Methods of In Ovo and Ex Ovo Ostrich Embryo Culture with Observations on the Development and Maturation of the Chorioallantoic Membrane.

Culture of shell-free and windowed eggs for drug testing and other experiments has been perfected for smaller eggs such as those of chickens, where the developing blood vessels of the chorioallantoic membrane (CAM) become accessible for manipulative studies. However, due to the thickness and hardness of the ostrich egg shell, such techniques are not applicable. Using a tork craft mini rotary and a drill bit, we established windowed egg, in-shell-membrane windowed egg, and in-shell-membrane shell-free methods in the ostrich egg, depending on whether the shell membranes were retained or not. Concomitant study of the developing CAM revealed that at embryonic day 16 (E16), the three layers of the CAM were clearly delineated and at E25, the chorionic capillaries had fused with the epithelium while the CAM at E37 had reached maturity and the chorion and the allantois were both 3-4 times thicker and villous cavity (VC) and capillary-covering cells were well delineated. Both intussusceptive and sprouting angiogenesis were found to be the predominant modes of vascular growth in the ostrich CAM. Development and maturation of the ostrich CAM are similar to those of the well-studied chicken egg, albeit its incubation time being twice in duration.

Slight volume changes in the duck lung do not imply a fundamental change in the structure of the parenchyma.

Slight changes in lung volume have previously been reported in ducks. We studied the functional structure of the lung of the domestic duck using classical anatomical techniques as well as ultrasound monitoring to unravel the causes of such changes. Later dorsal and medioventral secondary bronchi were superficially positioned and covered with a thin transparent and collapsible membrane, internally lined with a cuboidal to squamous epithelium. The lung parenchyma was rigid, with atria well supported by septa containing smooth muscles, interparabronchial septa reinforced by collagen fibres, and blood capillaries supported by epithelial plates. On ultrasound monitoring, an outward and inward movement of the lung surface during inspiration and expiration, respectively, was evident at the region where the airways were covered by the thin membranes. The movements plausibly facilitated air movement in the lung just like the air sacs. We conclude that volume changes in the duck lung occur due to a slight morphological adaptation rather than a change in the archetypical design of the avian lung parenchyma.

Membrane mediated development of the vertebrate blood-gas-barrier.

During embryonic lung development, establishment of the gas-exchanging units is guided by epithelial tubes lined by columnar cells. Ultimately, a thin blood-gas barrier (BGB) is established and forms the interface for efficient gas exchange. This thin BGB is achieved through processes, which entail lowering of tight junctions, stretching, and thinning in mammals. In birds the processes are termed peremerecytosis, if they involve cell squeezing and constriction, or secarecytosis, if they entail cutting cells to size. In peremerecytosis, cells constrict at a point below the protruding apical part, resulting in fusion of the opposing membranes and discharge of the aposome, or the cell may be squeezed by the more endowed cognate neighbors. Secarecytosis may entail formation of double membranes below the aposome, subsequent unzipping and discharge of the aposome, or vesicles form below the aposome, fuse in a bilateral manner, and release the aposome. These processes occur within limited developmental windows, and are mediated through cell membranes that appear to be of intracellular in origin. In addition, basement membranes (BM) play pivotal roles in differentiation of the epithelial and endothelial layers of the BGB. Laminins found in the BM are particularly important in the signaling pathways that result in formation of squamous pneumocytes and pulmonary capillaries, the two major components of the BGB. Some information exists on the contribution by BM to BGB formation, but little is known regarding the molecules that drive peremerecytosis, or even the origins and composition of the double and vesicular membranes involved in secarecytosis.

Avian area vasculosa and CAM as rapid in vivo pro-angiogenic and antiangiogenic models.

Angiogenesis, the development of new blood vessels from preexisting ones, is driven by coordinated signaling pathways governed by specific molecules, hemodynamic forces, and endothelial and periendothelial cells. The processes involve adhesion, migration, and survival machinery within the target endothelial and periendothelial cells. Factors that interfere with any of these processes may therefore influence angiogenesis either positively (pro-angiogenesis) or negatively (antiangiogenesis). The avian area vasculosa (AV) and the avian chorioallantoic membrane (CAM) are two useful tools for studying both angiogenesis and antiangiogenesis since they are amenable to both intravascular and topical administration of target, agents, are relatively rapid assays, and can be adapted very easily to study angiogenesis-dependent processes, such as tumor growth. Both models provide a physiological setting that permits investigation of pro-angiogenic and antiangiogenic agent interactions in vivo.

