacquired in the next repetition time. The self-gated SPACE sequence was validated on eight healthy volunteers and two patients and, when compared with the conventional SPACE sequence, proved to be more resistant to swallowing motion and significantly improved image quality as well as the sharpness of carotid artery wall boundaries.

The vertebral venous plexuses: the internal veins are muscular and external veins have valves.

The internal and external vertebral venous plexuses (VVP) extend the length of the vertebral column. Authoritative sources state that these veins are devoid of valves, permitting bidirectional blood flow and facilitating the hematogenous spread of malignant tumors that have venous connections with these plexuses. The aim of this investigation was to identify morphologic features that might influence blood flow in the VVP. The VVP of 12 adult cadavers (seven female, mean age 79.5 years) were examined by macro- and micro-dissection and representative veins removed for histology and immunohistochemistry (smooth muscle antibody staining). A total of 26, mostly bicuspid, valves were identified in 19 of 56 veins (34%) from the external VVP, all orientated to promote blood flow towards the internal VVP. The internal VVP was characterized by four main longitudinal channels with transverse interconnections; the maximum caliber of the longitudinal anterior internal VVP veins was significantly greater than their posterior counterparts (P < 0.001). The luminal architecture of the internal VVP veins was striking, consisting of numerous bridging trabeculae (cords, thin membranes and thick bridges) predominantly within the longitudinal venous channels. Trabeculae were composed of collagen and smooth muscle and also contained numerous small arteries and nerve fibers. A similar internal venous trabecular meshwork is known to exist within the dural venous sinuses of the skull. It may serve to prevent venous overdistension or collapse, to regulate the direction and velocity of venous blood flow, or is possibly involved in thermoregulation or other homeostatic processes.

Reduced vessel elasticity alters cardiovascular structure and function in newborn mice.

Elastic blood vessels provide capacitance and pulse-wave dampening, which are critically important in a pulsatile circulatory system. By studying newborn mice with reduced (Eln(+/)(-)) or no (Eln(-)(/)(-)) elastin, we determined the effects of altered vessel elasticity on cardiovascular development and function. Eln(-)(/)(-) mice die within 72 hours of birth but are viable throughout fetal development when dramatic cardiovascular structural and hemodynamic changes occur. Thus, newborn Eln(-)(/)(-) mice provide unique insight into how a closed circulatory system develops when the arteries cannot provide the elastic recoil required for normal heart function. Compared with wild type, the Eln(-)(/)(-) aorta has a smaller unloaded diameter and thicker wall because of smooth muscle cell overproliferation and has greatly reduced compliance. Arteries in Eln(-)(/)(-) mice are also tortuous with stenoses and dilations. Left ventricular pressure is 2-fold higher than wild type, and heart function is impaired. Newborn Eln(+/)(-) mice, in contrast, have normal heart function despite left ventricular pressures 25% higher than wild type. The major vessels have smaller unloaded diameters and longer lengths. The Eln(+/)(-) aorta has additional smooth muscle cell layers that appear in the adventitia at or just before birth. These results show that the major adaptive changes in cardiovascular hemodynamics and in vessel wall structure seen in the adult Eln(+/)(-) mouse are defined in late fetal development. Together, these results show that reduced elastin in mice leads to adaptive remodeling, whereas the complete lack of elastin leads to pathological remodeling and death.

Imaging of vascular smooth muscle cells with soft X-ray spectromicroscopy.

Using X-ray microscopy and spectromicroscopy, vascular smooth muscle cells (VSMCs) were imaged, prepared without using additional embedding material or staining, but by applying simple, noncryo fixation techniques. The cells were imaged with a compact source transmission X-ray microscope and a scanning transmission X-ray microscope (STXM). With the STXM, spectromicroscopy was performed at the C K-edge and the Ca L(III,II)-edges. VSMCs were chosen because of their high amount of actin stress fibers, so that the actin cytoskeleton should be visible. Other parts of the cell, such as the nucleus and organelles, were also identified from the micrographs. Both in the spectra and the images, the effects of the different preparation procedures were observable. Furthermore, Ca hotspots were detected and their density is determined.

Structure and functional significance of the transverse vesical fold.

