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Capillary network characteristics are invaluable for diagnostics of muscle diseases. Biopsy material is limited in size and mostly not accessible for intensive research. Therefore, especially in human tissue, studies are performed on autopsy material. To approach the problem whether it is reliable to deduce hypotheses from autopsy material to explain physiological and pathological processes, we studied capillarity in pig soleus muscle 1 and 24 hr after death. Capillaries and muscle fibers were immunofluorescently marked, and images were acquired with a confocal microscope. Characteristics of the capillary network were estimated by image analysis methods using several plugins of the Ellipse program. Twenty-four hours after death, the measured characteristics of the capillary network differ by up to 50% when compared with samples excised 1 hr after death. Muscle fiber diameter, the measured capillary length, and tortuosity were reduced, and capillary network became more anisotropic. The main postmortem change that affects capillaries is evidently geometric deformation of muscle tissue. In conclusion, when comparing results from biopsy samples with those from autopsy samples, the effect of postmortem changes on the measured parameters must be carefully considered.

Vasa vasorum supply both the tunica adventitia and the tunica media of major arteries with nutrients and oxygen. We estimated the density of von Willebrand factor-positive profiles of vasa vasorum visible in transversal histological sections of 123 tissue samples collected from five anatomical positions in the porcine aortae of growing pigs (n=25). The animals ranged in age from 0 to 230 days. The tunica media of the thoracic aorta had a greater vasa vasorum density, with microvessels penetrating deeper towards the lumen than in the abdominal aorta. The density of vasa vasorum gradually decreased with age in both the media and the adventitia. The relative depth into which the vasa vasorum penetrated and where they branched remained constant during the ageing and growth of the media. The ratio of the tunica media and tunica adventitia thicknesses did not change in the single aortic segments during ageing. The media of older animals received fewer but equally distributed vasa vasorum. A greater density of vasa vasorum in the media was correlated with greater media thickness and a greater elastin fraction (data on elastin taken from another study on the same samples). Immunohistochemical quantification revealed deeper penetration of vasa vasorum towards the adluminal layers of the tunica media that were hitherto reported to be avascular. The complete primary morphometric data, in the form of continuous variables, have been made available as a supplement. Mapping of the vasa vasorum profile density and position has promising illustrative potential for studies on atherosclerotic and inflammatory neovascularization, aortic aneurysms, and drug distribution from arterial stents in experimental porcine models.

The effect of ageing on the capillary network in skeletal muscles has produced conflicting results in both, human and animals studies. Some of the inconsistencies are due to non-comparable and biased methods that were applied on thin transversal sections, especially in muscles with complicated morphological structures, such as in human masseter muscle. We present a new immunohistochemical method for staining capillaries and muscle fibres in 100 µm thick sections as well as novel approach to 3D visualization of capillaries and muscle fibres. Applying confocal microscopy and virtual 3D stereological grids, or tracing capillaries in virtual reality, length of capillaries within a muscle volume or length of capillaries adjacent to muscle fibre per fibre length, fibre surface or fibre volume were evaluated in masseter muscle of young and old subjects by an unbiased approach. Our findings show that anatomic capillarity is well maintained in masseter muscle in old subjects; however, vascular remodelling occurs with age, which could be a response to changed muscle function and age-related muscle fibre type transformations.

During pregnancy, oxygen diffuses from maternal to fetal blood through villous trees in the placenta. In this paper, we simulate blood flow and oxygen transfer in feto-placental capillaries by converting three-dimensional representations of villous and capillary surfaces, reconstructed from confocal laser scanning microscopy, to finite-element meshes, and calculating values of vascular flow resistance and total oxygen transfer. The relationship between the total oxygen transfer rate and the pressure drop through the capillary is shown to be captured across a wide range of pressure drops by physical scaling laws and an upper bound on the oxygen transfer rate. A regression equation is introduced that can be used to estimate the oxygen transfer in a capillary using the vascular resistance. Two techniques for quantifying the effects of statistical variability, experimental uncertainty and pathological placental structure on the calculated properties are then introduced. First, scaling arguments are used to quantify the sensitivity of the model to uncertainties in the geometry and the parameters. Second, the effects of localized dilations in fetal capillaries are investigated using an idealized axisymmetric model, to quantify the possible effect of pathological placental structure on oxygen transfer. The model predicts how, for a fixed pressure drop through a capillary, oxygen transfer is maximized by an optimal width of the dilation. The results could explain the prevalence of fetal hypoxia in cases of delayed villous maturation, a pathology characterized by a lack of the vasculo-syncytial membranes often seen in conjunction with localized capillary dilations., Comment: Final version of manuscript

