A simple tool for stereological assessment of digital images: the STEPanizer.

STEPanizer is an easy-to-use computer-based software tool for the stereological assessment of digitally captured images from all kinds of microscopical (LM, TEM, LSM) and macroscopical (radiology, tomography) imaging modalities. The program design focuses on providing the user a defined workflow adapted to most basic stereological tasks. The software is compact, that is user friendly without being bulky. STEPanizer comprises the creation of test systems, the appropriate display of digital images with superimposed test systems, a scaling facility, a counting module and an export function for the transfer of results to spreadsheet programs. Here we describe the major workflow of the tool illustrating the application on two examples from transmission electron microscopy and light microscopy, respectively.

Functional respiratory morphology in the newborn quokka wallaby (Setonix brachyurus).

A morphological and morphometric study of the lung of the newborn quokka wallaby (Setonix brachyurus) was undertaken to assess its morphofunctional status at birth. Additionally, skin structure and morphometry were investigated to assess the possibility of cutaneous gas exchange. The lung was at canalicular stage and comprised a few conducting airways and a parenchyma of thick-walled tubules lined by stretches of cuboidal pneumocytes alternating with squamous epithelium, with occasional portions of thin blood-gas barrier. The tubules were separated by abundant intertubular mesenchyme, aggregations of developing capillaries and mesenchymal cells. Conversion of the cuboidal pneumocytes to type I cells occurred through cell broadening and lamellar body extrusion. Superfluous cuboidal cells were lost through apoptosis and subsequent clearance by alveolar macrophages. The establishment of the thin blood-gas barrier was established through apposition of the incipient capillaries to the formative thin squamous epithelium. The absolute volume of the lung was 0.02 +/- 0.001 cm(3) with an air space surface area of 4.85 +/- 0.43 cm(2). Differentiated type I pneumocytes covered 78% of the tubular surface, the rest 22% going to long stretches of type II cells, their precursors or low cuboidal transitory cells with sparse lamellar bodies. The body weight-related diffusion capacity was 2.52 +/- 0.56 mL O(2) min(-1) kg(-1). The epidermis was poorly developed, and measured 29.97 +/- 4.88 microm in thickness, 13% of which was taken by a thin layer of stratum corneum, measuring 4.87 +/- 0.98 microm thick. Superficial capillaries were closely associated with the epidermis, showing the possibility that the skin also participated in some gaseous exchange. Qualitatively, the neonate quokka lung had the basic constituents for gas exchange but was quantitatively inadequate, implying the significance of percutaneous gas exchange.

Morphometry and allometry of the postnatal marsupial lung development: an ultrastructural study.

An utrastructural morphometric study of the postnatally remodelling lungs of the quokka wallaby (Setonix brachyurus) was undertaken. Allometric scaling of the volumes of the parenchymal components against body mass was performed. Most parameters showed a positive correlation with body mass in all the developmental stages, except the volume of type II pneumocytes during the alveolar stage. The interstitial tissue and type II cell volumes increased slightly faster than body mass in the saccular stage, their growth rates declining in the alveolar stage. Conversely, type I pneumocyte volumes increased markedly in both the saccular and alveolar stages. Both capillary and endothelial volumes as well as the capillary and airspace surface areas showed highest rates of increase during the alveolar stage, at which time the rate was notably higher than that of the body mass. The pulmonary diffusion capacity increased gradually, the rate being highest in the alveolar stage and the adult values attained were comparable to those of eutherians.

Morphometry and allometry of the postnatal lung development in the quokka wallaby (Setonix brachyurus): a light microscopic study.