Effects of anticancer drug docetaxel on the structure and function of the rabbit olfactory mucosa.

Docetaxel (DCT) is an anticancer drug which acts by disrupting microtubule dynamics in the highly mitotic cancer cells. Thus, this drug has a potential to affect function and organization of tissues exhibiting high cellular turnover. We investigated, in the rabbit, the effects of a single human equivalent dose (6.26 mg/kg, i.v.) of DCT on the olfactory mucosa (OM) through light and electron microscopy, morphometry, Ki-67 immunostaining, TUNEL assay and the buried food test for olfactory sensitivity. On post-exposure days (PED) 5 and 10, there was disarrangement of the normal cell layering in the olfactory epithelium (OE), apoptotic death of cells of the OE, Bowman's glands and axon bundles, and the presence (including on PED 3) of blood vessels in the bundle cores. A decrease in bundle diameters, olfactory cell densities and cilia numbers, which was most significant on PED 10 (49.3%, 63.4% and 50%, respectively), was also evident. Surprisingly by PED 15, the OM regained normal morphology. Furthermore, olfactory sensitivity decreased progressively until PED 10 when olfaction was markedly impaired, and with recovery from the impairment by PED 15. These observations show that DCT transiently alters the structure and function of the OM suggesting a high regenerative potential for this tissue.

Casting materials and their application in research and teaching.

From a biological point of view, casting refers to filling of anatomical and/or pathological spaces with extraneous material that reproduces a three-dimensional replica of the space. Casting may be accompanied by additional procedures such as corrosion, in which the soft tissue is digested out, leaving a clean cast, or the material may be mixed with radiopaque substances to allow x-ray photography or micro computed topography (µCT) scanning. Alternatively, clearing of the surrounding soft tissue increases transparency and allows visualization of the casted cavities. Combination of casting with tissue fixation allows anatomical dissection and didactic surgical procedures on the tissue. Casting materials fall into three categories namely, aqueous substances (India ink, Prussian blue ink), pliable materials (gelatins, latex, and silicone rubber), or hard materials (methyl methacrylates, polyurethanes, polyesters, and epoxy resins). Casting has proved invaluable in both teaching and research and many phenomenal biological processes have been discovered through casting. The choice of a particular material depends inter alia on the targeted use and the intended subsequent investigative procedures, such as dissection, microscopy, or µCT. The casting material needs to be pliable where anatomical and surgical manipulations are intended, and capillary-passable for ultrastructural investigations.

Morphofunctional adaptations of the olfactory mucosa in postnatally developing rabbits.

Rabbits are born blind and deaf and receive unusually limited maternal care. Consequently, their suckling young heavily rely on the olfactory cue for nipple attachment. However, the postnatal morphofunctional adaptations of olfactory mucosa (OM) are not fully elucidated. To clarify on the extent and the pattern of refinement of the OM following birth in the rabbit, morphologic and morphometric analysis of the mucosa were done at neonatal (0-1 days), suckling (2 weeks), weanling (4 weeks), and adult (6-8 months) stages of postnatal development. In all the age groups, the basic components of the OM were present. However, proliferative activity of cells of the mucosal epithelium decreased with increasing age as revealed by Ki-67 immunostaining. Diameters of axon bundles, packing densities of olfactory cells, and cilia numbers per olfactory cell knob increased progressively with age being 5.5, 2.1, and 2.6 times, respectively, in the adult as compared with the neonate. Volume fraction values for the bundles increased by 5.3% from birth to suckling age and by 7.4% from weaning to adulthood and the bundle cores were infiltrated with blood capillaries in all ages except in the adult where such vessels were lacking. The pattern of cilia projection from olfactory cell knobs also showed age-related variations, that is, arose as a tuft from the tips of the knobs in neonates and sucklings and in a radial pattern from the knob bases in weanlings and adults. These morphological changes may be attributed to the high olfactory functional demand associated with postnatal development in the rabbit.