Terrestrial isopods moult first the posterior and then the anterior half of the body, allowing for storage and recycling of CaCO3. We used synchrotron-radiation microtomography to estimate mineral content within skeletal segments in sequential moulting stages of Porcellio scaber. The results suggest that all examined cuticular segments contribute to storage and recycling, however, to varying extents. The mineral within the hepatopancreas after moult suggests an uptake of mineral from the ingested exuviae. The total maximum loss of mineral was 46% for the anterior and 43% for the posterior cuticle. The time course of resorption of mineral and mineralisation of the new cuticle suggests storage and recycling of mineral in the posterior and anterior cuticle. The mineral in the anterior pereiopods decreases by 25% only. P. scaber has long legs and can run fast; therefore, a less mineralised and thus lightweight cuticle in pereiopods likely serves to lower energy consumption during escape behaviour. Differential demineralisation occurs in the head cuticle, in which the cornea of the complex eyes remains completely mineralised. The partes incisivae of the mandibles are mineralised before the old cuticle is demineralised and shed. Probably, this enables the animal to ingest the old exuviae after each half moult.

Maternal diabetes is associated with changes of the placental structure. These changes include great variability of vascularity manifested by strikingly hypovascular as well as hypervascular terminal villi. In this paper, normal placental terminal villi and pathological villi of type 1 diabetic placentas were compared concerning the structure of villous stroma, spatial arrangement of villous capillary bed and quantitative assessment of capillary branching pattern. Formalin fixed and paraffin embedded specimens of 14 normal and 17 Type 1 diabetic term placentas were used for picrosirius staining, vimentin and desmin immunohistochemistry and confocal microscopy. 3D models of villi and villous capillaries were constructed from stacks of confocal optical sections. Hypervascular as well as hypovascular villi of diabetic placenta displayed changed structure of villous stroma, i.e. the collagen envelope around capillaries looked thinner and the network of collagen fibers seemed less dense. The desmin immunocytochemistry has shown that stromal cells of hypervascular as well as hypovascular villi appeared nearly or completely void of desmin filaments. In comparison with normal villi, capillaries of hypovascular villi had a smaller diameter and displayed a markedly wavy course whereas in hypervascular villi numerous capillaries occurred in reduced stroma and often had a large diameter. The quantitative assessment of capillary branching has shown that villous capillaries are more branched in diabetic placentas. It is concluded that type 1 maternal diabetes enhances the surface area of the capillary wall by elongation, enlargement of diameter and higher branching of villous capillaries and disrupts the stromal structure of terminal villi.

The microvascular bed of the human term placenta is located in the mature intermediate and terminal villi. Despite repeated studies, a commonly accepted model of its spatial organization is not yet available. In the present study, the 3-D arrangement of vessels in mature intermediate and terminal placental villi was examined using the combination of confocal microscopy and image processing. Fixed and paraffin embedded placental samples were cut into 120 μm thick sections. Serial optical sections were captured by a confocal microscope and used as an initial data for 3-D reconstruction. The reconstructed segments of the villous tree show parallel vessels of mature intermediate villi giving rise to the capillaries of terminal villi. The signs of longitudinal and sprouting capillary growth as well as growth of new villi were observed. The presented method allowed us to visualize large parts of peripheral placental villi, which is useful for elucidating the arrangement of their vascular bed.