The postnatally developing lungs of the quokka wallaby, Setonix brachyurus, were investigated macroscopically and by light microscopic morphometry. Lung, parenchymal and non-parenchymal volumes as well as the components of the latter two were analysed by regression analysis. The lungs comprised a single undivided left lung and a right lung with an adherent accessory lobe. Septal tissue growth was most remarkable in the canalicular and saccular stages. Between mid-canalicular stage and the saccular stage, the lung volume increased 2-fold, mainly due to airspace expansion, coupled with septal tissue thinning. The non-parenchymal vascular volume increase accelerated in the successive developmental stages while the airway and connective tissue volumes progressed in a decreasing order, being highest in the canalicular and saccular stages and lowest in the alveolar stage. Growth and remodelling of the alveolar septa occurred simultaneously with airspace subdivision. Airspace expansion accelerated during the stage of microvascular maturation, when most other parameters showed the least rate of increase.

Chorioallantoic membrane capillary bed: a useful target for studying angiogenesis and anti-angiogenesis in vivo.

The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane that is commonly used in vivo to study both angiogenesis and anti-angiogenesis. This review 1) summarizes the current knowledge about the structure of the CAM's capillary bed; 2) discusses the controversy about the existence of a single blood sinus or a capillary plexus underlying the chorionic epithelium; 3) describes a new model of the CAM vascular growth, namely the intussusceptive mode; 4) reports findings regarding the role played by endogenous fibroblast growth factor-2 in CAM vascularization; and 5) addresses the use and limitations of the CAM as a model for studying angiogenesis and anti-angiogenesis.

Morphological analysis of the postnatally developing marsupial lung: The quokka wallaby.

We investigated the events that take place during the postnatal morphogenesis of the lung of the quokka wallaby, Setonix brachyurus, using the light microscope and both the scanning and transmission electron microscopes. The lung of term, newborn babies (joeys) at 3-days of postnatal life was at late canalicular stage and comprised large airways and tubules separated by thick mesenchymal interstitium. The tubules were lined by a low cuboidal epithelium but had few portions with true gas exchange barrier where capillaries came into close contact with squamous type of epithelium. By the fifth day postpartum, the lung entered the early saccular stage characterised by large air sacs, thinner septa, a better developed double capillary system and conversion of the cuboidal epithelium into a squamous one of type I cells interrupted by groups of cuboidal type II cells with lamellar bodies. Transitory respiratory bronchioles were recognisable toward the end of this stage. Formation of secondary septa started by Day 15, dividing the saccules into several generations of smaller air spaces. There were alternating and concurrent periods of tissue proliferation and air space expansion, followed by septal thinning. Alveolization started from about 125 days postpartum when the first burst of small sized air spaces bounded by septa with a single capillary layer were encountered. By Day 180 the process of alveolization was completed with only occasional septa showing a double capillary system and by Day 210 postnatally, the lung resembled that of an adult. For the first time in a mammal, the canalicular stage was encountered postnatally during lung development.

Intussusceptive arborization contributes to vascular tree formation in the chick chorio-allantoic membrane.

Various reports indicate that the process of intussusceptive microvascular growth (IMG) plays a crucial role in capillary network formation of the chorio-allantoic membrane (CAM). In the present study we demonstrate by methylmethacrylate (Mercox) casting and in vivo time-lapse observations that intussusception, i.e. insertion of transcapillary tissue pillars, is also strongly involved in vascular tree formation, a process we refer to as intussusceptive arborization (IAR). From day 7 to day 14 of incubation, several arterial and venous branching generations arise from the capillary plexus. The process is initiated by pillar formation in rows, which are demarcating future large vessels in the capillary meshwork. In a subsequent step the pillars undergo reshaping to form narrow tissue septa that successively merge, which results in the production of new generations of blood vessels. This is followed by growth and maturation of all vascular components. The process of IAR in the CAM is very active at days 10 and 11 of incubation and takes place in preferentially perfused capillary regions determining "dynamic areas". The process of intussusception may be preceded by endothelial division, but the transcapillary pillar formation itself occurs primarily by rearrangement and attenuation of the endothelial cells without local endothelial cell proliferation. We conclude that after the early sprouting phase, the process of intussusception is the basic mechanism of CAM vascularization. It leads to capillary network growth and expansion (IMG) and, at the same time to feed vessel formation with several branching generations (IAR).

Intussusceptive angiogenesis: its role in embryonic vascular network formation.