The plica vesicalis transversa or transverse vesical fold (TVF) is a peritoneal fold extending from the lateral side of the bladder to the side of the lesser pelvis near the deep inguinal ring. It is an important landmark in laparoscopic surgery of the pelvis but is variably observed in the embalmed cadaver. We investigated the gross anatomy of this structure in the cadaver and confirmed that its medial portion corresponds to the location of the superior vesical artery(ies), thus supporting the idea that the TVF is "mesovesical." However, no large vessels were observed grossly in the lateral portion of the TVF. The hypothesis that the lateral TVF has a suspensory function was tested histologically by comparison with the suspensory ligament of the duodenum and the phrenicocolic ligament, both of which have smooth muscle contributing to their inferred suspensory function. Microscopic examination of prepared samples from 20 cadavers shows that the TVF evinces no smooth muscle in either its lateral or medial segments. The TVF is demonstrated to be a mesentery-like reflection of peritoneum raised by branches of the superior vesical artery which provides no demonstrable structural support for the bladder. Implications of these findings include avoidance of sectioning of medial TVF during laparoscopic surgery because of its vascular nature, and inadvisability of utilizing any portion of theTVF for an anchor in reconstruction of the anterior pelvic floor within the paravesical fossae.

Epicardial course of the musculature related to the great cardiac vein: Anatomical considerations and clinical implications for mitral isthmus block after vein of Marshall ethanol infusion.

BACKGROUND: Mitral isthmus gaps have been ascribed to an epicardial musculature anatomically related to the great cardiac vein (GCV) and the vein of Marshall (VOM). Their lumen offers an access for radiofrequency application or ethanol infusion, respectively. OBJECTIVE: The purpose of this study was to evaluate the frequency of mitral isthmus gaps accessible via the GCV lumen, to assess their location around the GCV circumference, and to propose an efficient ablation strategy when present. METHODS: One hundred consecutive patients underwent VOM ethanol infusion (step 1) and endocardial linear ablation from the mitral annulus to the left inferior pulmonary vein (step 2). In cases of mitral isthmus gap, endovascular ablation of the GCV anchored wall facing the left atrium was systematically performed (step 3), while the opposite GCV free wall was targeted in case of block failure only (step 4). RESULTS: After VOM ethanol infusion and endocardial ablation, mitral isthmus block occurred in 51 patients (51%). Pacing maneuvers and activation sequences demonstrated an epicardial gap via the VOM in 2 patients (2%) and via the GCV in 47 patients (47%). In the latter case, block was achieved at the GCV anchored wall in 42 patients (89%) and the GCV free wall in 5 patients (11%). Global success rate of mitral isthmus block was 98%. No tamponade occurred. CONCLUSION: With the advent of VOM ethanol infusion, residual mitral isthmus gaps are mostly eliminated within the first centimeter of the GCV. Thorough mapping of the entire circumference of the GCV wall can help identify these epicardial gaps.

Validation of canine uterine and testicular arteries for the functional characterisation of receptor-mediated contraction as a replacement for laboratory animal tissues in teaching.

Teaching practicals for receptor physiology/pharmacology in medical and veterinary schools have involved the use of in vitro experiments using tissues from laboratory animals, which have been killed for isolated vascular strip or ring preparations. However, the use of scavenged tissues has been advocated to reduce animal use. Utilising discarded tissues from routine surgical procedures, such as canine neutering, has not previously been investigated. Canine testicular and uterine tissues (discarded tissues) were obtained from routine neutering procedures performed by the veterinary team at a local animal neutering clinic for stray dogs. Rings of uterine and testicular artery were dissected and mounted on a Mulvany-Halpern wire myograph in order to characterize the adrenergic and serotonergic receptors mediating vasoconstriction. Cumulative contractile concentration-response curves were constructed for the alpha adrenoceptor agonists epinephrine (α1 and α2 receptors), phenylephrine (α1 selective) and UK14304 (α2 selective). Pre-treatment with the α1-selective antagonist, prazosin, was also investigated. The response to serotonin (5-HT) receptor agonists were also investigated, including 5-HT (acting at both 5-HT1 and 5-HT2 receptors), 5-carboxamidotryptamine (5-CT; 5-HT1 selective) and α-methyl 5-HT (5-HT2 selective). A contractile response was observed in both canine uterine and testicular arteries to epinephrine and phenylephrine, and prazosin caused a dose-dependent parallel rightward shift in the phenylephrine dose-response curve (pA2 values of 7.97 and 8.39, respectively). UK14304 caused a contractile response in canine testicular arteries but very little appreciable contractile response in uterine arteries. The maximum responses produced by the uterine arteries to 5-HT was significantly lower than those of the testicular arteries. In the testicular artery, the 5-HT2 receptor selective agonist, α-methyl 5-HT, produced a similar contractile response to 5-HT but the administration of 5-CT failed to produce a response in either the testicular or uterine artery segments. These results validate the use of discarded tissue from routine canine neutering procedures as a useful source of vascular tissue for pharmacological teaching, for characterizing alpha and 5-HT receptor contractile responses.