A well developed capillary bed is essential for proper function of skeletal muscles. We present for the first time a triple immunofluorescent method suitable for staining capillaries and muscle fibre outlines in thick sections of human skeletal muscle, applying antibodies against collagen IV (in red) and F8 (in green) as well as Ulex europaeus lectin, visualized in green fluorescence. Further, we present possibilities for quantitative evaluation of the capillary network which implies the length of capillaries per unit volume of muscle tissue (Lcap/Vmuscle) and the length of capillaries supplying individual muscle fibres per unit fibre length (Lcap/Lfib), per surface area (Lcap/Sfib) and per volume (Lcap/Vfib) as well as the course of capillaries in the muscle. The latter can be described by the tortuosity, orientation and mean capillary length. To get reasonable results we met the following requirements: i) high quality thick tissue sections, from which 3D image data were acquired; ii) immunofluorescent methods suitable for confocal microscopy; iii) penetration of the fluorescent dyes throughout the tissue section; iv) proper 3D image analysis methods for performing reliable measurements and v) control over relevant tissue deformations. The developed methodology is illustrated by results obtained from autopsy or biopsy samples of three human muscles, i.e. vastus lateralis, multifidus and masseter muscle that exhibit differences in genetic background, innervation, tasks and functional activity.

Three consecutive pairs of head cavities (premandibular, mandibular, and hyoid) found in elasmobranchs have been considered as remnants of preotic 'head' somites-serial homologues of the myotomic compartments of trunk somites that give rise to the extraoccular musculature. Here, we study a more derived vertebrate, and show that cavitation is more complex in the head of Crocodylus niloticus, than just the occurrence of three pairs of cavities. Apart from the premandibular cavities, paired satellite microcavities, and unpaired extrapremandibular microcavities are recognized in the prechordal region as well. We observed that several developmental phenomena occur at the same time as the formation of the head cavities (premandibular, satellite, extrapremandibular, mandibular, and hyoid) appear temporarily in the crocodile embryo. These are 1) rapid growth of the optic stalk and inflation of the optic vesicle; 2) release of the intimate topographical relationships between the neural tube, notochord and oral gut; 3) tendency of the prechordal mesenchyme to follow the curvature of the forebrain; and 4) proliferation of the prechordal mesenchyme. On the basis of volumetric characters, only the hyoid cavity and hyoid condensation is comparable to the trunk somitocoel and somite, respectively.

Summary Although kidney trauma is a relatively common injury, its microscopic biomechanics are poorly understood. Experimental low-grade trauma in pig kidneys was studied using optical microscopy. We observed ruptures in the cortex as well as in the medulla. Both parts of the renal parenchyma were damaged, even in areas of the kidneys that were free of macroscopic cracks on the surface. To determine which constituents of the renal cortex and medulla, i.e. tubular parts of the nephron or the interstitial connective tissue, were less resistant to injury during the drop shatter test, we applied a simple stereological method to discriminate between random and tissue-specific rupture propagation. The ruptures propagated predominantly through the interstitial connective tissue of the renal cortex and medulla. The volume fraction of the tubules assessed by the Cavalieri principle was 90.4% within the renal cortex and 52.4% within the medulla. The most frequently affected blood vessels were the arcuate and interlobular veins, followed by the arcuate and interlobular arteries. No disruptions of the renal calyces were found.

We postulated that, in rat extensor digitorum longus muscle (EDL), the length of capillaries per fibre surface area (Lcap/Sfib) and per fibre volume (Lcap/Vfib) could reflect fibre-type transformations accompanied by changes in oxidative metabolic profile and selective fibre-type atrophy. We excised rat EDL muscle 2 weeks after the sciatic nerve was cut (acute denervation; DEDL) and 4 weeks after the nerve was crushed (early reinnervation; REDL) and characterised muscle fibre-type transformation by the expression of myosin heavy-chain isoforms and by succinate dehydrogenase (SDH) and nicotinoamide adenine dinucleotide-tetrazolium reductase (NADH-TR) reactions. The numerical percentage (N/N) and area percentage (A/A) of pure and hybrid fibres and their diameter were determined, as was the A/A of SDH- and NADH-TR-positive fibres. The length of capillaries per fibre length (Lcap/Lfib), Lcap/Sfib and Lcap/Vfib were estimated in REDL and Lcap/Vfib in DEDL. In DEDL, the type 2x and 2b fibres evidently atrophied, with the N/N of type 2x fibres being lower and that of hybrid fibres higher. In REDL, the N/N of hybrid fibres was even higher, consequent to a lower N/N of type 2b fibres; however, fibre diameters approached values of the control EDL. Compared with control EDL, denervated and reinnervated muscles exhibited a higher A/A of oxidative fibres. This is probably the result of fibre-type transformation and selective fibre atrophy. We conclude that capillary length does not change during acute denervation and early reinnervation. The obtained higher values of Lcap/Sfib and Lcap/Vfib are related to changes in muscle fibre cross-sectional area.