Intussusceptive angiogenesis is a novel mode of blood vessel formation and remodeling, which occurs by internal division of the preexisting capillary plexus without sprouting. In this study, the process is demonstrated in developing chicken eye vasculature and in the chorioallantoic membrane by methylmethacrylate (Mercox) casting, transmission electron microscopy, and in vivo observation. In a first step of intussusceptive angiogenesis, the capillary plexus expands by insertion of numerous transcapillary tissue pillars, ie, by intussusceptive microvascular growth. In a subsequent step, a vascular tree arises from the primitive capillary plexus as a result of intussusceptive pillar formation and pillar fusions, a process we termed "intussusceptive arborization." On the basis of the morphological observations, a 4-step model for intussusceptive arborization is proposed, as follows: phase I, numerous circular pillars are formed in rows, thus demarcating future vessels; phase II, formation of narrow tissue septa by pillar reshaping and pillar fusions; phase III, delineation, segregation, growth, and extraction of the new vascular entity by merging of septa; and phase IV, formation of new branching generations by successively repeating the process, complemented by growth and maturation of all components. In contrast to sprouting, intussusceptive angiogenesis does not require intense local endothelial cell proliferation; it is implemented primarily by rearrangement and attenuation of the endothelial cell plates. In summary, transcapillary pillar formation, ie, intussusception, is a central and probably widespread process, which plays a role not only in capillary network growth and expansion (intussusceptive microvascular growth), but also in vascular plexus remodeling and tree formation (intussusceptive arborization).

Do mature pulmonary lobes grow after transplantation into an immature recipient?

BACKGROUND: The use of reduced-size adult lung transplants could help solve the profound pediatric donor lung shortage. However, adequate long-term function of the mature grafts requires growth in proportion to the recipient's development. METHODS: Mature left lower lobes from adult mini-pigs (age: 7 months; mean body weight: 30 kg) were transplanted into 14-week-old piglets (mean body weight: 15 kg). By the end of the 14-week holding period, lungs of the recipients (n = 4) were harvested. After volumetric measurements, the lung morphology was studied using light microscopy, scanning, and transmission electron microscopy. Changes of alveolar airspace volume were determined using a computer aided image analysis system. Comparisons were made to age- and weight-matched controls. RESULTS: Volumetric studies showed no significant differences (p = 0.49) between the specific volume (mL/kg body weight) of lobar grafts and left lower lobes of adult controls. Morphologic studies showed marked structural differences between the grafts and the right native lungs of the recipients, with increased average alveolar diameter of the grafts. On light microscopy and scanning electron microscopy, alveoli appeared dilated and rounded compared to the normal polygonal shape in the controls. The computer generated semi-quantitative data of relative alveolar airspace volume tended to be higher in transplanted lobes. CONCLUSIONS: The mature pulmonary lobar grafts have filled the growing left hemithorax of the developing recipient. Emphysema-like alterations of the grafts were observed without evidence of alveolar growth in the mature lobar transplants. Thus, it can be questioned whether mature pulmonary grafts can guarantee sufficient long-term gas exchange in growing recipients.

Structural considerations. Early intervention in childhood asthma.

Any therapeutic treatment applied to a developing and/or growing organism is complicated by the fact that the treatment window may hit a critical stage of a developing organ. This means that unlike administration in adults, dosage and timing of drug applications are important issues because of their possible damaging effects. In view of the timing of lung developmental phases, it appears that perinatal interferences by diseases, environmental influences or drugs will affect specifically alveolar development and microvascular maturation. Numerous publications report, that the developing lung can be disturbed by a variety of factors, such as temperature, oxygen tension, cigarette smoke, malnutrition, drugs and hormones, in particular glucocorticoids. In well-documented animal experiments perinatal glucocorticoid treatment, even in very low doses, inhibited alveolization. This effect has been analysed in more detail and a hypothesis for its mechanism has been presented. The above effects on lung development may be extremely difficult to assess in human infants by noninvasive techniques. The message, however, remains that potent drugs should be used with great reservation.