Escape mechanisms after antiangiogenic treatment, or why are the tumors growing again?

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after cessation of treatment. In numerous preclinical studies, angiogenesis inhibitors were shown to be efficient in the treatment of many pathological conditions, including solid cancers. In most clinical trials, however, this approach turned out to have no significant effect, especially if applied as monotherapy. Recovery of tumors after therapy is a major problem in the management of cancer patients. The mechanisms underlying tumor recovery (or therapy resistance) have not yet been explicitly elucidated. This review deals with the transient switch from sprouting to intussusceptive angiogenesis, which may be an adaptive response of tumor vasculature to cancer therapy that allows the vasculature to maintain its functional properties. Potential candidates for molecular targeting of this angioadaptive mechanism are yet to be elucidated in order to improve the currently poor efficacy of contemporary antiangiogenic therapies.

Spatial and functional relationships between air conduits and blood capillaries in the pulmonary gas exchange tissue of adult and developing chickens.

The documented data regarding the three-dimensional structure of the air capillaries (ACs), the ultimate sites of gas exchange in the avian lung is contradictory. Further, the mode of gas exchange, described as cross-current has not been clearly elucidated. We studied the temporal and spatial arrangement of the terminal air conduits of the chicken lung and their relationship with the blood capillaries (BCs) in embryos as well as the definitive architecture in adults. Several visualization techniques that included corrosion casting, light microscopy as well as scanning and transmission electron microscopy were used. Two to six infundibulae extend from each atrium and give rise to numerous ACs that spread centrifugally. Majority of the ACs are tubular structures that give off branches, which anastomose with their neighboring cognates. Some ACs have globular shapes and a few are blind-ending tapering tubes. During inauguration, the luminal aspects of the ACs are characterized by numerous microvillus-like microplicae, which are formed during the complex processes of cell attenuation and canalization of the ACs. The parabronchial exchange BCs, initially inaugurated as disorganized meshworks, are reoriented via pillar formation to lie predominantly orthogonal to the long axes of the ACs. The remodeling of the retiform meshworks by intussusceptive angiogenesis essentially accomplishes a cross-current system at the gas exchange interface in the adults, where BCs form ring-like patterns around the ACs, thus establishing a cross-current system. Our findings clarify the mode of gas exchange in the parabronchial mantle and illuminate the basis for the functional efficiency of the avian lung.

Intussusceptive angiogenesis and its role in vascular morphogenesis, patterning, and remodeling.

New blood vessels arise initially as blood islands in the process known as vasculogenesis or as new capillary segments produced through angiogenesis. Angiogenesis itself encompasses two broad processes, namely sprouting (SA) and intussusceptive (IA) angiogenesis. Primordial capillary plexuses expand through both SA and IA, but subsequent growth and remodeling are achieved through IA. The latter process proceeds through transluminal tissue pillar formation and subsequent vascular splitting, and the direction taken by the pillars delineates IA into overt phases, namely: intussusceptive microvascular growth, intussusceptive arborization, and intussusceptive branching remodeling. Intussusceptive microvascular growth circumscribes the process of initiation of pillar formation and their subsequent expansion with the result that the capillary surface area is greatly enhanced. In contrast, intussusceptive arborization entails formation of serried pillars that remodel the disorganized vascular meshwork into the typical tree-like arrangement. Optimization of local vascular branching geometry occurs through intussusceptive branching remodeling so that the vasculature is remodeled to meet the local demand. In addition, IA is important in creation of the local organ-specific angioarchitecture. While hemodynamic forces have proven direct effects on IA, with increase in blood flow resulting in initiation of pillars, the preponderant mechanisms are unclear. Molecular control of IA has so far not been unequivocally elucidated but interplay among several factors is probably involved. Future investigations are strongly encouraged to focus on interactions among angiogenic growth factors, angiopoetins, and related receptors.

The structural design of the bat wing web and its possible role in gas exchange.