The confocal microscope can image a specimen in its natural environment forming a 3D image of the whole structure by scanning it and collecting light through a small aperture (pinhole), allowing in vivo and in vitro observations. So far, the confocal fluorescence microscope (CFM) is considered a true volume imager because of the role of the pinhole that rejects information coming from out-of-focus planes. Unfortunately, intrinsic imaging properties of the optical scheme presently employed yield a corrupted image that can hamper quantitative analysis of successive image planes. By a post-image collection restoration, it is possible to obtain an estimate, with respect to a given optimization criterium, of the true object, utilizing the impulse response of system or Point Spread Function (PSF). The PSF can be measured or predicted so as to have a mathematical and physical model of the image-formation process. Further modelling and recording noise as an additive Gaussian process has used the regularized Iterative Constrained Tykhonov Miller (ICTM) restoration algorithm for solving the inverse problem. This algorithm finds the best estimate iteratively searching among the possible positive solutions; in the Fourier domain, such an approach is relatively fast and elegant. In order to compare the effective improvement in the quantitative image information analysis, we measured the volume of reference objects before and after image restoration, using the isotropic Fakir method.

Computer-based visualization of large tissue volumes with high resolution based on composing series of high-resolution confocal images is presented. GlueMRC and LinkMRC programs are introduced, implementing composition of overlapping series of optical sections cap- tured by a confocal microscope, registration and subsequent composition of successive confocal stacks. Both programs are using an interactive approach in combination with automatic algorithms for image registration. Further, the method for obtaining surface renderings of microscopical structure under study is described. For this purpose, structure contours visible in the sections are interactively digitized using a Colon plug-in module running in Ellipse environment. Then the coordinates of the contours are processed by special modules in the graphic programming environ- ment IRIS Explorer and the structure surface is rendered. The method is shown on the 3-D reconstruction of the capillary bed of human placental villi and chick embryonic gut and its vascular bed. Microsc. Res. Tech. 62:415- 422, 2003. © 2003 Wiley-Liss, Inc.

Three-dimensional (3D) study of capillary network of individual muscle fibres in rat extensor digitorum longus (EDL) and soleus (SOL) muscles is presented. Stereology and 3D reconstruction techniques were applied to stacks of serial optical sections recorded by a confocal microscope from thick muscle slices. The results suggest that SOL muscle fibres have a larger surface area and volume as well as a larger length of capillaries per fibre length than EDL. On the other hand, these two muscles have a similar ratio of capillary length to fibre surface area. The 3D approach to evaluation of muscle fibre capillarization brings many advantages over traditional measurements made on single muscle sections and could also be applied to the study of angiogenesis in other tissues.