Programmed cell death contributes to postnatal lung development.

The rat lung undergoes the phase of maturation of the alveolar septa and of the parenchymal microvascular network mainly during the third postnatal week. Speculating that programmed cell death may contribute to the thinning of the alveolar septa, we searched for the presence of DNA fragmentation in rat lungs between postnatal days 6 and 36 using the TUNEL procedure. The number of positive nuclei was compared at different days. We observed an 8-fold increase of programmed cell death toward the end of the third week as compared to the days before and after this time point. The precise timing of the appearance of the peak depended on the size of the litter. Double-labeling for DNA fragmentation (TUNEL) and for type I and type II epithelial cells (antibodies E11 and MNF-116), as well as morphologic studies at electron microscopic level, revealed that during the peak of programmed cell death mainly fibroblasts and type II epithelial cells were dying. While both dying cell types were TUNEL-positive, nuclear fragments and apoptotic bodies were exclusively observed in the dying fibroblasts. We conclude that programmed cell death is involved in the structural maturation of the lung by reducing the number of fibroblasts and type II epithelial cells in the third postnatal week. We observed that the dying fibroblasts are cleared by neighboring fibroblasts in a later stage of apoptosis, and we hypothesize that type II epithelial cells are cleared by alveolar macrophages in early stages of the programmed cell death process.

Protein cross-linking mediated by tissue transglutaminase correlates with the maturation of extracellular matrices during lung development.

At birth, the mammalian lung is still immature. The alveoli are not yet formed and the interairspace walls contain two capillary layers which are separated by an interstitial core. After alveolarization (first 2 postnatal weeks in rats) the alveolar septa mature: their capillary layers merge, the amount of connective tissue decreases, and the mature lung parenchyma is formed (second and third week). During the first 3 wk of life the role of tissue transglutaminase (tTG) was studied in rat lung by immunostaining of cryostat and paraffin sections, by Northern and Western blotting, and by a quantitative determination of gamma-glutamyl-epsilon-lysine. While enzyme activity and intracellular tTG were already present before term, the enzyme product (gamma-glutamyl-epsilon-lysine-crosslink) and extracellular tTG appeared between postnatal days 10 and 19 in the lung parenchyma. In large blood vessels and large airways, which mature earlier than the parenchyma, both the enzyme product and extracellular tTG had already appeared at the end of the first postnatal week. We conclude that tTG is expressed and externalized into the extracellular matrix of lung shortly before maturation of an organ area. Because tTG covalently and irreversibly crosslinks extracellular matrix proteins, we hypothesize that it may prevent or delay further remodeling of basement membranes and may stabilize other extracellular components, such as microfibrils.

A stereological comparison of villous and microvillous surfaces in small intestines of frugivorous and entomophagous bats: species, inter-individual and craniocaudal differences.