Arterial internal elastic lamina holes: relationship to function?

Internal elastic lamina (IEL) hole (fenestration) characteristics and myoendothelial gap junction (MEGJ) density were examined in selected resistance and conduit arteries of normal and diseased rat and mouse models, using conventional, ultrastructural and confocal microscopy methods. Selected vessels were those commonly used in functional studies: thoracic aorta, proximal and distal mesenteric, caudal, saphenous, middle-cerebral and caudal cerebellar artery. Rat and mouse strains and treatment groups examined were Dahl, Sprague Dawley, Wistar Kyoto, Wistar, spontaneously hypertensive (SHR), deoxycorticosterone (DOC) treated rat; and apolipoprotein E knockout, C57/BL6 and BALB/c mice. Vessel size (as IEL circumference), IEL hole and MEGJ density were quantified. In mesenteric arteries, the width of IEL holes and the percent of IEL occupied by holes were also determined. IEL hole density varied significantly within and between mesenteric artery beds, even among normotensive rat strains. Among the hypertensive rats (SHR and DOC), hole density in some vessels was higher in the normotensives than in the hypertensives within each strain, whereas in Dahl rats, hole density was similar between hypertensives and normotensives. Hole density was not correlated with the formation of intimal lesions in superior mesenteric artery. There was no positive general correlation between IEL hole and MEGJ density in resistance and conduit vessels. However, there was a positive correlation between the size of some resistance arteries and MEGJ density, although such a relationship did not hold for conduit vessels or during development, and there was no such relationship between vessel size and IEL hole density. Whilst IEL holes are obviously required for MEGJ communication, their presence is not an indication of contact-mediated communication, but rather may be related to the presence of sites for the low resistance passage of diffusion-mediated release of vasoactive endothelial and smooth muscle substances.

A blood vessel model constructed from collagen and cultured vascular cells.

A model of a blood vessel was constructed in vitro. Its multilayered structure resembled that of an artery and it withstood physiological pressures. Electron microscopy showed that the endothelial cells lining the lumen and the smooth muscle cells in the wall were healthy and well differentiated. The lining of endothelial cells functioned physically, as a permeability barrier, and biosynthetically, producing von Willebrand's factor and prostacyclin. The strength of the model depended on its multiple layers of collagen integrated with a Dacron mesh.

Adaptive response of pulmonary arterial smooth muscle to length change.

Hypervasoconstriction is associated with pulmonary hypertension and dysfunction of the pulmonary arterial smooth muscle (PASM) is implicated. However, relatively little is known about the mechanical properties of PASM. Recent advances in our understanding of plastic adaptation in smooth muscle may shed light on the disease mechanism. In this study, we determined whether PASM is capable of adapting to length changes (especially shortening) and regain its contractile force. We examined the time course of length adaptation in PASM in response to step changes in length and to length oscillations mimicking the periodic stretches due to pulsatile arterial pressure. Rings from sheep pulmonary artery were mounted on myograph and stimulated using electrical field stimulation (12-16 s, 20 V, 60 Hz). The length-force relationship was determined at L(ref) to 0.6 L(ref), where L(ref) was a reference length close to the in situ length of PASM. The response to length oscillations was determined at L(ref), after the muscle was subjected to length oscillation of various amplitudes for 200 s at 1.5 Hz. Release (or stretch) of resting PASM from L(ref) to 0.6 (and vice versa) was followed by a significant force recovery (73 and 63%, respectively), characteristic of length adaptation. All recoveries of force followed a monoexponential time course. Length oscillations with amplitudes ranging from 5 to 20% L(ref) caused no significant change in force generation in subsequent contractions. It is concluded that, like many smooth muscles, PASM possesses substantial capability to adapt to changes in length. Under pathological conditions, this could contribute to hypervasoconstriction in pulmonary hypertension.

On the in-series and in-parallel contribution of elastin assessed by a structure-based biomechanical model of the arterial wall.