The structure of the skin in the epauletted fruit bat (Epomophorus wahlbergi) wing and body trunk was studied with a view to understanding possible adaptations for gas metabolism and thermoregulation. In addition, gas exchange measurements were performed using a respirometer designed for the purpose. The body skin had an epidermis, a dermis with hair follicles and sweat glands and a fat-laden hypodermis. In contrast, the wing web skin was made up of a thin bilayered epidermis separated by a connective tissue core with collagen and elastic fibres and was devoid of hair follicles and sweat glands. The wings spanned 18-24 cm each, with about 753 cm2 of surface exposed to air. The body skin epidermis was thick (61 +/- 3 microm, SEM), the stratum corneum alone taking a third of it (21 +/- 3 microm). In contrast, the wing web skin epidermis was thinner at 9.8 +/- 0.7 microm, with a stratum corneum measuring 4.1 +/- 0.3 microm (41%). The wing capillaries in the wing web skin ran in the middle of the connective tissue core, with a resultant surface-capillary diffusion distance of 26.8 +/- 3.2 microm. The rate of oxygen consumption (VO2) of the wings alone and of the whole animal measured under light anaesthesia at ambient temperatures of 24 masculineC and 33 masculineC, averaged 6% and 10% of the total, respectively. Rate of carbon dioxide production had similar values. The membrane diffusing capacity for the wing web was estimated to be 0.019 ml O2 min(-1) mmHg(-1). We conclude that in Epomophorus wahlbergi, the wing web has structural modifications that permit a substantial contribution to the total gas exchange.

Microvascular growth, development, and remodeling in the embryonic avian kidney: the interplay between sprouting and intussusceptive angiogenic mechanisms.

Embryonic development is associated with extensive vascular growth and remodeling. We used immunohistochemical, light and electron microscopical techniques, as well as vascular casting methods to study the developing chick embryo kidney with special attention to the interplay between sprouting and intussusceptive vascular growth modes. During inauguration at embryonic day 5 (E5), the early mesonephros was characterised by extensive microvascular sprouting. By E7, the vascular growth mode switched to intussusception, which contributed to rapid kidney vasculature growth up to E11, when the first obvious signs of vascular degeneration were evident. The metanephros underwent similar phases of vascular development inaugurating at E8 with numerous capillary sprouts and changing at E13 to intussusceptive growth, which was responsible for vascular amplification and remodeling. A phenomenal finding was that future renal lobules arose as large glomerular tufts, supplied by large vessels, which were split into smaller intralobular feeding and draining vessels with subsequent formation of solitary glomeruli. This glomerular duplication was achieved by intussusception, i.e., by formation of pillars in rows and their successive merging to delineate the vascular entities. Ultimately, the maturation of the vasculature was achieved by intussusceptive pruning and branching remodeling. An interesting finding was that strong VEGF expression was associated with the sprouting phase of angiogenesis while bFGF was upregulated during the phase of intussusceptive microvascular growth. We conclude that microvascular growth and remodeling in avian kidney follows an adroitly crafted pattern, which entails a precise spaciotemporal interplay between sprouting and intussusceptive angiogenic growth modes supported partly by VEGF and bFGF.

Effects of neonatal high-dose short-term glucocorticoid treatment on the lung: a morphologic and morphometric study in the rat.

Glucocorticoids are often applied in neonatology and perinatology to fight the problems of respiratory distress and chronic lung disease. There are, however, many controversies regarding the adverse side effects and long-term clinical benefits of this therapeutic approach. In rats, glucocorticoids are known to seriously impair the formation of alveoli when applied during the first two postnatal weeks even at very low dosage. The current study investigates short-term and long-term glucocorticoid effects on the rat lung by means of morphologic and morphometric observations at light and electron microscopic levels. Application of a high-dosage protocol for only few days resulted in a marked acceleration of lung development with a precocious microvascular maturation resulting in single capillary network septa in the first 4 postnatal days. By postnatal d 10, the lung morphologic phenotype showed a step back in the maturational state, with an increased number of septa with double capillary layer, followed by an exceptional second round of the alveolarization process. As a result of this process, there was an almost complete recovery in the parenchymal lung structure by postnatal d 36, and by d 60, there were virtually no qualitative or quantitative differences between experimental and control rats. These findings indicate that both dosage and duration of glucocorticoid therapy in the early postnatal period are very critical with respect to lung development and maturation and that a careful therapeutic strategy can minimize late sequelae of treatment.