In contrast to limb muscles where neonatal myosin (MyHC-neo) is present only shortly after birth, adult masseter muscles contain a substantial portion of MyHC-neo, which is coexpressed with mature MyHC isoforms. Changes in the numerical and area proportion of muscle fibers containing MyHC-neo in masseter muscle with aging could be expected, based on previously reported findings that (i) developmental MyHC-containing muscle fibers exhibit lower shortening velocities compared to fibers with exclusively fast MyHC isoforms and (ii) transformation toward faster phenotype occurs in elderly compared to young masseter muscle. In this study, we detected MyHC isoforms in the anterior superficial part of the human masseter muscle in a sufficiently large sample of young, middle-aged, and elderly subjects to reveal age-related changes in the coexpression of MyHC-neo with adult MyHC isoforms. MyHC isoforms were visualized with immunoperoxidase method and the results were presented by (i) the area proportion of fibers containing particular MyHC isoforms and (ii) the numerical proportion of fiber types defined by MyHC-1, -2a, -2x, and -neonatal isoform expression from a successive transverse sections. We found a lower numerical and area proportion of fibers expressing MyHC-neo as well as a lower area proportion of fibers containing MyHC-1 in elderly than in young subjects. We conclude that the diminished expression of MyHC-neo with age could point to a lower regeneration capacity of masseter muscle in the elderly.

The distribution of air-filled structures in the craniofacial and neurocranial bones of the oviraptorid ZPAL MgD-I/95, discovered at the Hermiin Tsav locality, Mongolia, is restored. Based on the complete obliteration of most of the cranial sutures, the specimen is identified as an adult individual of Conchoraptor gracilis Barsbold 1986. Except for the orbitosphenoids and epipterygoids, the preserved bones of the neurocranium are hollow. Three types of tympanic recess are present in Conchoraptor, a characteristic shared with troodontids, dromaeosaurids, and avian theropods. The contralateral middle ear cavities are interconnected by the supraencephalic pathway that passes through the dorsal tympanic recesses, the posterodorsal prootic sinuses and the parietal sinus. The spatial arrangements of the middle ear cavity and a derived neurocranial pneumatic system in Conchoraptor indicate enhancements of acoustic perception in the lower-frequency registers and of auditory directionality. We further speculate that this improvement of binaural hearing could be explained as an adaptation required for accurate detection of prey and/or predators under conditions of low illumination. The other potentially pneumatic structures of the Conchoraptor cranium include (1) recessus-like irregularities on the dorsal surface of the nasal and frontal bones (a putative oviraptorid synapomorphy; pos); (2) a subotic recess; (3) a sub-condylar recess; and (4) a posterior condylar recess (pos).

A confocal laser scanning microscope (CLSM) enables us to capture images from a biological specimen in different depths and obtain a series of precisely registered fluorescent images. However, images captured from deep layers of the specimen may be darker than images from the topmost layers because of light loss distortions. This effect causes difficulties in subsequent analysis of biological objects. We propose a solution using two approaches: either an online method working already during image acquisition or an offline method assisting as a postprocessing step. In the online method, the gain value of a photomultiplier tube of a CLSM is controlled according to the difference of mean image intensities between the reference and currently acquired image. The offline method consists of two stages. In the first stage, a standard histogram maintaining relative frequencies of gray levels and improving brightness and contrast is created from all images in the series. In the second stage, individual image histograms are warped according to this standard histogram. The methods were tested on real confocal image data captured from human placenta and rat skeletal muscle specimens. It was shown that both approaches diminish the light attenuation in images captured from deep layers of the specimen.

Spatial arrangement and complexity of the capillary bed of placental terminal villi were analyzed in 9 normal and 11 diabetic placentas. Specimens were taken by systematic random sampling, fixed and stained in toto, and embedded in paraffin. Fifteen fields of view were sampled systematically from 120-µm-thick sections of specimens and examined using a confocal laser scanning microscope. Series of thin optical sections of terminal villi and their developmental forms were recorded by the confocal microscope and used as initial data for three-dimensional visualization of the spatial arrangement of villous capillaries. Vascular topology and branching were studied by focusing through the villus, making a schematic drawing of the villous capillary bed and counting redundant capillary connections. It was found that the basic arrangement of villous capillaries is similar in both normal and diabetic placentas. Nevertheless, the proportion of simple forms of the capillary bed without redundant connections is significantly higher in normal placentas and the mean number of redundant connections per villus is significantly higher in diabetic placentas. It is concluded that both the longitudinal growth and branching of capillaries contribute to the increase in the placental capillary bed in late gestation and that the capillary bed of diabetic villi is more complicated due to more intense capillary branching.