Earlier experimental work on decellularized arteries revealed the existence of significant residual stresses within the arterial wall, which are released upon chemical removal of vascular smooth muscle in normal arteries causing substantial radial expansion. Hence, the often-used Hill's model describing active and passive stresses within the wall does not hold true, because the existence of prestresses precludes the fundamental assumption of zero active stress when the vascular smooth muscle is inactive. We have, therefore, developed a new mathematical model based on a modified Hill's model, where the total wall elastin is partitioned in two parts: one in-parallel to vascular smooth muscle and collagen and one connected in-series with vascular smooth muscle. Based on experimental evidences, compressive prestresses were assumed to exist on the parallel elastic component and tensile prestresses on the series elastic component. Further, we assumed that the elastic constants of elastin and collagen and the statistical description of collagen engagement are not affected by decellularization. Excellent fits of the pressure-diameter curves of normal and decellularized arteries were obtained. The model predicts that the majority of elastin is in-series with the vascular smooth muscle (74 +/-8%) and thus only about one-fourth of elastin acts in parallel to the vascular smooth muscle. We conclude that correct biomechanical modeling of the arterial wall requires the knowledge of the zero stress state of both the series and parallel elastic components.

Quantitative analysis of the microstructure of human umbilical vein for assessing feasibility as vessel substitute.

We explored the feasibility of human umbilical vein (HUV) as a small-caliber vessel substitute. HUVs of 50 fetuses were collected on spontaneous miscarriage or labor with the pregnant women's permission. Gestational age ranged 24-42 weeks, and parturients were 20-30 years old. Each sample was sliced into 5 mum frozen transverse sections and stained with hematoxylin-eosin (HE), Weigert, aniline blue, and orange yellow G. The geometric morphological indexes and microstructural component were measured by a computer image analysis system. The media thickness was 0.186, 0.203, 0.237, 0.264, and 0.268 mm at 24-27, 28-32, 33-36, 37-40, and 41-42 weeks, respectively (F = 133.35, p < 0.01); diameters were 1.861, 1.962, 2.303, 2.464, and 2.465 mm (F = 37.35, p < 0.01), respectively. The media thickness and diameter of HUVs increased with gestational age. The elastin content of media increased at 24-40 weeks, but the collagen content and collagen/elastin (C/E) ratio decreased. Elastin content in the proximal segment was higher than in the distal segment [10.16, 6.36 Aa%, (Aa% is the unit of relative content, ie, the ratio of absolute areas to the total tested area of smooth muscle, collagen and elastin in the vascular wall) F = 5.77-12.3, p < 0.05], with the collagen to elastin (C/E) ratio increasing from the proximal to the distal segment (F = 7.63-13.4, p < 0.05). Our results suggest that the microstructural component of HUVs (2.0-3.0 mm caliber) at 37-40 weeks of gestation was similar to that of the small-caliber arteries and had moderate amounts of collagen and elastin and good elasticity, i.e., a good C/E ratio; therefore, HUV may be a substitute for small-caliber arteries (e.g., brachial, ulnar, radial, right coronary, anterior tibial, and posterior tibial). HUV is one of several graft materials that may be used when autogenous saphenous vein is absent or inadequate.

Pressure equilibration in the penguin middle ear.

CONCLUSIONS: King penguins have a venous structure in the form of a corpus cavernosum (CC) in their middle ear (ME) submucosa. The CC may be viewed as a special organelle that can change ME volume for pressure equilibration during deep-sea diving it is a pressure regulating organelle (PRO). A similar CC and muscles also surround the external ear (EE) and may constrict it, isolating the tympanic membrane from the outside. A CC was previously found also in the ME of marine diving mammals and can be expected to exist in other deep diving animals, such as marine turtles. OBJECTIVES: Marine animals require equalization of middle ear (ME) pressure when diving hundreds or thousands of meters to catch prey. We investigated what mechanism enables king penguins to protect their ME when they dive to great depths. MATERIALS AND METHODS: Biopsies and serial sections of the ME and the EE of the deep diving king penguin (Aptenodytes patagonicus) were examined microscopically. RESULTS: It was demonstrated that the penguin ME has an extensive network of small and large submucosal venous sinuses. This venous formation, a corpus cavernosum, can expand and potentially 'flood' the ME almost completely on diving, thus elevating ME pressure and reducing the ME space. The EE has a similar protective mechanism.

Junction between the great cerebral vein and the straight sinus: an anatomical, immunohistochemical, and ultrastructural study on 25 human brain cadaveric dissections.