The extents of functional surfaces (villi, microvilli) have been estimated at different longitudinal sites, and in the entire small intestine, for three species of bats belonging to two feeding groups: insect- and fruit-eaters. In all species, surface areas and other structural quantities tended to be greatest at more cranial sites and to decline caudally. The entomophagous bat (Miniopterus inflatus) had a mean body mass (coefficient of variation) of 8.9 g (5%) and a mean intestinal length of 20 cm (6%). The surface area of the basic intestinal tube (primary mucosa) was 9.1 cm2 (10%) but this was amplified to 48 cm2 (13%) by villi and to 0.13 m2 (20%) by microvilli. The total number of microvilli per intestine was 4 x 10(11) (20%). The average microvillus had a diameter of 8 nm (10%), a length of 1.1 microns (22%) and a membrane surface area of 0.32 micron 2 (31%). In two species of fruit bats (Epomophorus wahlbergi and Lisonycteris angolensis), body masses were greater and intestines longer, the values being 76.0 g (18%) and 76.9 g (4%), and 73 cm (16%) and 72 cm (7%), respectively. Surface areas were also greater, amounting to 76 cm2 (26%) and 45 cm2 (8%) for the primary mucosa, 547 cm2 (29%) and 314 cm2 (16%) for villi and 2.7 m2 (23%) and 1.5 m2 (18%) for microvilli. An increase in the number of microvilli, 33 x 10(11) (19%) and 15 x 10(11) (24%) per intestine, contributed to the more extensive surface area but there were concomitant changes in the dimensions of microvilli. Mean diameters were 94 nm (8%) and 111 nm (4%), and mean lengths were 2.8 microns (12%) and 2.9 microns (10%), respectively. Thus, an increase in the surface area of the average microvillus to 0.83 micron 2 (12%) and 1.02 microns 2 (11%) also contributed to the greater total surface area of microvilli. The lifestyle-related differences in total microvillous surface areas persisted when structural quantities were normalised for the differences in body masses. The values for total microvillous surface area were 148 cm2g-1 (20%) in the entomophagous bat, 355 cm2g-1 (20%) in E. wahlbergi and 192 cm2g-1 (17%) in L. angolensis. This was true despite the fact that the insecteater possessed a greater length of intestine per unit of body mass: 22 mm g-1 (8%) versus 9-10 mm g-1 (9-10%) for the fruit-eaters.

Quantitative study of intussusceptive capillary growth in the chorioallantoic membrane (CAM) of the chicken embryo.

In an attempt to sort out the respective contributions of sprouting and intussusceptive microvascular growth (IMG) during chicken chorioallantoic membrane (CAM) development, we analyzed the morphology and the quantitative growth of the capillary bed of the CAM by light microscopy. By perfusing the CAM microvasculature with highly concentrated colloidal gold particles, the capillaries could be unambiguously distinguished from the surrounding unlabelled tissue. This allowed us to identify, count and measure the intercapillary tissue profiles. By means of morphometric analysis we could show that CAM angiogenesis undergoes three phases of development. In an early phase, from Day 5 to Day 7, the major mechanism of capillary network growth is sprouting. In an intermediate phase, from Day 8 to Day 12, IMG is prevailing, and at Days 13 and 14, CAM structure is undergoing expansion with only a small increase in complexity. These findings are important in view of experimental protocols using the CAM as a model for testing angiogenetic factors. Indeed, care has to be taken not to misinterpret normal age-dependent alterations of the CAM vascular architecture as specific responses to tested agents.

Implementation of intussusceptive microvascular growth in the chicken chorioallantoic membrane (CAM).

Intussusceptive microvascular growth (IMG) is a new mechanism of capillary growth: The vascular network expands by insertion of newly formed columns of interstitial tissue (interstitial tissue structures) into the vascular lumen called tissue pillars or posts (diameter: 0.5-2.5 microm). IMG has so far been described during organ development and growth and in tumor angiogenesis. Different modes of its implementation could be demonstrated in the rat lung and the chicken chorioallantoic membrane (CAM). In the present investigation a further mechanism of IMG is reported in the chicken CAM: tissue pillars form by splitting of larger interstitial tissue structures and intercapillary walls located between neighboring capillary segments which will consecutively fuse. Splitting is dependent on the existence of a pillar's core composed of a bundle of collagen fibrils ensheathed by extensions of endothelial-like cells inside these structures. Pillar cores thus represent the smallest unit of interstitial tissue around which the vascular lumen might expand. This mode of IMG is obviously connected to physiological remodeling of the capillary network and appears to be dominant during later stages of CAM development.

Influence of bilirubin on surface tension properties of lung surfactant.