The cerebral venous system is poorly understood, and best appreciated under macroscopic anatomical considerations. We present an anatomical and immunohistochemical studies to better define the morphological characteristics of the junction between the great cerebral vein and the straight sinus. Twenty-five cadaveric specimens from the anatomy laboratory of the University Victor Segalen of Bordeaux were studied. The observation of the venous junctions with the straight sinus was performed under an operating microscope. The smooth muscular actin immunohistochemical staining was performed for 18 veno-sinosal junctions. Five venous junctions were observed using an electron microscope. We observed 3 different anatomic aspects: type 1 was a junction with a small elevation in its floor and a posterior thickening (14 cases); type 2 was a junction with an outgrowth on the floor like a cornice (7 cases); and type 3 was a junction presenting a nodule. Microscopic study of type 1 and 2 junctions showed a positive coloration to orceine attesting the presence of elastic fibers. Immunohistochemistry revealed the presence of smooth muscular actin and S 100 protein attesting the presence of smooth muscular fibers and nervous fibers. We observed in the ultrastructural study, a morphological progression of the endothelium. The venous orifice of the great cerebral vein into the straight sinus could be anatomically assimilated as a true "sphincter." Its function in the regulation of the cerebral blood flow needs further exploration.

The Roach muscle bundle and umbilical cord coiling.

OBJECTIVE: To determine if presence of the Roach muscle, a small muscle bundle lying just beside the umbilical artery, contributes to umbilical cord coiling. METHODS: 251 umbilical cords were examined. The umbilical coiling index (UCI) was calculated as the number of coils divided by the cord length in cm. Cords were classified as hypocoiled (UCIp90). On microscopic examination of a cross section of the cord, absence or presence of a Roach muscle was determined. The t-test for independent samples and logistic regression were used for statistical analysis. RESULTS: A Roach muscle was observed in 101 cords. The mean UCI was higher in cords with the muscle bundle (0.23 coils/cm) than in cords without a muscle (0.18 coils/cm). Difference in mean: 0.05 coils/cm (95% C.I. 0.01-0.09). OR for hypercoiling in presence of the muscle was 2.98 (95% C.I. 1.57-5.64). OR for hypocoiling in the presence of the muscle was 1.49 (95% C.I. 0.79-2.81). CONCLUSIONS: Our results suggest that presence of a Roach muscle bundle contributes to umbilical cord coiling. Given the divergence in umbilical cord coiling within subgroups with or without this muscle, other factors must play a more dominant role.

Experimental system for ex vivo measurement of murine aortic stiffness.

While vascular stiffness is universally studied using pulse wave velocity, this method overestimates the stiffness of small calibre blood vessels. We have developed and rigorously validated an ex vivo system for measuring stiffness of the mouse aorta. The system consists of a temperature-controlled tissue bath, a pressurization loop and a helium-neon laser micrometer. We harvested thoracic aortas from 8 (n = 56), 11 (n = 6) and 14 (n = 6) week male C57BL/6J mice, mounted them within a tissue chamber and applied an intraluminal pressure waveform while measuring mid vessel outer diameter. Vessel stiffness (E(p), mmHg) was calculated from the pressure-diameter response. Vessels were then stained for endothelial cells, smooth muscle cells, elastin fibres and collagen. The data indicate highly reproducible stiffness measurements in 8 week mice (E(p) = 602.4 +/- 160.2; p = 0.934), age-related stiffening between 11 and 14 week mice (11 week E(p) = 646.9 +/- 62.4, 14 week E(p) = 795.4 +/- 87.5, p = 0.008), and a morphologically intact vessel wall. These results represent the first ex vivo measurements of murine aortic stiffness and illustrate that our methods are feasible and reliable. Since we demonstrate that the system is sensitive to age-related stiffening and does not damage the vessel, this approach is useful for investigating the pathophysiology of vascular disease from biomechanical and histological perspectives.

How many Layers has the Adventitia? - Structure of the Arterial Tunica Externa Revisited.

Tunica adventitia or tunica externa is the outer layer of the blood vessel wall. It consists of connective tissue with vasa and nervi vasorum and plays a key role in vascular health. The aim of our study was to compare the wall layers beyond tunica media in arteries of different type and location. The following arteries of pig, dog and cat were processed histologically and analysed by light microscopy: aorta ascendens, arcus aortae, aorta thoracica, aorta abdominalis, arteria (a.) femoralis, a. tibialis cranialis, a. carotis communis, a. lingualis, a. basilaris, a. cerebralis media, a. testicularis and aa. jejunales. We found two layers of connective tissue outside the media: (1) a compact layer with many elastic fibres in muscular and few in elastic arteries and (2) an outer layer of loose connective tissue. The compact layer was missing in aorta ascendens, arcus aortae and intracranial vessels. Adventitial stripping removed only the loose connective tissue layer. In spite of the still present compact layer, stripped arteries were very flimsy. We suggest using the term 'tunica externa' for the compact connective tissue layer and 'tunica adventitia' for the outermost loose connective tissue layer as in other organs. The presence of the tunica externa differs between species, arteries and arterial side, as well as the removability of tunica adventitia and tunica externa by anatomical dissection.