AIM: To investigate the influence of bilirubin on the surface tension activity of a porcine derived (Curosurf) and synthetic (Exosurf) surfactant. METHODS: The captive bubble surfactometer at phospholipid doses of 0.5 mg/ml (low dose) and 1 mg/ml (high dose) in solutions of increasing bilirubin concentrations (0.25, 0.5, and 1.0 mg/ml) was used. RESULTS: Curosurf (without bilirubin) showed a higher surface f1p4ion activity than Exosurf, as shown by area compression of 30 (SD 0.6)% compared with 76(1.4)% at low surfactant dose and 25 (0.9)% compared with 85 (0.5)% at high dose (P < 0.01). Bilirubin showed negligible surface activity at the concentrations studied. At low phospholipid dose (0.5 mg/ml Curosurf), bilirubin increased film area compression of lipid extract surfactant from 30 (0.6)% to 55 (1.6)%, 59 (0.1)%, and 68 (0.5)% at the three studied bilirubin concentrations, respectively (P < 0.01). At high phospholipid dose (1 mg/ml Curosurf), bilirubin had the same adverse, although less pronounced, effect on film area compression of porcine lipid extract surfactant (25 (0.9)% vs 26 (0.9)%, 39 (1.3)%, and 44 (1.1)%, respectively) (P < 0.01). Using synthetic surfactant (Exosurf), with a much lower original surface activity, bilirubin did not further inhibit its surface tension properties at any of the phospholipid doses studied. CONCLUSION: These results indicate that in vitro bilirubin impairs the surface tension activity of porcine lipid extract surfactant, but does not affect synthetic surfactant activity.

Formation of interalveolar pores in the rat lung.

BACKGROUND: The aim of this morphological investigation was to obtain more information about the structural and cellular mechanisms of interalveolar pore formation in postnatal lung development. Assuming that alveolar pore formation is related to the general thinning of interalveolar walls observed in the postnatal period, we have focused our attention on the topographical relationship between epithelial cells and connective tissue in the septum. Thereby we tried to formulate a uniform concept of pore formation. METHODS: After fixation with glutaraldehyde and osmiumtetroxide, tissue blocks of rat lungs aged 44 days were embedded in Epon. Serial sections were obtained in order to analyse precisely pores and supposed sites of pore formation (type II cells and thin spots in transsections of interalveolar walls). RESULTS: We made the following observations: there are pores with or without type II cells in the neighbourhood, and "pre-pores" with either fully transseptal granular pneumocytes, or thin spots in the interalveolar wall consisting of one or two layers of type I cell epithelium or of type II and type I cells without intervening connective tissue. CONCLUSIONS: From these findings we deduce that there is a general principle of interalveolar pore formation which consists in the formation of transseptal interepithelial cell contacts (i.e., between cells of type II and type I or type I and type I), promoted by the thinning of interalveolar walls in the stage of microvascular maturation. Within the zone of contact the cells thin out and give way to form an interalveolar opening.

Development of the human fetal airway tree: analysis of the numerical density of airway endtips.

BACKGROUND: It has been long agreed that the lung contained, at the end of the pseudoglandular stage, mostly the future conductive airway tree and that the intra-acinar structures developed during the subsequent canalicular stage. Recent immunocytochemical investigations have disclosed that the epithelial cells lining the distal airways in the pseudoglandular stage have the characteristics of alveolar epithelial cells. The purpose of this study was to quantitatively evaluate the progress of fetal airway branching. METHODS: The numerical density of endtips of the airway tree was estimated in three human fetal right lungs after 3D reconstruction of airways from serial histologic sections. The CRLs of the three fetuses were 103 mm, 132 mm, and 145 mm. The former two corresponded to the late pseudoglandular stage and the last to the early canalicular stage. Three-dimensional reconstructions were performed on two portions of the upper and lower lobes in each lung. The numbers of endtips of the airway tree contained in the reconstructed portions were counted an the numerical densities calculated. RESULTS: The numerical densities in the parenchyma of the three right lungs were 1.2 x 10(3)/mm3, 1.7 x 10(3)/mm3, and 2.4 x 10(3)/mm3, respectively. The estimated total numbers of endtips in the whole right lung were 0.85 x 10(6), 2.25 x 10(6), and 5.17 x 10(6), which were equal to 2(19.8), 2(21.2), and 2(22.3), respectively. CONCLUSIONS: Since this suggests that 20 generations of branching were completed, we concluded that the airway branching has already reached the level of the future alveolar ducts in the late pseudoglandular stage.